Elevated plasma concentrations of soluble VEGFA isoforms are associated with poor prognosis in parallel with improved response to treatment with the anti-VEGFA antibody bevacizumab. To uncover the underlying mechanism to these observations, we administered anti-VEGFA therapy to mice bearing luminescent mouse fibrosarcomas expressing single VEGFA isoforms or their wild-type counterparts expressing all isoforms (fs120, fs164, fs188, or fsWT). Expression of the more soluble isoforms conferred an advantage for lung metastasis from subcutaneous tumors (fs120/164 vs. fs188/WT); fs120 cells also produced more lung colonies than fs188 cells when injected intravenously. Metastasis from subcutaneous fs120 tumors was more sensitive than fs188 to treatment with the anti-VEGFA antibody B20-4.1.1. Despite elevated plasma levels of VEGFA in fs120 tumor-bearing mice and a dependence on VEGF receptor 1 activity for metastasis to the lung, B20-4

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<p>The supplementary data file contains the following data and figures: Fig S1- CD31 and CD34 co-localize to the vasculature in fs120-LS and fs188-LS subcutaneous tumors. Fig S2 - VEGFR1 activity has no effect on tumor growth or vascular density of subcutaneous fs120-LS or fs188-LS tumors. Fig S3 - fs120-LS and fs188-LS cells metastasize to the lung early during subcutaneous tumor growth. Fig S4 - fs120-LS cells express higher levels of PlGF2 than fs188-LS cells in vitro, with no difference in levels of PLGF2 detected in subcutaneous tumors. Fig S5 - Expression of laminin and collagen-I in lysates of fs120-LS and fs188-LS subcutaneous tumors. Fig S6 - Quantification of single cell migration using live video microscopy. Fig S7 - Localization of CD11b cells with respect to intravenously injected fibrosarcoma cells within the lung. Fig S8 - Summary of results showing key differences between fibrosarcomas expressing VEGF120 and VEGF188 and the effects of B20-4.1.1 and stromal VEGFR1 activity. Fig S9 - Effect of cediranib on survival of fs120-LS cells in the lung 48 h after iv injection.</p>

<p>The supplementary methods file contains additional detailed information regarding: 1. The production of cell lines stably expressing luciferase. 2. Mouse strains and pre-clinical models of tumor growth and metastasis. 3. Acquisition and analysis of bioluminescence data from preclinical and in vitro studies. 4. Acquisition and analysis of in vitro migration data. 5. Antibodies and immuno-techniques used within this study.</p>

<div>Abstract<p>Elevated plasma concentrations of soluble VEGFA isoforms are associated with poor prognosis in parallel with improved response to treatment with the anti-VEGFA antibody bevacizumab. To uncover the underlying mechanism to these observations, we administered anti-VEGFA therapy to mice bearing luminescent mouse fibrosarcomas expressing single VEGFA isoforms or their wild-type counterparts expressing all isoforms (fs120, fs164, fs188, or fsWT). Expression of the more soluble isoforms conferred an advantage for lung metastasis from subcutaneous tumors (fs120/164 vs. fs188/WT); fs120 cells also produced more lung colonies than fs188 cells when injected intravenously. Metastasis from subcutaneous fs120 tumors was more sensitive than fs188 to treatment with the anti-VEGFA antibody B20-4.1.1. Despite elevated plasma levels of VEGFA in fs120 tumor-bearing mice and a dependence on VEGF receptor 1 activity for metastasis to the lung, B20-4.1.1 did not affect survival in the lung on intravenous injection. B20-4.1.1 inhibited subcutaneous tumor growth and decreased vascular density in both fs120 and fs188 tumors. However, migration of fs120, but not fs188 cells, <i>in vitro</i> was inhibited by B20-4.1.1. The greater survival of fs120 cells in the lung was associated with VEGFR1-dependent accumulation of CD11b-positive myeloid cells and higher expression of the VEGFR1 ligand, PlGF2, by the fs120 cells <i>in vitro</i> and in the plasma and lungs of fs120 tumor-bearing mice. We conclude that soluble VEGFA isoform expression increases fibrosarcoma metastasis through multiple mechanisms that vary in their sensitivity to anti-VEGF/VEGFR inhibition, with VEGFA-targeted therapy suppressing metastasis through effects on the primary tumor rather than the metastatic site. <i>Cancer Res; 77(10); 2633–46. ©2017 AACR</i>.</p></div>