Model of collective detachment in high-grade serous ovarian cancer demonstrates that tumor spheroids produce ECM to support metastatic processes

Abstract

High-grade serous ovarian cancer (HGSOC) metastasizes through transcoelomic spread, with both single cells and spheroids of tumor cells observed in patient ascites. These spheroids may form through single cells that detach and aggregate (Sph-SC) or through collective detachment (Sph-CD). We developed an in vitro model to generate and separate Sph-SC from Sph-CD to enable study of Sph-CD in disease progression. In vitro-generated Sph-CD and spheroids isolated from ascites were similar in size (mean diameter 51 vs 55 μm, p > 0.05) and incorporated multiple ECM proteins. Using the in vitro model, nascent protein labeling, and qRT-PCR, we determined that ECM was produced after detachment. As fibronectin plays a key role in many cell adhesion events, we confirmed that inhibiting RGD-based adhesion or fibronectin assembly reduced Sph-CD-mesothelial adhesion strength under shear stress. Our model will enable future studies to determine factors that favor formation of Sph-CD, as well as allow investigators to manipulate Sph-CD to better study their effects on HGSOC progression.

The role of Piezo1 mechanotransduction in high-grade serous ovarian cancer: Insights from an in vitro model of collective detachment

“…We observed changes in MMP-1 and/or MMP-10 in a subset of conditions, as well as decreases in Col I and fibronectin for all cell lines on stiffer substrates. This finding may seem in conflict with our prior study demonstrating a role for fibronectin in Sph-CD for reattachment; however, in that study, we demonstrated that the ECM was primarily produced by cells after they detached rather than taken from the substrate ( 9 ). Consistent with the proposed MMP-based mechanism, Piezo1 is linked to increased MMP-2 in endothelial cells ( 54 ) and human trabecular meshwork cells ( 55 ).…”

“…Last, we considered the cell types to incorporate into our model. Our prior analysis of spheroids isolated from patient ascites suggested that they are composed primarily of epithelial cells ( 9 ). However, it is possible that the originating site has more cellular diversity.…”

“…We previously developed an in vitro model using a filter to separate Sph-CD from single cells and any spheroids that form by aggregation (Sph-SC). However, the physical setup of the model with a solid substrate under the cells means that it is possible that some Sph-CD form from single cells that quickly aggregated in solution before reaching the filter ( 9 ). Our results here support that most of the spheroids captured in the top filter are produced by collective detachment.…”

“…Tumor cells that have detached can be isolated from ascites as both single cells and spheroids of cells on the order of 50 to 100 μm in diameter ( 9 , 10 ). While most studies of HGSOC metastasis have used single cells, spheroids are believed to disproportionately contribute to metastasis due to their resistance to apoptosis and chemotherapy ( 11 ).…”

“…Unfortunately, animal models are not easily adapted to address what controls the extent of detachment and whether a tumor cell releases as a single cell or in a spheroid. Therefore, we recently developed an in vitro model that isolates tumor cells that detach as spheroids from cells that detach as single cells ( 9 ). In this model, ovarian cancer cells are cultured on top of a polyacrylamide (PAA) gel that rests within a cell strainer (Fig.…”