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Fibrinogen-like protein 2 (FGL2) expression is associated with tumour progression and poor survival in a number of different cancers. In these cancers, FGL2 promotes tumour progression via an immunosuppression-mediated mechanism or by promoting angiogenesis. Querying single-cell RNA sequencing (scRNA-seq) human cancer data shows FGL2 is produced by immune and stromal cells in the tumour microenvironment (TME), while cancer cells have minimal expression of FGL2. We therefore studied the role of FGL2 produced by cells in the TME in tumour progression in two models of cancer in which the role of FGL2 has not been previously studied: epithelial ovarian cancer (EOC) and melanoma. EOC is the most lethal gynecologic cancer with an imperative need for new treatments. Before testing novel immunotherapies, we first characterized the TME of six syngeneic ovarian cancer models. ID8-p53-/- and ID8-C3 tumours were most highly infiltrated by T cells, whereas STOSE and MOE-PTEN/KRAS tumours were primarily infiltrated by tumour associated macrophages and were unique in MHC class I and II expression. This panel of well-defined murine EOC models can serve as a valuable resource for studies that aim to test immunotherapies. We next studied the role of FGL2 in tumour progression in melanoma and ovarian cancer models. Using wild-type and FGL2 knockout mice, we found that absence of FGL2 led to a more activated TME, including activated dendritic cells (DCs - CD86+, CD40+) and T cells (CD25+, TIGIT+), as well as demonstrating for the first time that the absence of FGL2 led to more activated Natural Killer (NK) cells (DNAM-1, NKG2D) in the TME. The absence of FGL2 prolonged survival in the B16F10 model of melanoma, and synergized with oncolytic virus to prolong survival in the ID8-p53-/-Brca2-/- model of ovarian cancer.

Vascular hyperpermeability is a common characteristic among many tumour types, especially those that grow in ascites form. With these, the exudate that flows out of the circulation collects as ascites fluid in the cavities within which these tumours are growing. In the past, this hyperpermeability has been attributed to the production of vascular permeability factor (VPF) by tumours. VPF has been found to bind to endothelial cells and lead to an increased vascular permeability. In the present study, the role of polymorphonuclear leukocytes (neutrophils) in tumour vascular hyperpermeability was investigated. Hey-3 tumour cells were grown into masses on the chick embryo chorioallantoic membrane (CAM). Interstitial neutrophilia was found to be a common feature at the tumour-host interface. Horseradish peroxidase was injected into the circulation and allowed to perfuse for five minutes. The density of labelled vesicles within the endothelial cell cytoplasm was calculated to be 0.99 +/-0.28 vesicles/μm². This vesicular density was comparable to that of N-formyl-methionine-leucine-phenylalanine (a chemotactic peptide for neutrophils)-treated CAM (1.04 +/-0.09 vesicles/μm²), but very different from control CAM (0.51 +/- 0.09 vesicles/μm²). In order to rule out any immune response to foreign cells, immune hepatocyte masses were grown on the CAM and vesicular density was calculated to be 0.54 +/-0.03 vesicles/μm². Through chemotaxis assays with the Boyden chamber, it was observed that Hey-3 tumour cells in culture were producing a chemotactic factor that is an attractant for human neutrophils. Once in the area, neutrophils do possess the potential to increase vascular permeability. Thus, neutrophils play a role in vascular endothelial hyperpermeability in tumours. / Thesis / Master of Science (MS)

Colorectal cancers (CRCs) rely heavily on host-derived structures to support tumour development and maintain growth. The tumour microenvironment (TME) includes contributions from underlying vascular endothelial cells to maintain nutrient and oxygen availability, as well as a delicate interplay with effector and suppressor immune cells to ensure effective immune evasion. Despite previous attempts to target these TME components through direct, individual therapeutic interventions targeting pro-angiogenic signaling and blocking immune suppression, in many cases the CRC TME remains supportive of tumour growth. Here, I have identified a loci-specific Autism Spectrum Disorder-linked 16p11.2 microdeletion, that demonstrates a negative impact on tumour growth. Initially, I show that a constitutive 16p11.2 deficiency (16p11.2df) alters tumour growth, although has no impact on tumourigenesis. Moreover, I demonstrate that a 16p11.2df in host- derived structures of the TME but not tumour cells is sufficient to slow tumour growth. Further, I demonstrate that vascular networks are altered in tumours derived from a 16p11.2df TME. While I present systemic alterations to the immunological landscape of 16p11.2df individuals at the transcriptional level, there is no detectable alteration in cytotoxic immune cell infiltration into 16p11.2df-derived tumours despite increased expression of T cell activating cytokines. In addition to altered growth characteristics, tumours derived from 16p11.2df mice show an enrichment in apoptosis of tumour cells, despite no change in proliferation. These findings suggest the potential for a 16p11.2df-mediated induction of tumour dormancy, through which volumetric tumour growth is slowed. Together, this study demonstrates the need for further investigation of the 16p11.2 microdeletion as a critical regulator of tumour growth.

