Evaluation of a novel 68Ga radiolabeled ligand targeting glutamate carboxypeptide II in an animal model of breast cancer J Mahapane orcid.org/ 0000-0003-4347-628x Dissertation accepted in fulfilment of the requirements for the degree Master of Science in Pharmaceutical Science at the North-West University Supervisor: Prof JR Zeevaart Co-supervisor: Dr R Hayeshi Co-supervisor: Dr T Ebenhan Graduation: May 2020 Student number: 29884322 PREFACE Submission of this dissertation is in fulfilment of the requirements for the degree Master of Science in Pharmaceutical Science at the North-West University. This dissertation is composed of six chapters in total. Chapter one - introduction, Chapter two - literature review, Chapter three - all relevant material and methods. The study results are divided into three chapter: Chapter four - xenografts model development of breast and prostate cancer, Chapter five - radiolabelling of [68Ga]Ga-DKFZ-PSMA-11, Chapter six - in vivo micro-PET/CT imaging. And the last Chapter is about the study outcome, limitations and recommendations. The figures cited in this study are used after obtaining permission from the copyright holder (Appendix C). The referencing style used throughout this dissertation is Harvard style, which appears at the end of each chapter. I ACKNOWLEDGEMENTS From the beginning of exciting times through to the very tough, trying and challenging times of my dissertation work, God’s presence has been with me. I found myself extremely troubled by the study challenges but God over and again gave me strength and courage to the end of my project. My sincere gratitude goes to the following people for granting me the opportunity to enroll in this project: Prof Jan Rijn Zeevaart, Prof Anne Grobler, Prof Mike Sathekge, Prof Rose Hayeshi and Dr Thomas Ebenhan To Dr Thomas Ebenhan and Prof Rose Hayeshi, you have been the best mentors I have had. Thank you for walking me through this educational process and allowing me to learn above all things. I am grateful to have you overseeing my project. Dr June Serem you are one of a kind. Cell work was new to me but over time you changed that for me. I am sincerely thankful for the time you took to teach and assist me in many ways pertaining to cell culture at the University of Pretoria. Dr Ambrose Okem and Mr. Tumelo Kgoe your kindness and assistance in cell culture work and animals inoculations has not gone unnoticed, I am grateful. To Dr Cathryn Driver, thank you so much for all the help in coordinating the animal imaging experiment and biodistribution analysis, and with the dissertation write-up. Dr Janke Kleynhans you were so easy to work with, you have been of great help in coordinating the animal imaging experiment and assisting with radiolabelling. Mrs. Palesa Koatale, a support I needed just in time, thank you so much for the support you gave me when I was at breaking point. To Mrs. Delene Van Wyk, thank you for all the dedicated times you assisted with animal imaging and processing, and teaching me imaging processing. It has been a privilege to work with you. To Mrs. Antoinette Fick, Mr. Cor Bester, Dr Nico Minaar, Mr. Jacob Mabena, Mr. Kobus Venter, Mrs. Jillene Visser, many thanks for all the assistance about animal availability, handling and inoculations. The Department of Nuclear Medicine staff, I am honored to work with people of such understanding, all of you have been of great support and encouraging in you unique ways. Getting time off work put enormous pressure on everyone, many times, but you have never ceased to understand and support me. To my dear family, husband and beautiful daughter, it was your love, support, patience and sacrifices that uplifted me and carried me through these years, right to the end, what more can I ask? I am humbled by your love. II ABSTRACT Aim: This study aims to pre-clinically investigate the accumulation of [68Ga]Ga-DKFZ-PSMA-11, a glutamate carboxypeptidase II ligand that was previously reported in advanced stages of breast cancer in humans, in different breast cancer xenograft mice, in order to better understand the accumulation of [68Ga] Ga-DKFZ-PSMA-11. Material and Methods: MCF-7, MDA-MB-231 and LNCaP athymic nude mice xenografts were developed by inoculating either MCF-7 cells suspension (DMEM/ Matrigel (1:1)) subcutaneously in the hind-right flank of the mice, MDA-MB-231 cells (PBS/ Matrigel (1:1)) inoculated into the mammary fat pad in the abdominal region or LNCaP cell suspension (PBS/ Matrigel (1:1)) subcutaneously into the hind-right flank of the mice. [68Ga]Ga-DKFZ-PSMA-11 was optimised for suitable administration into mice. On day one, MCF-7 and MDA-MB-231 female athymic nude mice were imaged with [18F]FDG-micro-PET/CT, and day two, with [68Ga]Ga-DKFZ-PSMA-11-micro- PET/CT followed by ex vivo biodistribution. Results: The radiolabelled [68Ga]Ga-DKFZ-PSMA-11 was purified by solid phase extraction using Sep-Pak C18-light cartridge, an ethanol concentration of 25% in saline with a volume of 0.3 ml demonstrated a radiochemical yield of ~ 69% and radiochemical purity of >96.9% (n = 5). Radiochemical yield (n = 7) was 165 ± 70 MBq [68Ga]Ga-DKFZ-PSMA-11. Female athymic nude mice (n = 4) with MCF-7 tumours measured 136 ± 100 mm3 prior to [18F]FDG-micro-PET/CT and 167 ± 83 mm3 prior to [68Ga]Ga-DKFZ-PSMA-11-micro-PET/CT. The MCF-7 xenografts performed micro-PET/CT following injection with [18F]FDG-day 1 (7 ± 2 MBq) and [68Ga]Ga-DKFZ-PSMA-11- day 2 (14 ± 4 MBq). Female athymic nude mice xenografts (n = 5) with MDA-MB-231 tumours measured 150 ± 31 mm3 prior to [18F]FDG-micro-PET/CT and 191 ± 19 mm3 prior to [68Ga]Ga-DKFZ- PSMA-11-micro-PET/CT. The MDA-MB-231 xenografts performed micro-PET/CT following injection with [18F]FDG-day 1 (11 ± 2 MBq) and [68Ga]Ga-DKFZ-PSMA-11-day 2 (14 ± 2 MBq). There were no LNCaP xenografts imaged due to failure to develop the model. MCF-7 tumours did not show accumulation of both [18F]FDG and [68Ga]Ga-DKFZ-PSMA-11. MDA-MB-231 tumours accumulated [18F]FDG and did not accumulate [68Ga]Ga-DKFZ-PSMA-11. Conclusion: The study reports on MCF-7 and MDA-MB-231 xenografts imaged with [68Ga]Ga- DKFZ-PSMA-11 or [18F]FDG. There was no accumulation of [68Ga]Ga-DKFZ-PSMA-11 in both the MCF-7 and MDA-MB-231 tumour. Enhanced permeability and retention effects might be responsible for tracer uptake, since clinical studies shown that accumulation of [68Ga]Ga-DKFZ-PSMA-11 correlates to pathologic neo-vasculature found in solid tumours. III Keywords: Breast cancer imaging, Glutamate carboxypeptidase II, MCF-7, MDA-MB-231, LNCaP, micro-PET/CT, [18F]FDG, [68Ga]Ga-DKFZ-PSMA-11. IV TABLE OF CONTENTS PREFACE .....................................................................................................................................I ACKNOWLEDGEMENTS ............................................................................................................II ABSTRACT.................................................................................................................................III 1 INTRODUCTION...................................................................................................1 1.1 Breast Cancer Types and Classification...........................................................1 1.2 Breast Cancer Molecular Subtypes ...................................................................2 1.3 Diagnosis and Therapy Management of Breast Cancer Disease ...................3 1.4 Nuclear Medicine Imaging ..................................................................................4 1.5 Glutamate Carboxypeptidase II..........................................................................5 1.6 Research Problem...............................................................................................6 1.7 Research Aim and Objectives............................................................................6 1.7.1 Aims ......................................................................................................................6 1.7.2 Objectives..............................................................................................................7 REFERENCES .............................................................................................................................8 2 LITERATURE REVIEW ......................................................................................11 2.1 Breast cancer: A Clinical Challenge................................................................11 2.2 Diagnostic Imaging Applications in Breast Cancer .......................................11 2.2.1 Anatomical Imaging.............................................................................................11 2.2.2 Computed Tomography Imaging Principle ..........................................................12 2.3 Nuclear Imaging ................................................................................................13 V 2.3.1 Positron Emission Tomography Imaging Principle..............................................13 2.3.2 Positron Emission Tomography/Computed Tomography....................................15 2.3.3 Dedicated Breast Positron Emission Tomography/Computed Tomography .......15 2.3.4 Pre-clinical Imaging using Dedicated micro-Positron Emission Tomography/Computed Tomography .................................................................15 2.4 Positron Emission Tomography Radiopharmaceuticals for Breast Cancer Imaging...............................................................................................................16 2.5 Glutamate Carboxypeptidase II........................................................................18 2.5.1 Molecular Structure .............................................................................................18 2.5.2 The Function and Role of Glutamate Carboxypeptidase II in Cancer .................19 2.5.3 Glutamate Carboxypeptidase II: A New Target in Nuclear Medicine ..................19 2.6 Research Tools and Narrative..........................................................................21 2.6.1 [68Ga]Ga-DKFZ-PSMA-11 ...................................................................................21 2.6.2 Human Breast Cancer Cells................................................................................22 2.6.3 Cell Line - Xenografts Model ...............................................................................23 2.7 Research Narrative............................................................................................24 REFERENCES ...........................................................................................................................25 3 MATERIALS AND METHODS............................................................................30 3.1 Materials and Equipment..................................................................................30 3.2 Ethics..................................................................................................................32 3.3 Cell Culture ........................................................................................................32 VI 3.4 Animal Preparation for Inoculation .................................................................33 3.5 Breast and Prostate Cancer Xenograft Model Establishment ......................33 3.6 Radiopharmaceuticals ......................................................................................34 3.6.1 [18F]FDG ..............................................................................................................34 3.6.2 [68Ga]Ga-DKFZ-PSMA-11 ...................................................................................34 3.7 Instant Thin-Layer Chromatography ...............................................................35 3.8 Micro-Positron Emission Tomography / Computed Tomography Imaging.36 3.8.1 Animal Preparation..............................................................................................36 3.8.2 Intravenous Tracer Administration ......................................................................36 3.8.3 Computed Tomography and Positron Emission Tomography Image acquisition37 3.8.4 Image Reconstruction and Analysis ....................................................................37 3.9 Biodistribution...................................................................................................37 3.10 Statistical Analysis............................................................................................37 REFERENCES ...........................................................................................................................39 4 XENOGRAFT MODEL DEVELOPMENT OF BREAST AND PROSTATE CANCER .............................................................................................................40 4.1 Development of suitable Cell Cultures............................................................40 4.2 Choice of Animals .............................................................................................41 4.3 Xenograft Model Development.........................................................................41 4.3.1 MCF-7 Xenografts ...............................................................................................41 4.3.2 MDA-MB-231 Xenografts ....................................................................................42 VII 4.3.3 LNCaP Xenografts ..............................................................................................43 4.4 Recommended Procedures to Warrant Tumours Growth for Imaging Purposes ............................................................................................................44 4.5 Discussion .........................................................................................................44 4.5.1 Athymic Nude Mice MCF-7 Xenograft.................................................................44 4.5.2 MDA-MB-231 Athymic Nude Mice Xenograft ......................................................46 4.5.3 LNCaP Athymic Nude Mice Xenograft ................................................................46 4.6 Conclusion.........................................................................................................47 REFERENCES ...........................................................................................................................48 5 RADIOLABELLING OF [68GA]GA-DKFZ-PSMA-11..........................................50 5.1 Results and Discussion....................................................................................50 5.1.1 Choice of DKFZ-PSMA-11 ..................................................................................50 5.1.2 Choice of Radioisotope .......................................................................................50 5.1.3 Elution of Gallium-68-Radioactivity for Radiolabelling.........................................51 5.1.4 Repeated Generator Elution................................................................................51 5.1.5 Eluate Fractionation ............................................................................................51 5.1.6 Testing [68Ga]Ga-DKFZ-PSMA-11 Radiolabeling Parameters............................51 5.1.7 Testing Quality of Radiolabelling.........................................................................52 5.1.8 [68Ga]Ga-DKFZ-PSMA-11 Purification ................................................................52 5.1.9 Preparation of a safe-to administer [68Ga]Ga-DKFZ-PSMA-11 Formulation.......56 5.2 Conclusion.........................................................................................................57 VIII REFERENCES ...........................................................................................................................58 6 PRE-CLINICAL IMAGING OF GCP II EXPRESSION IN BREAST CANCER USING [68GA]GA-DKFZ-PSMA-11 MICRO-PET/CT..........................................59 6.1 Results and Discussion....................................................................................59 6.1.1 Animal Preparation and Tracer Administration....................................................59 6.1.2 Image Acquisition ................................................................................................60 6.1.3 PET/CT Imaging and Analysis ............................................................................61 6.1.3.1 [18F]FDG Images .................................................................................................64 6.1.3.2 [68Ga]Ga-DKFZ-PSMA-11 Images ......................................................................65 6.1.4 Ex vivo [68Ga]Ga-DKFZ-PSMA-11 Biodistribution...............................................66 6.2 Conclusion.........................................................................................................67 REFERENCES ...........................................................................................................................68 7 STUDY OUTCOMES, LIMITATIONS AND RECOMMENDATIONS ..................69 7.1 Research Outcomes..........................................................................................69 7.2 Research Limitations ........................................................................................69 7.3 Research Recommendations ...........................................................................70 REFERENCES ...........................................................................................................................72 Appendix A: Summary of all [68Ga]Ga-DKFZ-PSMA-11 radiosynthesis ..............................73 Appendix B: [68Ga]Ga-DKFZ-PSMA-11 ex vivo biodistribution............................................74 Appendix C: Journal right of permission of the figures used in