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Immune cells play an important role in tumour growth and progression, as well as establishment at metastatic sites. Although inherently, immune system is designed to locate, target and eliminate malignant cells, evolutionary processes within a host allow tumourigenic cells to develop mechanisms and pathways to avoid immune recognition. There is a substantial amount of knowledge on how particular immune cell subtypes contribute to cancer growth and progression. Specifically, macrophages play an important role in mitigating immune response and induction of anti-inflammatory response. Due to this reason, macrophages can become potential new therapeutic targets. However, the knowledge of underlying mechanisms is limited due to the absence of robust tools for studying transient cell-cell interactions between cancer cells and macrophages at tumour microenvironment. Recent advances in synthetic biology have introduced a vast array of tools, particularly synthetic receptors, which have reported a broad range of applications in biosensing. One of such tools is synNotch receptor, which is derived from the core of the Notch receptor and is activated by cell-cell contact. Both extracellular and intracellular domains of synNotch can be substituted with custom sensing and signal transduction domains to carry out custom input/output circuits. In this thesis, the aim is to repurpose synNotch to detect interactions between cancer cells and macrophages in aims to develop a robust tool to aid in studying the mechanisms of metastasis development and bring insights into potential therapeutic targets.

Many anti-cancer drugs are hydrophobic, and this requires the use of organic solvents for their clinical administration, resulting in inefficient therapies and side effects including cardiotoxicity, nephrotoxicity, neurotoxicity and hypersensitivity when injected into the bloodstream. To tackle these issues, there has been tremendous research on a variety of carrier-based nanoparticles (NPs), but such strategies often fail to encapsulate drugs efficiently and require significant amounts of inorganic and/or organic nanocarriers with potential toxicity in the long term. The preparation of nanoformulations without using carriers for the delivery of anti-cancer drugs with poor water solubility is thus desired, requiring elegantly designed strategies for nanoproducts with high performance and stability. These strategies include direct physical self-assembly or chemical modifications where drug molecules are coupled or conjugated together via various functional molecules. Facile tuning of hydrophobic drug molecule properties by combining them with different chemotherapeutics, immunotherapeutic agents and functional molecules enables the preparation of nanodrugs with improved functional performance. This thesis presents three different novel approaches for the preparation of carrier-free therapeutic anti-cancer agents as nanodrugs. Initial work focussed on the direct physical self-assembly of two anti-cancer agents, paclitaxel (PTX) and curcumin (CUR). While PTX fails to form stable NPs on its own, the addition of CUR into the drug molecule enabled NP formation. An investigative study on the preparation of carrier-free PTX-CUR NPs was performed to understand the stability of the resulting NPs. It was found that different variables, such as PTX:CUR weight ratio and the purity of CUR, which can be obtained as curcuma longa (turmeric) extract (>65% curcumin) or as high purity (>98%) powders, have significant effects on the self-assembly process. The latter observation was further investigated using both experimental and computational methods and it was discovered that the naturally existing curcuminoids inside the turmeric extract help with the selfassembly process and are essential for the formation of monodisperse, spherical nanodrugs. Later stages of the work focussed on preparing conjugate drugs to form multifunctional nanosystems. First, NLG919, an immunotherapy agent which inhibits the over-expressed indoleamine dioxygenase (IDO) in many types of cancer, was covalently bonded with PTX to form PTX-NLG919 conjugates linked by an esterase-sensitive chemical bond. The conjugates were then nanoprecipitated to form carrier-free NPs in a single step to achieve potential chemo-immunotherapy agents. This strategy directly tackled the issues associated with the poor water solubility of both PTX and NLG919, where two drug molecules incapable of forming stable NPs on their own in the absence of excipient carrier molecules were able to do so when they were conjugated. This was attributed to the folding properties of the linker bridge as investigated in literature previously. Finally, another novel prodrug molecule was prepared by conjugating DOX, another FDA-approved chemotherapy drug with dehydroepiandrosterone (DHEA), an inhibitor of glucose-6 phosphate dehydrogenase (G6PD) which causes energy loss which is required for cell proliferation. Given that both DOX and DHEA affect the cell replication cycle, their combinational use may improve their respective anti-cancer properties. The molecules were conjugated via different chemical linkages - ester and thioether bonds. While the prodrug synthesis was successful, the preparation of stable NPs was challenging using the nanoprecipitation method. The anti-cancer efficacies of all the reported formulations were investigated in vitro. PTX-CUR NPs demonstrated comparable cell viabilities to free drug PTX, whereas PTX-NLG919 NPs and DOX-DHEA prodrug exhibited relatively milder cytotoxicity due to the presence of the linker bridge, prolonging the release of the anti-cancer agents. It must be noted that similar observations were also made with different prodrug conjugates in vitro, but such systems' cytotoxicities improved drastically in in vivo studies due to the tumour redox environment and the presence of over-expressed enzymes as well as the improved delivery enabled the nanodrugs. The results described in this thesis demonstrate the strong potential of the prepared NPs for in vivo and clinical studies as well as the versatility of the carrier-free nanodrug systems for cancer therapy. With more synthetic approaches for the preparation of carrier-free nanodrugs than ever, we expect a range of new carrier-free formulations to be developed and employed in safe and effective cancer therapy in the future.