Tumour angiogenesis is a hallmark of cancer. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in endothelial cells (ECs) survival, proliferation and migration. FAK has several tyrosine phosphorylation sites thought to be involved in FAK function but the requirement of phosphorylation of these residues in vivo is unknown. We have generated mice where endogenous FAK is deleted simultaneously with the expression of nonphosphorylatable FAK-Y397F or FAK-Y861F mutated or wild type forms of FAK in adult endothelium in order to test this. My data show that EC-FAK-Y397FKI mice present with decreased tumour angiogenesis (in sygeneic B16F0, CMT19T and LLC) but impaired B16F0 and CMT19T tumour growth only, with increased tumour hypoxia. FAK-Y397F tumour endothelium is not perfusion, leakage or vascular maturation defective. This mutation affects VEGF-, PlGF- and bFGF-driven angiogenesis in vivo and VEGF+Ang2 administration is able to partially rescue this phenotype ex vivo. In contrast, endothelial FAK-Y861F mutation leads to an initial delay in B16F0 tumour angiogenesis, that subsequently resolves, and does not affect B16F0 tumour growth. LLC and CMT19T tumour growth and angiogenesis are not affected by the endothelial FAK-Y861F mutation; neither are tumour blood vessel perfusion, leakage, vascular maturation or tumour hypoxia. VEGF-, PlGFand bFGF-driven angiogenesis in vivo and ex vivo was not affected by the endothelial FAK-Y861F mutation, whereas increased in vivo angiogenesis was triggered by Ang2 administration. Lastly, to understand whether cytokine profiles that might affect angiocrine signalling are affected differentially in FAK-Y397F vs FAK-Y861F endothelial cells, I show that CCL1 and CCL2 are increased in FAK-Y397F but IL- 13, IL-1F3, CCL4, IL-1F1, CCL2 and others are increased in FAK-Y861F endothelial cells. Overall my data indicates that endothelial-specific FAK mutations on two phosphorylation sites has different effects on tumour angiogenesis, tumour growth, growth factor stimulated angiogenesis in vivo and ex-vivo and cytokine production.

Tumour necrosis has long been associated with poor prognosis and reduced survival in cancer. Hypotheses to explain this include the idea that as aggressive tumours tend to grow rapidly, they outgrow their blood supply leading to areas of hypoxia and subsequently necrosis. However whilst this and similar hypotheses have been put forward to explain the association, the biological significance of the cells which make up necrotic tissue has been largely ignored. This stems from the belief that because a tumour is more aggressive and fast growing it develops areas of necrosis, rather than, the tumour is more aggressive because it contains areas of necrosis. Which came first like the egg and chicken is yet to be determined, however to date most research has only considered the possibility of the former. Viable cells were found in the necrotic core of Multicellular Tumour Spheroids. When examined these cells were found to be different to the original cell line in terms of proliferation, migration, and chemosensitivity. A proteomic analysis showed that these phenotypical changes were accompanied by changes in a large number of proteins within the cells, some of which could be potential therapeutic targets. Furthermore this has led to a new hypothesis for tumour necrosis and its association with poor prognosis. Necrotic tissue provides a microenvironemental niche for cells with increased survival capabilities. Protected from many chemotherapeutics by their non-proliferative status once conditions improve these cells can return to proliferation and repopulate the tumour with an increasingly aggressive population of cells.

