Ramray Bhat

Downregulation of Decorin in ovarian cancer cells and colonization microenvironment drives progression

Epithelial ovarian cancer is a gynecological disease in which transformed cells, upon dissemination into the peritoneum colonize locales such as omenta and form metastatic foci. Colonization is an emergent outcome of the interactions between the invading cancer cells and extracellular matrix (ECM) of the peritoneal serosa. Although ECM is known to be remodeled in cancer, the dynamics in ovarian cancer of a major class of ECM-remodeling factors: the proteoglycans remain understudied. Here, we focus on Decorin, a proteoglycan with binding activity to the principal stromal ECM protein Collagen I and investigate its regulation of ovarian cancer colonization. We observe that Decorin is depleted in cancer deposits within omenta of cancer patients. The spreading of suspended spheroids of the ovarian cancer line SK-OV-3 on engineered Collagen I scaffolds is impaired when the latter is polymerized in the presence of Decorin. Decorin-supplemented Collagen I shows poorer fibrillar organization, which has been associated with slower kinetics of cancer cell migration. To our surprise, Decorin was also found to be depleted in primary tumor cells as well as in ovarian cancer cell lines compared with their controls. Overexpression of wild type Decorin, but not its glycosaminoglycan (GAG)-removed mutant in cancer cells decreased mean spheroid size, invasion through Collagen I matrix, and migration on fibronectin matrix scaffolds. Our results suggest that downregulation of an extracellular inhibitor of colonization occurs both in the seed and soil components of the metastatic toolkit; in addition, the GAG chains of Decorin may be crucial to its carcinomatosis-inhibiting functions.

Rheological transition driven by matrix makes cancer spheroids resilient under confinement

Cancer metastasis through confining peritoneal microenvironments is mediated by spheroids: clusters of disseminated cells. Ovarian cancer spheroids are frequently cavitated; such blastuloid morphologies possess an outer ECM coat. We investigated the effects of these spheroidal morphological traits on their mechanical integrity. Atomic force microscopy showed blastuloids were elastic compared with their prefiguring lumenless moruloid counterparts. Moruloids flowed through microfluidic setups mimicking peritoneal confinement, exhibited asymmetric cell flows during entry, were frequently disintegrated, and showed an incomplete and slow shape recovery upon exit. In contrast, blastuloids exhibited size-uncorrelated transit kinetics, rapid and efficient shape recovery upon exit, symmetric cell flows, and lesser disintegration. Blastuloid ECM debridement phenocopied moruloid traits including lumen loss and greater disintegration. Multiscale computer simulations predicted that higher intercellular adhesion and dynamical lumen make blastuloids resilient. Blastuloids showed higher E-cadherin expression, and their ECM removal decreased membrane E-cadherin localization. E-cadherin knockdown also decreased lumen formation and increased spheroid disintegration. Thus, the spheroidal ECM drives its transition from a labile viscoplastic to a resilient elastic phenotype, facilitating their survival within spatially constrained peritoneal flows.

The senescent mesothelial matrix accentuates colonization by ovarian cancer cells

Abstract Ovarian cancer is amongst the most morbid of gynecological malignancies due to its diagnosis at an advanced stage, a transcoelomic mode of metastasis, and rapid transition to chemotherapeutic resistance. Like all other malignancies, the progression of ovarian cancer may be interpreted as an emergent outcome of the conflict between metastasizing cancer cells and the natural defense mounted by microenvironmental barriers to such migration. Here, we asked whether senescence in coelom-lining mesothelia, brought about by drug exposure, affects their interaction with disseminated ovarian cancer cells. We observed that cancer cells adhered faster on senescent human and murine mesothelial monolayers than on non-senescent controls. Time-lapse epifluorescence microscopy showed that mesothelial cells were cleared by a host of cancer cells that surrounded the former, even under sub-confluent conditions. A multiscale computational model predicted that such colocalized mesothelial clearance under sub-confluence requires greater adhesion between cancer cells and senescent mesothelia. Consistent with the prediction, we observed that senescent mesothelia expressed an extracellular matrix with higher levels of fibronectin, laminins and hyaluronan than non-senescent controls. On senescent matrix, cancer cells adhered more efficiently, spread better, and moved faster and persistently, aiding the spread of cancer. Inhibition assays using RGD cyclopeptides suggested the adhesion was predominantly contributed by fibronectin and laminin. These findings led us to propose that the senescence-associated matrisomal phenotype of peritoneal barriers enhances the colonization of invading ovarian cancer cells contributing to the metastatic burden associated with the disease.