Bladder cancer (BC) is the 12th most common cancer worldwide, with approximately 5,300 deaths every year in the UK. Currently, diagnosis is only possible through the assessment of the tissue by invasive techniques such as cystoscopy and transurethral resection of bladder tumour (TURBT). Depending on its invasiveness in muscularis propria, it is clinically divided into non-muscle invasive BC (NMIBC) and muscle-invasive BC (MIBC). Low-grade NMIBCs usually undergo surveillance for many years with recurrent cystoscopies and TURBTs, while high-grade tumours are treated with BCG (Bacillus Calmette-Guérin) instillations in addition to life-long surveillance. Although the efficiency and quality of the surgical techniques have improved over the years, BC, even at early stages, continues to have a distinctively high incidence of recurrence and progression to invasive stages. This project addresses the two main unmet needs of bladder cancer clinical management. First, the need to identify patients who are likely going to recur: all patients must undergo years of surveillance, which is not only extremely unpleasant but also a burden for the health system. Identifying markers that predict recurrence would help clinicians to avoid unnecessary surveillance and promptly provide treatments to those who will recur. Second, there is the necessity to identify patients who will not respond to BCG treatment. In 40% of cases, patients fail to respond to BCG and can have serious side effects. Being able to predict BCG would help nonresponders to have access to alternative treatment straightaway and avoid side effects. To address these aims, the tumour immune microenvironment was investigated at the RNA and protein levels. Clinical collaborators identified a discovery set of sixty-five archival Formalin-Fixed Paraffin-Embedded (FFPE) biopsies, composed mostly of NMIBC but containing also MIBC in order to represent the spectrum of stages and grades. Genes of interest were identified through literature or RNA analysis using the NanoString PanCancer IO 360 panel. To be able to quickly translate our results into the clinic, interesting markers were selected and stained on biopsy's sections. Antibodies were optimised in immunohistochemistry and multiplex immunofluorescence with Quantum Dots or Tyramide Signal Amplification fluorophores. Images were analysed with machine learning image analysis and data-driven analysis using a newly identified analysis pipeline. The composition of the immune cell population in NMIBC showed higher CD4+ and CD8+ T-cells and lower CD68+ macrophages when compared with MIBC. Interestingly, the density of CD8+ cells was also statistically higher in patients that did not recur compared to patients who recurred. RNA analysis of 27 NMIBCs identified four genes that were differentially expressed in patients who recurred, KIR3DL1, S100A8, CCNA1 and KIR2DL3, while CCND1, LAMB3, SERPINB5, COL17A1 were linked to patients who did no recurred. KIR2DL3, CCNA1, CCND1 and LAMB3 were further evaluated at the protein level in 53 NMIBCs, however, the results did not confirm the earlier discovery. Interestingly, when comparing the response to BCG treatment in NMIBC, two genes were identified and confirmed to be differentially expressed at the protein level: MMP7 and IFI27. IFI27, also known as ISG12a, was linked to a positive response to BCG, possibly through the regulation of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and was identified as the most promising marker. IFI27 could identify 78% of BCG responders with a specificity of 76% on a cohort of forty-three patients treated with BCG. Further evaluation of IFI27 immediately before the start of BCG therapy and afterwards could provide a more comprehensive view of its role in the response to BCG.

