A class of extracellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis

Non-classical secretory vesicles, collectively referred to as extracellular vesicles (EVs), have been implicated in different aspects of cancer cell survival and metastasis. Here, we describe how a specific class of EVs, called microvesicles (MVs), activates VEGF receptors and tumour angiogenesis through a unique 90 kDa form of VEGF (VEGF90K). We show that VEGF90K is generated by the crosslinking of VEGF165, catalysed by the enzyme tissue transglutaminase, and associates with MVs through its interaction with the chaperone Hsp90. We further demonstrate that MV-associated VEGF90K has a weakened affinity for Bevacizumab, causing Bevacizumab to be ineffective in blocking MV-dependent VEGF receptor activation. However, treatment with an Hsp90 inhibitor releases VEGF90K from MVs, restoring the sensitivity of VEGF90K to Bevacizumab. These findings reveal a novel mechanism by which cancer cell-derived MVs influence the tumour microenvironment and highlight the importance of recognizing their unique properties when considering drug treatment strategies.

Relative # of blood vessels that formed with HCI-001 p = 0.002

Amounts of endothelial cells in the angioreactors
E.
F.  Figure 5. A, MDAMB231 cells transfected with siRNAs targeting VEGF that yielded a partial knock-down of VEGF expression were analyzed by immunofluorescent (IF) microscopy using antibodies against VEGF and DAPI (to label nuclei) to validate the specificity of the VEGF antibody. Cells in which VEGF expression was knocked down were not labeled by VEGF antibody (compare cells expressing VEGF (arrows "a") with cells where VEGF expression was not detected due to the knock-down (arrows "b")). Scale bar: 20 µm. B, rVEGF165 (30 ng) was incubated with recombinant tTG (100 ng, lane 2) under optimal protein crosslinking conditions to generate VEGF 90K . MVs shed from serum-starved SKBR3 (lane 3) or HeLa (lane 4) cells were isolated and lysed. Immunoprecipitations (IPs) using an Hsp90 antibody (lanes 3 and 4) were performed on these samples, followed by Western blotting with antibodies against VEGF and Hsp90. Lanes 1 and 2 represented negative controls. The MV lysate inputs were Western blotted with antibodies against VEGF, Hsp90, and flotillin-2.

MV protein inputs
anti-VEGF VEGF 90K anti-Hsp90 anti-flotillin-2 Page 1 Continuation Format Page S1. Cool-1 (Y442F) inhibits the cell surface localization of VEGF in v-Src-expressing cells. Using immunofluorescence, I have detected VEGF on the surface of v-Src-expressing cells [ Figure S1, arrow a]. However, when the phosphorylation-defective Cool-1 (Y442F) mutant was ectopically expressed in these v-Src cells, the surface localization of VEGF was nearly complete blocked [ Figure S1, arrow b].

S2. Knocking down Cool-1/!-Pix or Cdc42 in human umbilical vein endothelial cells (HUVEC) resulted in reduced tube formation.
To examine the role of Cool-1/!-Pix in angiogenesis, I performed a tube formation assay, which is a process mimicking sprouting during angiogenesis (150). This assay was performed as described (148, 150). The HUVEC transfected with scrambled RNAi differentiated into well-defined tube-like structures [ Figure  S2A]. However, the tube formation was inhibited when Cool-1/!-Pix was knocked down by RNAi [ Figure S2B]. ! ! Figure S1.

Cool-1 (Y442F) inhibits the cell surface localization of VEGF in v-Src cells. v-Src cells transiently over-expressing
Cool-1 (Y442F) were serum-starved for 18 hours. Cells were fixed with 3.7% formaldehyde without Triton X-100 (i.e. under these conditions the cells were not permeabilized). Anti-VEGF antibody was used to detect VEGF (Red in A and B) on the cell surface by immunofluorescence. Hoechst stain is used to label cell nucleus (Blue in B). onal critical supplemental information ve generated several additional preliminary results, which further sal. I would like these findings to be considered when reviewing the cell surface localization of VEGF in v-Src-expressing nce, I have detected VEGF on the surface of v-Src-expressing However, when the phosphorylation-defective Cool-1 (Y442F) ssed in these v-Src cells, the surface localization of VEGF was re S1, arrow b].

/!-Pix or Cdc42 in human umbilical vein endothelial cells ed tube formation.
To examine the role of Cool-1/!-Pix in tube formation assay, which is a process mimicking sprouting is assay was performed as described (148, Table 1) were immunoblotted using a pan VEGF antibody to detect VEGF 90K (top panel) and a smaller VEGF species. Tumor cell lysates (WCL) were probed with an actin antibody to confirm the conditioned medium was from equivalent amounts of tumor cells (bottom panel). B, Cell lysates from PDX samples HCI-001 to HCI-003, and HCI-005 to HCI-010 were probed with antibodies against tTG, and Hsp90. Supplementary Figure 8. A, MDAMB231 cells were cultured in CoCl2 (100 mM) containing medium (hypoxia) or medium without CoCl2 (normoxia). Left: MVs prepared from the same amount of MDAMB231 cells, treated with or without CoCl2, were immunoblotted with antibodies against VEGF and the MV marker flotillin-2 (top two panels). Lysates of the MDAMB231 cells were immunoblotted with antibodies against HIF-1a and actin (bottom two panels). Right: Plots of the relative VEGF secretion from the MDAMB231 cells, treated with or without CoCl2, were measured by VEGF ELISA. B, HCI-002 primary tumor cells were cultured in CoCl2 (100 mM) containing medium (hypoxia) or medium without CoCl2 (normoxia). Left: MVs prepared from the same amount of HCI-002 primary tumor cells, treated with or without CoCl2, were immunoblotted with antibodies against VEGF and the MV marker flotillin-2 (top two panels). Lysates of the HCI-002 primary tumor cells were immunoblotted with antibodies against HIF-1a and actin (bottom two panels). Right: Plots of the relative VEGF secretion from the HCI-002 primary tumor cells were measured by VEGF ELISA. C, MDAMB231 cells were lysed after being exposed to 17AAG for the indicated periods of time (lanes 1-4). The lysates were then immunoblotted with antibodies that recognize VEGF or actin.