Abstract 274: Resistance to VEGFR inhibitors converges from molecular diversity in angiosarcoma cells

Angiosarcomas are aggressive soft tissue sarcomas that form malignant endothelium. Standard care treatment for angiosarcoma patients includes surgery, radiation, and chemotherapy, but outcomes remain poor, especially in unresectable or metastatic cases. These endothelial malignancies activate angiogenic and vascular pathways, implying VEGF/VEGFR and Tie2 signaling as key drivers of tumor progression. However, therapeutic antibodies, such as bevacizumab (Avastin), and chemical inhibitors targeting VEGF pathways have shown limited clinical benefits in angiosarcomas. The molecular mechanisms that establish resistance to VEGF inhibition in angiosarcomas are virtually unknown. In this study, we used five human angiosarcoma cell lines - KU-CAS3, KU-CAS5, HAMON, ISO-HAS-B, and AS5 - for functional characterization. We first determined cell surface expression of four endothelial markers: CD31, neurophilin-1, VEGFR2, and Tie2. Our flow cytometry data revealed that CD31 expression was markedly elevated in nearly 100% of cells in four of the five angiosarcoma cell lines, with the exception of AS5, which showed 26.1% expression. Neurophilin-1 expression varied across the cell lines: 5.6% in KU-CAS3, 11.8% in AS5, 31.1% in ISO-HAS-B, 60.0% in HAMON, and 99.7% in KU-CAS5. We also found that three cell lines - HAMON (95.2%), KU-CAS3 (72.6%) and KU-CAS5 (92.7%) - showed high expression of VEGFR2, while two cell lines - ISO-HAS-B (14.0%) and AS5 (14.8%) - exhibited low expression. The expression levels of Tie2 were abundant, ranging from 74.8% to 96.4% in four cell lines, with modest expression in AS5 (47.8%). Then, we examined cellular response to VEGFR2 (Regorafenib, Sorafenib) and Tie2 inhibitors (Rebastinib, Pexmetinib) using cell growth inhibition assays. All five angiosarcoma cell lines exhibited IC50 values higher than 1 uM against both VEGFR2 inhibitors, Regorafenib and Sorafenib, except for KU-CAS5, which showed an IC50 of 0.44 uM. Both Tie2 inhibitors, Rebastinib and Pexmetinib, were also ineffective in decreasing cell proliferation in most of the cell lines, with two exceptions showing submicromolar IC50 values of 0.91 uM and 0.25 uM in KU-CAS3 and AS5, respectively. In summary, our data revealed that the robust expression of endothelial and vascular markers in angiosarcoma cells indicates a conserved endothelial phenotype under in vitro conditions. However, none of the cell lines exhibited significant sensitivity to VEGFR and Tie2 inhibitors in terms of clinical potential. It demonstrates broad resistance to VEGFR2 and Tie2 inhibition, independent of VEGFR2 and Tie2 expression in angiosarcoma cells. Furthermore, our results suggest angiosarcoma cells establish a functional convergence that potentiates the signaling activation of VEGF and Tie2 pathways, despite their molecular divergence. Our ongoing work aims to explore the molecular mechanisms promoting resistance to VEGF and Tie2 inhibitors. Citation Format: Eslenur Nipa, Donghee Lee, Sun Hee Choi, Jong Hyuk Kim. Resistance to VEGFR inhibitors converges from molecular diversity in angiosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 274.