Dr. Melissa Conroy

This proposal seeks to significantly upgrade the infrastructure in the Anatomy Research Lab (ARL), co-run by three independent PIs; Dr Melissa Conroy, Dr Daniel Johnston and Dr Denis Barry

The global health burden of obesity is of grave concern, affecting over half a billion adults worldwide, and directly attributable to approximately 3.5 million deaths each year. In addition, WHO figures show that 1 in 3 European children are overweight or obese and of these, 60% are predicted to be obese in adulthood. Furthermore, despite national and international interventions to promote a healthy diet and lifestyle, recent reports predict that global obesity rates show no signs of abating and therefore large numbers of adults and children are at serious risk of developing obesity associated diseases such as cancer. Obesity induces organ and whole body chronic inflammation. It is the active infiltration of immune cells into expanding visceral adipose tissue (VAT) that drives this inflammation in obesity and studies have shown that T cells are key players in this process. VAT surrounds abdominal organs and its expansion is closely associated with obesity-related pathologies. Therefore, we propose that preventing T cells from infiltrating the VAT will prevent or reduce local and systemic inflammation and as a result the associated comorbidities. Proteins known as chemokines are responsible for guiding the migration of immune cells during normal immune responses and it is now well documented that chemokines mediate the natural cycling of T cells to the liver. However, previous studies have suggested excessive chemokine production from obese VAT results in the dysregulated recruitment of inflammatory cells. Our recent work in patients with obesity-associated cancer, has revealed preferential movement of T cells to human VAT and liver and have identified macrophage inflammatory protein 1" (MIP-1") as a key chemokine guiding this movement. Therefore, we propose that a therapeutic strategy to specifically block MIP-1"-guided inflammatory T cell trafficking to the VAT and liver using novel chemokine receptor antagonists will attenuate local and systemic chronic inflammation in obesity. We have already demonstrated in vitro that a novel CCR1 antagonist (Chemocentryx) can effectively block human T cell chemotaxis to secreted factors from VAT and liver. However, anti-inflammatory effects of the drug cannot be tested in such ex vivo settings using tissue explants and therefore the use of an in vivo model is crucial to the advancement of this research. For the first time, the proposed study would validate a novel immunotherapeutic strategy in an in vivo model of obesity. Non-obese and diet-induced obese C57BL/6 mice will be administered with a CCR1 antagonist (Chemocentryx). This treatment regime would determine the effects of CCR1 blockade on T cell infiltration to VAT and liver and its ability to alleviate pre-existing obesity-associated inflammation. This would indicate whether such a therapeutic intervention can successfully control inflammation and whether it can be used to treat/prevent obesity-associated disease. In addition, C57BL/6 mice will be administered with a CCR1 antagonist (Chemocentryx) during the development of diet induced obesity. This prophylactic treatment regime would determine the effects of CCR1 blockade on obesity associated inflammation during the development of obesity and will indicate whether intervention can successfully prevent inflammation and disease. Professor Thomas Wunderlich is an internationally renowned principle investigator at the Max Planck Institute (MPI) for metabolic research with major interest in the area of obesity and cancer. Prof. Wunderlich is well-published in the area of obesity with a multitude of publications in high-impact journals. His team routinely use C57BL/6 mice to generate mouse models of obesity and cancer. Therefore, his research group is an ideal choice for generating the mouse models during the project and shall be the catalyst in propelling our study toward clinical trials.

Obesity is a global health problem affecting millions of adults and children worldwide. In Ireland, approximately 66% of adults and 25% of children are overweight or obese and are therefore at serious risk of developing obesity-associated diseases. Obesity induces organ and whole body chronic inflammation. It is the active infiltration of immune cells into expanding visceral adipose tissue (VAT) that causes this inflammation in obesity and studies have shown that T cells are predominantly responsible for this. VAT surrounds abdominal organs and its expansion is closely associated with obesity-related pathologies including many types of cancer and liver disease. Therefore, we propose that preventing T cells from infiltrating the VAT will prevent or reduce local and systemic inflammation and as a result the associated comorbidities. Proteins known as chemokines are responsible for guiding the migration of immune cells during normal immune responses and it is now well documented that chemokines mediate the natural cycling of T cells to the liver. However, previous murine studies have implicated dysregulated chemokine production in the recruitment of inflammatory cells to the VAT in obesity. Our recent work has revealed preferential movement of T cells to human VAT and liver and have identified two key chemokines guiding this movement; macrophage inflammatory protein 1α (MIP-1α) and interferon-inducible protein 10 (IP-10). Therefore, we propose that a therapeutic strategy specifically blocking MIP-1α- and IP-10-guided inflammatory T cell trafficking to the VAT and liver using novel chemokine receptor antagonists will prevent and reduce both local and systemic chronic inflammation in obesity. For the first time, this innovative study will validate a novel immunotherapeutic strategy targeting immune cell trafficking, which has significant potential to prevent and reduce the detrimental consequences of obesity-associated inflammation, as a major driver of carcinogenesis and liver disease, in the rapidly growing number of obese individuals worldwide.