this study .......................75 IX Appendix D: Language editing certificate..............................................................................79 X LIST OF FIGURES Figure 1.1: Breast cancer classification according to hormone receptor expression, as determined by immune-histochemistry and gene expression profile (microarray expression) (Uscanga-Perales et al., 2016). Permission to reprint the figure for dissertation purpose obtained from author Lopez et al., 2016. .....................................................................3 Figure 2.1: Schematic representation of CT image acquisition; X-ray emitted from the rotating X- ray tube and attenuated in the patient, and the remnant beam attenuation is recorded and measured on a ring of detectors (Goldman, 2007). ............................................12 Figure 2.2: Coincidence events, positronium annihilation yields photons of equal energy (511 keV) emitted and travel in the opposite direction (180 degrees) towards the detector ring (Turkington, 2001)......................................................................................................14 Figure 2.3: Demonstration of a ring of detectors around the patients. After an annihilation has occurred, the resultant two photons each of energy 511keV, are detected simultaneously along the line of response through coincidence events (Turkington, 2001), which forms the basis for three-dimensional image reconstruction used for PET. 14 Figure 2.4: Overview of GCP II (ED-extracellular domain, TM-transmembrane, CD-cytoplasmic domain and F-enzyme active site) (Rajasekaran et al., 2005b).................................18 Figure 2.5: Chemical structure of [68Ga]PSMA-HBED-CC, Glu-NH-CO-NH-Lys (Ahx)- is targets PSMA-binding motif and N, N'-bis [2-hydroxy-5-(carboxyethyl)benzyl] ethylenediamine- N, N'- diacetic acid [HBED-CC] allows the chelation of the [68Ga]Ga-(III)3+ chelator (Eder et al., 2012). ...............................................................................................................22 Figure 4.1: The MCF-7 F (female) ANM (athymic nude mice) (Xen) xenograft, MDA-MB-231 female athymic nude mice xenograft, and LNCaP M (male) athymic nude mice xenograft model development. .............................................................................................................44 Figure 5.1: Representative radio-chromatograms showing 68Ga-radioactivity showing counts related to free 68Ga-species retention at the origin (OR) and/or counts for [68Ga]Ga- DKFZ-PSMA-11 at the solvent front (SF) following 5-7 min exposure of the ITLC-SG strips to the same mobile phase (Methanol/Saline 80/20 v/v). Strips were incubated as follows: (A) [68Ga]GaCl3, (B) HPLC verified 100% pure [68Ga]Ga-DKFZ-PSMA-11, (C) XI radiolabelling mixture incubated for 5 min (3.5 nmol DKFZ-PSMA-11, pH 4, 95°C), (D) radiolabelling mixture incubated for 2 min (3.5 nmol DKFZ-PSMA-11, pH 4, room temperature), (E) SPE-purified [68Ga]Ga-DKFZ-PSMA-11 before evaporation of ethanol and (F) sample E after evaporation of ethanol...........................................................55 Figure 5.2: Increasing concentration of ethanolic saline applied to recover the purified [68Ga]Ga- DKFZ-PSMA-11 from a Sep-Pak light C-18 cartridge (n=4). .....................................55 Figure 5.3: Graphical overview of a standardised [68Ga]Ga-DKFZ-PSMA-11 radiosynthesis, 68Ge/68Ga generator elution (blue arrow), 68Ga-radioactivity eluted is added into the DKFZ-PSMA-11 kit immediately (red arrow)..............................................................56 Figure 6.1: PET/CT MIP images of the same MCF-7-female athymic nude mouse day 1- [18F]FDG imaging at 45 minutes (A) and 2 hour (C); and day 2: [68Ga]Ga-DKFZ-PSMA-11 imaging at 45 min (B) and 2 hour (D). The white arrow indicated the tumour. ........................62 Figure 6.2: PET/CT MIP images of the same MDA-MB-231 female athymic nude mouse xenograft day 1: [18F]FDG imaging at 45 minutes (E) and 2 hour (G); and day 2: [68Ga]Ga-DKFZ- PSMA-11 imaging at 45 min (F) and 2 hour (H). The white arrow indicates the tumour. 63 Figure 6.3: Post mortem organ and tissue biodistribution of [68Ga]Ga-DKFZ-PSMA-11 in MCF-7 /MDA-MB-231 tumour cell bearing mice following 2 hours micro-PET/CT imaging acquisition (n ≥ 3).......................................................................................................67 XII LIST OF TABLES Table 3.1: List of equipment and materials.................................................................................30 Table 4.1: Experiments conducted to develop female athymic nude mice bearing MCF-7 xenografts. .................................................................................................................42 Table 4.2: Experiments conducted to develop female athymic nude mice bearing MDA-MB-231 xenografts. .................................................................................................................43 Table 5.1: Summary of the [68Ga]Ga-DKFZ-PSMA-11 purification ............................................54 Table 5.2: Summary of results from repeated [68Ga]Ga-DKFZ-PSMA-11 radiolabelling and preparation of the safe-for-administration formulation (n ≥3).....................................57 Table 6.1: Comparison of parameters addressed for athymic nude mice xenograft for [18F]FDG and [68Ga]Ga-DKFZ-PSMA-11 micro-PET/CT imaging. ...................................................60 XIII Abbreviations β+ Beta positive/ positron [68Ga]Ga-DKFZ-PMSA-11 68Ga-labelled Glu-NH-CO-NH-Lys-HBED-CC CT Computed Tomography [18F]FDG 2-deoxy-2-[18F]Fluoro-D-glucose DMEM Dulbecco’s Modified Eagles Medium DMEM/ F12 DMEM/ Ham’s Nutrient Mixture F12 EtOH Ethanol Ex Experiment FBS/ FCS Foetal bovine serum/ Foetal calf serum 18F Fluorine-18 [18F]FES 16α-[18F]Fluoro-17β-estradiol [18F]FMISO [18F]Fluoromisonidazole [18F]FLT 18F]Fluorothymidine 68Ga Gallium-68 68Ge/68Ga Germanium-68/Gallium-68 G1 1850 MBq 68Ge/68Ga generator G2 1110 MBq 68Ge/68Ga generator GCP II Glutamate carboxypeptidase II HER2 Human epidermal growth factor receptor 2 HCL Hydrochloric acid 111In Indium-111 ITLC Instant thin-layer chromatography L Length MeV Mega electron volt XIV NWU North-West University ECs Estradiol cypionate ER Estrogen receptor PRTT Peptide radioligand-targeted therapy PBS Phosphate-buffered saline PET Positron Emission Tomography PET/CT Positron Emission Tomography/ Computed Tomography PCDDP Pre-Clinical Drug Development Platform Necsa South African Nuclear Energy Corporation PR Progesterone receptor RCY Radiochemical yield Rf Retention factor SPE Solid-phase extraction SUV Standard uptake value s.c. Subcutaneous TCs Testosterone Cypionate TNBC Triple-negative breast cancer UP University of Pretoria W Width XV 1 INTRODUCTION 1.1 Breast Cancer Types and Classification Breast cancer is a common cancer leading to high mortality in the female population in the world (He et al., 2016; Health, 2017a; Vanderpuye et al., 2017). Breast cancer disease is remarkably diverse in nature in that despite originating from cells of the mammary gland it displays morphological and molecular diversities. The breast cancer diversities as mentioned above are taken into consideration when predicting the disease prognosis and selection of therapy (Chiu et al., 2018; Makki, 2015; Uscanga-Perales et al., 2016). About 95% of breast cancer is classified adenocarcinoma; this is cancer that originates from the epithelial cells of the milk-duct and milk- producing lobule (Makki, 2015). Adenocarcinoma tumours are generally confined to the duct and lobule and known as ductal and lobular cancer respectively. However, these breast cancer types can also be invasive and are then known as invasive ductal carcinoma and invasive lobular carcinoma. Unlike localized carcinoma, the invasive carcinoma migrates from the tissue of origin to the surrounding tissue of the breast. Invasive ductal carcinoma constitutes 70 to 80% of all breast cancer and can be sub- divided into rarer types of breast carcinoma, namely tubular carcinoma, medullary carcinoma, mucinous carcinoma, papillary carcinoma and cribriform carcinoma (American Cancer Society, 2019; Makki, 2015). Inflammatory breast cancer and triple-negative breast cancer (TNBC) are other types of invasive breast cancer. Inflammatory breast cancer is a rare type of cancer (1 to 5 % of all invasive ductal and lobule breast cancer) in which cancer cells block the lymph vessels in the skin and leave the breast looking inflamed; it is aggressive (grows and spread faster), has a poor prognosis and at diagnosis it always presents at locally advanced stages (American Cancer Society, 2019). Therapy constitutes a combination of chemotherapy, surgery and radiation. TNBC (10 to 30% of all invasive breast cancer) does not express the hormone receptors, estrogen (ER) or progesterone (PR) and only very little expression of human epidermal growth factor 2 (HER 2). This type of breast cancer is also aggressive, with therapy being limited to chemotherapy, and even then therapy response is inadequate (Hon et al., 2016; Uscanga- Perales et al., 2016). Further uncommon types of breast cancer are Paget disease of the breast, angiosarcoma and phyllodes tumours. Paget disease of the breast originates in the milk-ducts and affects the skin of the nipple and the areola, and exists along with either ductal cancer or invasive ductal carcinoma. This disease is treated by resection of the tumours/lump (lumpectomy) as well as resection of the nipple and areola. Follow-up therapy includes radiation of the entire 1 breast and if no improvements are seen then removal of the whole breast (mastectomy).The disease prognosis is good if cancer has not spread. Angiosarcoma accounts for 1% of breast cancer and arises from the cells of the blood and lymph vessels. It easily spreads to the skin and tissue of the breast and mastectomy is almost the only therapy option for this type of cancer. Phyllodes tumours arise from the connective tissue of the breast. Most of these tumours are benign but they can be malignant. The cancer is treated with lumpectomy or a partial or complete mastectomy followed by radiation therapy (American Cancer Society, 2019; Health, 2017b; Makki, 2015). 1.2 Breast Cancer Molecular Subtypes The molecular diversities of breast cancer cells are classified according to the expression or non- expression of hormone receptors and the genetic profile. The following hormone receptors are assayed by immune-histochemistry (Figure 1.1): ER, PR and HER2. Furthermore, breast cancer can be then classified into four intrinsic molecular subtypes. Luminal A subtype is positive for ER and or PR, but negative HER2. Luminal B subtype is positive for ER and or PR and HER2. HER2- enriched subtype is positive for HER2, but negative for ER and PR. Basal-like breast cancer subtype is negative for all the three receptors; ER, PR and HER2 and is also known as TNBC. Luminal A, and B, as well as HER2-enriched subtype are responsive to targeted therapy, while therapy for basal-like breast cancer subtype is only limited to chemotherapy (Hon et al., 2016; Uscanga-Perales et al., 2016). 2 Figure 1.1: Breast cancer classification according to hormone receptor expression, as determined by immune-histochemistry and gene expression profile (microarray expression) (Uscanga-Perales et al., 2016). Permission to reprint the figure for dissertation purpose obtained from author Lopez et al., 2016. 1.3 Diagnosis and Therapy Management of Breast Cancer Disease Clinical breast examination is the gold standard routinely used as a screening tool for breast cancer assessment. Furthermore, breast cancer diagnosis is made using ultrasound or mammography, or both imaging modalities, as well as biopsy (Health, 2017b; Lince-Deroche et al., 2017). The therapy options available for breast cancer are mainly surgery, systemic therapy (chemotherapy and hormonal therapy) and radiation therapy (Health, 2017b; Lince-Deroche et al., 2017). Surgery is the primary therapy in managing early-stage breast cancer (stage 0) when the tumours is confined to the breast. Stage 1 and 2 refers to when the tumours is confined to the breast with only the involvement of a few lymph nodes and these stages require surgery along with another therapy form. Surgery involves lumpectomy or mastectomy and limited lymph nodes resection, and when integrated with systemic therapy has improved success in the therapy of locally and advance metastatic breast cancer. 3 Systemic therapy consists of the use of chemotherapy, either before or after tumours resection and hormonal therapy. A large tumours is initially treated with chemotherapy to reduce the size before resection. Chemotherapy might be administered post-surgery to reduce the chance of the disease recurrence. Hormonal therapy is indicative in breast cancer that is hormone receptor- positive (ER, PR and HER 2). Radiation, an alternative therapy for breast cancer disease (Health, 2017b; Lince-Deroche et al., 2017), make use of high focused, external doses of radiation to treat and kill cancer (Institude, 2019). As already explained, breast cancer is a complex and diverse disease at the morphological and molecular level. The diversities are exhibited between tumours or possibly even within a single tumour, and a single patient can exhibit differing features between the primary tumour and its metastases. (Aleskandarany et al., 2018; Cheng et al., 2013). These diversities could pose a dilemma in the diagnosis and selection of available standard therapy for breast cancer disease resulting in poor prognosis and response to therapy (He et al., 2016; Ottaviano et al., 1994; Ulaner et al., 2016). 1.4 Nuclear Medicine Imaging Nuclear medicine is a specialised field that utilises radioactive substances for disease diagnosis and therapy. Nuclear medicine is part of the multidisciplinary team towards management of breast cancer. Targeted nuclear imaging is imaging of a specific molecular biomarker (e.g. receptor, enzyme etc.) that is overexpressed by a particular cancer. Patients with a particular type of cancer that overexpress a specific molecular biomarker are administered a synthesised target ligand conjugated with a radioisotope that is able to bind to the targeted molecular biomarker on the disease. The tumours can then be imaged and detected using different nuclear imaging modalities, depending on the type of radioisotope conjugated (Dalm et al., 2017). Positron emission tomography/ computed tomography (PET/CT) imaging is an advanced hybrid imaging modality. PET component can demonstrate functional or abnormal metabolic activity at the molecular level while CT component provides with morphological information. PET/CT imaging co-registers functional and morphological in order to detect and location of unusual metabolic activity (Almuhaideb et al., 2011; Kapoor et al., 2004). The following are examples of breast cancer targeting PET tracers that are used for diagnostic and/ or therapeutic purposes and are 2- deoxy-2-[18F]Fluoro-D-glucose ([18F]FDG), [18F]Fluoromisonidazole ([18F]FMISO), [18F]Fluorothymidine ([18F]FLT) and 16 α –[18F]-Fluoro-17 β –estradiol ([18F]FES). Among these 4 tracers, [18F]FDG is the mostly used tracer. It is a glucose analogue and accumulates in cancer and non-cancer tissues that have high glucose metabolism (glycolysis). Increased glycolysis activity in non-cancer areas, such as inflammation and or infection areas, cardiology and neurology render [18F]FDG to be a non-specific radio-pharmaceutical (He et al., 2016; Pahk et al., 2015; Vercher-Conejero et al., 2015). These tracers are explained further in the literature chapter. 1.5 Glutamate Carboxypeptidase II Glutamate carboxypeptidase II (GCP II) is a type II transmembrane glycoprotein, known also as N-acetyl-α-linked acidic dipeptidase I, protein specific membrane antigen (PSMA) or folate hydrolyse (Barinka et al., 2012; Kabasakal & Demirci, 2015; Milowsky et al., 2007). Application of GCP II in nuclear medicine is as a targeted molecular biomarker by GCP II ligand tracers such as the monoclonal antibody based tracers Indium-111 [111In]-labelled 7E11-C5/ CYT-356 ([111In]- Capromab pendetide) commercially known as ProstaScintTM and [111In]/ Technetium-99m [99mTc]- labelled J591. Currently the major development in GCP II targeting is using gallium-68 (68Ga) radiolabelled GCP II ligand, which has proved to be a GCP II-binding tracer. It is composed of 68Ga (positron emitter radioisotope) and small molecule Urea-based inhibitor of GCP II/ PSMA known as (Glu-NH-CO-NH-Lys (Ahx)-HBED. 68Ga radio-labelled GCP II ligand is called [68Ga]Ga- DKFZ-PSMA-11 or [68Ga]PSMA-HBED-CC (Ebenhan et al., 2015; Eder et al., 2012), but for the purpose of the study we will refer to 68Ga radio-labelled GCP II ligand as [68Ga]Ga-DKFZ-PSMA- 11). GCP II is found to be overexpressed in prostate cancer and as a results is used for imaging and therapy in this cancer (Foss et al., 2012; Kabasakal & Demirci, 2015). GCP II was also confirmed by immuno-histochemistry to be overexpressed in vascular endothelial cells of the solid tumours (breast, bladder, lung, colon, kidney, renal, gastric cancers, transitional cell, neuroendocrine and pancreas) (Foss et al., 2012; Liu et al., 2011). To date, a few cases exist where GCP II ligand ([68Ga]Ga-DKFZ-PSMA-11) was used for targeting GCP II over-expression in solid tumours of breast (Sathekge et al., 2016; Sathekge et al., 2015) and renal cell cancer (Demirci et al., 2014). In a case study of a metastatic breast cancer patient, a PET/CT scan was performed on the patient following injection of [68Ga]Ga-DKFZ-PSMA-11; the same patient was imaged with 18[F]FDG-PET/CT for comparison. The purpose was to restage the disease and gain other prognostic information as well as to evaluate the possibility of peptide radio-ligand targeted therapy (PRTT) (Sathekge et al., 2016; Sathekge et al., 2015). The information gained was that 5 metastatic breast cancer lesions that are found to overexpress this GCP II could be suitable for therapy with GCP II-PRTT. Despite the immuno-histochemistry, confirmation of over-expression of GCP II by vascular endothelial cells of the solid tumours, a limited number of in vivo studies and no pre-clinical study exists investigating the effectiveness of GCP II ligand targeted imaging. Hence in this study, we sought to better understand the molecular accumulation of [68Ga]Ga-DKFZ-PSMA-11 PET/CT tracer in human breast cancer by clarifying if any variation in cellular accumulation of [68Ga]Ga- DKFZ-PSMA-11 exists between different forms of breast cancer with varying hormone receptors (ER-positive and TNBC). 