Mechanotransduction of cancer cells in the solid tumor environment is an active area of research, yet far less work has been done to examine the biological behavior of cancer cells in the blood flow environment. Recently, mechanical stimuli such as shear stress have received attention for their effects on cancer progression. For instance, studies have shown that shear stress has been associated with enhanced metastasis and cancer cell death. In the applicant’s laboratory, the synergistic effect of shear stress on tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis of circulating tumor cells (CTCs) was demonstrated, as well as the unique ability of cancer cells to survive extremely high pulses of shear stress, comparable to blood cells. These mechanical cues can be translated into biochemical responses in cells through the process of mechanotransduction. It is proposed to subject cell suspensions to repeated shear stress pulses in a multiwell plate format to study shear stress response and to develop “mechanoresistant” cell lines that will be phenotypically and genotypically characterized with the goal of identifying the drivers that enable cancer cells to survive in circulation. Moreover, given that the presence of CTC aggregates in the blood signal more aggressive and metastatic disease, multicellular aggregates modeled after aggregates isolated and characterized from prostate cancer patient blood samples will be tested in vitro for their mechanical responses, and also used to guide the development of model cells and spheroids to be injected into experimental mouse models of bloodborne metastasis. This research is organized around three specific aims: Specific Aim 1: To develop a new high throughput device to study the effect of fluid shear stress on cancer cell responses. A multiwell plate configuration based on a BioJet printer will enable direct analysis with multiwell plate-capable flow cytometers and spectrophotometers. Calcium influx, membrane and mitochondrial damage, and apoptosis of cancer cells in response to shear stress signals will be examined, and “mechanoresistant” prostate cancer cells developed and characterized. Specific Aim 2: To develop the shear flow device and culture conditions to study shear stress responses modulated by interactions with stromal cells. Circulating tumor cell aggregates isolated from prostate cancer patient blood samples will be characterized, and used to develop model aggregates for further study. The stability and survival of heterogeneous tumor cell aggregates in shear flow will then be studied. Specific Aim 3: To examine the roles of cancer cell mechanosensitization and mechanoresistance on metastatic tumor burden in vivo. Orthotopic metastasis studies using cells with modulated shear sensitivity will be performed. Mechanoresistant cancer cells vs. parental cancer cells will be compared in an experimental mouse model of metastasis, and the fate of injected cell aggregates studied as well.

In many types of cancer, cancer cells from the primary tumor can intravasate into the peripheral circulation as circulating tumor cells (CTCs). Cancer cells respond to the forces in blood flow in ways that are not yet fully understood. This project will develop new technology to facilitate new understanding of the response of cancer cells to fluid flow.