Exploring the Synergistic Effect between Mechanical Stimulation and VEGFR-2 Expression in TNBC

Introduction: : Triple negative breast cancer (TNBC) is a subtype of breast cancer lacking estrogen, progesterone, and HER2 receptors. TNBC is characterized by both its increased aggressiveness, chemoresistance, and high incidence of metastasis. The combination of this, with the absence of receptors, leads to limited treatment options for this disease. High levels of vascular endothelial growth receptor 2 (VEGFR-2) in TNBC which have been correlated to a decreased survival. Many anti-VEGFR-2 therapies, aimed at inhibiting angiogenesis, have been developed to treat cancers. However, these drugs tend to show limited efficacy in clinical trials. We believe this is due to the fact that most of these drugs are created to inhibit ligand/receptor interactions, without considering mechanosignaling. Specifically, we believe that understanding how biomechanical forces within the tumor microenvironment (TME) affect tumor growth, proliferation, migration, and response to chemotherapies is crucial in working towards advancing treatment options. Our previous study shows that VEGFR-2 activation is increased in endothelial cells when exposed to biomechanical strains, and this behavior was not blocked even in the presence of a VEGFR-2 inhibitor. Stromal components of the TME, such as cancer-associated fibroblasts (CAFs), have been shown to generate strains of similar magnitude through matrix deformation resulting from increased contractile activity. Our hypothesis is that strain regulates VEGFR-2 activation in TNBC, leading to increased migration and proliferation.

Materials and

Methods: : We utilized a 3D-engineered microfluidic model of the TME to investigate strain regulation of TNBC migration and proliferation. The device consists of 3 tissue chambers, all of which can be loaded independently (Figure 1A). The chambers are connected via 20mm ports that allow for communication. The MDA-MB-231 model TNBC line was modified with shRNA to knockdown VEGFR-2 (shVEGFR-2), with ~90% less expression compared to scramble controls (SCR). Cells were loaded into the center chambers in a 10mg/mL fibrin gel. Normal breast fibroblasts (NBFs) and CAFs were loaded into opposing side chambers; prior studies have established the CAFs drive larger matrix deformations, and therefore higher strains, than NBFs. The devices were cultured for 14 days before they were fixed and stained for Ki-67. Devices were fed in an “outward” manner, in the sense that media was supplied to the center chamber and flowed out to the sides. This limited the opportunity of secreted factors from CAFs and NBFs to influence migration at the interfaces. A second set of studies used magnetic beads to act as pseudo-cells to introduce strains without secreted factors. To measure proliferation, cells were embedded in fibrin gels and separated into the following treatment groups: Vehicle (VEH, control), SU5416 (25 ng/mL), Pseudo-cells, and no Pseudo-cells. SU5416 is a tyrosine kinase inhibitor used to block VEGFR-2 activation. Rings were fed with EGM-2 every other day and fixed and stained for Ki-67 and DAPI after 7 days in culture.

Results, Conclusions, and Discussions:: The purpose of these experiments was to determine how strain alters TNBC migration and proliferation in 3D TME models. Results show that in both the SCR and KD (shVEGFR-2) samples, the TNBC cells migrated more towards the side chambers containing CAFs compared to NBFs (Figure 1B-D). More specifically, the normalized migration towards the chamber containing CAFs was highest in the SCR devices, demonstrating the highest migration response in VEGFR-2 expressing cells. Additionally, there was an overall decrease of migration in the devices containing shVEGFR-2 cells suggesting that the VEGFR-2 knockdown had a partial effect on TNBC migration (Figure 1D). Additionally, we quantified proliferation in the side chambers for both TNBC and fibroblasts. From this, we observed that proliferation in the CAF-containing chamber was higher compared to the NBF chamber, exhibited by increased Ki-67 expression (Figure 1E,F). This data suggests that while VEGFR-2 may regulate overall migration of TNBC, CAF generated matrix deformations may also direct migration toward areas of higher contractile activity. In proliferation studies, SU5416 was shown to inhibit proliferation compared to the vehicle controls in the SCR samples (Figure 2A,B). The shVEGFR-2 showed no changes in proliferation across treatment groups. Together, this data suggests that mechanical stimulation, resulting from cell-induced or non-cellular induced strains, may promote enhanced proliferation of TNBC. Future studies will work to decouple proliferation and migration in side chambers to determine which is the primary driver of these observed behaviors. Furthermore, by knocking down VEGFR-2 expression in the cells, this response is decreased. Overall, these results display enhanced tumor progression demonstrated by the increased proliferation and migration towards a higher contractile area within the microtissues. Additionally, the results help highlight the importance of understanding the role that CAFs and VEGFR-2 play in progressing TNBC. As a result, this interaction can be targeted in effort to develop better therapies, advancing TNBC treatments.

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