1.6 Research Problem Given the challenges with targeting increased glycolysis activity in cancer patients, [68Ga]Ga- DKFZ-PSMA-11 could be explored as a valuable diagnostic alternative to [18F]FDG for breast cancer. Before this is possible, a better understanding of the cellular accumulation mechanism of [68Ga]Ga-DKFZ-PSMA-11 in breast cancer is required and more specifically, the possible variation in cellular accumulation between breast cancers that differ in hormone receptor (ER positive and TNBC) expression. A further opportunity beyond diagnosis using [68Ga]Ga-DKFZ- PSMA-11, is presented by way of GCP II-targeted therapy. This therapy could potentially be useful in patients with breast cancer that expresses GCP II, thereby leading to a more personalised form of therapy. 1.7 Research Aim and Objectives 1.7.1 Aims This study aims to pre-clinically investigate the accumulation of [68Ga]Ga-DKFZ-PSMA-11 in different breast cancer xenograft mice to better understand the accumulation of [68Ga]Ga-DKFZ- PSMA-11, which was previously reported in advanced stages of breast cancer in humans. 6 1.7.2 Objectives A) To develop an athymic nude mice (ANM) xenografts model, with actively growing tumours of MCF-7 and MDA-MB-231 breast cancer (female mice), as well as LNCaP prostate cancer (female and male). B) To develop a [68Ga]Ga-DKFZ-PSMA-11 radiolabelling procedure suitable for safe administration into ANM. C1) To determine [18F]FDG and [68Ga]Ga-DKFZ-PSMA-11 accumulation by MDA-MB-231 tumours (MDA-MB-231 female ANM xenografts) C2) To determine [18F]FDG and [68Ga]Ga-DKFZ-PSMA-11 accumulation by MCF-7 tumours (MCF-7 female ANM xenografts). C3) To determine [18F]FDG and [68Ga]Ga-DKFZ-PSMA-11 accumulation by LNCaP tumours (LNCaP female and male ANM xenografts). 7 REFERENCES Aleskandarany, M.A., Vandenberghe, M.E., Marchiò, C., Ellis, I.O., Sapino, A. & Rakha, E.A. 2018. Tumour Heterogeneity of Breast Cancer: From Morphology to Personalised Medicine. Pathobiology, 85(1-2):23-34. Almuhaideb, A., Papathanasiou, N. & Bomanji, J. 2011. 18F-FDG PET/CT imaging in oncology. Annals of Saudi medicine, 31(1):3-13. American Cancer Society. 2019. Understanding a breast cancer diagnosis. https://www.cancer.org/cancer/breast-cancer/understanding-a-breast-cancer-diagnosis.html Date of access: 25 May 2019. 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The use of fluorine-18 fluorodeoxyglucose positron emission tomography for imaging human motor neuronal activation in the brain. Experimental and Therapeutic Med, 10(6):2126-2130. 9 Sathekge, M., Lengana, T., Modiselle, M., Vorster, M., Zeevart, J., Maes, A. & al., e.a.e. 2016. Ga-68-PSMA-HBED-CC PET imaging in breast carcinoma patients. Eur J Nucl Med Mol Imaging(44):689-694. Sathekge, M., Modiselle, M., Vorster, M., Mokgoro, N., Nyakale, N., Mokaleng, B. & Ebenhan, T. 2015. 68Ga-PSMA imaging of metastatic breast cancer. Eur J Nucl Med and Mol Imaging, 42(9):1482-1483. Ulaner, G.A., Riedl, C.C., Dickler, M.M., Jhaveri, J.K., Pandit-Taskar, N. & Weber, W. 2016. Molecular imaging of biomarkers in breast cancer. J Nucl Med, 57(1):53-59. Uscanga-Perales, G.I., Santuario-Facio, S.K. & Ortiz-Lopez, R. 2016. Triple negative breast cancer: Deciphering the biology and heterogeneity. Medicina Universitaria, 17(71):105-114. Vanderpuye, V., Grover, S., Hammad, N., PoojaPrabhakar, Simonds, H., Olopade, F. & Stefan, D.C. 2017. An update on the management of breast cancer in Africa. Infectious Agents and Cancer, 12(1):13. Vercher-Conejero, J.L., Pelegri-Martinez, L., Lopez-Aznar, D. & Cozar-Santiago, M.D. 2015. Positron emmision tomography in breast cancer. Diagnostics,, 5(1):61-83. 10 2 LITERATURE REVIEW 2.1 Breast cancer: A Clinical Challenge Breast cancer is one of the complex and diverse diseases that presents challenges in disease management. A particular therapy might be applicable in treating the initiation of the disease but, may not be appropriate to treat the same illness at recurrence or advanced stages. This is because of the differences in the disease characteristics that exist between the primary disease and at the local and distance disease recurrence (Aleskandarany et al., 2018). This, for example, is seen in oestrogen receptor (ER)-positive breast cancer. ER-positive breast cancer is sensitive to hormonal therapy initially, but in time, when the disease progresses and aggressively grow independently from oestradiol, making the disease non-responsive to the therapy (Ottaviano et al., 1994). Other types of breast cancer, such as triple negative breast cancer (TNBC), can only be managed and treated by radiation and/ or chemotherapy. Therapy limitations arise when the disease stops responding to the therapy and also at the stage of recurrence (Uscanga-Perales et al., 2016). The challenges mentioned above call for continues investigations to find more appropriate diagnostic and therapy tools for better care and management of breast cancer disease. 2.2 Diagnostic Imaging Applications in Breast Cancer 2.2.1 Anatomical Imaging Morphology imaging involves non-invasive imaging modalities, such as mammogram, computed tomography (CT), ultrasound and magnetic resonance imaging (Vercher-Conejero et al., 2015b). These modalities are utilised in the screening, detection and diagnosis of the disease, and additionally for staging and follow-up (Kapoor et al., 2004; Lince-Deroche et al., 2017). They predominately focus on the detection of abnormal changes in the morphology due to primary breast cancer. However, cancer cells often undergo metabolic activity changes before any patho- morphological changes occur (Kapoor et al., 2004; Vercher-Conejero et al., 2015a). 11 2.2.2 Computed Tomography Imaging Principle As CT imaging will form part of the investigations herein, the principle will be briefly reviewed. CT image formation in contrast to positron emission tomography (PET) uses the external radiation source. CT scanners have an X-ray tube equipped with a cathode on the end of the tube and an anode on the opposite side; the cathode has a filament and a focusing cup. The electrons emitted from the cathode are accelerated by high voltage towards the target on the anode and X-rays are formed. The X-ray beam traverses through the patient in many directions and is attenuated. The attenuated X-ray beam is recorded and measured on the detectors. The measurement is further reconstructed mathematically to produce an image that can be analysed in three-dimensional slices (Michael, 2001) (Figure 2.1). Figure 2.1: Schematic representation of CT image acquisition; X-ray emitted from the rotating X- ray tube and attenuated in the patient, and the remnant beam attenuation is recorded and measured on a ring of detectors (Goldman, 2007). 12 2.3 Nuclear Imaging Nuclear medicine currently uses PET and single-photon emission computed tomography as non- invasive technologies to obtain images of the body function. This can be achieved by injecting small amounts of a biological molecule that is radiolabelled with a medicinal, usually short-lived radioisotope. As it is used in this investigation, only PET will be further reviewed going forward I this chapter. Historically, PET was a standalone technology and has limitations in providing morphological information connected to a disease. 2.3.1 Positron Emission Tomography Imaging Principle Positron emission tomography image acquisition is a combination of a series of activities from the injection of the PET radiopharmaceutical, also known as a tracer, into the patient to ultimately performing image acquisition of the in vivo distribution of the radiopharmaceutical in the patient. Through the decay process of the PET, isotopes in a radiopharmaceutical positrons are emitted from within the body. A positron is a positively charged electron. After travelling a minute distance and losing almost its energy, a positron will than interact with the electron in close proximity. Due to the positron and electron interaction, a latent positronium forms. Positronium then annihilates emitting two photons of equal energy (511 keV) in opposite 180º direction. These are then detected simultaneously through a line joining (line of response) the two detectors. The detectors are arranged in a ring-form around the patient, which are designed to detect two photons through coincidence events (Figure 2.2 & Figure 2.3); these events are assayed within 10 to 20 nanoseconds. The coincidence events produce raw data in the form of a sonogram, a two- dimensional matrix, which is used to provide a projection of data to do image reconstruction (Omami et al., 2014; Turkington, 2001). 13 Figure 2.2: Coincidence events, positronium annihilation yields photons of equal energy (511 keV) emitted and travel in the opposite direction (180 degrees) towards the detector ring (Turkington, 2001). Figure 2.3: Demonstration of a ring of detectors around the patients. After an annihilation has occurred, the resultant two photons each of energy 511keV, are detected simultaneously along the line of response through coincidence events (Turkington, 2001), which forms the basis for three-dimensional image reconstruction used for PET. 14 2.3.2 Positron Emission Tomography/Computed Tomography Currently, nuclear imaging uses a hybrid modality of PET combined with CT in the same equipment (PET/CT) as an advanced diagnostic tool for investigating metabolic and biological conduct, as well as morphological changes, for example due to organ pathology, infection or cancer. The advantage of this is that functional and morphological (e.g. cancer-related) changes can be investigated within a single examination - a whole-body PET/CT scan (Kapoor et al., 2004). Concerning cancer, PET/CT imaging is, for example, successfully used for re-staging and monitoring of therapy outcome in patients with known or suspected cancer recurrence (Koolen et al., 2012; Tokes et al., 2013; Torii & Toi, 2018). The acquired PET and CT images are co-register, and the resultant fused PET/CT images can be analysed qualitatively and/or semi-qualitatively. Fused PET/CT image qualitative and semi- quantitative analysis involves visual interpretation and standard uptake value (SUV) measurements respectively. The analysis distinguish the normal from the disease accumulation of the tracer being studied. SUV is measured by the activity per unit volume of the region of interest to the activity per whole-body volume ratio (Kinahan & Fletcher, 2010; Thie, 2004). 2.3.3 Dedicated Breast Positron Emission Tomography/Computed Tomography Dedicated breast PET is a specialised nuclear medicine modality dedicated to evaluating primary breast tumours. Dedicated PET modality can detect the smallest (sub-centimetre) avid lesions and intra-tumours heterogeneity, respectively. There are numerous challenges regarding the use of whole-body PET/CT imaging in assessing primary breast tumours. Whole-body PET/CT imaging is hindered by limited spatial resolution, which introduces complications in quantifying tiny lesions due to the significant partial volume effect. Whole-body PET/CT imaging is acquired when the patient is lying supine on the bed, and this position makes the volume of the breast collapse which makes it difficult to assess the breast (Jones et al., 2019; Koolen et al., 2012; Torii & Toi, 2018). 2.3.4 Pre-clinical Imaging using Dedicated micro-Positron Emission Tomography/Computed Tomography Micro-positron emission tomography/ computed tomography (micro-PET/CT) is sensitive tomographic equipment employed for image acquisition and quantitative analyses of diseased animal models, e.g. the study of xenografted cancer cells in vivo (Yao et al., 2012). The principle 15 of micro-PET/CT image acquisition is the same as the clinical PET/CT scanner. This modality is suitable for imaging of mice, due to their weight which a magnitude less compared to that of the human (Kuntner & Stout, 2013). Micro-PET/CT modalities features spatial resolution in the sub- millimeter range to allow visualize tracer distribution in small animals. Spatial resolution is affected by the positron range in the tissue connected to the radioisotope of interest. The detector ring of micro-PET/CT has a diameter of 150 mm compared to 800 mm diameter for the clinical PET/CT scanner (Liang et al., 2007; Yao et al., 2012). A small detector ring has an advantage of better geometric detection efficiency (Yao et al., 2012). The MCF-7, MDA-MB-231 and LNCaP athymic nude mice xenografts developed will be imaged with micro-PET/CT to investigate the in vivo biodistribution of [68Ga]Ga-DKFZ-PSMA-11 compared to [18F]FDG. 2.4 Positron Emission Tomography Radiopharmaceuticals for Breast Cancer Imaging The tracer 2-deoxy-2-[18F]Fluoro-D-glucose ([18F]FDG) is the most commonly used radio- diagnostic agent for PET/CT imaging (IAEA, 2008; Tokes et al., 2013). Fluorine-18 (18F) is a PET standard radioisotope with a physical half-life of 109.8 minutes. There are over 20 methods to produce 18F using a cyclotron, and these can either yield a high or low specific activity 18F. About 96.7% of this radioisotope decays by beta positive/ positron (β+) emission and maximum energy of 0.634 mega electron volt (MeV), the remaining 3% is emitted by electron capture and no gamma emission (Conti & Erikisson, 2016). [18F]FDG, is a glucose analogue, which provides metabolic activity based on the increased cellular demand for glucose due to the elevated glycolysis in cancer cells (IAEA, 2008; Vercher-Conejero et al., 2015b). Therefore, [18F]FDG- PET/CT imaging mostly provides important information regarding cancer diagnosis, staging, the guidance of appropriate therapy, monitoring of therapy response and for the detection of cancer recurrence. Studies have however, revealed limitations about the role of PET/CT imaging with [18F]FDG in diagnosing primary breast cancer due to poor sensitivity in the detection of small lesions and or carcinomas in situ. [18F]FDG-PET/CT in breast cancer patients is appropriately indicated for the examination of recurrence and monitoring of therapy response (Vercher- Conejero et al., 2015a). Early changes in the metabolic activity of cancer cells have positioned PET/CT imaging with [18F]FDG as a superior technique over morphological imaging modalities, especially in asymptomatic patients presenting with rising tumours markers or where morphological imaging findings are uncertain and negative (Vercher-Conejero et al., 2015a). However, there is also a considerable accumulation of [18F]FDG in non-malignant tissue (inflammation and or infection areas) and energetic tissues such as the brain, muscles and brown 16 fat tissue). The above attributes render [18F]FDG to be a non-specific radio-pharmaceutical for breast cancer (He et al., 2016; Vercher-Conejero et al., 2015b). Apart from [18F]FDG, other PET/CT tracers that have been previously utilised at pre-clinical and clinical settings are as follows: [18F]Fluoromisonidazole ([18F]FMISO), [18F]Fluorothymidine ([18F]FLT) and the modified ER-ligand/substrate 16α-[18F]fluoro-17β-estradiol ([18F]FES). [18F]Fluoromisonidazole ([18F]FMISO) is a PET/CT radioactivity analogue of nitroimidazole. It binds to hypoxic cells with functional nitro-reduced enzyme, and it is not taken up by necrotic tissues. Hypoxia is a tissue-associated process in solid tumours due to tumours proliferation and tumour hypo-vascularisation (Cheng et al., 2013). Its biomarker is the hypoxia-induced factor. Conditions such as tumours growth, the progression of cancer and resistance to therapy are associated with hypoxia-induced factor expression (Cheng et al., 2013). [18F]FMISO-PET/CT imaging helps to assess the status of tumour oxygenation (Cheng et al., 2013). [18F]Fluorothymidine ([18F]FLT) is indicative of monitoring early chemotherapy response (Li et al., 2012). The accumulation of [18F]FLT by breast cancer cells is based on cell proliferation and biomarkers, such as the expression of equilibrative nucleoside transporters and thymidine kinase- 1 activity (Li et al., 2012). 16 α –[18F]-Fluoro-17 β –estradiol ([18F]FES) is a modified oestrogen molecule that specifically targets the ER; it assists in the evaluation of the ER expression on recurrent breast cancer cells and metastasis and forecasts the tumours response to hormone therapy (Jones et al., 2019; Salem et al., 2018; Vercher-Conejero et al., 2015a). [18F]FES tracer accumulation in the detection of ER-positive breast depends greatly on the receptor-ligand binding mechanism instead of the amount of the receptor expression present, as a result, it has high detection efficiency and high specificity (Salem et al., 2018). However, the limitations with ([18F]FMISO, [18F]FLT and [18F]FES are that they are not peptide ligand-based radiopharmaceuticals and as such, they cannot further develop for PRTT applications. 