R18 - Exploring the Effects of Contractility Inhibitors and Strain on VEGF Secretion and VEGFR-2 Activation

Introduction: : Angiogenesis is a key driver of tumor progression, where new blood vessels branch and grow from existing vascular networks. Several anti-cancer therapies currently attempt to block tumor progression by inhibiting angiogenesis, which deprives the growing tumor of needed nutrients. Two primary targets of these therapies are vascular endothelial growth factor (VEGF) and its receptor, vascular endothelial growth factor receptor-2 (VEGFR-2) [1]. In clinical settings, these drugs are used to interrupt interactions of VEGF/VEGFR-2 to ultimately decrease angiogenesis and stop tumor progression. Inefficiencies in these therapies may be due to increased mechanical changes in the tumor microenvironment (TME) such as strain. In the TME, mechanical changes such as tensile strains or increased matrix stiffness have been shown to alter how cells proliferate [2]. In these models, strain can come from the contractility of stromal cells. Prior work in our lab has identified how different tyrosine residues become phosphorylated to activate VEGFR-2 signaling in response to mechanical stimulation from CAFs. However, it is unclear if blocking CAF contractility also alters VEGF secretion. We hypothesize that inhibiting contractility does not alter VEGF secretion by CAFs and therefore that these strategies only alter mechanical activation of VEGFR-2.

Materials and

Methods: : A 3D microtissue model was made by loading cells, thrombin, and fibrinogen into a poly(dimethylsiloxane) (PDMS) ring. For these studies, human umbilical vein endothelial cells immortalized with TERT2 (HUVEC TERT2s) and breast cancer associated fibroblasts (CAFs) were loaded in single or co-cultures into the 3D TME models. Models were then given 20µM Blebbistatin, 50µM Blebbistatin, or a vehicle control (Veh); some samples received no treatment (NT) as a negative control. Conditioned media samples were collected to quantify VEGF expression using an ELISA. To determine if Blebbistatin had cytotoxic effects at these levels, cells were then stained with Hoescht 33342 and Sytox for 1h prior to imaging. Images were collected on an Olympus IX83 inverted microscope. Dead cell count was normalized to the total cell count.
HUVEC TERT2 cells were modified with CRISPR (Synthego) to generate a spCas9 control line and mutant line with a non-phosphorylatable residue of interest. For the mutant line, Y1054 was substituted with a phenylalanine (F) to block activation at this site (Y1054F). To study how VEGFR-2 was altered in these cells with mechanical stimulation, we utilized a Flexcell system. Cells were plated on Flexcell plates (Uniaxial, Collagen-1 coated) and strained for 0 ,5, or 15 min (9% elongation, 0.3 Hz), with some groups receiving treatment with exogeneous VEGF (25ng/mL) with controls in 0ng/mL VEGF media. A Western Blot quantified total VEGFR-2 relative to β-actin loading controls. All samples were run in triplicate, and statistical comparisons were completed with Kruskal-Wallis tests, followed by Dunn’s tests.

Results, Conclusions, and Discussions:: For this study, we investigated if Blebbistatin has the potential to decrease VEGF secretion when blocking contractility of CAFs. Results show that CAFs secrete significantly more VEGF in comparison to HUVEC samples (Figure 1A). Importantly, Blebbistatin did not increase cell death for CAF only or CAF+HUVEC cultures; indicating that the contractility inhibitor did not alter viability or VEGF secretion. As an additional study, we are investigating how inducing strains, in the absence of stromal cells, with the Flexcell system affects total VEGFR-2 expression in HUVEC TERT-2 spCas9 cells and Y1504F cells. When normalizing VEGFR-2 to β-actin, the total expression of VEGFR-2 increased in Y1504F cells when compared to spCas9 cells of the same treatment groups (Figure 2). . It is seen that there are no changes in overall levels of VEGFR-2 within the same cell type regardless of treatment, however, more studies will need to be done to explore the effects of strain on this trend. It is possible the CRISPR-induced mutations have altered additional activation dynamics. Overall, our studies suggest that decoupling VEGF signaling and CAF-induced mechanical strains may be useful in informing novel strategies to target VEGFR-2 in anti-cancer therapies.

Acknowledgements and/or References (Optional):: [1] R Lugano, Cell Mol Life Sci, 2020, v77, p1745-1770. [2] AC. Shieh, Annals of Biomedical Engineering, 2011, v39, 1379-89.