Since the introduction of Auranofin in 1985 there has been no new clinically approved gold containing drugs introduced. Although promising results were achieved with a gold(I) phosphine complex [Au(dppe) 2]Cl (Hoke et al., 1991; Mckeage et al., 2002), this compound was never entered into clinical trials due to its toxicity to normal tissue such as the liver and heart (Smith et al.,1989). Six novel derivatives of [Au(dppe) 2]Cl were developed and synthesized to identify possible new candidates with improved tumour specificity compared to [Au(dppe) 2]Cl and cisplatin. Human cervical carcinoma cells (HeLa) were used for an initial toxicity screening. IC50’s obtained for [Au(dppe) 2]Cl and cisplatin were 0.661 and 0.710 µM respectively. Three mixed novel derivatives (MM4, MM5 and MM6) displayed IC50’s ranging between 0.026 and 0.103 µM. These compounds were then selected to be tested further for selectivity and cytotoxicity on various malignant and normal cell lines. MM4 showed selectivity for ovarian, prostate, cervical and breast cancer cells, while MM5 was the most effective against ovarian, colon, prostate, cervical and breast cancer cells. MM6 was most active against ovarian, colon, prostate, cervical and breast cancer cells. The experimental compounds had much higher IC50’s when tested on the normal cells, which indicates selectivity for cancer cells. The octanol/water partition coefficient (lipophilicity) of all the experimental compounds was measured to determine the lipophilicity of the compounds. [Au(dppe) 2]Cl was found to be strongly lipophilic; while the novel compounds had varying degrees of lipo- and hydrophilicity. The octanol/water partition coefficient (lipophilicity) was also used to establish whether there is a correlation between the lipophilicity, IC50 and tumour specificity. In this study no correlation was found between these parameters. [Au(dppe) 2]Cl is known to have an effect on the mitochondrial membrane potential of cells. MM4, MM5, MM6 and cisplatin were compared to [Au(dppe) 2]Cl for effects on mitochondrial membrane potential. PHA stimulated human lymphocytes and a human undifferentiated leukemia T-cell line (Jurkat cells) were used in these experiments. [Au(dppe) 2]Cl, MM4, MM5 and MM6 depolarized the mitochondrial membranes of PHA stimulated lymphocytes significantly, while only [Au(dppe) 2]Cl depolarized the mitochondrial membranes of the Jurkat cells significantly, indicating that a different mechanism of action might be operational. MM4, MM5, MM6 and cisplatin were compared to [Au(dppe) 2]Cl for effects on plasma membrane potential. PHA stimulated human lymphocytes and Jurkat cells were used in these experiments. [Au(dppe) 2]Cl and MM6 depolarized the plasma membranes of both PHA stimulated lymphocytes and the Jurkat cells significantly. In order to determine whether the depolarization of mitochondrial and plasma membranes was a precursor for apoptosis, experiments were done to determine whether MM4, MM5 and MM6 induce apoptosis in Jurkat cells. [Au(dppe) 2]Cl and cisplatin were added for comparison. [Au(dppe) 2]Cl, cisplatin, MM4 and MM6 did induce apoptosis in Jurkat cells, but MM5 did not. The effect of [Au(dppe) 2]Cl, cisplatin, MM4, MM5 and MM6 on the cell cycle of Jurkat cells was determined to establish whether the experimental compounds altered this process. [Au(dppe) 2]Cl, MM4, MM5 and MM6 arrested the cell cycle in the G1 phase and cisplatin did so in the S phase. In order to determine whether the inhibition of cell growth and partition coefficient of the experimental compounds is related to the uptake of the drug, radio labeled drug uptake experiments were carried out with 198Au labeled [Au(dppe) 2]Cl, MM5 and MM6. Two different types of ovarian cancer cells were used for these studies. One cell line was sensitive to cisplatin (A2780) and the other was resistant to cisplatin (A2780 cis). Results obtained from these experiments showed that the uptake of these experimental compounds was dependent on their octanol/water partition coefficient. However, the inhibition of cell growth did not correlate with the uptake of these compounds by the cells that were tested. To confirm the octanol/water partition coefficient and drug uptake results, 198Au labelled [Au(dppe) 2]Cl, MM5 and MM6 were testedin vivo for bio distribution in rats. [Au(dppe) 2]Cl (lipophilic) had higher bio distribution compared to MM5 and MM6 (hydrophilic). Conclusion The experimental compounds show low IC50’s combined with increased tumour specificity. This indicates that these compounds have great potential to target tumour cells selectively and should be investigated further as anti-cancer agents. / Dissertation (MSc)--University of Pretoria, 2008. / Pharmacology / unrestricted

Tumour necrosis has long been associated with poor prognosis and reduced survival in cancer. Hypotheses to explain this include the idea that as aggressive tumours tend to grow rapidly, they outgrow their blood supply leading to areas of hypoxia and subsequently necrosis. However whilst this and similar hypotheses have been put forward to explain the association, the biological significance of the cells which make up necrotic tissue has been largely ignored. This stems from the belief that because a tumour is more aggressive and fast growing it develops areas of necrosis, rather than, the tumour is more aggressive because it contains areas of necrosis. Which came first like the egg and chicken is yet to be determined, however to date most research has only considered the possibility of the former. Viable cells were found in the necrotic core of Multicellular Tumour Spheroids. When examined these cells were found to be different to the original cell line in terms of proliferation, migration, and chemosensitivity. A proteomic analysis showed that these phenotypical changes were accompanied by changes in a large number of proteins within the cells, some of which could be potential therapeutic targets. Furthermore this has led to a new hypothesis for tumour necrosis and its association with poor prognosis. Necrotic tissue provides a microenvironemental niche for cells with increased survival capabilities. Protected from many chemotherapeutics by their non-proliferative status once conditions improve these cells can return to proliferation and repopulate the tumour with an increasingly aggressive population of cells. / Yorkshire Cancer Research

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