17 2.5 Glutamate Carboxypeptidase II 2.5.1 Molecular Structure Glutamate carboxypeptidase II has a molecular weight of 90 to 110 kilo-Dalton (Barinka et al., 2007). GCP II consists of a short amino-terminus-NH2-terminus (A) in the cytoplasmic domain- CD (1-19 amino acids), a hydrophobic transmembrane domain-TM (20-24 amino acids) and extracellular domain-ED (45-750 amino acids) at the carboxyl-terminus-COOH-terminus (Figure 2.4). The amino-terminus-NH2-terminus is involved with the interaction of several proteins and has a significant role in the localisation and molecular properties of GCP II. The extracellular domain has three distinct areas (amino acid): protease domain (B and D), apical domain (C) and carboxyl-terminus-COOH-terminus (E to G) (Figure 2.4) (Bařinka et al., 2012; Mesters et al., 2006; Rajasekaran et al., 2005b). The collaboration of the three subdomains of the extracellular domain are involved in the GCP II substrate binding and the recognition of the ligand. Figure 2.4: Overview of GCP II (ED-extracellular domain, TM-transmembrane, CD-cytoplasmic domain and F-enzyme active site) (Rajasekaran et al., 2005b). 18 2.5.2 The Function and Role of Glutamate Carboxypeptidase II in Cancer GCP II is a type II transmembrane glycoprotein, well known as prostate-specific membrane antigen (PSMA) for diagnostic and therapeutic interventions of prostate cancer (Barinka et al., 2012; Kabalaskal & Demerci, 2015; Milowsky et al., 2007b). There is a high expression of GCP II in all types of prostate cancer (Foss et al., 2012; Kabasakal & Demirci, 2015). Apart from prostate cancer, GCP II was seen overexpressed by neovascular endothelial cells of nearly all solid tumours cells. GCP II expression was absent in the normal vascular endothelium (Denmeade et al., 2012; Milowsky et al., 2007a; Rajasekaran et al., 2005a; Sathekge et al., 2016). Neovasculature of the solid tumours cells of the breast, bladder, lung, colon, kidney, renal, gastric cancers, transitional cell, neuroendocrine and pancreas were found positive for GCP II expression through immune-histochemical assays (Liu et al., 2011). Over-expression of GCP II is attributed to the aggressiveness of the tumours. Thus GCP II over-expression in the solid tumours may enable PET/CT imaging (Rajasekaran et al., 2005a). Furthermore, the expression of GCP II was seen in non-cancer tissues, such as secretory cells of the salivary glands, the proximal tubules of the kidney and the jejunal brush border membrane of the small intestine. However, the expression by these non-cancer tissues is up to 1000-fold less than that of the prostate tissue (Abdel aziz et al., 2015; O'Keefe et al., 2018; Wustemann et al., 2016a). Therefore, prostate cancer cells were used as the control for this study due to the high expression of GCP II. 2.5.3 Glutamate Carboxypeptidase II: A New Target in Nuclear Medicine Molecular imaging in the field of oncology is utilised to explore, amongst other cellular processes, receptor expression. GCP II receptor expression in advanced prostate cancer and neovasculature of most solid tumours is a target of interest in diagnosis and therapy of metastatic disease (Foss et al., 2012). The 7E11-C5/ CYT-356 is the first mouse monoclonal antibody developed to target GCP II expressed by prostate cancer. This monoclonal antibody was further developed as 111Indium [111In]-labelled 7E11-C5/ CYT-356 ([111In]-Capromab Pendetide), commercially known as ProstaScintTM and was approved by the Food and Drug Administration for prostate cancer imaging (Foss et al., 2012; O'Keefe et al., 2018). Antibody J591 was developed (O'Keefe et al., 2018), and contrary to 7E11-C5, this antibody binds to the extracellular domain of the GCP II. Unlike ProstaScintTM J591 demonstrated higher target to background ratios. Radioisotopes 111In and 99mTechnetium have been used to label antibody J591 for diagnostic imaging, and 90Yttrium and 177Lutetium labelled with antibody J591 for therapy purposes. The third monoclonal antibody generation under development is 3/A12, 3F11 and 3E7, in this case, radiolabelled with 64Copper, 19 a positron emitter and imaging performed on PET/CT imaging (Foss et al., 2012). Clinical data emerged, confirming the detection and visualisation of GCP II by the solid tumours. A case study reported a 65-year-old woman diagnosed with renal cell cancer post-nephrectomy, underwent [68Ga]Ga-DKFZ-PSMA-11 and [18F]FDG-PET/CT imaging for restaging. [68Ga]Ga-DKFZ-PSMA- 11-PET/CT images demonstrated the positive lesions-renal cell cancer, and positive lesions of the axial and appendicular skeleton. The [68Ga]Ga-DKFZ-PSMA-11 positive lesions were compared to the [18F]FDG once, through the maximum SUV. It was found that the SUV max for [68Ga]Ga-DKFZ-PSMA-11 was higher in bone lesion than for [18F]FDG, which provided a lower diagnostic power of the bone metastasis (Demirci et al., 2014). Another case study was of a 33- year-old female with metastatic breast cancer, where [68Ga]Ga-DKFZ-PSMA-11-PET/CT was compared with [18F]FDG-PET/CT for restaging purpose and possible evaluation of PRTT option. Both radiopharmaceuticals showed intense and extensive accumulation by the axial and appendicular skeleton and liver metastasis (Sathekge et al., 2015). The prospective study had 19 patients, some of them already diagnosed with metastatic disease and others with recurrence disease. The type of breast cancer dealt with in the study was of ductal, lobular and neuroendocrine origin. Based on the results, six out of 19 patients were known to be PR positive, and only seven were PR negative. The resultant PET/CT images detected and visualised GCP II positive breast cancer lesions, in other words, they were accumulation and retention of [68Ga]Ga- DKFZ-PSMA-11 in the tumours lesions seen in the primary site or loco-recurrences, lymph nodes and metastatic sites. Furthermore, the authors were able to compare tracer accumulation between the PR positive and PR negative breast cancer lesions. The difference in the tracer accumulation was found to be not statistically significant (Sathekge et al., 2016; Sathekge et al., 2015). The value of PET/CT imaging using radio-ligands targeting the neovascular endothelial cells that are GCP II-positive may be a novel clinical biomarker in nuclear medicine. The diagnostic potential of [68Ga]Ga-DKFZ-PSMA-11 imaging will be compared to [18F]FDG imaging to understand the relationship between this GCP II ligand and the malignancy of breast cancer. As indicated above, only clinical data exists of studies done by Sathekge et al. (2015 and 2016) on [68Ga]Ga-DKFZ-PSMA-11 imaging of breast cancer patients. No studies to the researcher’s knowledge have been done pre-clinical. Therefore, a pre-clinical setup is required to help to understand the molecular mechanism accumulation of [68Ga]Ga-DKFZ-PSMA-11 by human breast cancer. 20 2.6 Research Tools and Narrative 2.6.1 [68Ga]Ga-DKFZ-PSMA-11 Figure 2.5 shows a chemical structure of 68Ga-labelled Glu-NH-CO-NH-Lys-HBED-CC, further referred to as [68Ga]Ga-DKFZ-PSMA-11 (Ebenhan et al., 2015; Eder et al., 2012). This chemical structure is composed as follows: a urea-based peptidomimetic (Glu-NH-CO-NH-Lys-Ahx) is conjugated to N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC), a chelator for complexation with radio-metal [68Ga]Ga(III)3+ (Eder et al., 2012). Therefore, as a new imaging agent, [68Ga]Ga-DKFZ-PSMA-11 targets tissues that specifically express GCP II. The mechanism of action for [68Ga]Ga-DKFZ-PSMA-11 includes targeting the enzyme active site in the extracellular compartment (Figure 2.4) followed by tracer internalisation into the cytoplasmic domain (Rajasekaran et al., 2005c). [68Ga] is a PET radio-metal isotope with a physical half-life of 68 minutes and is conveniently extracted from a 68Germanium/68Gallium ([68Ge]/[68Ga]) generator. About 89% of this radioisotope decay by β+ emission and the remaining 11% by electron capture. Only 1.2% decays to the excited state and decays further to the ground state releasing gamma-energy of 1.077 MeV (Conti & Erikisson, 2016). In comparison to 18F radioisotope (97% β+ emission and 0.633 MeV) 68Ga (89% β+ emission and 1.900 MeV) has lower positron yield and longer positron range which degrade the image resolution quality (Kuntner & Stout, 2013; Sanchez-Crespo, 2012). 21 Figure 2.5: Chemical structure of [68Ga]PSMA-HBED-CC, Glu-NH-CO-NH-Lys (Ahx)- is targets PSMA-binding motif and N, N'-bis [2-hydroxy-5-(carboxyethyl)benzyl] ethylenediamine-N, N'- diacetic acid [HBED-CC] allows the chelation of the [68Ga]Ga-(III)3+ chelator (Eder et al., 2012). 2.6.2 Human Breast Cancer Cells Human breast cancer cells isolates are a common research tool to study breast cancer in vitro or in a pre-clinical setup. Both MDA-MB-231 and MCF-7 are used as experimental cell lines for the study. The MDA-MB-231 cell line is a human breast cancer cell line (Cailleau et al., 1978) derived from a metastatic site. The MDA-MB-231 cell line is negative for all three hormone receptors, which are ER, PR and HER2 representing TNBC (Foulkens et al., 2010). The MCF-7 cell line is a human invasive breast ductal carcinoma cell line (luminal) (Soule, 1973), derived from a metastatic site. The MCF-7 cell line is positive for the expression of ER and PR and negative for HER2 (labs, 2017). Estrogen hormone supplementation is required to stimulate tumours growth when establishing a xenograft model with MCF-7 (Dall et al., 2015; Fleming et al., 2010). LNCaP prostate cancer cells was used as the positive control for the study because of their high expression of GCP II. The LNCaP cell line is an androgen-sensitive human prostate adenocarcinoma derived from a lymph node metastasis. It is known from literature that GCP II is overexpressed in LNCaP (Lutje et al., 2015; Wustemann et al., 2016b). Athymic nude mice are 22 envisaged as the recipient strain for an LNCaP cell line inoculum. Since LNCaP is an androgen- sensitive human prostate adenocarcinoma cell line, it is clear that this tumour growth may dependent on androgen hormone supply in the environment (Horoszewicz et al., 1983). 2.6.3 Cell Line - Xenografts Model Murine models are widely utilised to mimic not only the process of development and progression of breast cancer, but also the efficacy of therapy strategies (Zhang et al., 2018). The following are three murine models of cancer utilised to date: xenograft model, syngeneic model and genetically engineered animals (Kim et al., 2004). The syngeneic murine model involves implantation of the cells/ tissue to the recipient strain, which are the same as the cells/tissue in the origin strain (e.g. mouse cells implanted into the recipient (mouse strain)). The common application of this model is studying the mechanism of tumours growth and metastasis under the condition of an intact immune system (Zhang et al., 2018). The genetically engineered murine model was established through transgenic and knockout processes, which is the process of foreign genes been introduced into the species of interest and therefore altering the genome of that species. These allow to better understand the formation of cancer cells and the efficacy of therapy strategies (Holen et al., 2017; Holliday & Speirs, 2011; Zhang et al., 2018). Xenograft models involve inoculation of human cell lines/tissue into immune incompetent animals (Holen et al., 2017; Holliday & Speirs, 2011; Puchalapalli et al., 2016). This model is broadly practiced since it provides a microenvironment that allows for tumours growth and progression, and permits the assessment of cancer biological processes (Holen et al., 2017; Holliday & Speirs, 2011). The immune incompetent mouse strains commonly used are athymic nude mice, severe combined immune deficiency and non-obese diabetic severe combined immune deficiency animals (Puchalapalli et al., 2016). Solid tumours formation in xenograft models depends on the extent of strain immune incompetence. Athymic nude mice lack a fully functioning thymus, and as a result, they are T cell deficient. This deficiency makes this strain immune incompetent, which allows tumours growth. However, the maturity of the lymphocytes and increasing activity of the natural killing cells as the animal ages brings limitations to this strain (Puchalapalli et al., 2016). In most cases, the ectopic xenograft model is utilised for validation and assessment in oncology studies. It involves subcutaneous (s.c.) injection of human cancer cells into the hind leg or back of a mouse. In comparison, in the orthotopic xenograft model, cancer cells are injected into the same origin site as that of the tumours. For example, the inoculation site for human breast cancer 23 cells will be in the mammary fat pad in the abdominal region of the mouse (Clarke, 1996; Fleming et al., 2010; Jung, 2014). The latter is considered physiologically superior to the s.c. injection site (Holliday & Speirs, 2011). Advantages of orthotopic inoculation are increased tumours take rate and growth and tumours that are well vascularised compared to ectopic tumours (Jung, 2014). However, well-trained and certified personnel in surgical operations are required to perform orthotopic cancer cells injection. Tumours visible to the eye are palpable and measured with caliper; and tumours not visible to the eye are measured with optical imaging using fluorescence or bioluminescence signals. Morphological imaging, such as CT and magnetic imaging resonance, are commonly used methods to monitor orthotopic tumours growth (Jung, 2014). Athymic nude mice were used in developing the xenografts model for this study. Due to non- existing MCF-7, MDA-MB-231 and LNCaP athymic nude mice xenografts models, the study first attempted to developed all these xenograft models for prospective [68Ga]Ga-DKFZ-PSMA-11 and [18F]FDG in vivo micro-PET/CT imaging. 2.7 Research Narrative Athymic nude mice were used to develop the cancer xenograft model for this study. The study first developed athymic nude mouse xenograft models bearing tumours of MCF-7, MDA-MB-231 and LNCaP. The MCF-7, MDA-MB-231 and LNCaP mice xenografts was used to study tracer accumulation of [68Ga]Ga-DKFZ-PSMA-11 compared to [18F]FDG by way of non-invasive whole body micro-PET/CT imaging. 24 REFERENCES Abdel aziz, A.M., Gabal, S.M., Salem, M.S. & Amer, S.L. 2015. Prostate specific membrane antigen expression in neovasculature associated with glioblastoma multiforme and other astrocytic neoplasms; immunohisto chemical and histopathological study. Middle-East journal of scientific research, 23(8):1851-1861. 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Table 3.1: List of equipment and materials Item Supplier Cell culturing Dulbecco’s Modified Eagle’s Medium Lasec SA (PTY) LTD, Midrand, South Africa Gibco Ham's F-12 nutrient mixture Foetal Bovine Serum Foetal Calf Serum Penicillin / Streptomycin L-Glutamine Trypsin /Ethylendiaminetetraacetic acid Phosphate-buffered saline Cell lines LNCaP (ATCC® CRL-1740TM) American Type Culture Collection, Virginia, USA MDA-MB-231 (ATCC® HTB 26TM) MCF-7 Council of Scientific and Industrial Research, Cape Town, South Africa Cell inoculation Syringes and needles Isigidi Medical Suppliers, Centurion, South Africa Phosphate-buffered saline (sterile) Adcock Ingram Critical Care (Pty) Ltd, Johannesburg, South Africa Corning® Matrigel® basement membrane Discovery Labware, Inc., Bedford, USA matrix Biochemicals ISOFOR inhalation anaesthetic Safeline Pharmaceuticals, Roodepoort, South Africa Estradiol-17-cypionate Kyron Labs, Benrose, South Africa 30 Testosterone cypionate V-Tech (Pty) Ltd, Johannesburg, South Africa Radiolabelling and tracer formulation DKFZ-PSMA-11 ABX advanced biochemical compounds GmbH, Germany [18F]FDG Cyclotope, Pelindaba, South Africa Saline (sterile) Adcock Ingram Critical Care (Pty) Ltd, Johannesburg, South Africa Hydrochloric acid (32%), suprapure grade Thermo Fischer Scientific, Massachusetts, USA Sodium Acetate Trihydrate Merck KGaA, Darmstadt, Germany Ethanol Demineralised water Millipore, Millisep, Johannesburg, South Africa SPE Sep-Pack C18 units Waters Corporation, Massachusetts, USA Instant thin-layer chromatography silica- Agilent, Forrest Lake, USA gel paper Equipment Germanium-68/Gallium-68 generator iThemba LABS, Somerset West, South Africa Thin layer chromatography scanner Raditec Medical AG, Bellikon, Switzerland Automated Heater Cleaver Scientific Ltd, Rugby, United Kingdom Balance Sartoriuos Lab Instruments GmbH and co, Goettingen, Germany CRC-25R dose calibrator CH® Capintec, Florham Park, USA Small animal PET/CT Mediso Ltd, Budapest, Hungary Animal monitoring system (Prepacell®) Anaesthesia station Instant blood glucose monitoring system Clicks, Pretoria, South Africa (ACCU-CHECK®) Automated gamma counter Hidex AMG Lablogic, Turku, Finland ESCO Laminar flow cabinets Labotec (Pty) Ltd, Midrand, South Africa NU-5510/E – air-jacketed DHD CO2 Marshall Scientific, Hampton, USA incubator SCO5W – water-jacketed CO2 Incubator Sheldon Mfg. Inc., Cornelius, USA Centrifuge Harmonic series MERK Laboratory Supplier (Pty) Ltd, Germiston, South Africa Centrifuge Hermle Z300 Lasec SA (PTY) LTD, Midrand, South Africa 31 Small rodent guillotine In house, PCDDP Vivarium, Potchefstroom, South Africa Individual ventilated cages Techniplast, Buguggiante, Italy 3.2 Ethics The animal experiments conducted in this study were approved by the AnimCare Ethics Committee on Animal Care, Health and Safety in Research (AnimCareREC-130913-015), at North-West University (NWU) (NWU-00289-17-A5). 