Ovarian cancer cells exhibit diverse migration strategies on stiff collagenous substrata

In homoeostasis, the shape and sessility of untransformed epithelial cells are intricately linked together. Variations of this relationship in migrating cancer cells as they encounter different microenvironments are as yet ill understood. Here, we explore the interdependency of such traits in two morphologically distinct invasive ovarian cancer cell lines (OVCAR-3 and SK-OV-3) under mechanically variant contexts. We first established a metric toolkit that assessed traits associated with cell motion and shape, and rigorously measured their dynamical variation across trajectories of migration using a Shannon entropic distribution. Two stiffness conditions on polymerized collagen I with Young's moduli of 0.5 kPa (soft) and 20 kPa (stiff) were chosen. Both the epithelioid OVCAR-3 and mesenchymal SK-OV-3 cells on soft substrata exhibited slow and undirected migration. On stiff substrata, SK-OV-3 showed faster persistent directed motion. Surprisingly, OVCAR-3 cells on stiffer substrata moved even faster than SK-OV-3 cells but showed a distinct angular motion. The polarity of SK-OV-3 cells on stiff substrata was well correlated with their movement, whereas, for OVCAR-3, we observed an unusual "slip" behavior, wherein the axes of cell shape and movement were poorly correlated. Whereas SK-OV-3 and OVCAR-3 showed greater mean deformation on stiffer substrata, the latter was anticorrelated with variation in angular motion or the mean deviation between shape and motility axis for SK-OV-3 but poorly correlated for OVCAR-3. Moreover, on softer substrata OVCAR-3 and SK-OV-3 were relatively rigid but showed greater shape variation (with OVCAR-3 showing a higher fold change) on stiffer substrata. Our findings suggest that greater deformability on stiffer milieu allow epithelioid cells to overcome constraints on the congruence in axis of shape and motion seen for mesenchymal cells and display distinct motile behaviors across this phenotypic spectrum.

Extracellular matrix mediates moruloid-blastuloid morphodynamics in malignant ovarian spheroids

Ovarian cancer metastasizes into peritoneum through dissemination of transformed epithelia as multicellular spheroids. Harvested from the malignant ascites of patients, spheroids exhibit startling features of organization typical to homeostatic glandular tissues: lumen surrounded by smoothly contoured and adhered epithelia. Herein, we demonstrate that cells of specific ovarian cancer lines in suspension, aggregate into dysmorphic solid “moruloid” clusters that permit intercellular movement, cell penetration, and interspheroidal coalescence. Moruloid clusters subsequently mature into “blastuloid” spheroids with smooth contours, a temporally dynamic lumen and immotile cells. Blastuloid spheroids neither coalesce nor allow cell penetration. Ultrastructural examination reveals a basement membrane-like extracellular matrix coat on the surface of blastuloid, but not moruloid, spheroids. Quantitative proteomics reveals down-regulation in ECM protein Fibronectin-1 associated with the moruloid-blastuloid transition; immunocytochemistry also confirms the relocalization of basement membrane ECM proteins: collagen IV and laminin to the surface of blastuloid spheroids. Fibronectin depletion accelerates, and enzymatic basement membrane debridement impairs, lumen formation, respectively. The regulation by ECM dynamics of the morphogenesis of cancer spheroids potentially influences the progression of the disease.