Combination therapy, often referring to the administration of two or more therapeutic agents with different functional sites in cancer cells, has been used to address the efficacy of chemotherapy in clinical application. However, the clinical outcomes of conventional combination therapies with the cocktail mixture of multiple free drugs often suffers from unideal therapeutic outcomes due to the variations in drug properties such as different solubility, individual pharmacokinetic fate, as well as inconsistent tissue distribution and cell permeability. The work presented in this thesis aimed to promote the synergism of the therapeutics with carrier-free co-delivery nanoplatforms. To achieve this purpose, three different nano-delivery systems (nanoDDSs) were designed and fabricated with different combinations of anti-tumour therapeutic agents. Physicochemical characterisations and in vitro anti-tumour activity of each nanoDDS were performed to evaluate their suitability for the drug co-delivery to realise an augmentation in the anti-tumour efficacy. In the first part of this work, a self-assembled carrier-free nanoDDS was prepared with cisplatin (CDDP, an alkylating agent) and dactolisib (BEZ, a PI3K inhibitor). The suitable formulation process for the nanoDDS was firstly explored by the preparation with different drug ratios, from which a modified formulation was selected through the comparison of particle size and morphological features. The as-prepared nanoparticles, denoted as CDDP-BEZ nanoparticles (NPs), were uniform spheres with a dynamic diameter of 136.5 ± 0.7 nm. Based on the coordination bond between the hydrophilic CDDP and BEZ, the driving force for the formation of CDDP-BEZ NPs was majorly hydrophobic interaction and electrostatic interaction, which further led to a pH-responsive drug release profile. By simultaneously affecting DNA and inhibiting PI3K signalling axis, CDDP-BEZ NPs exhibited potent anti-proliferative effect on cancer cells. Besides, the increased lipophilicity due to the coordination with BEZ facilitated the accumulation of CDDP in mitochondrion, resulting in the activation of mitochondrion-dependent intrinsic apoptosis and subsequently leading to a further elevated synergistic efficacy in killing cancer cells. Additionally, CDDP-BEZ NPs successfully restraint the cell migration and invasion induced by chemo therapy. With a 3-dimensional (3D) tumour spheroid model, we further confirmed that CDDP-BEZ NPs maintained all the superiorities in combating tumour development and progression under complicated physiological condition. In the second part of this work, the feasibilities of addressing the negative insulin feedback by the co-administration of PI3K inhibitors with anti-hyperglycaemia agents were explored. To realise this goal, two nanoDDSs were designed and fabricated, one for the co-delivery of BEZ and glucose oxidase (GOx), and the other one of the co-delivery of BEZ and metformin. An albumin-based nanoDDS was prepared for the co-delivery of BEZ and GOx through the hydrophobic interaction-induced co-assembly of chemically modified BEZ (Lx-BEZ), GOx, and bovine serum albumin (BSA). For the modification of the formulation process, nanoparticles prepared with different ratios among BEZ, GOx, and BSA at various mixing time were compared in the aspect of particle size and morphology, from which a nanoDDS denoted as LBGB was selected. The as-prepared LBGB was uniform quasi-sphere with a diameter of around 70 nm, with preservation of the catalytic activity of GOx and a pH-responsive drug release profile of BEZ. Enhanced synergy between GOx and BEZ was observed with LBGB in cancer cells compared with the physical mixture of dual agents, even with the existence of insulin. Besides, LBGB exhibited a more potent restraint on the migration property of cancer cells compared with the mixed administration of GOx and BEZ. Consistent anti-tumour activity of LBGB was also observed on 3D tumour spheroids, indicating its excellent therapeutic efficacy even under complicated environment. For the co-delivery of BEZ and metformin, a nanoDDS based on Lx-BEZ and the polymeric prodrug of metformin (pMet) was proposed and established through the π-π stacking interaction. The modified formulation process was