3.3 Cell Culture Cell culture work was conducted at two different facilities. The MCF-7 cells were cultured in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 20% foetal bovine serum (FBS), 1% penicillin/streptomycin and 0.2 mg/ml of L-Glutamine at the Laboratories of the DST/NWU Pre-clinical Drug Development Platform (PCDDP), NWU. The MDA-MB-231 cells were cultured in DMEM supplemented with 20% foetal calf serum (FCS) and 1% penicillin/streptomycin. The LNCaP cells were cultured in DMEM/Ham’s Nutrient Mixture F12 supplemented with 20% FCS and 1% penicillin/streptomycin. These two cell lines were cultured and maintained at the laboratory of the Department of Pharmacology, University of Pretoria (UP). Aseptic techniques were used throughout all cell culture methods, working in sterile ESCO laminar low cabinet. Routine, checks for contamination were performed. Cell seeding from frozen stock was done immediately once defrosted and placed into a 25 cm2 flask with warm media containing 20% FBS or FCS; subsequent confluent cells were split and further maintained in 75 cm2 flask with 10% of FBS or FCS. All cell incubation were kept at 37ºC and at 5% carbon dioxide using an air-jacketed auto-flow incubator (UP) and water-jacketed incubator (NWU). MDA-MB-231 or LNCaP cell cultures of approximately 60% confluency were transported to NWU for further culturing and inoculation into the mice, which was performed at the 80% confluency. Cells were harvested by discarding the culture media, followed by exposure to 2 ml of 0.25% (w/v) trypsin-0.53 mM EDTA under 37ºC and 5% CO2 incubation for 3 to 5 minutes. When the cells showed detachment, 8 ml of complete medium was added to deactivate the trypsin. The cell were transferred to a sterile 15 ml falcon centrifuge tube and spun at 2000 revolution per minutes for four minutes (centrifuges- UP) and (centrifuge-NWU). The cell pellet was re-suspended in 1 ml of fresh medium. A 50 µl sample of cells was mixed with 50 µl of trypan blue into an Eppendorf tube for cell counting and 32 to determine cell viability. Cell count was conducted using a haemocytometer, counting at least 50 cells in 4 squares. A cell viability of at least 90% was determined preceding cell re-suspension. Cell viability less than 90% did not qualify for cell inoculation into the mice however further culturing done to check cell growth, and upon cultured cells reaching the acceptable cell viability is then the cells qualify for inoculations. A desired amount of cells (MCF-7, MDA-MB-231 and LNCaP) was re-suspended into PBS or plain media with PBS (1:1), or PBS with Matrigel (1:1), or media with Matrigel (1:1). 3.4 Animal Preparation for Inoculation Athymic nude mice bearing MCF-7 or MDA-MB-231 or LNCaP tumours were used in the study. Breeding of mice stock took place at the PCDDP Vivarium, NWU. Athymic nude mice at the age of 4 to 6 or 6 to 8 weeks-old were subjected to cell inoculations. A week before cell inoculation mice were randomised into groups of six or eight and housed in individual ventilated cages, the system provided controlled ventilation temperature (23 to 26ºC) and humidity (50 to 60%). Cages were equipped with bedding, paper towels and plastic tubes for the mice’s environmental enrichment. Mice were kept on a twelve-hour light/dark cycle, and fed on a normal diet and given water ad libitum. Prior to cell inoculation mice were randomly grouped and given time to rest and acclimatise for a one-week period. A 1 ml syringe fixed with a 25 gauge needle was used to inject 100µl cell suspension into the mice. 3.5 Breast and Prostate Cancer Xenograft Model Establishment A week before MCF-7 cell line inoculation mice were treated with a dose of estradiol cypionate (ECs) (3mg/kg) (Johnson et al., 2013; Varricchio et al., 2007). Female mice to be receiving LNCaP inoculum were pre-treated with a bolus of testosterone cypionate (TCs) (200µg) three days before inoculation (Day et al., 2013). Both, female athymic nude mice bearing MDA-MB-231 cells and male athymic nude mice bearing LNCaP cells did not receive any hormone supplement. Mice were inoculated with total cell counts ranging from 1 x 106 to 5 x 106. Mice subjected to orthotopic MDA-MB-231 inoculation were anaesthetised for a short period using an inhalation of 1.5 - 3% Isoflurane/ oxygen mixture. The fourth nipple of the animal was lifted using sterile tweezers, and a 100 µl of MDA-MB-231 cell suspension injected directly into the fourth mammary fat pad tissue (Zhang et al., 2018). A subcutaneous area in the hind-right flank (MCF-7 and LNCaP) was 33 targeted for internal placement of the cells. This procedure was performed on restrained mice without the need of anesthesia. Post inoculation mice were closely monitored two to three times a week for their general health and tumours growth. Monitoring included observing body weight, appearance, clinical signs, natural behaviour, provoked behaviour. Going forward, the latter mice bearing MCF-7 and LNCaP cells were treated with the respective hormone supplements once weekly. The tumours volume was measured using digital caliper; measurement of the width (W) and length (L) were taken to calculate the volume using the equation 3.1. (𝑾 × 𝑾 × 𝑳) Equation 3.1: Tumours volume (mm3) = 𝟐 The tumours take rate and day of tumours onset (i.e. first time palpable) was determined for each batch of mice and recorded for each inoculation procedure. At approximately two and half weeks post inoculations, mice were transferred to the Pre-clinical Imaging Facility located at the South African Nuclear Energy Corporation (Necsa) for acclimatisation, further general monitoring and measurement of tumour growth, followed by nuclear imaging and post mortem tracer biodistribution. 3.6 Radiopharmaceuticals 3.6.1 [18F]FDG The doses required per animal were directly prepared in-house from a certified ready-to-use [18F]FDG. Sterile saline was used to dilute the doses to the desired radioactivity concentration. 3.6.2 [68Ga]Ga-DKFZ-PSMA-11 Two germanium-68/ gallium-68 (68Ge/68Ga) generators, one loaded with 1110 MBq and the other loaded with 1850 MBq were used to provide the 68Ga-radioactivity for in-house radio-syntheses of [68Ga]Ga-DKFZ-PSMA-11 performed at the Radiochemistry at Necsa. The 1850 MBq 68Ge/68Ga generator were used mostly during phase 1: the establishment of the [68Ga]Ga-DKFZ- PSMA-11 radiolabelling procedure to achieve a safe-for-administration formulation suitable for mice. The 1110 MBq 68Ge/68Ga generator was used for routine radiolabelling of [68Ga]Ga-DKFZ- PSMA-11 to prepare doses for micro-PET/CT imaging. 34 An eluate fractionation method was employed to assemble the 68Ga-activity from the generator in a volume of 1.2 ml using 0.6 N hydrochloric acid (HCl) (Sudbrock et al., 2014). A ready to use freeze-dried kit with 3.5 nmol DKFZ-PSMA-11 and 325 µg of Sodium Acetate Trihydrate (NaOAc) was brought to room temperature and supplemented with the 1.2 ml of 68Ga-radioactivity. The reaction mixture was placed in a temperature-controlled heating block and incubated >95°C for 5 to 10 minutes. The radiolabelled mixture was allowed to cool down and was subsequently purified by a solid-phase extraction (SPE) method, which was optimised from a previously published method (Ebenhan et al., 2015) to suit the requirements for pre-clinical tracer administration into mice. Briefly, the radioactivity mixture containing [68Ga]Ga-DKFZ-PSMA-11 was loaded on a preconditioned Sep-Pak C18-light cartridge, then rinsed with saline to flush out uncomplexed [68Ga]GaCl3, [68Ga]Ga[Cl -4] and [68Ga]Gax(H2O)x-species. The C18 matrix stripping conditions to gain purified [68Ga]Ga-DKFZ-PSMA-11 involved different concentration and volumes of ethanol/saline (EtOH/saline) mixture (25% concentration in 0.3 ml, 25% in 0.5 ml, 15% concentration in 0.5 ml) followed by EtOH solvent evaporation. EtOH solvent evaporated from the radiolabelled [68Ga]Ga-DKFZ-PSMA-11 product vial at 75°C for 5 minutes. The maximum allowed volume was 300 µL of ethanolic saline (e.g. 250 MBq), to strip the purified products from the cartridge. It had to be ensured that the final ethanol content in the formulation for intravenous injection was lower than 5%. The pH of the formulation was required to measure at pH level of 6 - 7.5 for safe injection into mice. 3.7 Instant Thin-Layer Chromatography Thin-layer chromatography and instant thin-layer chromatography (ITLC) quality control methods have been tested and verified by high performance liquid chromatography for their reliability in determining radiochemical yield (RCY) of [68Ga]Ga-DKFZ-PSMA-11 complex (Eppard et al., 2017). Thin-layer chromatography-silica gel plates based stationary phase require longer time to develop them in the mobile phase to efficiently separate the free 68Ga and the [68Ga]Ga-DKFZ- PSMA-11 labelled complex compared to ITLC-SG paper based stationary phase (Ebenhan et al., 2015; Eppard et al., 2017). The use of ITLC-SG paper based stationary phase will be beneficial to reduce the development times and results in less loss of product radioactivity. The study made used of ITLC quality control to ascertain radiochemical purity and stability of [68Ga]Ga-DKFZ- PSMA-11 before administration into the mice. ITLC-SG paper based stationary phase developed 35 in (Methanol/Saline 20/80 v/v) was routinely performed ahead of tracer injection (Ebenhan et al., 2015). A 5µL sample of the product radiolabelled [68Ga]Ga-DKFZ-PSMA-11 was dotted on the ITLC-SG paper dried and incubated in a mobile phase before and following EtOH evaporation from the product radioactivity. ITLC-SG stationary phase was developed until the solvent front reached the 90% of the ITLC-SG stationary phase. The radioactivity was counted by way of radio- detection and recording on a radio-chromatogram (Veenstra, the Netherlands); maximum peak identification and percentage quantification was performed by integration of the “area under the curve.” 3.8 Micro-Positron Emission Tomography / Computed Tomography Imaging 3.8.1 Animal Preparation After the arrival of the tumour-bearing athymic nude mice at Necsa, the mice were given a week to acclimatise to a new environment. During acclimatisation, the mice were monitored every day until in vivo micro-PET/CT imaging occurred. On the day of [18F]FDG imaging, mice were fasted for 4 to 6 hours, only allowing water ad libitum. A drop of venous blood was required to record the blood glucose level (g/ml) aligned with the [18F]FDG administration using the Accu-Check® Instant blood glucose monitoring system. No particular animal preparation was required leading up to the administration of [68Ga]Ga-DKFZ-PSMA-11. 3.8.2 Intravenous Tracer Administration Before tracer administration, animal weight and tumours size was recorded. Whilst a 150 µL bolus containing a isotonic [18F]FDG solution was prepared the animal was sedated by inhalation of a 5% Isoflurane/ oxygen mixture (medium flow) using a dedicated animal monitoring station (Prepacell®) and further maintained at 2%, [18F]FDG was then injected into the lateral tail vein (Yao et al., 2012). The animal remained under anesthesia for a 45 minutes tracer accumulation period. [68Ga]Ga-DKFZ-PSMA-11 doses (Chapter 6, No 6.1.1) were prepared in a similar fashion to [18F]FDG; no anesthesia was required, mice were merely restrained during tracer injection which was also given intravenously. These mice were only sedated shortly before and during image acquisition to manage and maintain the mouse position throughout the scanning period (Yao et al., 2012). 36 3.8.3 Computed Tomography and Positron Emission Tomography Image acquisition On two consecutive days, mice underwent non-invasive micro-PET/CT imaging with [18F]FDG (day 1) and [68Ga]DKFZ-PSMA-11 (day 2). For each tracer, scan 1 and scan 2 were performed 45 minutes and 2 hours after the tracer injection, respectively. Mice were allowed to recover overnight from day one’s activities ([18F]FDG imaging). A topogram was acquired with a mouse prone positioned on the MulticellTM imaging bed oriented with the nose first to map out the subsequent CT acquisition. CT was performed in semi-circular mode within 5 minutes using a 50 KVp X-ray tube energy and exposure time of 300 ms; this was followed by whole-body PET acquisition (one-bed position, full field of view, 15-20 minutes) (Mediso, 2016). 3.8.4 Image Reconstruction and Analysis After image acquisition with both [18F]FDG and [68Ga]Ga-DKFZ-PSMA-11 tracer; PET and CT images were co-registered proceeded by reconstruction, (using Terra-Toma 3D ordered subset expectation maximisation to yield axial, sagittal, and coronal slices (matrix size 128 x 128). All images were evaluated using a Nuclide Nano-Scan and InterViewTM Fusion software (Mediso, 2016). The qualitative analysis included visual inspection of images reconstructed into coronal, axial and sagittal planes and as maximum intensity projection. Organ tracer concentration was analysed quantitatively using calculation of the SUV. 3.9 Biodistribution Immediately following the 2-hour image acquisition with [68Ga]Ga-DKFZ-PSMA-11 on day 2, mice were euthanised by decapitation whilst under anesthesia. Animal dissection was performed providing samples of tumours, organs and other tissue of interest, which were subsequently assayed using automatic gamma counter. The radioactivity accumulation in the organs/ tissue of interest were analysed quantitatively and expressed as a percentage of the injected dose per gram (% ID/g). 3.10 Statistical Analysis If nor stated otherwise, experiments were performed at least in triplicate, results are expressed as mean and SD of mean; outliers were identified using Grubbs Test. Power analysis is performed 37 with time dependent data sets, valued by the regression coefficient (R2). Levels of significance were determined by non-parametric comparison of two data sets. Student-t test was used to calculate p-values with p<0.05, p<0.01 and p<0.001 are the considered thresholds, respectively. 38 REFERENCES Day, J.M., Foster, P.A., Tutill, H.J., Schmidlin, F., Sharland, C.M., Hargrave, J.D., Vicker, N., Potter, B.V., Reed, M.J. & Purohit, A. 2013. STX2171, a 17beta-hydroxysteroid dehydrogenase type 3 inhibitor, is efficacious in vivo in a novel hormone-dependent prostate cancer model. Endocr Relat Cancer, 20(1):53-64. Ebenhan, T., Vorster, M., Marjanovic-Painter, B., Wagener, J., Suthiram, J., Modiselle, M., Mokaleng, B., Zeevaart, J.R. & Sathekge, M. 2015. Development of a Single Vial Kit Solution for Radiolabeling of 68Ga-DKFZ-PSMA-11 and Its Performance in Prostate Cancer Patients. Molecules, 20(8):14860-14878. Eppard, E., Homann, T., Fuente, A., Essler, M. & Roesch, F. 2017. Optimization of labeling PSMAHBED with 68Ga and its quality control systems. J Nucl Med, 58. Johnson, C.H., Manna, S.K., Krausz, K.W., Bonzo, J.A., Divelbiss, R.D., Hollingshead, M.G. & Gonzalez, F.J. 2013. Global metabolomics reveals urinary biomarkers of breast cancer in a mcf-7 xenograft mouse model. Metabolites, 3(3):658-672. Mediso. 2016. nanoScan systems for preclinical applications imaging http://www.mediso.com/products.php?fid=2,11 Date of access: 7 September 2018. Sudbrock, F., Fischer, T., Zimmermanns, B., Guliyev, M., Dietlein, M., Drzezga, A. & Schomäcker, K. 2014. Characterization of SnO2-based 68Ge/68Ga generators and 68Ga- DOTATATE preparations: radionuclide purity, radiochemical yield and long-term constancy. EJNMMI Res, 4(1):36. Varricchio, L., Migliaccio, A., Castoria, G., Yamaguchi, H., de Falco, A., Di Domenico, M., Giovannelli, P., Farrar, W., Appella, E. & Auricchio, F. 2007. Inhibition of Estradiol Receptor/Src Association and Cell Growth by an Estradiol Receptor α Tyrosine-Phosphorylated Peptide. Mol Cancer Res, 5(11):1213-1221. Yao, R., Lecomte, R. & Crawford, E.S. 2012. Small-animal PET: what is it, and why do we need it? J Nucl Med Technol, 40(3):157-165. Zhang, Y., Zhang, G.L., Sun, X., Cao, K.X., Ma, C., Nan, N., Yang, G.W., Yu, M.W. & Wang, X.M. 2018. Establishment of a murine breast tumor model by subcutaneous or orthotopic implantation. Oncol Lett, 15(5):6233-6240. 39 4 XENOGRAFT MODEL DEVELOPMENT OF BREAST AND PROSTATE CANCER Regardless of the available diagnostic and treatment management tools for breast cancer, evident morphological and molecular breast cancer diversities present challenges in the overall management of these disease. Pre-clinical investigations using imaging have shown value and the ability to measure the distribution property of new targeting agents (Clarke, 2009; Jung, 2014). This study is investigating GCP II, potentially overexpressed by solid tumours of the breast, as a possible new cancer target to allow diagnosing breast cancer by nuclear imaging. This part of the study was required to develop an estrogen-positive (MCF-7) and a triple receptor negative (MDA-MB-231) breast cancer xenograft. In parallel to this project, another study at the Department of Pharmacology, UP, is investigating the expression of GCP II in these cell lines (in- vitro). Prostate cancer (LNCaP) xenograft is used as a positive control, since it is proven to have an overexpressed GCP II (Foss et al., 2012; Kabalaskal & Demerci, 2015). The in-house establishment of the human breast cancer xenografts in mice will allow the study of the accumulation of [68Ga]Ga-DKFZ-PSMA-11 and may led to visualising the tumours in vivo by way of micro-PET/CT imaging. Therefore this section will report the results on development of MCF-7 and MDA-MB-231 athymic nude mice xenograft model and LNCaP athymic nude mice xenograft. Every successful xenograft model was later reproduced and used for micro-PET/CT imaging and ex vivo biodistribution analysis. 