Mechanogrowth factor (IGF-1Ec) and fluorescent nanoparticles in colorectal cancer Swethan Alagaratnam A thesis submitted to the University College London for the degree of Doctor of Medicine (Research) 2020 UCL Center for Nanotechnology and Regenerative Medicine Division of Surgery & Interventional Science University College London Declaration of originality I, Swethan Alagaratnam, confirm that the work presented in this thesis is my own. I have outlined all contributions by others to this work in the acknowledgement sections. Information derived from other sources is indicated in the thesis. i Abstract The IGF-1 axis was an area of significant interest in cancer therapy following promising preclinical studies but led to disappointing clinical trials. Further scrutinization of this pathway is, therefore, war- ranted. The IGF-1 axis has been demonstrated to inhibit autophagy via the Akt/PI3K pathway and in- duce autophagy via the ERK pathway. Autophagy has been associated with chemotherapy resistance in tumour cells. My work in this thesis involved investigating the expression of an isoform of IGF-1 referred to as IGF1-Ec or Mechanogrowth Factor (MGF) in colorectal cancer tissues and polyps with immunohistochemistry. Further work was done with fluorescent nanoparticles which have exciting potential to improve the diagnostic yield of investigations including colonoscopy, improve immuno- histochemistry assessment of tissue biopsies and help in surgery with intraoperative delineation of tumours. In addition, I investigated the relationship between autophagy and apoptosis with a view towards developing a model for further work in investigating the effect of MGF in autophagy. Semi-quantitative immunohistochemistry for MGF on colonic tissues including normal, polyp and can- cer tissues demonstrated a significantly higher expression of MGF in colonic polyps (with higher ex- pression with worsening dysplasia, p=0.001) and cancer compared to normal colon tissues (p<0.001). Semiconductor CdTe quantum dots and gold nanoparticles were synthesised and conjugated to the MGF peptide and antibody. Gold nanoparticles were successfully characterised with immunodots and applied to the HT29 and SW620 colorectal cancer cell lines and to tissues including normal, colon can- cer and polyp tissues reflecting the results from conventional immunohistochemistry. Autophagy inducers were administered to the cell lines HT29 and SW620 and inhibited with the use of Bafilomycin and 3-MA. Immunohistochemistry for LC3B was used to confirm the induction of au- tophagy, and cell viability studies were used to demonstrate significantly increased cell viability with autophagy induction and significant reduction of cell viability with inhibition of autophagy (p<0.01 at 24 hours). This model can be subjected to the application of MGF and assess its effects on cell viability. MGF is overexpressed in colonic polyps and cancer with low levels of expression in normal colon tis- sues offering an opportunity for the use of fluorescent gold nanoparticles to augment polyp and can- cer detection in colonoscopy and intraoperative tumour delineation. ii Impact statement Colorectal cancer is the third most prevalent cancer diagnosed worldwide with an early diagnosis associated with better outcomes and patients presenting with features such as involvement of draining lymph nodes demonstrating higher rates of cancer recurrence. Chemotherapy can reduce the risk of recurrence, but its efficacy is limited by toxicity due to interactions with healthy tissues. This emphasises the importance of ‘targets’ expressed at significantly higher levels in tumour tissues compared to healthy tissues thereby enabling drug delivery agents to accumulate chemotherapy drugs in tumours. In addition, the efficacy of chemotherapy can be affected by the expression of factors such as KRAS, emphasising the importance of accurate quantitative immunohistochemistry techniques to guide treatments. Chemotherapy agents have also been demonstrated to induce autophagy in colorectal cancer tissues, which acts as a protective mechanism for cancer cells at the later stages of cancer pathogenesis and a cause for resistance to chemotherapy. My work has identified the overexpression of Mechanogrowth factor (MGF) expression in primary colorectal cancer tissues and polyps compared to normal colon tissues, thereby offering a target for drug delivery and imaging agents in colorectal cancer. MGF is expressed at low levels in healthy tissues in the body, and therefore, this would reduce the systemic distribution of targeting drug delivery agents. Further research work in this area would include the application of fluorescent gold nanoparticles conjugated to MGF antibody in xenograft models of colorectal cancer in vivo and investigating the efficacy of drug delivery and systemic toxicity. The advantage of fluorescent gold nanoparticles is that it provides a dual capability of tumour imaging and drug delivery alongside its low inherent toxicity. This can be further applied to prostate cancer and osteosarcomas, which have been reported in the literature to overexpress MGF. There is also a potential for application of MGF antibodies conjugated to gold nanoparticles in reducing missed lesion rates in colonoscopy, which has recently been estimated as high as 25%. The benefit of intravenously or locally administering this nanoparticle and performing colonoscopy with appropriate excitation energy will help identify these lesions with fluorescence. Finally, improving understanding of autophagy and apoptosis is essential to help further improve the efficacy of chemotherapy agents. IGF-1 has been demonstrated to induce autophagy via the ERK pathway and inhibit autophagy via the Akt/PI3K pathway. My work demonstrating the successful induction of autophagy and inhibition in colorectal cancer cell lines provides the groundwork for investigating this with the administration of the isoforms of IGF-1. iii Acknowledgements I am very grateful to my supervisors Dr Shi-Yu Yang and Professor Barry Fuller for their guidance, help and patience throughout this research project, mainly due to the trying circumstances during my experiments with changes in my supervisors and their job roles. I am particularly grateful to Professor Loizidou for her time and help with my thesis and submission for this degree despite her busy schedule. I would like to thank and acknowledge Dr Bala Ramesh for his invaluable support, expertise, guidance and constant encouragement without whom this may never have been possible. In particular, his work in synthesis, functionalization and conjugation of the fluorescent nanoparticles, which was a crucial part of my thesis. I would also like to acknowledge the work done by Dr Jessica Broni and Dr Francesca Launchbury with the immunohistochemistry experiments at the UCL Institute of Neurology. In addition, I would like to acknowledge Professor Fuller’s MSc student, Ajit Abraham Nirmal, for his help with the autophagy experiments and Dr Richard Thorogate for his help with atomic force microscopy. I would like to extend my gratitude to my clinical supervisors over the years including Mr David Stoker, Mr Saswata Banerjee, Mr Michael Dworkin and Mr Chetan Bhan for their persistent belief, understanding and support through this process, and Professor Marc Winslet for his guidance over the years. Finally, I would like to dedicate this thesis to my family for their unwavering support over the years. iv Table of Contents Declaration of originality ......................................................................................................................... i Abstract ................................................................................................................................................... ii Impact statement ................................................................................................................................... iii Acknowledgements ................................................................................................................................ iv Table of Contents .................................................................................................................................... v List of Figures ......................................................................................................................................... xi List of tables ......................................................................................................................................... xiii Abbreviations ........................................................................................................................................xiv 1 CHAPTER 1: Introduction into molecular pathways in colorectal cancer, biomarkers and the Mechanogrowth factor ........................................................................................................................... 1 1.1 Colorectal cancer .................................................................................................................... 2 1.1.1 Introduction/demographics ............................................................................................ 2 1.1.2 Pathology ........................................................................................................................ 2 1.1.2.2 Chromosomal instability (CIN) ........................................................................................ 4 1.1.3 Management of colorectal cancer .................................................................................