obtained by the screening of nanoparticle prepared with different ratios of Lx-BEZ and pMet at different mixing time, from which a suitable nanoDDS (pMet-LB) was selected by the comparison of particle size and morphology. Despite unsatisfactory pH-responsive drug release profile, pMet-LB exhibited excellent anti-tumour efficacy in 2-dimensional (2D) monolayer-cultured cancer cells, and maintained its potency in killing cancer cells with the presence of insulin. However, no superiority was observed in the anti-tumour activity from pMet-LB on the 3D tumour spheroids, likely due to its lack of pH-responsiveness. This failed intention emphasised the importance of using suitable 3D tumour models to better simulate the actual tumour microenvironment, through which an outcome better reflecting the real physiological process could be obtained. In the third part of this work, the feasibilities of establishing a nanoDDS for the steady delivery of Ca2+ and other anti-tumour therapeutic agents was explored. Both CaCO3- and CaO2-based nanoparticles were explored for this purpose, through which a CaO2-based nanoDDS modified with curcumin-grafted-hyaluronic acid and transferrin (CaO2@HC-Fe-Tf) was selected for further study. With excellent lyophilisation and serum stability, CaO2@HC-Fe-Tf was uniform sphere of around ~250 nm in diameter with a clear core-shell structure, holding the capacity to enter cancer cells, release H2O2, and initiate Ca2+ overloading, and resulting in its anti-cancer activity. Despite the relatively mild potency in killing cancer cells, CaO2@HC-Fe-Tf exhibited obvious suppression on the migration of cancer cells from the spheroid. Results from this experiment suggested that further combination with other therapeutic agents might be necessary for an ideal therapeutic outcome. Collectively, this work demonstrated that carrier-free co-delivery nanoDDSs represented a promising strategy in addressing the issues related with the combination therapy currently applied in clinical. With the precise delivery of both therapeutic agents into the same cell with minimum assistance of vector materials, the carrier-free co-delivery nanoDDSs implemented a way for anti-tumour agents with different targets to simultaneously exert their functions, thus achieving a facilitated anti-tumour efficacy. Through the design and fabrication of three nanoDDSs based on different combinations of therapeutic agents, this work explored the feasibility of building stable carrier-free co-delivery nanoDDSs via different strategies, while evaluating the potential of each nanoDDSs in the aspects of physicochemical properties such as particle size and morphology, as well as in vitro anti-tumour efficacy on 2D monolayer-cultured cells or 3D tumour spheroids. With the discrepancies observed between the results from 2D monolayer study and those from the 3D tumour spheroid study, this work also proposed the application of tumour spheroids as in vitro model in evaluating the therapeutic profile of the established nanoDDSs, which could better simulate the complicated in vivo environment for the actual pharmacokinetic evaluation. The results obtained from this work suggested that with proper design, the carrier-free co-delivery nanoDDS held the potential to serve as versatile platform, realising the simultaneous delivery of a wide range of anti-tumour agents including chemotherapeutics, enzymes, as well as inorganic substance. Apart from the augmentation in the anti-tumour efficacy, carrier-free co-delivery nanoDDS also provided a cost-friendly alterations, for there was a significant cut-down in the development lifecycle with the combination of two FDA-approved therapeutic agents. In addition, the recently emerged computational screening greatly accelerated the progress of the rational design of optimal combination, paving the way for the industrialisation of the carrier-free co-delivery nanoDDSs and the subsequent clinical translation., Collectively, the work presented in this thesis aimed to exploit the possibility of using carrier-free co-delivery nanoDDS to augment the therapeutic efficacy and minimise the vector-driven side effects in the anti-tumour combination therapies, exploring a potent and affordable way to benefit the cancer patients worldwide.

