Abstract 4514: Low release of exosomal miR-663a from hypoxic tumor cells and poor tumor response to neoadjuvant radiotherapy in rectal cancer patients

Introduction: Hypoxia is an important hallmark of the tumor microenvironment and contributes significantly to radiotherapy resistance in solid tumors. Exosomes are nanosized vesicles actively released from tumor cells, and recent studies support their central role in the aggravated biology caused by tumor hypoxia through their miRNA cargo. In this study, we aimed to characterize miRNAs of exosomes from hypoxic colorectal cancer (CRC) cell lines and investigate these miRNAs in circulating exosomes from rectal cancer patients with poor response to neoadjuvant radiotherapy. Methods: Five CRC cell lines were cultured in RPMI-1640 medium supplemented with 1% bovine serum albumin under normoxia (21% O 2 ) or hypoxia (0.2% O 2 ) for 24 hours. Exosomes were isolated from conditioned media by differential ultracentrifugation, and integrity and size were determined by cryo-electron microscopy and NanoSight tracking analysis and further characterized by Western blot and flow cytometry analyses. In a prospective biomarker study, plasma samples were collected from 29 patients with rectal cancer at the time of diagnosis, and histologic tumor response (tumor regression grade, TRG) to neoadjuvant radiotherapy was evaluated. Exosomes were isolated from plasma using the miRCURY™ Exosome Isolation Kit (Exiqon). Expression profiling of exosomal miRNAs of the CRC cell lines and the patients’ plasma samples was conducted using the miRCURY LNA™ Universal RT microRNA PCR Human panel I (Exiqon). Data normalization was performed based on global array mean. Results: Normoxic and hypoxic CRC cells released vesicles, 30-150 nm in size, that expressed proteins known to be enriched in exosomes (CD9, CD63, CD81, Alix) and with absence of markers of the endoplasmic reticulum (GRP78, Calnexin) and the Golgi apparatus (GM130). Exosomes from the CRC cell lines harbored strong cell line-specific miRNA profiles; however, when comparing the hypoxic cell lines collectively with the normoxic counterparts, four exosomal miRNAs (miR-195-5p, miR-423-3p, miR-622, miR-663a) were significantly down-regulated by hypoxia. Of these, circulating exosomal miR-663a was lower (p = 0.033) in rectal cancer patients with poor tumor response (TRG 2-3) to neoadjuvant radiotherapy. Conclusion: Vesicles released from CRC cell lines were characterized as exosomes carrying miRNAs that were retrieved in the circulation of rectal cancer patients, where low level of exosomal miR-663a at the time of diagnosis was associated with poor tumor response to neoadjuvant radiotherapy. These findings suggest that repressed release of miR-663a from tumor cells into the circulation may be a marker of radiotherapy resistance caused by tumor hypoxia. The results are currently under validation in an independent patient cohort. Trial registration: NCT01816607 Citation Format: Tonje Bjornetro, Karianne R. Handeland, Sebastian Meltzer, Rampradeep Samiappan, Lars G. Lyckander, Caroline Jegerschold, Linda Sonstevold, Nirujah S. Thusyanthan, Kathrine R. Redalen, Anne H. Ree. Low release of exosomal miR-663a from hypoxic tumor cells and poor tumor response to neoadjuvant radiotherapy in rectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4514. doi:10.1158/1538-7445.AM2017-4514