4.1 Development of suitable Cell Cultures Culturing MCF-7 in standard medium led to a moderate growth behaviour. The MCF-7 cell line is a human invasive breast ductal carcinoma cell line (luminal) (Soule et al., 1973), derived from a metastatic site. The MCF-7 cell line is positive for the expression of ER and PR, and negative for HER2 (Labs, 2017). The culturing of the cells under routine condition followed the expected population doubling time. The cells were cultured in-house at NWU which negated any transport that might hamper cell viability. Cell viability in batches ready for inoculation was > 95 %. MDA- MB-231 cell line was cultured as prescribed and showed rapid growth in-vitro. The MDA-MB-231 cell line is a human invasive breast ductal carcinoma (basal) cell line (Cailleau et al., 1978) derived from a metastatic site, and negative for all three hormones which are ER, PR and HER2 (Foulkes et al., 2010). The cells showed a >95% viability ahead of passaging and following transport to 40 NWU, which was expected. The LNCaP cell line is an androgen-sensitive human prostate adenocarcinoma derived from a lymph node metastasis. It is known from literature that GCP II is overexpressed in LNCaP (Lutje et al., 2015; Wustemann et al., 2016). The culturing of the cells showed expected growth behaviour and sufficient cell differentiation and viability. The cells showed a >90% viability ahead of passaging or relocation to NWU, which was considered normal. 4.2 Choice of Animals Xenograft mice models are broadly used; since they provide a micro-environment that allows for tumours growth and progression and permits the assessment of cancer biological processes (Holen et al., 2017; Holliday & Speirs, 2011). Athymic nude mice are widely used particularly in developing breast cancer xenografts model, hence in this study immune compromised intact athymic nude mice were used in developing MCF-7, MDA-MB-231 and LNCaP xenografts model. The mice were successfully bred in-house at the PCDDP Vivarium, NWU, and used at the ages of 4 to 6 and 6 to 8 weeks-old. The recorded mice weight at age 4 to 6 was 17.50 ± 1.24 g and at age 6 to 8, 20.72 ± 2.77 g. 4.3 Xenograft Model Development 4.3.1 MCF-7 Xenografts Four experiments (Ex), were conducted to develop the MCF-7 tumours xenograft model (Table 4.1). Mice were administered s.c. with ECs (3 mg/kg) mid-scapular, six days in Ex 1, and seven days in Ex 2, Ex 3 and Ex 4 before mice were inoculated. As a result of inoculating 1 x 106 cells in PBS, Ex 1 mice had poor tumours take (33%) and showed too small tumours (3.51 ± 6.11 mm3, n = 2). In Ex 2 about 67% showed MCF-7 tumours three days after cell inoculation due to the use of plain DMEM/ PBS mixture for the inoculation. Tumours volume was significantly improved to 73.63 ± 60.26 mm3 (n = 4). In Ex 3, the number of cells inoculated per mouse was increased from 1 x 106 to 2.4 x 106. The tumours take rate improved to 75% with tumours showing at three days of cell inoculation. At the end of the Ex at week 3.9, mice had higher tumours volume (91.32 ± 84.45 mm3, n = 6). Doubling the amount of cells and changing cell suspended to plain DMEM /Matrigel in Ex 4 showed a 75% tumours take rate and 82.22 ± 39.03 mm3 (n=6) tumours growth within a shorter period (2.7 weeks) compared to Ex 3. 41 Table 4.1: Experiments conducted to develop female athymic nude mice bearing MCF-7 xenografts. Ex Cell inoculum Site Formulation Tumour Tumour take take (%) 1 1 x 106 s.c. PBS 2/6 33 2 1 x 106 s.c. DMEM: PBS 4/6 67 3 2.4 x 106 s.c. DMEM: PBS 6/8 75 4 5 x106 s.c. DMEM: Matrigel 6/8 75 Footnotes s.c. = subcutaneous; PBS = phosphate buffered saline; DMEM = Dulbecco’s Modified Eagles Medium 4.3.2 MDA-MB-231 Xenografts In Ex 1, 2 and 3 mice were inoculated with 1 x 106, 2 x 106 and 5 x 106 MDA-MB-231 cells, respectively. In all the three Ex the tumours take rate ranged from 60 to 83% (Table 4.2), however, tumours growth was very sporadic, and depicted tumours growth below 60 mm3. After 15 days, regardless of the increased number of cells in Ex 2 and 3, all tumours seem to have disappeared. The mice continued to be monitored (Ex 1, 2 and 3) from week 3.7 to 4.8 weeks, with no sign of tumours. Ex 3 mice were inoculated with cell suspension 5-fold of Ex 1, however, a trend of poor growth continued. Conditions used for Ex 4 improved the tumours take rate to 100% (Table 4.2).and significantly increased the tumours volume to 167.62 ± 73.46 mm3. From two days post inoculation, the tumours in these mice were palpable and measurable. Tumours growth was measured and recorded up to week 3. These mice were inoculated with 5 x 106 cells, re- suspended into PBS with Matrigel, into the mammary fat pad area for better nutrition. 42 Table 4.2: Experiments conducted to develop female athymic nude mice bearing MDA-MB-231 xenografts. Ex Animal age Cell Site Formulation Tumour Tumour (weeks) inoculum take take (%) 1 6-8 1 x 106 s.c. PBS 3/5 60 2 6-8 2 x 106 s.c. DMEM:PBS 5/6 83 3 6-8 5 x 106 s.c. DMEM:PBS 2/3 67 4 4-6 (n=1), 5 x 106 m.f.p. PBS: Matrigel 5/5 100 6-8 (n=4) Footnotes s.c. = subcutaneous; m.f.p. = mammary fat pad; PBS = phosphate buffered saline; DMEM = Dulbecco’s Modified Eagles Medium 4.3.3 LNCaP Xenografts Three times the LNCaP cells inoculations were done in female athymic nude mice without TCs supplementation. Ex 1, (1 x 106 in PBS), Ex 2 (1 x 106 in plain DMEM/ F12: PBS (1:1)) and Ex 3 (2 x 106 in PBS/ Matrigel (1:1)) LNCaP cells per mouse were injected s.c. In all the three Ex mice failed to develop tumours throughout 3 to 4.5 weeks of monitoring post the inoculation. Only two mice were inoculated in each of the three Ex. In Ex 4 a pre-treatment (three days prior) was used with TCs in the mid-scapular s.c. on two female athymic nude mice. Subsequently the mice were inoculated with the LNCaP cells (2 x 106 cells PBS/ Matrigel (1:1)) and maintained on the hormone weekly. In parallel, one male athymic nude mouse was inoculated with LNCaP cells (2 x 106 cells PBS/ Matrigel (1:1)) without TCs. LNCaP female athymic nude mice xenografts failed to develop the tumours over 6 weeks post inoculations. The male mouse bearing LNCaP xenograft developed a tumours after 4 weeks post inoculation; the LNCaP tumours grew rapidly, from 268.71 mm3 to 1823.5 mm3 over three day. Following dissection, the tumor was excised to determine the cause of the rapid growth. The tumours appeared bloody and the tumours rapid growth was suspected to be due to a cyst that appeared to be around the tumours. The ex vivo tumours measured 1020.4 mm3. 43 4.4 Recommended Procedures to Warrant Tumours Growth for Imaging Purposes The focus of this section was to develop female athymic nude mice xenografts bearing MCF-7 tumours and MDA-MB-231 tumours, and female and/or male athymic nude mice xenografts bearing LNCaP tumours. Figure 4.1 gives a recommendation to follow regarding optimal tumours growth. Figure 4.1: The MCF-7 F (female) ANM (athymic nude mice) (Xen) xenograft, MDA-MB-231 female athymic nude mice xenograft, and LNCaP M (male) athymic nude mice xenograft model development. 4.5 Discussion 4.5.1 Athymic Nude Mice MCF-7 Xenograft There were challenges regarding the development of some of the xenografts model (Holen et al., 2017; Mullen et al., 1996). Immune incompetent athymic nude mice are broadly utilised to develop xenograft model (Jung, 2014), however it was suspected that using these animals at 6 to 8 weeks old, maturity of the lymphocytes and increasing activity of the natural killing cells as the animal ages brings limitations to this strain. (Puchalapalli et al., 2016). The research had to consider 44 some conditions that could enhances the tumours incidence, growth and take rate as the study progressed. In the study the researcher developed ER-positive human breast cancer cell line (MCF-7) and TNBC cell line (MDA-MB-231) xenograft model to investigate the molecular mechanism accumulation of [68Ga]Ga-KZFZ-PSMA-11. The MCF-7 cell line is estrogen dependent, hence nude mice, prior to cell inoculation, were supplemented with ECs to stimulate in vivo tumours growth (Dall et al., 2015; Mullen et al., 1996). Many studies have made use of estradiol pellet (0.72 mg), which are reported to have been successful in xenograft model development. A study done by Dall et al. (2015) has shown that mice suffer a great deal of urosepsis. This imposes challenges regarding reference tracer ([18F]FDG) accumulation in areas with inflammation due to urosepsis side effect of the estradiol pellet. [18F]FDG is a glucose analogue, which provides metabolic activity based on the increased cellular demand for glucose due to the elevated glycolysis in cancer cells (Association, 2008; Vercher-Conejero et al., 2015). Furthermore there are considerable accumulation levels of [18F]FDG in non-cancer tissue (inflammation and or infection areas) and also in physiological processes (energetic tissues such as brain, muscles and also in brown fat tissue) (Vercher-Conejero et al., 2015). In this study the researcher have made use of the injectable ECs (3mg/kg) that were pre-injected and further injected weekly into the mice throughout the Ex and resulted in variable tumours take and size (Table 4.1). In this study, the tumours growth and take did not quite reflect the trend of the studies done by Behzadi et al (2015) and Johnson et al (2013). Injectable estradiol supplement has been studied. One study investigated the effect of injectable estradiol Valerate (2mg/kg) on different concentration of MCF-7 cells (5 x 106, 10 x 106, 20 x 106) to grow tumours and the rate of tumours growth, and the findings confirmed that this form of estradiol supplements can be used as a substitute to successfully promote MCF-7 tumours growth and rate in developing a xenograft mice model (Behzadi et al., 2015). In another study, athymic nude mice were injected with MCF-7 cells only compared to the group that was injected with both MCF-7 cells and ECs. At the end of week 10, mice injected with both MCF-7 cells and ECs had formed tumours with the average tumours weight of 700 ± 240 mg. Tumour weight was calculated using a prolate ellipsoid equation, assuming a density of 1 g/cm3. In the group that was injected with MCF-7 cells only, mice showed a tiny lump at the injection side, which disappeared over time (Johnson et al, 2013).In this study the MCF-7 cell concentration was increased to 5 x 106, (Table 4.1) and Ex 3 showed a slight improvement of tumours take after the mice were inoculated with 2 .4 x 106 of cells. However, the tumour growth and progression was poor. In Ex 4 mice were able to develop tumours in just over 45 two weeks after inoculations after increasing the cells to 5 million and introducing matrigel to the cell formulation. matrigel is an extracellular basement membrane matrix, rich in extracellular matrix proteins and has been reported to improve the tumours take and growth in establishing xenograft model of various cancer cells (Mullen et al., 1996). 4.5.2 MDA-MB-231 Athymic Nude Mice Xenograft MDA-MB-231 is a human TNBC cell line that grows easily in vitro or in vivo without hormonal supplementation (Kaza et al., 2018). MDA -MB-231 cells were inoculated s.c. without matrigel; there was tumours onset as early as three days and after day 15, the tumour stopped growing and disappeared. Several conditions were considered to increase the chance of developing the MDA-MB-231 xenograft successfully. Injection mode and site were changed and cells were inoculated into the m.f.p. in the abdomen. A study comparing the potential injection site that will result in improved breast cancer tumour incidence, tumour growth e.tc between the thorax and abdomen region and observed that mice injected into the abdominal region developed tumours compare to the thorax as a site of injection. The average tumours growth was bigger in the abdomen region compared to the thorax (Fleming et al., 2010). The cells was inoculated via non- invasive procedure into the m.f.p. in the abdomen to eliminate any chance of inflammatory response as a result of the invasive procedure (Fleming et al., 2010) which could have a negative effect when the mice undergo [18F]FDG in vivo micro PET/CT imaging. TNBC cells injected orthotopically have shown tumours growth significantly greater than when injected s.c., furthermore tumours are well vascularised (Fleming et al., 2010; Jung, 2014). In Table 4.2, in Ex 4, after the MDA-MB-231 cells were inoculated non-invasive orthotopically into the fourth m.f.p, with 5 x 106 cells re-suspended in PBS with Matrigel, all the mice injected by week 3 showed tumours and the average tumours size measured 167.62 ± 72.46 mm3. 4.5.3 LNCaP Athymic Nude Mice Xenograft LNCaP cell line is an androgen-sensitive human prostate adenocarcinoma cell line. Horoszewics et al. (1983) were able to compare the LNCaP tumours appearance and tumours incidence in both genders of athymic nude mice. Both male and female athymic nude mice were of the same age as used in this study, the difference was that female mice were ovariectomised and supplemented with 2 mg pellet of TCs prior to the cell inoculation, the mice had the hormone pellet throughout the experiment. They reported that tumours appearance occurred just above three and half weeks in males compared to almost five weeks in the females; a low tumours 46 incidence in female compared to male athymic nude mice. In Ex 1, Ex 2 and Ex 3 of developing LNCaP female athymic nude mice xenografts the researcher suspected failure to grow LNCaP tumours cells in female athymic nude mice was related to the absence of the growth stimulating androgen hormone. Conducting further Ex in developing the model, TCs was administered into the female athymic nude mice. Since LNCaP is an androgen-sensitive human prostate adenocarcinoma cell line, growth is dependent on androgen hormone in the environment (male athymic nude mouse), the lack thereof in female mice will compromise tumours growth (Horoszewicz et al., 1983). LNCaP male mice xenografts developed a tumour similarly to what the authors reported. The LNCaP female athymic nude mice, even after supplemented with TCs, failed to develop the tumour post six weeks of animal monitoring. LNCaP cell culture was very sporadic, and was a great limitation in developing the model. This study Ex used injectable TCs and it was not established at which TCs dosage or level female athymic nude mice inoculated with LNCaP cells would develop the LNCaP tumours. 4.6 Conclusion Both MCF-7 and MDA-MB-231 female athymic nude mice xenograft models were successfully developed. After numerous challenges with LNCaP xenograft model development, the researchers were able to grow a tumour in one male mouse. Further work is needed to achieve this model before subjecting it for nuclear imaging procedures. 47 REFERENCES International Atomic Energy Association. 2008. A guide to clinical PET in oncology: Improving clinical management of cancer patients. https://www- pub.iaea.org/MTCD/Publications/PDF/te_1605_web.pdf Date of access12 August 2017. Behzadi, R., Fattahi, S., Momtaz, M.R., Kavoosian, S., Asouri, M. & H., A.-N. 2015. Injectable Estradiol Valerate, as a Substitute for Estradiol Pellets in Breast Cancer Animal Model. Inter Biological and Biomed Journal, 1(1):35-38. Cailleau, R., Olive , M. & Cruciger, Q.V. 1978. Long-term huma breast carcinoma cell lines of metastatic origin: preliminary characterization. In Vitro, 14(11):911-915. Clarke, R. 2009. The role of preclinical animal models in breast cancer drug development. Breast Cancer Res, 11 Suppl 3(Suppl 3):S22-S22. Fleming, J.M., Miller, T.C., Meyer, M.J., Ginsburg, E. & Vonderhaar, B.K. 2010. 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Theranotics, 6(8). 49 5 RADIOLABELLING OF [68GA]GA-DKFZ-PSMA-11 [68Ga]Ga-DKFZ-PMSA-11 may target tissues that have overexpressed levels of GCP II, which may be detected and visualised by positron emission tomography/computed tomography (PET/CT) imaging. Therefore, the focus of this section was on the development of a [68Ga]Ga- DKFZ-PSMA-11 radiolabelling procedure suitable for safe tracer administration into tumour- bearing mice. As part of a previous study, a general [68Ga]Ga-DKFZ-PSMA-11 radiolabelling protocol was achieved for human application (Ebenhan et al., 2015b). Herein we report on further optimisation of this radiosynthesis considering the following limitations for animals use: restricted injection volume, physiological pH and isotonicity, optimal specific activity, and the lower tracer molarity compared to humans (Vanhove et al., 2015). To yield 68Ga-radioactivity, a fractionation method was adopted to see how an optimal amount of 68Ga-radioactivity from a generator may be collected in 1.2 ml, opting for a 0.6 N hydrochloric acid (HCl) concentration that falls into the suggested range (0.1-1 M HCL) (Sudbrock et al., 2014). 5.1 Results and Discussion 5.1.1 Choice of DKFZ-PSMA-11 DKFZ-PSMA-11 is frequently used as a [68Ga]Ga-DKFZ-PSMA-11-radiolabelled PET tracer for diagnosis of PSMA/GPC II-expressing cancer. As a urea-based inhibitor of PSMA-11, it is conjugated to the acyclic, metal chelating agent HBED-CC, which will allow complexation with 68Ga (Eder et al., 2012). DKFZ-PSMA-11 was chosen as the targeting agent as there are defined radiolabelling methods to prepare the tracer with high specific activity and purity for application in humans (Ebenhan et al., 2015a; Eder et al., 2012); however, refinements to these methods are required to make them suitable for preclinical application in small animals. 5.1.2 Choice of Radioisotope Gallium-68 was considered suitable for imaging purposes as about 89% will decay by β+ emission and the remaining 11% by electron capture. The 89% β+ emission is further divided into two: 87.7% will decay by pure β+ emission with maximum energy of 1.899 MeV, which enable PET imaging and the residual and 1.2% will ultimately decay to the ground state by gamma emission with an energy of 1.077 MeV (Conti & Eriksson, 2016). 68Ga-radioactivity can be conveniently be made available by elution of a 68Ge/68Ga-generator. 