Cancer Technologies
INVESTIGATING THE EFFECT OF PRO-LONGED TENSILE STRAIN ON THE VASCULAR ENDOTHELIAL GROWTH RECEPTOR 2 AND THE MECHANICAL MEMORY OF ENDOTHELIAL CELLS
Michael D. Heim (he/him/his)
Undergraduate Student
The University of Alabama at Birmingham, Alabama, United States
.jpg "Bronte Miller, MS (she/her/hers) photo")
Bronte Miller, MS (she/her/hers)
Doctoral Student
The University of Alabama at Birmingham
Birmingham, Alabama, United States
MK Sewell-Loftin (she/her/hers)
Assistant Professor
The University of Alabama at Birmingham, United States
While much attention has been devoted to the study of the biochemical signaling processes in the tumor microenvironment (TME) as targets for anti-cancer therapies, few studies investigate the mechanical factors involved in supporting tumor progression. As tumors grow, they require additional nutrients and oxygen which triggers a signaling cascade to drive angiogenesis, or the growth of blood vessels from previously existing vasculature. [1] Recent studies have shown that endothelial cells (ECs) that make up blood vessels are mechanically sensitive. Vascular endothelial growth factor receptor 2 (VEGFR-2) on ECs and the ligand vascular endothelial growth factor (VEGF) are critical regulators of angiogenesis. Strain occurs in the TME and is a result of mechanically active cells such as cancer-associated fibroblasts (CAFs) deforming an extracellular matrix that is stiffer than normal tissue. [2] On VEGFR-2, Y1054/Y1059 is a phosphorylation site on the intracellular domain, and phosphorylation of this tyrosine is necessary for the process of angiogenesis. Another phosphorylation site, Y1214, can be activated via mechanical stimulation [3]. Cells have a mechanical memory, which is an ability to react to a mechanical stimulus and then adjust cell behavior future cell behavior as a result of the stimulus [4]. We hypothesize that a mechanical memory is observable via prolonged changes to phosphorylation when ECs are exposed to prolonged tensile strain treatment in combination with exogenous VEGF treatment.
Human microvascular endothelial cells (HMECs) were seeded onto FlexCell plates with a collagen I coating. In each well, 5x105 cells were seeded and allowed to grow for 48hr prior to strain exposure. The FlexCell (FX-6000T) system was used to treat samples with cyclic strain at 10% and 0.3Hz for 24hr or 72hr; control cells were plated on the same FlexCell plates but did not receive mechanical stimulation (0hr). The strain treatment was designed to mimic CAF induced matrix distortions [3,5] and the frequency mimics normal respiratory rates. After strain treatments, cells were harvested and seeded into 24 well plates at 5x104 cells per well. The cells were cultured with or without exogeneous VEGF. After 48hr, the cells were fixed and stained via immunofluorescence techniques for pY1054/Y1059 and DAPI. Samples were imaged and were analyzed in FIJI to determine area of positive staining relative to total cell area. A second study was performed with the same conditions, but the cells were lysed and analyzed via Western Blot. Blots were stained for VEGFR-2, pY1054/Y1059, pY1214, and b-actin as a loading control. Statistical significance in both studies was determined using ANOVA followed by post-hoc Tukey testing or by student’s t-tests assuming unequal variance.
The immunofluorescence study of Y1054/Y1059 demonstrated a statistical increase in pY1054/Y1059 after cells were pre-treated with 24hr strain when VEGF was added compared to samples that did not receive exogenous VEGF (Figure 1). For the Western blot data, the amount of pY1054/Y1059 trends upward when no exogenous VEGF is present after 24hr of strain treatment (Figure 2A). With exogeneous VEGF, pY1214 seems to trend upwards after 24hr of strain as compared to just a 24hr strain treatment, and 72hr strain seems to keep pY1214 constant even without VEGF (Figure 2B). Finally, total VEGFR-2 increases significantly as the length of strain treatments increase when exogenous VEGF is not present, but when exogenous VEGF is present, a 72hr strain treatment seems to decrease overall levels of VEGFR-2 (Figure 2C).
The strain treatments affected phosphorylation levels in ECs in both the immunofluorescence study and the Western blot, verifying that the effects of strain and a strain memory can potentially alter prolonged activation of VEGFR-2. Specifically, strain treatment increased the phosphorylation levels of VEGFR-2 when exogenous VEGF was added as compared to the ECs that were not strained. The decrease in total VEGFR-2 after 72hr strain treatment and exogenous VEGF may represent enhanced activation; after the receptor is activated, it becomes internalized and degrades to prevent feedforward signaling [6]. Since some increases in both Y1054/Y1059 and Y1214 were observed with strain, these cells may have a higher angiogenic potential compared to non-strained cells. An understanding of the mechanoactivation of VEGFR-2 is critical in designing new cancer treatments. Previous anti-angiogenic drugs have attempted to inhibit VEGFR-2 by blocking VEGF to stop tumors from growing vasculature; however, the limited success of these drugs may have been due to the ability of the angiogenic pathway to be activation by forces exerted on the ECs by the TME [7]. Future studies will examine angiogenic vessel growth in a 3D microtissue model using ECs that have been treated with strain and/or VEGF.