This multi-investigator collaboration will integrate the cancer immunology and treatment resistance expertise of Dr Conroy and Dr Barr, together with the clinical expertise of Dr Sinead Cuffe, to generate novel, clinically-relevant data on the therapeutic utility of Olaparib in combination with NK cell therapy for NSCLC. This multidisciplinary study will assess the use of novel treatment combinations to address the clinical challenge of treatment-resistant NSCLC tumours. Innovative and transformative treatments are urgently needed to overcome abysmal survival rates in NSCLC and we propose that Olaparib holds significant potential to boost the efficacy of NK cell therapy and promote tumour eradication. The proposed study is based on compelling published by Dr Conroy demonstrating that treatment-resistant tumours are less susceptible to NK cells [1], together with published work by others [2] and pilot data by Dr Barr [Fig.1] that Olaparib can sensitise such tumours to NK cells. Here, we will examine whether Olaparib can sensitise treatment-resistant NSCLC cells to NK cells and to the FDA approved NK cell therapy, NK92. By assessing the effects of Olaparib on NK cell therapy, this first-of-its-kind study in drug-resistant NSCLC, has the additional potential to elucidate new mechanisms of action which may have consequences for enhancing the anti-tumour immune response and improving the efficacy of immunotherapies in other malignancies.

Oesophagogastric adenocarcinomas (OAC) encompass a group of inflammation-driven and obesity-associated cancers. Poor 5-year survival rates of less than 20% and treatment response rates of less than 30% place these as poor prognosis cancers, in urgent need of new therapeutic options. As immunotherapy emerges as the fourth pillar of cancer treatment and immune dysregulation in OAC has been uncovered, we propose that novel immunotherapeutic approaches can improve treatment efficacy and outcomes in the growing number of OAC patients. Natural killer (NK) cells are lymphocytes of the innate immune system which function as cytotoxic mediators and key cytokine producers. However, NK cells are severely dysregulated in obesity with phenotypic and functional alterations compromising their anti-tumour abilities. Furthermore, a number of studies have shown that low infiltration of NK cells into solid tumours is associated with poorer prognosis. Therefore, NK cell-based immunotherapies hold great promise to overcome immune dysfunction in cancer. However, two key issues are known to compromise their efficacy: namely successful infiltration of solid tumours and the immunosuppressive tumour microenvironment (TME). In the context of OAC, we have reported active recruitment of NK cells to extratumoural tissues namely the omentum and believe that this compromises their infiltration of the tumour. We have previously reported that such erroneous migration is mediated by the Fractalkine:CX3CR1 axis and propose that CX3CR1 antagonism may be employed to limit this. In this thesis, we have explored the therapeutic applicability of CX3CR1 antagonism to boost NK cell infiltration of tumours in OAC. We have profiled NK cells in OAC patient-derived blood, omentum and tumour samples unearthing unique phenotypes within these compartments and identifying immune dysregulation in the most viscerally obese OAC patients. Importantly, we have identified that the frequencies of intratumoural NK cells are lowest in the most viscerally obese OAC patients, highlighting how detrimental visceral obesity is for anti-tumour immunity in these patients. Furthermore, we have explored the impact of the soluble microenvironments of adipose and tumour tissues derived from viscerally obese and non-obese OAC patients and unveiled divergent effects of these microenvironments on NK cell phenotype and function. Interestingly, our data have revealed that the obese TME impairs NK cell cytotoxicity, thus emphasising the importance of immunotherapeutic approaches to overcome immunosuppressive signalling in OAC. Fractalkine has been previously reported as a driver of obesity-association inflammation, both by our group and others. Our group have previously reported that fractalkine is a key driver of NK and CD8+ T cell migration to the omentum in OAC and uncovered its non-chemotactic effects on CD8+ T cells. Here, we examined the effects of physiologically relevant levels of fractalkine on the phenotype and function of NK cells. Our data revealed that fractalkine mediates the endocytosis of CX3CR1 on the surface of NK cells, increased CD27 expression and shifts NK cell phenotype towards a pro-inflammatory cytokine profile, thus further implicating this chemokine in the potentiation of omental inflammation in obesity. Furthermore, we identified a role for fractalkine in antagonising IL-15-mediated activation of NK cells, highlighting the potential multi-faceted benefits that CX3CR1 antagonism could confer in OAC. Finally, in this thesis, we have developed a novel OAC patient-derived ex vivo model to study immune cell migration between different tissue compartments. Using this platform our data showed that when simultaneously exposed to the chemotactic cues of OAC patient-derived omentum and tumour, NK cells preferentially migrate towards the soluble chemotactic cues of the omentum.