50 5.1.3 Elution of Gallium-68-Radioactivity for Radiolabelling The two generators loaded with either 1110 or 1850 MBq were used in-house to provide the 68Ga- radioactivity to label with DKFZ-PSMA-11. The 1850 MBq 68Ge/68Ga generator (G1) was mostly used during phase 1: the establishment of the [68Ga]Ga-DKFZ-PSMA-11 radiolabelling procedure to achieve a formulation suitable and safe for administration to mice. Once the radiolabelling procedures were established, the 1110 MBq 68Ge/68Ga generator (G2) was used for routine radiolabelling of [68Ga]Ga-DKFZ-PSMA-11 to prepare doses for micro-PET/CT imaging. The radioactivity (200 MBq and 1500 MBq (n = 40 elutions)) for radiolabelling was eluted in a 0.6 M ultrapure HCl solution. Due to the different amount of 68Ge loaded on the two generators, the elutable activity (n = 5; G1 and G2) at three months post manufacturing was significantly higher for G1 (1341 ± 25 MBq) than for G2 (935 ± 13 MBq). 5.1.4 Repeated Generator Elution The generator can be eluted up to 300 times over a nine-month period before the quality of the eluate deceases (Decristoforo et al., 2007). Getting to the equilibrium, i.e. the maximal amount of 68Ga-radioactivity, will take 10 to 12 hours. For the maintenance of the system, there has been the practice of pre-eluting the generator on a daily basis, and in preparation for the experiments, the generator is pre-eluted 2 to 3 hours before “fresh 68Ga-radioactivity for use” is eluted. This procedure resulted in an improved quality of eluate, but of lower specific activity as only 75 to 80% “fresh” 68Ga was generated during this time compared to letting the generator remain un- eluted overnight. 5.1.5 Eluate Fractionation Typically, the elutable radioactivity is yielded in 5-10 ml but for this study a method was adapted to collect it in 0.9-1.2 ml (Ebenhan et al., 2015a). This eluate fractionation resulted in collecting a slightly lower 66-75% of elutable activity but the marked reduction in volume improved the activity concentration by 4 to 5 times (610 ± 25 MBq/ml) as compared to (139 ± 5 MBq/ml routine elution) from the same generator. 5.1.6 Testing [68Ga]Ga-DKFZ-PSMA-11 Radiolabeling Parameters Previously (Ebenhan et al., 2015b) it was suggested to use a concentration of 5 nmol/ml DKFZ- PSMA-11 to perform radiosynthesis with <5% free 68Ga, however the high volume and salt content (325 µg NaOAc /kit) had to be investigated for this study. Firstly, a full-scale radiolabelling (i.e. 51 using all elutable radioactivity collected in 2 ml, using 10 nmol DKFZ-PSMA-11 (4.35 µM) dissolved in 0.3 ml of 2.5 M NaOAc at pH 2 was performed; ITLC analysis showed low radiolabelling efficiencies (Appendix A, No 1 and 2) due to insufficient pH adjustment. Reducing the DKFZ-PSMA-11 molarity to 3.82 µM and increasing the buffering agent improved radiolabelling to approximately 77% (at pH 4, Appendix No 3a/b); however, further reduction of the molarity (pH 4) made the radiolabelling variable (33% and 60% radiolabelling efficiencies in Appendix No 4a/b). More robust radiolabelling was achieved keeping the pH between 4.5 - 5 and the peptide molarity at 3.5 - 3.88 µM (68.6 - 72.2%, n = 4; Appendix No 5 - 8). Further attempts to lower the peptide molarity (2.91 - 3.18 µM) to add less buffering agent (resulting in pH 3.5 - 4) did not improve any of the radiolabelling (Appendix No 9 - 12). Repeated and more standardised radiosynthesis was performed using 1.2 ml 68Ga-radioactivity with 3.5 nmol DKFZ- PSMA-11 containing buffer with pH adjusted to 4 - 4.5, while the highest radiolabelling efficiencies occurred when heated for 5 to 10 minutes at >95°. 5.1.7 Testing Quality of Radiolabelling Chromatography was required to ascertain radiochemical purity and stability of [68Ga]Ga-DKFZ- PSMA-11. As previous reports (Ebenhan et al., 2015a) concluded, ITLC-silica gel (SG) paper should be used instead of aluminium-based silica gel stationary phase (TLC) to decrease the time required for analysis. Radioactivity was counted and displayed as a radio-chromatogram. The change of stationary phase did not compromise the analysis and quantification of [68Ga]Ga-DKFZ- PSMA-11 as summarised in Figure 5.1. The results from using ITLC-SG together with a known mobile phase (Methanol/Saline 20/80 v/v) to differentiate [68Ga]Ga-DKFZ-PSMA-11 from free 68Ga-radioactivity is shown in Figure 5.1A (retention profile of [68Ga]GaCl3; Rf = 0.05-0.01) and Figure 5.1B (retention profile of pure [68Ga]Ga-DKFZ-PSMA-11; Rf = 0.75-0.90). The difference in the Rf values clearly shows [68Ga]Ga-DKFZ-PSMA-11 sufficiently interacting with the mobile phase to separate it from the unreactive [68Ga]GaCl3. Samples analysed at either room temperature or heated can be compared for the success of radiolabelling as displayed in Figure 5.1C/D. 5.1.8 [68Ga]Ga-DKFZ-PSMA-11 Purification Post radiolabelling purification was done by solid phase extraction (SPE): [68Ga]Ga-DKFZ-PSMA- 11 was loaded onto the Sep-Pak C18-light cartridge, washed with saline and then desorbed off the cartridge using a mixture of EtOH in saline (Figure 5.2); which correlated well with (R2 = 52 0.957). Purification led to a product of 95 -100% radiochemical purity (Figure 5.1 E). Further refinements were done with the aim of obtaining a more concentrated product activity, post purification in the smallest-possible volume. A previous published [68Ga]Ga-DKFZ-PSMA-11 labeling method (Ebenhan et al., 2015a) has been the bases for the process of modifying and optimising this compound to make it suitable for use in mice. Purification using SPE Sep-Pak C-18 light cartridge was implemented routinely as a crucial step to obtain concentrated [68Ga]Ga-DKFZ-PSMA-11 activity (Table 5.1). The yield of a radiolabelled compound was around 80% following a routine cartridge stripping procedure with 1 ml of 50% ethanolic saline (n = 3). However, the final product required dilution to get to < 5% EtOH in the formulation (Table 5.1, No 1 - 3). Testing of a 15% ethanolic solution (Table 5.1, No 4 - 5) and a volume of 0.5 ml (n = 2) 25% ethanol concentration in 0.5 ml (n = 2) caused the yields to vary inefficiently between 33-77%. A slight improvement was achieved by using 25% ethanolic saline, but this restricts the maximum volume to 0.5 ml (applied in small fractions: 0.1 ml/0.2 ml to the SPE cartridge) (Table 5.1, No 6 - 7). After repeated radiolabelling and purification with 25% ethanolic saline solution, the volume was reduced to 0.3 ml (3 X 0.1 ml – 15 sec incubation/0.1 ml) (Table 5.1, No 8 - 13), and it was concluded that stripping procedure recovered enough [68Ga]Ga-DKFZ-PSMA-11 in the smallest practicable volume (46 – 72% n = 6). Thereafter, the EtOH content was removed (as best as possible) by evaporations. ITLC analysis (Figure 5.1E/F) showed that this step did not affect the purity of the product. The final product was reconstituted with at least 0.3 ml of saline to prepare suitable injections. This product formulation is warranted for up to three mice, considering injection interval of 20 to 25, which was deemed sufficient. It should be noted that the scope of this optimised radiolabelling method of [68Ga]Ga-DKFZ-PSMA- 11 for application in mice imaging has lower product yield compared to the former method established. The radioactivity losses to SPE and reaction vials were accepted, as the total SPE recovery volume and the activity concentration were the limiting factor for preclinical application. 53 Table 5.1: Summary of the [68Ga]Ga-DKFZ-PSMA-11 purification Ex E / S Total volume Loaded SPE Empty SPE Product Yield Product (%) for SPE (ml) Unit (MBq)** Unit (MBq) (MBq)* Recovery (%)* 1 50 1.0 310.6 35.2 251.7 80.0 2 50 1.0 386.2 18.7 307.8 79.7 3 50 1.0 555.0 25.1 440.3 79.3 4 15 0.5 143.9 34.4 109.5 76.7 5 15 0.5 130.2 72.5 42.9 33.1 6 25 0.5 208.2 33.3 162.8 78.3 7 25 0.5 73.4 24.6 43.7 59.9 8 25 0.3 218.9 33.3 157.6 72.2 9 25 0.3 129.8 18.5 89.0 68.7 10 25 0.3 195.6 29.6 126.0 64.3 11 25 0.3 474.2 172.8 220.9 46.6 12 25 0.3 427.3 182.0 196.1 45.9 13 25 0.3 118.4 14.8 85.1 71.9 Footnotes A list of single experiments is presented, dashed lines emphasised the stages in the development. E/S (%) = percentage of an ethanolic saline solution (v/v) used; SPE = solid-phase extraction (C18 SepPak light); *) all purifications took about 7-9 min **) total radioactivity trapped after removal of free 68Ga-species; 54 Figure 5.1: Representative radio-chromatograms showing 68Ga-radioactivity showing counts related to free 68Ga-species retention at the origin (OR) and/or counts for [68Ga]Ga-DKFZ-PSMA- 11 at the solvent front (SF) following 5-7 min exposure of the ITLC-SG strips to the same mobile phase (Methanol/Saline 80/20 v/v). Strips were incubated as follows: (A) [68Ga]GaCl3, (B) HPLC verified 100% pure [68Ga]Ga-DKFZ-PSMA-11, (C) radiolabelling mixture incubated for 5 min (3.5 nmol DKFZ-PSMA-11, pH 4, 95°C), (D) radiolabelling mixture incubated for 2 min (3.5 nmol DKFZ-PSMA-11, pH 4, room temperature), (E) SPE-purified [68Ga]Ga-DKFZ-PSMA-11 before evaporation of ethanol and (F) sample E after evaporation of ethanol. 100 80 60 40 20 0 1 2 4 8 16 32 64 Percentage Ethanol /Saline (v/v) Figure 5.2: Increasing concentration of ethanolic saline applied to recover the purified [68Ga]Ga- DKFZ-PSMA-11 from a Sep-Pak light C-18 cartridge (n=4). 55 % RCY [68Ga]Ga-DKFZ-PSMA-11 5.1.9 Preparation of a safe-to administer [68Ga]Ga-DKFZ-PSMA-11 Formulation The optimised radiolabelling approach (Figure 5.3) was practised for its robustness and repeatability. The results are summarised in Table 5.2. Figure 5.3: Graphical overview of a standardised [68Ga]Ga-DKFZ-PSMA-11 radiosynthesis, 68Ge/68Ga generator elution (blue arrow), 68Ga-radioactivity eluted is added into the DKFZ- PSMA-11 kit immediately (red arrow). 56 Table 5.2: Summary of results from repeated [68Ga]Ga-DKFZ-PSMA-11 radiolabelling and preparation of the safe-for-administration formulation (n ≥3) Parameter Specification Radiosynthesis yield ndc (MBq) 165 ± 70 Radiolabelling efficiency dc (%) 73 – 89 Losses to material (SPE unit, vial, syringe) dc (%) 10 – 19 Final product volume (ml) 0.28 – 0.33 Molar activity (MBq/ nmol) 29 – 64 Radiochemical purity (ITLC) - crude (%) > 60 - SPE-purified (%) > 97 Percentage SPE recovery (25 % E/S) 69 – 97 Time (68Ga-elution final product) (min) 46 – 60 Footnotes ndc: not decay-corrected; dc: decay-corrected; E/S: ethanol –saline mixture 25/75 v/v; ITLC =Instant thin- layer chromatography 5.2 Conclusion This section reports on the modification and optimisation of a [68Ga]Ga-DKFZ-PSMA-11 radiolabelling procedure. [68Ga]Ga-DKFZ-PSMA-11, in this final formulation was safe and suitable for administration into mice to perform non-invasive micro-PET/CT imaging. 57 REFERENCES Conti, M. & Eriksson, L. 2016. Physics of pure and non-pure positron emitters for PET: a review and a discussion. EJNMMI Physics, 3(1):8. Decristoforo, C., Knopp, R., Von Guggenberg, E., Rupprich, M., Dreger, T., Hess, A., Virgolini, I. & Haubner, R. 2007. A fully automated synthesis for the preparation of /ga-68-labelled peptides. Nucl Med Communications, 28(11). Ebenhan, T., Vorster, M., Marjanovic-Painter, B., Wagener, J., Suthiram, J., Modiselle, M., Mokaleng, B., Zeevaart, J.R. & Sathekge, M. 2015a. Development of a Single Vial Kit Solution for Radiolabeling of 68Ga-DKFZ-PSMA-11 and Its Performance in Prostate Cancer Patients. Molecules, 20(8):14860-14878. Sudbrock, F., Fischer, T., Zimmermanns, B., Guliyev, M., Dietlein, M., Drzezga, A. & Schomäcker, K. 2014. Characterization of SnO2-based 68Ge/68Ga generators and 68Ga- DOTATATE preparations: radionuclide purity, radiochemical yield and long-term constancy. EJNMMI Res, 4(1):36. Vanhove , C., Bankstahl, J.P., Kramer, S.D., Visser, E., Belcari, N. & Vandenberghe, S. 2015. Accurate molecular imagign of samll animals taking into account aniaml models, handling, anaesthesia, quality control and imaging system performance. EJNMM Physics, 2(31):2-25. 58 6 PRE-CLINICAL IMAGING OF GCP II EXPRESSION IN BREAST CANCER USING [68GA]GA-DKFZ-PSMA-11 MICRO-PET/CT GCP II is a possible new cancer target to allow diagnosing of breast cancer by nuclear imaging. A clinical study by Sathekge et al. (2015 and 2016) reported on the PSMA/GCP II imaging agent [68Ga]Ga-DKFZ-PSMA-11 showing accumulation in breast cancer cells. In this section we used athymic nude mice bearing xenografts of an oestrogen receptor-positive human breast cancer cell line (MCF-7) and a triple receptor negative breast cancer cell line (MDA-MB-231) as experimental models. These xenograft models were initially developed as described in Chapter 4, and were herein used for [68Ga]Ga-DKFZ-PSMA-11-micro-PET/CT imaging, subsequently followed by biodistribution analysis. [18F]FDG-micro-PET/CT was used as a general (unspecific) procedure (Füger et al., 2006) to visualise the tumours by way of elevated glycolysis. Tracers were administered intravenously and mice were anaesthetised allowing high-resolution images. 6.1 Results and Discussion 6.1.1 Animal Preparation and Tracer Administration Mice showed suitable MCF-7 and MDA-MB-231 tumour xenografts and were subjected, at week 9 to 10, to consecutive [18F]FDG and [68Ga]Ga-DKFZ-PSMA micro-PET/CT imaging. Mice health checks and tumour measurement were recorded on a daily basis while animals were at Necsa. There were no significant differences in body weight between the two animal models at time of imaging however, averages for MCF-7-tumour-bearing mice were approximately 3 g lighter than MDA-MB-231-tumour-bearing mice due to being two weeks older in age. Results for injected doses are summarised in Table 6.1. Total [18F]FDG activity dosage per mouse to MCF-7 and MDA-MB-231 xenografts were 7 ± 2 MBq and 11± 2 MBq, respectively. Blood glucose levels at 4 to 6 hours of fasting were similar for the two groups (MCF-7 < MDA-MB-231; p>0.05). MCF-7- bearing mice received insignificantly less [18F]FDG (0.4 ± 0.1 MBq/g) than MDA-MB-231-bearing mice [18F]FDG (0.6 ± 0.2 MBq/g). The starving period resulted in slightly lower blood glucose levels in MCF-7-tumour bearing mice than in MDA-MB-231-tumour bearing mice. No particular mice preparation was followed leading to the [68Ga]Ga-DKFZ-PSMA-11 injection; MCF-7 and MDA-MB-231 xenografts [68Ga]Ga-DKFZ-PSMA-11 dosage per mouse was 14 ± 4 MBq and 14 ± 2 MBq, respectively. Similar [68Ga]Ga-DKFZ-PSMA-11 doses were administered to both MCF- 7-bearing mice (0.8 ± 0.2 MBq/g) and MDA-MB-231-bearing mice (0.7 ± 0.2 MBq/g). All mice 59 tolerated the two injections (injected volumes were kept similar) without any acute reactions. 6.1.2 Image Acquisition Micro-PET/CT imaging was performed as outlined in Chapter 3 (3.7.3 - 3.7.4). The anesthesia procedure required for the PET/CT scans (Isoflurane inhalation: 5% for induction, 2% for maintaining anesthetic plane) was tolerated well by all animals. The CT scan performance (parameters: acquisition mode - semi-circular, X-ray tube energy - 50 KVp, exposure time - 300 ms) resulted in sufficient CT images, which could be analysed further (CT-based SUV analysis). PET images were acquired for up to 15 minutes at 45 minutes and at 2 hour post [18F]FDG administration due to logistical challenges. Both [68Ga]Ga-DKFZ-PSMA-11 PET/CT image acquisitions were performed for 15 minutes at 45 minutes and 2 hour post [68Ga]Ga-DKFZ-PSMA- 11 administration, respectively. The protocols for animal positioning, image acquisition and reconstruction algorism used (6 cm field of view, 1 bed position, scatter correction, filter: cosine, 3D-OSEM, Tera-Toma) were sufficient to provide high-quality whole-body images. Table 6.1: Comparison of parameters addressed for athymic nude mice xenograft for [18F]FDG and [68Ga]Ga-DKFZ-PSMA-11 micro-PET/CT imaging. Mouse model MCF-7 (n=4) MDA-MB-231 (n=5) 18 Parameter measured [ F]FDG [ 68Ga]Ga-DKFZ- [18F]FDG [68Ga]Ga-DKFZ- PSMA-11 PSMA-11 Mice weight (g) 18 ± 1 18 ± 1 21 ± 3 21 ± 2 Tumour volume (mm3) 136 ± 100 167 ± 83 150 ± 31 191 ± 19 Blood glucose (mmol/l) 4 ± 1 N/A 5 ± 2 N/A Activity/mouse (MBq) 7 ± 2 14 ± 4 11 ± 2 14 ± 2 Dose volume (µl) 125 ± 50 142 ± 20 125 ± 50 113 ± 25 Injected dose (MBq/g) 0.4 ± 0.1 0.8 ± 0.2 0.6 ± 0.2 0.7 ± 0.2 Footnotes Parameter presented as mean ± SD 60 6.1.3 PET/CT Imaging and Analysis Qualitative assessment of the MCF-7 and MDA-MB-231 xenografts, using the corrected maximum intensity projection PET/CT images, displayed different whole body activity concentration (Figure 6.1 & Figure 6.2) for [18F]FDG compared to [68Ga]Ga-DKFZ-PSMA-11 as expected due to the different nature of both the tracers. 61 Figure 6.1: PET/CT MIP images of the same MCF-7-female athymic nude mouse day 1- [18F]FDG imaging at 45 minutes (A) and 2 hour (C); and day 2: [68Ga]Ga-DKFZ-PSMA-11 imaging at 45 min (B) and 2 hour (D). The white arrow indicated the tumour. 62 Figure 6.2: PET/CT MIP images of the same MDA-MB-231 female athymic nude mouse xenograft day 1: [18F]FDG imaging at 45 minutes (E) and 2 hour (G); and day 2: [68Ga]Ga-DKFZ- PSMA-11 imaging at 45 min (F) and 2 hour (H). The white arrow indicates the tumour. 