Poster S20 - Cancer Cell Glucose Metabolic Reprogramming via A Fluid Shear Stress Activated PI3K Pathway

Introduction: Cellular metabolic reprogramming is a defining hallmark of cancer due to its ability to attenuate apoptosis. Cancer cell metabolism is a sought-after target for chemotherapeutics, but effectiveness has been hindered by metabolic plasticity. Cells dynamically tune their bioenergetic needs in response to changes in the tumor microenvironment. In cancer, this metabolic plasticity aids cells as they traverse through the diverse environments presented in the metastatic cascade. Therefore, it is crucial to comprehend the metabolic alterations in cells at each metastatic step, understanding the transition from the primary site to the metastatic site. While numerous studies have identified roles for various chemical mediators of glucose metabolism and cell energetics, there is a gap regarding metabolism and mechanotransduction.

Circulatory fluid shear stress (FSS) modulates several cellular components that have been shown to also affect cell metabolism including the actin cytoskeleton, RhoA, YAP and PI3K. However, FSS-induced changes in metabolism and energetics in cancer cells undergoing circulating tumor cell (CTC)-like conditions have not yet been investigated directly. We have previously shown that mesenchymal subpopulations of CTCs are better equipped to survive FSS. Here, we aim to investigate the role of FSS-modulated metabolism on survival in CTC-like conditions. We utilized cone-and-plate viscometers to apply circulatory FSS to suspended cancer cells, and we monitored their metabolic activity via a Seahorse XF Analyzer and the PercevalHR probe of ATP:ADP. Our data indicates that FSS leads to an increase in metabolic activity through PI3K activity. Moreover, our data indicate that this induction aids in shear stress survival.

Materials and

Methods: MDA-MB-231 metastatic breast cancer cells were sheared for 2 hours at 12 dyne/cm2 using a cone and plate viscometer blocked with 5% BSA in HBSS to prevent non-specific binding. Metabolic activity of the population was measured using a Real Time ATP-Rate Assay in a Seahorse XF Analyzer 24 hrs post-shear. ATP:ADP in individual cells was measured using the genetically encoded PercevalHR probe on an Zeiss LSM800 live cell imaging platform. Quantification of the ATP:ADP ratio is normalized against a pH calibration probe.

To assess proliferation, cells were cultured for 48 hours post shear and fixed in 3.2% PFA and stained with DAPI and Ki67. Cells were imaged on an Zeiss LSM800 confocal microscope.

To test PI3K activity in response to FSS, we inhibited PI3K utilizing 3 μM of a pan class 1 PI3K inhibitor BKM120 for three hours. After BKM120 exposure cells were sheared and tested on the Seahorse XF Analyzer 24 hours post shear. In separate experiments, post-shear were immediately stained with Annexin V and propidium iodide to measure viability of the cell population.

Results, Conclusions, and Discussions: Our results indicate that 24 hours post-FSS exposure, glycolysis, oxidative phosphorylation, and ATP increase (Fig. 1A). To observe single-cell changes, we exposed cells expressing the PercevalHR probe to FSS and measured ATP:ADP every 24 hours for 4 days. Our data indicates that the bioenergetic increase due to FSS persisted for at least 96 hours post-FSS exposure (Fig. 1B), suggesting that FSS causes a prolonged period of energetic increase in cancer cells experiencing CTC-like conditions.

To assess if this increase in ATP production caused changes in cell behavior, we investigated if FSS exposure altered cellular proliferation. Colocalization of DAPI and Ki67 revealed that after shearing, the number of cells expressing nucleus-bound Ki67 (green) increased nearly three-fold (Fig. 1C). This suggests that FSS increases cancer cell proliferation for up to 48 hours after FSS.

Studies have shown that FSS increases PI3K activation, and PI3K is linked to metabolic regulation. Therefore, we assessed the effects of PI3K activity on FSS-induced metabolism. Inhibition of PI3K via BKM120 prevents the increase of glycolysis and mitochondrial respiration post-FSS exposure without affecting basal ATP levels in the static condition (Fig. 1D). This data indicates that the inhibitor is not metabolically harming the cells while preventing a metabolic increase in response to shear. Together, our data indicates that PI3K activity is required for FSS to mechanically induce increased bioenergetics.

Since metabolic reprogramming aids in cancer cell survival during metastasis, we aimed to determine if preventing FSS-induced metabolism reduced cell viability. Treatment with BKM120 leads to a significant 10% decrease in viability under shear (Fig. 1E). Our results indicate that there is an increase in late apoptotic cells, and a significant increase in necrotic cells (Fig. 1F) in the BKM120 group. Lack of cell death in the static conditions demonstrates that BKM120 alone is not inducing cell death. These findings suggest that PI3K inhibition under shear leads to immediate cell destruction that bypasses the initial steps of the canonical apoptotic pathway.

Overall, our study suggests that CTC-like FSS mechanotransduction increases cellular bioenergetics and survival through PI3K, while also increasing proliferation.

Acknowledgements (Optional):