ROLE OF STAT1 AND CXCL10 IN THE TUMOUR IMMUNE MICROENVIRONMENT OF HIGH-GRADE SEROUS OVARIAN CANCER

High-grade serous ovarian cancer (HGSC) is the most prevalent epithelial ovarian cancer characterized by late detection, metastasis and resistance to chemotherapy. Previous studies on the tumour immune microenvironment in HGSC identified STAT1 and CXCL10 as the most differentially expressed genes between treatment naïve chemotherapy resistant and sensitive tumours. Interferon-induced STAT1 is a transcription factor, which induces many genes including tumour suppressor genes and those involved in recruitment of immune cells to the tumour immune microenvironment (TME), including CXCL10. CXCL10 is a chemokine that recruits tumour infiltrating lymphocytes (TILs) and exhibits angiostatic function. The current study was performed to determine the effects of differential STAT1 and CXCL10 expression on HGSC disease progression and TME. STAT1 expression and intratumoural CD8+ T cells were evaluated as prognostic and predictive biomarkers via immunohistochemistry on 734 HGSC tumours accrued from the Terry Fox Research Institute-Canadian Ovarian Experimental Unified Resource. The combined effect of STAT1 expression and CD8+ TIL density was confirmed as prognostic and predictive companion biomarkers in the second independent biomarker validation study. Significant positive correlation between STAT1 expression and intratumoral CD8+ TIL density was observed. The effects of enforced CXCL10 expression on HGSC tumour growth, vasculature and immune tumour microenvironment were studied in the ID8 mouse ovarian cancer cell engraftment in immunocompetent C57BL/6 mice. Significant decrease in tumour progression in mice injected with ID8 CXCL10 overexpressing cells compared to mice injected with ID8 vector control cells was observed. Multiplexed cytokine analysis of ascites showed differential expression of IL-6, VEGF and CXCL9 between the two groups. Endothelial cell marker staining showed differences in tumour vasculature between the two groups. Immune transcriptomic profiling identified distinct expression profiles in genes associated with cytokines, chemokines, interferons, T cell function and apoptosis between the two groups. These findings provide evidence that STAT1 is an independent biomarker and in combination with CD8+ TIL density could be applied as novel immune-based biomarkers in HGSC. These results provide the basis for future studies aimed at understanding mechanisms underlying differential tumour STAT1 and CXCL10 expression and its role in pre-existing tumour immunologic diversity, thus potentially contributing to biomarker guided immune modulatory therapies.

IMMUNE-ANGIOGENESIS MECHANISMS ASSOCIATED WITH PORCINE PREGNANCY SUCCESS AND FAILURE

Spontaneous fetal loss (25-40%) leading to decrease in litter size is a significant concern to the pork industry. A deficit in the placental vasculature has emerged as one of the important factors associated with fetal loss. During early pig pregnancy, the endometrium becomes enriched with immune cells recruited by conceptus-derived signals including specific chemokine stimuli. These immune cells assist in various aspects of placental development and angiogenesis. Recent evidence suggests that microRNAs (miRNAs: small non-coding RNAs that regulate gene expression) regulate immune cell development and their functions. In addition, intercellular communication including exchange of biomolecules (e.g. miRNAs) between the conceptus and endometrium regulate key developmental processes during pregnancy. To understand the biological significance of immune cell enrichment, regulation of their functions by miRNAs and transfer of miRNAs across the maternal fetal-interface, we screened specific sets of chemokines and pro- and anti-angiogenic miRNAs in endometrial lymphocytes (ENDO LY), endometrium, and chorioallantoic membrane (CAM) isolated from conceptus attachment sites (CAS) during early, gestation day (gd)20 and mid-pregnancy (gd50). We report increased expression of selected chemokines including CXCR3 and CCR5 in ENDO LY and CXCL10, CXCR3, CCL5, CCR5 in endometrium associated with arresting CAS at gd20. Some of these differences were also noted at the protein level (CXCL10, CXCR3, CCL5, and CCR5) in endometrium and CAM. We report for the first time significant differences for miRNAs involved in immune cell-derived angiogenesis (miR-296-5P, miR-150, miR-17P-5P, miR-18a, and miR-19a) between ENDO LY associated with healthy and arresting CAS. Significant differences were also found in endometrium and CAM for some miRNAs (miR-17-5P, miR-18a, miR-15b-5P, and miR-222). Finally, we confirm that placenta specific-exosomes contain proteins and 14 select miRNAs including miR-126-5P, miR-296-5P, miR-16, and miR-17-5P that are of relevance to early implantation events. We further demonstrated the bidirectional exosome shuttling between porcine trophectoderm cells (PTr2) and porcine aortic endothelial cells (PAOEC). PTr2-derived exosomes were able to modulate the endothelial cell proliferation that is crucial for the establishment of pregnancy. Our data unravels the selected chemokines and miRNAs associated with immune cell-regulated angiogenesis and reconfirm that exosome mediated cell-cell communication opens-up new avenues to understand porcine pregnancy.