63 6.1.3.1 [18F]FDG Images Dual time point and delayed time point imaging have been utilized for the purpose of distinguishing between cancerous, inflammation and normal physiological processes (Houshmand et al., 2014). Studies have shown an increase [18F]FDG accumulation in cancerous cells on a second acquisition point time in comparison with the initial scan (baseline). On the other hand with inflammation there is equal or decrease [18F]FDG accumulation on the second acquisition point time in comparison with the baseline scan. Cancer cells and inflammation cells are known to contain low and high levels of glucose-6-phosphatase enzyme respectively. Glucose-6-phosphatase enzyme maybe responsible for dephosphorylating of FDG-6- phosphotase. Hence the distinct continuous accumulation of FDG-6-phosphatase in cancer cells over time compared to decreased accumulation in inflammation cells (Houshmand et al., 2014; Kumar et al., 2005). MCF-7 xenografts images taken at 45 minutes (A) and at 2 hours (C) (Figure 6.1) post administration of [18F]FDG injection could not visualise the MCF-7 tumour. Figure 6.2 demonstrated [18F]FDG accumulation in the MDA-MB-231 tumour as early as 45 minutes (E). The MDA-MB-231 tumour was also localised on the delayed image (2 hour) (G), with less tracer intensity compared to the 45-minute image. A decrease in [18F]FDG accumulation in the delayed MDA-MB-231 image (2 hour) (G) could be due to the presence of inflammation cells within the tumour. There are other in vivo micro-PET/CT imaging studies (Heidari et al., 2015; Li et al., 2016) done where [18F]FDG is used as a reference tracer. MCF-7 and MDA-MB-231 athymic nude mice xenografts were imaged with [18F]FDG-micro-PET/CT and indeed, both tumours demonstrated [18F]FDG accumulation. None [18F]FDG accumulation in the MCF-7 tumour could possibly be due to lack of glucose metabolism activity (Vercher-Conejero et al., 2015). However, for both MFC-7 and MDA-MB-231 xenografts imaging (Figure 6.1 & Figure 6.2) there was variable [18F]FDG accumulation in the normal physiological organs, such as the brain, lung, stomach, heart intestine, kidneys (A, C, E and G). Optical inspection revealed that the background activities significantly cleared in images C/G compared to these in the images A/E. During animal handling, tracer injection and image acquisition, some limitations occurred such as challenges considering the intravenous injection, particularly technically challenging on day one when [18F]FDG-imaging was performed; such data sets could not be included in the study but the minimum statistical requirement was not compromised. 64 6.1.3.2 [68Ga]Ga-DKFZ-PSMA-11 Images Tumour angiogenesis is the formation of new blood vessels supplying the tumour from resident endothelial cells. It is crucial for the development, survival and progression of solid tumours (Nguyen et al., 2016). Angiogenesis occurs from balance between pro-and anti-angiogenic factors. It is studied that GCP-II is selectively expressed in endothelial cells of neo-vasculature of solid tumors but not endothelial of normal vasculature. There was no MCF-7 and MDA-MB-231 tumour detected or visualised with [68Ga]Ga-DKFZ-PSMA-11 in both images (Figure 6.1B & Figure 6.2F) at 45 minutes and (Figure 6.1D & Figure 6.2H) at 2 hour post the tracer injection. Morgenroth A et al. (2019) were able to demonstrate in vivo accumulation of [68Ga]Ga-DKZF- PSMA-11 in MDA-MB-231 tumours. However, there was no positive GCP II expression reported on the endothelial and MCF-7 tumour cells (Morgenroth et al., 2019). The scope of a parallel project performed at University of Pretoria, Department of Pharmacology has confirmed these findings (results are subject to another MSc manuscript). Morgenroth et al. (2019) further analysed the MDA-MB-231 tumour with endothelial cell marker specific antibody (CD31) and a PSMA-specific antibody and demonstrated blood vessels tumour associated and also interestingly the tumor itself shown expression of PSMA on the endothelial. We suspect the absence of [68Ga]Ga-DKFZ-PSMA-11 accumulation in the MDA-MB-231 tumour could be due to the lack of viable angiogenic activity of the tumour. The purpose of the study was not to evaluate tumour angiogenic status. However non-invasive nuclear medicine studies make use of arginine- glycine-aspartic acid based tracer to investigate angiogenesis characteristics of the tumour. Image B, D, F and H showed high accumulation of [68Ga]Ga-DKFZ-PSMA-11 in the bladder, and in both the left and right kidney normal expected biodistribution of the tracer. All mice underwent [68Ga]Ga-DKFZ-PSMA-11 micro-PET/CT imaging, which demonstrated very high accumulation of the tracer in the kidneys and the bladder compared to the other organs. The kidneys accumulation of the tracer was partly due to GCP II receptor expression in the mouse kidney proximal tubules, but mainly due to tracer excretion through the kidneys, consequently, very high amounts of radioactivity were expected and were previously reported for the kidneys in early on- set imaging and delayed imaging (Ray Banerjee et al., 2016). This was confirmed by ex vivo biodistribution analysis (Figure 6.3). However, high tracer accumulation in the bladder is prominently seen on PET/CT images, contrary to the ex vivo biodistribution particularly of the MDA-MB-231 xenografts. A high tracer accumulation was expected in the bladder due to rapid clearance of the tracer from the kidneys. Furthermore, typical tracer accumulation was noted in the salivary glands ([68Ga]Ga-DKFZ-PSMA-11 images), and the spleen as part of the normal 65 biodistribution pattern for [68Ga]Ga-DKFZ-PSMA-11. 6.1.4 Ex vivo [68Ga]Ga-DKFZ-PSMA-11 Biodistribution Radioactivity quantification of [68Ga]Ga-DKFZ-PSMA-11 was performed by gamma counting all relevant organs, tissue and samples of blood and plasma (Appendix B). The total tracer amounts (%ID/g) in both kidneys of the MCF-7 xenografts was very high, but similar for the left and right kidney (70 ± 29 and 66 ± 26). Kidneys of the MDA-MB-231 xenografts showed significantly higher radioactivity than in MCF-7, with 149 ± 34 (right) and 131 ± 39 (left) %ID/g. This was not expected but renal tracer excretion can be influenced by several factors. Probably the water consumption caused the different rates of excretion (as water was not restricted or could not be standardised during the experiment). Due to being part of renal excretion, the bladder uptake of mice bearing MCF-7 was substantial larger (37 %ID/g) than that of the MDA-MB-231-bearing mice (<1 %ID/g). Moderate tracer uptake was quantified for the spleen, amounting to similar levels in MCF-7 (8 ± 6 %ID/g) and MDA-MB-231-bearing mice (7 ± 3 %ID/g). All other values ranged <5 %ID/g (Figure 6.3). [68Ga]Ga-DKFZ-PSMA-11 uptake in MCF-7 tumours (2.8 ± 1.3 %ID/g) was significant compared to MDA-MB-231 tumours (0.25 ± 0.12 %ID/g; p=0.026), about three times higher than uptake in muscle (p = 0.123), four times higher than liver uptake (p = 0.097) and five times higher than heart uptake (p = 0.082). 66 5 MCF-7 4 MDA-MB-231 3 2 1 0 od m art gs er ch ne ne cle ur ies in in or Blo er u S H e Lu n Liv aom tes ti tes ti us emF va r Sk a t M B r um S in in O T ma ll arg e S L Figure 6.3: Post mortem organ and tissue biodistribution of [68Ga]Ga-DKFZ-PSMA-11 in MCF-7 /MDA-MB-231 tumour cell bearing mice following 2 hours micro-PET/CT imaging acquisition (n ≥ 3). 6.2 Conclusion Insufficient [68Ga]Ga-DKFZ-PSMA-11-micro PET/CT imaging and different biodistribution was reported for MCF-7 and MDA-MB-231 tumours. The [68Ga]Ga-DKFZ-PSMA-11 uptake in both type of breast cancer cell xenografts lead to poor detection in PET/CT images. Correlations to the successful breast cancer visualisation that was clinically reported are not supported by the results of this study. This could be due to an enhanced permeability and retention effect present in the tumours, as studies shown [68Ga]Ga-DKFZ-PSMA-11 accumulation in breast cancer cells correlates to the degree of tumour neo-vasculature. 67 ORGAN UPTAKE (%ID/g) REFERENCES Füger, B., Czernin, J., Hildebrandt, I., Tran, C., Halpern, B., Stout, D., Phelps, M. & Weber, W. 2006. Impact of animal handling on the results of 18F-FDG PET studies in mice. J Nucl Med, 47: 999-1006. National Academies of Science. 2019. Recognition and alleviation of pain and distress in laboratory animals. https://www.nap.edu/read/1542/chapter/8 Date of access 5 November 2019. Mediso. 2016. Nano-Scan systems for preclinical applications imaging http://www.mediso.com/products.php?fid=2,11 Date of access 7 September 2018. 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The Synthesis and Evaluations of the 68Ga- Lissamine Rhodamine B (LRB) as a New Radiotracer for Imaging Tumors by Positron Emission Tomography. BioMed Res International, 2016:6. Morgenroth, A., Tinkir, E., Vogg, A.J., Sankaranarayanan, R.A., Baazaoui, F. & Mottaghy, F.M. 2019. Targeting of prostate-specific membrane antigen for radio-ligand therapy of triple- negative breast cancer. Breast Cancer Res, 21(1):116. Nguyen, D.P., Xiong, P.L., Liu, H., Pan, S., Leconet, W., Navarro, V., Guo, M., Moy, J., Kim, S., Ramirez-Fort, M.K., Batra, J.S. & Bander, N. 2016. Induction of PSMA and Internalization of an Anti-PSMA mAb in the Vascular Compartment. Mol Cancer Res, 14(11):1045. Vercher-Conejero, J.L., Pelegri-Martinez, L., Lopez-Aznar, D. & Cozar-Santiago, M. 2015. Positron emission tomography in breast cancer. Diagnostics, 5:61-83. 68 7 STUDY OUTCOMES, LIMITATIONS AND RECOMMENDATIONS The study intended utilising three cell lines: an estrogen receptor-positive breast cancer cell line (MCF-7), a triple negative breast cancer cell line (MDA-MB-231) and a prostate cancer cell line (LNCaP) to perform a xenograft development. A [68Ga]Ga-DKFZ-PSMA-11 radiolabelling optimisation for safe and suitable administration into mice was envisaged. Consequently, 18F]FDG and [68Ga]Ga-DKFZ-PSMA-11-micro-PET/CT imaging of these xenografts was performed. 7.1 Research Outcomes  An appropriate formulation of [68Ga]Ga-DKFZ-PSMA-11 was developed, and provided injectable doses for the mice suitable for non-invasive micro-PET/CT imaging.  MCF-7 and MDA-MB-231 nude mice xenografts were developed and enrolled for in vivo imaging and ex vivo biodistribution analysis.  MCF-7 and MDA-MB-231 cell xenografts successfully underwent micro-PET/CT imaging on consecutive days following injection with [18F]FDG or [68Ga]Ga-DKFZ-PSMA-11. [68Ga]Ga-DKFZ-PSMA-11 micro-PET/CT was followed by animal organs/tissue dissection, including measurement of the tracer accumulation in tumours.  The study findings reported poor MCF-7 and MDA-MB-231 tumour detection on [68Ga]Ga- DKFZ-PSMA-11-PET/CT images. [68Ga]Ga-DKFZ-PSMA-11-micro-PET/CT imaging results were confirmed by ex vivo biodistribution. Therefore correlations to the successful breast cancer visualisation that was reported clinically (Sathekge et al., 2016; Sathekge et al., 2015) were not supported by the results of this study. However, MDA-MB-231 tumours, contrary to MCF-7 tumours, demonstrated accumulation of [18F]FDG. 7.2 Research Limitations  LNCaP: The cell culturing process was challenging; LNCaP cellular growth in vitro using DMEM/Hams F12 was very sporadic, which resulted in limited cell material for mice inoculations; this restricted a maximum of two mice to be inoculated with 2 x 106 million cells. The micro-environment of female athymic nude mice hampered LNCaP tumour growth due to insufficient androgen hormone. These resulted in poor development of the LNCaP xenografts. Therefore, the LNCaP xenografts could not be included in the study.  Radiolabelling: Regardless of sufficient [68Ga]Ga-DKFZ-PSMA-11 product concerning the final formulation for tracer injection into mice. [68Ga]Ga-DKFZ-PSMA-11 optimisation 69 methods resulted in higher specific activity, activity concentration, only residual amounts of EtOH, but lower product yield compared to the former reports (Ebenhan et al., 2015).  There was no accumulation of [68Ga]Ga-DKFZ-PSMA-11 in both the MCF-7 and MDA- MB-231 tumours, which could be due to minimal or lack of expression of GCP II of these tumours. If this aspect is addressed and proof is provided that the expression is indeed occurring, repeat of these animal studies are warranted.  There was unexpected lack of [18F]FDG accumulation in the MCF-7 tumour xenografts which needs to be investigated before repeat testing in animals. 7.3 Research Recommendations  Further LNCaP cell culture optimisation could be carried out by using complete RPMI- 1640 media, which is suggested elsewhere (Day et al., 2013; Horoszewicz et al., 1983).  Growth of LNCaP, being an androgen-sensitive human prostate adenocarcinoma cell line, may depend more than expected on androgen hormone supplement in its environment. It is therefore mandatory that further development of LNCaP female athymic nude mice xenografts with maintained levels of androgen hormone to stimulate LNCaP tumor growth (Horoszewicz et al., 1983).  Some studies suggest the used of testosterone cypionate pellets (Gupta et al., 2010; Horoszewicz et al., 1983), which is proven to be support successful development of LNCaP cell xenografts in female mice. The use of injectable testosterone cypionate is suggested but more time is required to establish the quantity of the hormone supplement and frequency of the hormone supplement administration to grow the tumour successfully.  Supplementary to eluate fractionation method that provide high concentration of 68Ga radioactivity in a small volume. Eluate pre-concentration and pre-purification method can be applied to further reduce the volume (200 or 400 µl) and eliminate metal cation impurities mother isotope 68Ge. And this will improve the quality of 68Ga molarity prior to complexation with DKFZ-PSMA-11 (Banerjee et al., 2010; Eder et al., 2012; Velikyan, 2015).  Due to insufficient accumulation of [68Ga]Ga-DKFZ-PSMA-11 in MCF-7 / MDA-MB-231, tumours further in vitro investigation might help characterising the tumours and to better relate the GCP II tumour expression. It should be noted that the scope of this study did not include ex-vivo tumour characterisation. A research project running in parallel to this study determined the in vitro GCP II tumour expression in MCF-7, MDA-MB-231 and 70 LNCaP cells by way of confocal microscopy, immune-histochemistry and flow cytometry. The results are subject to another manuscript (data not shown). 71 REFERENCES Banerjee, S.R., Pullambhatla, M., Byun, Y., Nimmagadda, S., Green, G., Fox, J.J., Horti, A., Mease, R.C. & Pomper, M.G. 2010. 68Ga-labeled inhibitors of prostate-specific membrane antigen (PSMA) for imaging prostate cancer. J Med Chem, 53(14):5333-5341. Day, J.M., Foster, P.A., Tutill, H.J., Schmidlin, F., Sharland, C.M., Hargrave, J.D., Vicker, N., Potter, B.V., Reed, M.J. & Purohit, A. 2013. STX2171, a 17beta-hydroxysteroid dehydrogenase type 3 inhibitor, is efficacious in vivo in a novel hormone-dependent prostate cancer model. Endocr Relat Cancer, 20(1):53-64. Ebenhan, T., Vorster, M., Marjanovic-Painter, B., Wagener, J., Suthiram, J., Modiselle, M., Mokaleng, B., Zeevaart, J.R. & Sathekge, M. 2015. Development of a Single Vial Kit Solution for Radiolabeling of 68Ga-DKFZ-PSMA-11 and Its Performance in Prostate Cancer Patients. Molecules, 20(8):14860-14878. Eder, M., Schafer, M., Bauder-Wust, U., Hull, W.-E., Wangler, C., Mier, W., Haberkorn, U. & Eisenhut, M. 2012. Ga-68-complex lipophylicity and the targeting property of a urea-based PSMA inhabitor for PET imaging. Bioconjugate Chem, 23:688-697. Gupta, S., Wang, Y., R., R.-G., Shevrin, D., Nelson, J.B. & Wang, Z. 2010. Inhibition of 5alpha- reductase enhances testosterone-induced expression of U19/Eaf2 tumor suppressor during the regrowth of LNCaP xenograft tumor in nude mice. The Prostate, 70(14):1575-1585. Horoszewicz, J.S., Leong, S.S., Kawinski, E., Karr, J.P., Rosenthal, H., Chu, T.M., Mirand, E.A. & Murphy, G.P. 1983. LNCaP Model of Human Prostatic Carcinoma. Cancer research, 43(4):1809-1818. Sathekge, M., Lengana, T., Modiselle, M., Vorster, M., Zeevart, J., Maes, A. & al., e.a.e. 2016. Ga-68-PSMA-HBED-CC PET imaging in breast carcinoma patients. Eur J Nucl Med Mol Imaging(44):689-694. Sathekge, M., Modiselle, M., Vorster, M., Mokgoro, N., Nyakale, N., Mokaleng, B. & Ebenhan, T. 2015. 68Ga-PSMA imaging of metastatic breast cancer. Eur J Nucl Med and Mol Imaging, 42(9):1482-1483. Velikyan, I. 2015. 68Ga-Based radiopharmaceuticals: production and application relationship. Molecules (Basel, Switzerland), 20(7):12913-12943. 72 Appendix A: Summary of all [68Ga]Ga-DKFZ-PSMA-11 radiosynthesis PSMA- M / K [68Ga]GaCl3 DKFZ-PSMA-11 68[Ga] pH [68Ga]DKFZ EtOH (%)/ 11 /2.5M concentration added (mCi) -PSMA-11 Sal (µl) (nmol) NaOAc (µM) (% RCY) #) (µl) 1 10 M 2000/300 4.35 6.28 2.0 30.7 50/1000 2 10 M 2000/300 4.35 18.25 2.0 24.0 25/300 3a 5 M 1000/310 3.82 18.83 4.0 78.3 25/500 3b 5 M 1000/310 3.82 *) 4.0 76.7 25/500 4a 5 M 1900/620 2.07 14.92 4.0 33.1 15/500 4b 5 M 1200/400 3.13 *) 4.0 59.9 25/500 5 3.5 K 1000/- 3.50 5.0 72.2 25/300 6 3.5 K 1000/- 3.50 2.65 5.0 70.8 25/300 7 3.5 K 900/- 3.88 2.77 5.0 68.6 25/300 8 3.5 K 1000/- 3.50 4.16 4.5 68.6 25/300 9 3.5 K 1100/- 3.18 6.56 4.0 64.2 25/300 10* 3.5 K 1200/- 2.91 16.9 3.5 46.7 25/300 11* 3.5 K 1100/- 3.18 14.7 3.5 38.8 25/300 12 3.5 K 1200/ 2.91 16.2 4.0 66.6 25/300 13 3.5 K 1200/ 2.91 13.14 4.0 73.5 25/300 Footnotes #) Product was purified with different concentration of Ethanol (EtOH) in saline to obtain a concentrated final formulation within a small volume to warrant animals injection of activity ranging from 10 to 20 MBq contained in 100 to 200 µl. Synthesis 5 to 11: DKFZ-PSMA-11 is buffered with 325 µg NaoAc using a freeze dryed kit (voiding the volume of the buffering agent). *) 2.3 ml [68Ga]GaCl3 radioactivity added into the reaction kit (10 nmol DKFZ-PSMA-11), each ml was purified separately. M= manual labelling and quality control, K= kit-based radiolabelling 73 Appendix B: [68Ga]Ga-DKFZ-PSMA-11 ex vivo biodistribution Organ MCF-7 MDA-MB-231 Blood 0.14 ± 0.12 0.10 ± 0.10 Serum 0.18 ± 0.08 0.11 ± 0.06 Heart 0.32 ± 0.07 0.27 ± 0.08 Lungs 0.85 ± 0.18 0.81 ± 0.41 Liver 0.40 ± 0.08 0.32 ± 0.06 Left Kidney 66 ± 26 131 ± 39 * Right Kidney 70 ± 29 149 ± 34 * Spleen 5 ± 3 7 ± 3 Stomach 0.22 ± 0.06 0.29 ± 0.12 Small Intestines 0.57 ± 0.25 * 0.34 ± 0.10 Large Intestines 1.1 ± 0.3 0.87 ± 0.44 Muscles 0.64 ± 0.21 * 0.20 ± 0.19 Femur 0.36 ± 0.30 0.14 ± 0.11 Bladder 22± 19 * 3 ± 5 Ovaries 0.80 ± 0.16 0.72 ± 0.40 Skin 1.8 ± 1.8 6 ± 1.2 Brain 0.07 ± 0.04 0.03 ± 0 Tumours 2.0 ± 1.3 * 0.25 ± 0.12 Footnotes The values are presented as %ID/g (mean ± SD); *) p<0.05 comparing MCF-7 with MDA-MB-231 74 Appendix C: Journal right of permission of the figures used in this study 75 76 77 78 Appendix D: Language editing certificate. 79