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Cancer Technologies

Cancer Technologies - Poster Session E

Poster T1 - Mechanical Strains Alter VEGFR-2 Activation and Migration in Triple Negative Breast Cancer

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer that differs from others due to its poor prognosis and limited treatment options, due to the lack of hormone receptors on the tumor cells. Aberrant expression and signaling of vascular endothelial growth receptor 2 (VEGFR-2) in TNBC have been linked to decreases in overall survival. Anti-VEGFR2 therapies were initially designed to target angiogenesis in the tumor microenvironment, but these drugs show limited efficacy in breast cancer patients. We believe this is due to the limited knowledge of how biomechanical forces within the tumor microenvironment (TME) affect TNBC growth, migration, and response to chemotherapies. Our recent studies have shown that VEGFR-2 in endothelial cells can be activated by biomechanical strains, and that this behavior is not blocked with soluble inhibitors against VEGFR-2. Receptor activation occurs through the phosphorylation of tyrosine residues including Y1054/1059 and Y1214. Cancer-associated fibroblasts (CAFs) in the TME have been shown to generate high levels of contractile activity due to increased α-smooth muscle actin, which induces strains around tumor cells. Our objective is to determine how strains regulate VEGFR-2 activation in TNBC cells to promote tumor progression and migration.

Materials and

Methods:  First, we investigated how short-term strain (5 or 15 minutes) affected phosphorylation of VEGFR-2 and tyrosine residues Y1054/1059 and Y1214. A FlexCell system was used to apply a strain at 10% elongation at 0.3Hz, mimicking CAF induced strains as measured by our previous studies. MDA-MB-231, a human TNBC line, were separated into the following treatment groups: No Treatment (NT, control), exogenous VEGF only (+V, 25ng/mL), strain only (+), or strain with exogenous VEGF (+V). Post-strain treatments, these lysates were collected, and Western blots were conducted to measure the change in the levels of phosphorylation. To study cell migration, we utilized a multi-chambered microfluidic platform previously developed by our lab. TNBC cells were loaded into the middle chamber, with CAFs or normal breast fibroblasts in the side chambers. Media was supplied such that flow moved from the TNBC chamber to the side chambers, effectively washing out CAF and NBF secreted factors away from chamber interfaces. The cells were allowed to migrate for 14 days, and then they were fixed to evaluate the effect of CAF-induced strains on migration.

Results, Conclusions, and Discussions: The purpose of this study was to determine the impact of strains on VEGFR-2 signaling and migration behaviors. Results show that the 231 cell line had significantly increased phosphorylation of VEGFR-2 at Y1054/Y1059 with strain and exogenous VEGF at both 5 and 15 minutes, representing a synergistic trend with VEGF and strain (Fig. 1A). There is increased phosphorylation of Y1214 at 15 minutes compared to 5 minutes (Fig.1B). The levels of total VEGFR-2 were also significantly increased with VEGF, with or without strain, at 5 and 15 minutes (Fig. 1C). Our migration study results demonstrate that significantly more 231 cells toward CAFs than NBFs (Fig. 2). As media flow was setup to “wash out” CAF and NBF secreted factors, this indicates that mechanical interactions caused by the fibroblasts at the interfaces between chambers may prompt changes in migration behaviors. Overall, these results indicate that coupled ligand and mechanical stimulation drives moreVEGFR-2 activation in TNBC cells and that mechanical stimulation from CAFs can alter migration. Together, this suggests that mechanical stimulation of TNBC may be a key regulator of disease progression. Furthermore, this could potentially explain the failure of anti-VEGFR-2 drugs to improve survival rates for breast cancer patients. Future directions will include investigating how mechanical stimulation regulates VEGFR-2 activation, proliferation, and migration in TNBC cells using genetic modifications to inhibit VEGFR-2 signaling.

Acknowledgements (Optional):