Investigating Mechanical Properties of Suspended Ovarian Cancer Cells and Clusters

Abstract

The peritoneal cavity of a patient suffering from advanced epithelial ovarian cancer is filled with disseminated multicellular aggregates, commonly known as spheroids. These spheroids colonize abdominal organs leading to metastasis. Metastasizing spheroidal cancer cells frequently become resistant to chemotherapeutic drugs, thus demanding a rigorous investigation of mechanisms underlying their formation and stability. Spheroids obtained by tapping the malignant ascites of ovarian cancer patients show heterogeneous morphologies: some exhibit a dysmorphic ‘moruloid’ (mulberry-like) phenotype, and others show smooth compacted surfaces and an internal lumen, giving them a ‘blastuloid’ appearance. Additionally, blastuloid spheroids reveal the presence of a basement membrane coat surrounding them which was also raised the interest in understanding its role in their structure, and its contribution to their localized stiffness. These morphologies could represent consequences of phenotypically heterogenous cell types, or indicate progressive stages of metastasis with moruloid phenotypes maturing into blastuloid counterpart. There is a burgeoning body of literature on biophysical investigations of tumorigenic cellular ensembles. Of these, most studies focus on the migrational dynamics of spheroidal or tumoroidal cells within stromal-like extracellular matrix (ECM) microenvironments. In fluid microenvironments, the assembly of multicellular structures from suspended single cells likely employ distinct mechanisms. Although elegant theoretical models have been constructed recently to explain dynamical structural transitions, technical difficulties of efficiently imaging floating clusters have allowed few biophysical characterizations of spheroids. Notable experimental exceptions include efforts to mechanically analyze spheroids using microtweezers, wherein those constituted from breast cancer cells were found to be softer than from untransformed controls, and investigations using cavitational rheology to determine the cortical tension in spheroids of HEK293 cells. A pertinent study by Panwhar and coworkers recently describes a high throughput approach using virtual liquid-bound channels to show that the stiffness of multicellular spheroids is an order of magnitude lower than that of cells that constitute them. Although these investigations have not studied temporal topological transitions between multicellular morphologies, they lay the foundation for such studies within fluid microenvironments. In this dissertation, I try to investigate the mechanics and biophysics of ovarian cancer starting at the single cell level, thereby moving to spheroids. I used AFM as an elegant tool to characterize the stiffness of single cells in suspension. The difficulty in imaging suspended cells and 3D cultures was overcome using a novel technique employing noble agar as a substratum to immobilize them. Three different cell lines representing ovarian cancer were studied for this and compared to their respective adhered states. The technique was also used for spheroids of the mentioned morphologies to sense localized stiffness changes as they mature, thereby making it versatile and size independent.

Subsequently, we combine microfluidics with high- speed time-lapse videography and imaging analysis to investigate the consequences of lumen formation and the basement membrane (BM) coat on the architecture and integrity of ovarian cancer spheroids providing architectural robustness to the transitory ovarian cancer metastatic niche within constrained flow spaces. We investigate their time of transit, relaxation dynamics, disintegration statistics, particle image velocimetry (PIV), shape evolution, etc as tools to further understand their structural and temporal integrity. We further extended these techniques to study the effect of chemoresistant drugs and inter-cellular motion inhibitors, and their effect on the cluster mechanics and biophysics. In future, some design iterations can be tried to trap the clusters and monitor the long-term effect of shear on them. Keeping them trapped for a longer duration can give insight into the real-time changes in a fluidic environment.