Cancer metastasis remains the largest challenge in oncology accounting for over 90% of cancer-associated deaths. The bone is the most prevalent site of metastasis in many cancers, including breast cancer where 70% of patients with advanced disease exhibit bone metastasis. With a current lack of curative treatments, patients with metastasis to the bone have a 5-year survival rate of approximately 15%. Recent focus on the tumour microenvironment (TME) has now identified an important role in cancer development. By establishing interactions with the tumour cells, the TME promotes tumour growth, metastasis and treatment resistance. However, much of the research into the TME has been performed in primary tumour models and research into the role of the TME in bone metastasis is limited. Using in vivo experimental models of breast cancer bone metastasis complemented with in vitro assays, we investigated the role of the TME throughout breast cancer bone metastasis development, including early stage seeding, metastatic growth and during chemotherapy treatment. We first demonstrated that macrophages play a significant role during tumour cell seeding to the bone where the depletion of macrophages during tumour cell seeding resulted in reduced tumour cell number and delayed metastatic growth. Novel intravital imaging and imaging of cleared tissues were also developed and are currently being used to visualise the seeding process in real time. We next investigated the role of the TME in the growth of breast cancer bone metastasis. An increased infiltration of mesenchymal stem cells (MSCs) was observed in the bone metastasis, which when depleted using Nestin-GFP-CreERT2-iDTR mice resulted in significant decrease in bone metastatic growth in comparison to non-depleted controls. In vitro proliferation assays demonstrated that direct contact between bone metastatic breast cancer cells and MSCs promotes tumour cell growth. Furthermore, preliminary data demonstrates that the co-operation between MSCs and macrophages within the TME may also be important in tumour growth. Lastly, we investigated the effect of chemotherapy treatment on breast cancer bone metastasis. Whilst bone metastasis appears to be resistant to doxorubicin, paclitaxel treatment results in increased growth, indicating paclitaxel-induced metastatic growth. The doxorubicin resistance and paclitaxel-induced growth are both dependent on the microenvironment in the bone, therefore flow cytometry, immunostaining techniques and cell depletion models were performed to characterise the component of the TME responsible for the effects of chemotherapy treatment. Collectively, in this project we demonstrate the role of the TME during the development of breast cancer bone metastasis including the macrophages during tumour cell seeding and MSCs during bone metastasis growth. The TME also plays a pivotal role in chemotherapy resistance and induced growth of bone metastasis. The identification of the molecular mechanisms involved in these processes may offer insight into potential novel therapeutic targets to overcome breast cancer bone metastasis.

Prostate cancer is a leading cause of mortality among men worldwide. Targeting the androgen receptor (AR) via androgen deprivation therapy is the first line of treatment against advanced prostate cancer. Nonetheless, the majority of patients develop therapy resistance within 1-3 years. Therefore, there is an imminent need to explore new signalling pathways that could provide novel therapeutic targets. The Hippo signalling pathway is a master regulator of cell growth, proliferation, and cancer. The transcriptional coactivators of the Hippo pathway, Yes-associated protein (YAP) and its paralog; the transcriptional coactivator with PDZ-binding motif (TAZ) are central in various cancers. The role of YAP/TAZ in prostate cancer (PCa) has gained momentum recently. Recent reports suggest that the central kinases MST1/2 and LAST1/2 inhibit androgenic signalling in prostate epithelial cells. Additionally, exogenous expression of YAP in vitro induces tumorigenesis in prostate epithelial cells. Importantly, YAP and the androgen receptor (AR) colocalise in the nucleus in an androgen dependent manner to promote tumorigenesis. Nonetheless, the exact mechanism of how androgens and AR regulate YAP/TAZ is unexplored. The Hippo-YAP signalling pathway was also reported to exacerbate PCa inflammation via inducing the recruitment of myeloid derived suppressor cells to the PCa niche which modulate immune response evasion. Additional evidence from different cancers and multiple conditions indicates that Hippo-YAP/TAZ signalling is an important regulator of inflammatory processes in non-immune cells. However, our understanding remains limited as to how the Hippo pathway and YAP/TAZ regulate leukocytes in inflammatory and homeostatic settings. This thesis aimed to understand the mutual regulatory interaction