EXTRACELLULAR VESICLES TRANSFER RESISTANCE TO VINCRISTINE IN SONIC HEDGEHOG MEDULLOBLASTOMA

Abstract

Abstract AIMS • To determine the expression patterns of specific ATP-binding cassette (ABC) transporters in medulloblastoma • To assess whether ABC transporters play a role in acquired drug resistance in SHH medulloblastoma using drug-tolerant cell line models • To assess whether extracellular vesicles transfer drug-tolerance in SHH medulloblastoma and determine the mechanism by which they do this METHOD Drug-tolerant cell lines were generated by continuous culture in drug-containing culture medium. ABC trans- porter gene and protein expression was assessed via qPCR and western blot. EVs were isolated from EV-depleted medium using size-exclusion chromatography. Nanoparticle tracking analysis was used to quantify and deter- mine size. Cytotoxicity analysis was used to determine EV-mediated transfer of resistance in the presence or absence of an ABC transporter inhibitor. RNA sequencing of EVs from matched vehicle and drug tolerant cell lines was carried out. RESULTS Analysis of drug-tolerant cell lines revealed significant increases in specific drug transporter gene expression across medulloblastoma subgroups. ABCB1 protein expression was significantly higher in drug-tolerant sonic- hedgehog (SHH) lines and inhibition of ABCB1 in a drug-tolerant SHH line abolished acquired resistance to vincristine. Cytotoxicity assays on sensitive cells, after pre-treatment with drug-tolerant EVs, showed a significant increase in vincristine resistance, implicating EVs in the transfer of resistance. CONCLUSIONS Data to date supports the hypothesis that multidrug transporter carrying extracellular vesicles may transfer their multidrug resistant phenotype to surrounding cells in medulloblastoma. We are currently testing this using inhibitors and knockout lines, in addition to RNA sequencing, with the hopes of providing new therapeutic targets to patients with medulloblastoma.