miRNA-1246 in extracellular vesicles secreted from metastatic tumor induces drug resistance in tumor endothelial cells

Tumor endothelial cells (TECs) reportedly exhibit altered phenotypes. We have demonstrated that TECs acquire drug resistance with the upregulation of P-glycoprotein (P-gp, ABCB1), contrary to traditional assumptions. Furthermore, P-gp expression was higher in TECs of highly metastatic tumors than in those of low metastatic tumors. However, the detailed mechanism of differential P-gp expression in TECs remains unclear. miRNA was identified in highly metastatic tumor extracellular vesicles (EVs) and the roles of miRNA in endothelial cell resistance were analyzed in vitro and in vivo. In the present study, we found that treatment of highly metastatic tumor-conditioned medium induced resistance to 5-fluorouracil (5-FU) with interleukin-6 (IL-6) upregulation in endothelial cells (ECs). Among the soluble factors secreted from highly metastatic tumors, we focused on EVs and determined that miR-1246 was contained at a higher level in highly metastatic tumor EVs than in low metastatic tumor EVs. Furthermore, miR-1246 was transported via the EVs into ECs and induced IL-6 expression. Upregulated IL-6 induced resistance to 5-FU with STAT3 and Akt activation in ECs in an autocrine manner. These results suggested that highly metastatic tumors induce drug resistance in ECs by transporting miR-1246 through EVs.


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Dulbecco's modified eagle medium (Sigma, St Louis, MO, USA) supplemented with 10% 126 FBS. The OS-RC-2 cells were cultured in RPMI1640 medium (Sigma-Aldrich) with 10% 127 FBS. Normal human epidermal melanocytes were purchased from Kurabo Industries Ltd. 128 (Osaka, Japan) and cultured in melanocyte growth medium (PromoCell, Germany). 129 The TECs were isolated from tumors that were subcutaneously xenografted with A375 130 or A375SM. The NECs were isolated from the dermis of tumor-free nude mice, as 131 previously described 13 . Furthermore, ECs were isolated using a magnetic cell sorter   Committee, and consent was obtained from the patients before blood collection.  we focused on IL-6. The TECs showed a significantly higher level of IL-6 in A375SM-

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The size of the isolated EVs was approximately 98 nm in diameter, which was similar to 369 the reported EV size 35 (Fig. S1A). The particle numbers of each EV were almost the same 370 (Fig. S1B). Additionally, EV markers, such as CD63 and CD9, were detected in the 371 extracted protein from each EV, whereas cytochrome C, a cytoplasm protein, was not 372 detected (Fig. S1C). When the HMVECs were treated with A375SM-EVs, IL-6 mRNA 373 was upregulated ( Fig. 2A). Furthermore, the EVs isolated from A375 were added to the 374 HMVEC culture. A375SM-EV treatment induced IL-6 mRNA at a higher level than did 375 A375-EV treatment (Fig. 2B). and A375-EV through miRNA array, since we have found that A375SM-TEC showed a 385 higher level of IL-6 than did A375-TEC, as shown in Fig. 1A. Furthermore, miR-1246 386 was picked up because its level was higher in the CM from tumor cells than in that from 387 normal cells and also in A375SM-EV than in A375-EV (Fig. 3A). Indeed, it was 388 confirmed that miR-1246 was higher in A375SM-EV than in A375-EV or in HMVEC-

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EV, as determined through PCR (Fig. 3B). The endocytosis inhibitor dynasore canceled HMVECs; however, CTSC mRNA expression level did not (Fig. 4B). This suggested 409 that AR and UNC5B are targets of miR-1246 but not CTSC. Similarly, EV treatment 410 caused downregulated AR and UNC5B mRNA expressions (Fig. 4C). IL-6 mRNA 411 expression was analyzed after AR or UNC5B inhibition to examine the contribution of 412 these genes on IL-6 expression. IL-6 expression level was increased by the knockdown 413 of AR but not of UNC5B, suggesting that AR suppresses IL-6 expression in HMVECs 414 (Fig. 4D) the AR gene, suggesting that AR is an miR-1246 target (Fig. 4E, Fig. S3). Furthermore,

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AR knockdown caused resistance to 5-FU in HMVECs (Fig. 4F). It is well known that IL-6 activates STAT3, a substance known to be related to drug 422 resistance 36 . Tumor CM induced STAT3 activation, whereas its activation was canceled 423 by S3I-201, a STAT3 inhibitor (Fig. 5A). It was confirmed that tumor EVs (A375SM-

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STAT3 activation was also enhanced by AR knockdown but not by UNC5B knockdown 426 (Fig. 5D). Akt activation has been reported to be involved in cell survival or drug 427 resistance 38 . Both tumor EVs and miR-1246 activated Akt ( Fig. 5E and Fig. 5F).   Hokkaido, Japan), and written informed consent was obtained from each patient before 576 surgery. All procedures for animal care and experimentation adhered to institutional 577 guidelines and were approved by the local animal research authorities.     The predicted miR-1246 binding site in the 3′UTR of AR is shown. The bold font shows 841 the seed sequence of miR-1246 (6-mer). The 3′UTR assay was conducted using vectors 842 with the indicated sequences.