between AR and YAP/TAZ, as well as to understand how YAP/TAZ contribute towards prostate tumorigenesis. Finally, the thesis also describes the development and initial characterisation of an in vivo model system to study the role of YAP/TAZ in regulating tissue resident macrophages and leukocytes. Herein, androgens are shown to activate YAP/TAZ differentially via AR, where AR regulates YAP translation, while inducing transcription of the TAZ encoding gene, WWTR1. Serum Response Factor (SRF) regulates this AR driven YAP/TAZ activation in a feed forward manner. Importantly, SRF expression correlates with TAZ, CYR61 and CTGF expression in patients with different stages of prostate cancer. Work described in the thesis provide evidence that YAP/TAZ are not essential for sustaining AR activity, however, targeting YAP/TAZ or SRF sensitizes PCa cells to AR inhibition in anchorage independent growth conditions. The findings presented dissect the cellular roles of YAP, TAZ and SRF in prostate cancer cells, which indicates the interplay between these transcriptional regulators and their roles in prostate tumorigenesis and highlights how this insight might be exploited therapeutically. In the final section of this thesis, Yapfl;fl/Wwtr1fl;fl mice were crossed with mice harbouring the novel CD64.iCre allele, which is a newly developed cre recombinase that targets macrophages independently from other immune cells. Basic characterisation of the CD64.iCre.Yapfl;fl/Wwtr1fl;fl mouse model shows that YAP/TAZ are not essential for macrophages differentiation and proliferation. Nonetheless, preliminary data shows that Yap/Taz loss in macrophages results in lower numbers of neutrophils in liver and lungs, suggesting that Yap/Taz might be regulating neutrophils recruitment to the inflammatory niche. Initial analysis of the CD64.iCre.Yapfl;fl/Wwtr1fl;fl mouse model provides a solid ground for future experiments to dissect how Yap/Taz regulate macrophages and leukocytes in homeostatic and inflammatory settings.

Bone metastasis is the major cause of death in breast cancer. However, the underlying mechanisms of bone metastasis progression are still largely unknown. The role of the tumour microenvironment in metastatic progression has been increasingly appreciated in recent years. As one of the major immune components of the tumour microenvironment, macrophages promote breast tumour progression and lung metastasis. In patient bone metastasis samples and preclinical in vivo models, I found that macrophages abundantly infiltrate in bone metastasis. The macrophage depletion assays significantly inhibit bone metastasis growth, showing the critical role of macrophages in bone metastasis growth. I noticed that heterogeneous macrophage populations in bone metastatic lesions and a specific subset of macrophages infiltrated inside tumours were identified by high expression of CD204 and IL-4R. Lineage tracking of macrophages showed they were recruited from circulating monocytes via CCL2/CCR2 chemokine signalling. Blocking their recruitment led to a significant delay of bone metastasis onset and inhibition of tumour growth. Furthermore, genetic ablation of IL-4R inhibits bone metastasis. To better understand the CD204hi bone metastasis-associated macrophage (BoMAMs) subset, I performed RNA sequencing to compare the transcriptional change of macrophages with high and low expression of CD204 in bone metastasis, as well as macrophages from healthy bone marrow with high CD204 expression. The results revealed the changes of CD204hi BoMAMs at the molecular level including the increased expression of wound healing, immunosuppressive and tissue remodelling related genes. These results provided several candidate genes that may benefit drug design. Meanwhile, I profiled the change of haematopoietic cell populations in bone metastasis progression using single-cell RNA sequencing. The data showed a significant difference of immune cell populations in early and late bone metastasis stages compared with normal bone. Several immune cell populations showed remarkable reductions including B cells and T cells, while neutrophils increased during metastasis growth. Interestingly, erythroid progenitors largely increased in the early stage of bone metastasis, which may promote the initial growth of bone metastasis. In summary, these studies identified the critical role of a monocyte-derived macrophage subset in promoting breast cancer bone metastasis. The macrophage subset is regulated by the CCL2/CCR2 axis and IL-4R signalling pathway. The BoMAMs highly upregulated wound healing and immunosuppressive related genes. In addition, I found other haematopoietic cell populations were also affected in bone metastasis. Apart from macrophages, erythroid progenitors may also play an important role in tumour growth. Together, these results provide several potential therapeutic targets to treat breast cancer bone metastasis.