Abstract 192: The extracellular vesicles from lung cancer cells activates Stat3 and tumor angiogenesis
Authors: Lin Chien-Chung , Huang Yu-Ting , Hung Chun-Hua , Tsai Mei-Ling , Su Wu-Chou 
Published: August 2019

Abstract
Current research suggests that on tumor-related extracellular vesicles (EV) play a significant role in paracrine signaling pathways, thus potentially influencing cancer progression via multiple mechanisms including angiogenesis. Our previous study demonstrated that IL6 induced secretion of VEGF and contributing angiogenesis via activation of Stat3. To further clarify the biological activities of the lung cancer derived EVs, we analyzed the content of IL6 and VEGF in exosome using ELISA kit. We found that IL-6 and VEGF were more enriched in EVs than non-EVs in AS2 and CL1-5 cells. And the isolated EV from AS2 and CL1-5 cells increased vascular permeability in miles assay and promotes angiogenesis in tubular formation assay. We further investigated if the inhibition of exosome secretion affects the autocrine IL6/Stat3/VEGF pathway. GW4869 inhibits secretion of exosome by playing an inhibitor of neutral sphingomyelinase (nSMase). Treatment of either cell with GW4869 significantly decreased the levels of released exosomes in AS2 but not in CL1-5 cell. Consistent with the result, the treatment GW4869 dampened autocrine IL6/Stat3/VEGF pathway in AS2 but not in CL1-5 cell. Bevacizumab, the recombinant anti- VEGF monoclonal antibody, were approved for treating patients with advanced NSCLC, has been shown to be efficient in suppressing the accumulation of pleural fluid (malignant pleural effusion). To investigate if the exosomal VEGF has a different affinity for bevacizumab, we treated AS2, CL1-5 and MPE-derived cancer cells with bevacizumab and EV or non-EV part from cell-free medium. We found VEGF dimer form were more abundant in EV part and cannot be neutralized by bevacizumab. Conversely, VEGF mono-form was more abundant non-EV part and can be neutralized by bevacizumab. In animal model, AS2 was injected intraperitoneal and then malignant ascites developed as follow. We found the administration of bevacizumab after AS2 injection i.p. inhibited peritoneal seeding and ascites formation compared to control group. But the inhibition was reversed when we injected EV as well as bevacizumab in combination group. In breast cancer cell, microvesicles (MVs) activates VEGF receptors through a unique 90 kDa form of VEGF (VEGF90K) which has a weakened affinity for Bevacizumab, causing Bevacizumab to be ineffective in blocking MV-dependent VEGF receptor. And an Hsp90 inhibitor (17-AAG) was demonstrated to release VEGF90K from MVs without interaction with VEGF restoring the sensitivity of VEGF90K to Bevacizumab. However, our study found 17-AAG alone inhibited VEGF secretion in EV as well as the activity of upstream Stat3, and Akt pathway in lung cancer. Our preliminary data implied EV-VEGF and the existence of different isoform of VEGF in EV contribute to tumor angiogenesis and limit the effectiveness of bevacizumab therapy which is not dependent on Hsp90.
Citation Format: Chien-Chung Lin, Yu-Ting Huang, Chun-Hua Hung, Mei-Ling Tsai, Wu-Chou Su. The extracellular vesicles from lung cancer cells activates Stat3 and tumor angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 192.