Homo and Heterotypic Cellular Cross-Talk in Epithelial Ovarian Cancer Impart Pro-Tumorigenic Properties through Differential Activation of the Notch3 Pathway

SIMPLE SUMMARY: The omental metastatic spreading of epithelial ovarian cancer is spearheaded by complex cell–cell interactions present in the fluidic microenvironment (ascites), which is yet to be fully decoded. Using a unique co-culture model of SNFT (SKOV3 cells expressing a Notch3 luciferase reporter-sensor) and NIH3T3 cells (differentially overexpressing Jagged1 ligand), we demonstrated that incremental Jagged1 expression led to proportional Notch3 activation in SNFT. Differential Notch3 activation was also evident from co-culture of SNFT with other EOC cell lines and ascites-derived cancer-associated fibroblasts of HGSOC patients expressing varying levels of Jagged1. Amongst the top five modulated genes identified by the gene profiler array, both p21 and VEGFA showed enhanced expression (for p21)/secretion (for VEGFA) in SNFT when induced with Jagged1. Secreted VEGFA further reduced CSC differentiation in platinum-resistant A2780 cells. Pronounced VEGFA expression associated with Notch3 up-regulation in metastatic HGSOC tumors delineates an unknown role of the Notch3/VEGFA axis in EOC progression. ABSTRACT: An active fluidic microenvironment governs peritoneal metastasis in epithelial ovarian cancer (EOC), but its critical functional/molecular cues are not fully understood. Utilizing co-culture models of NIH3T3 cells (differentially overexpressing Jagged1) and SKOV3 cells expressing a Notch3 luciferase reporter-sensor (SNFT), we showed that incremental expression of Jagged1 led to proportional Notch3 activation in SNFT. With no basal luciferase activity, this system efficiently recorded dose-dependent Notch3 activation by rh-Jag1 peptide and the non-appearance of such induction in co-culture with NIH3T3(Δjag1) cells indicates its sensitivity and specificity. Similar Notch3 modulation was shown for the first time in co-cultures with HGSOC patients’ ascites-derived cancer-associated fibroblasts and Jagged1-expressing EOC cell lines. NIH3T3(J1-A) and OVCAR3 co-cultured SNFT cells showed maximum proliferation, invasion, and cisplatin resistance among all the heterotypic/homotypic cellular partners. VEGFA and CDKN1A are the two most upregulated genes identified across co-cultures by the gene profiler array. Co-culture induced VEGFA secretion from SNFT cells which also reduced cancer stem cell differentiation in platinum-resistant A2780 cells. rh-Jag1-peptide promoted enhanced nuclear-cytoplasmic p21 expression. Additionally, metastatic HGSOC tumors had higher VEGFA than corresponding primary tumors. This study thus demonstrates the tumoral and non-tumoral cell-mediated differential Notch3 activation imparting its tumorigenic effects through two critical molecular regulators, VEGFA and p21, during EOC progression.