Background: It is a challenge that cancer cells evolves in to become more refractory to the chemotherapeutic agents when a patient receives multiple lines of chemotherapy. Extracellular vesicles (EV) are small membranous vesicles with a diameter of ~30-100 nm, which are released into extracellular environment from the parental cells. EV is also involved in drug resistance of cancer cells by exchanging signals between cells by delivering responsible biomolecules including proteins and miRNAs. EV has been reported to be able to transfer drug resistance from drug-resistant tumor cells to adjacent drug-sensitive cells. The main goal of this study is to analyze EV proteome to investigate EV-meidated drug- resistance using cell lines that were induced resistance sequentially by treatment with one or more anticancer agents.

Methods: We isolated EV from drug resistant cancer cell lines including gastric cancer (SNU620-5FU/R) and cololectal cancer cell lines (HT29-OXA/R, HCT116-OXA/R) and its parental cell lines (SNU620, HT29, HCT116) using the ExoLutE isolation kits based on a combination of multiple isolation techniques. Quantitative protein analysis of prepared EVs derived from gastric cancer cell lines (SNU620, SNU620-5FU/R) was performed using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). Base on these data, we are preparing for analysis of exosomal profiling in oxaliplatin resistance cell lines(HT29, HT29-OXA/R, and HT29-OXA/SN38/R & HCT116, HCT116-OXA/R, and HCT116-OXA/SN38/R).

Results: LC-MS/MS analysis identified with high confidence (false discovery rate < 0.05, fold change ≥2) more than 200 EV proteins enriched in EV derived from SNU620-5FU/R cells, which are resistant to 5-FU. Bioinformatic network analyses suggested Ubiquitin-conjugating enzyme E2 R2 (UBE2R2), which is involved in ubiquitination, was most prominently expressed in the resistant cell lines compared to control, whereas ANXA1 was expressed the lowest. Interestingly, in the other preliminary analysis of real-time quantitative PCR in the intracellular level obtained from a different resistant cell line treated with oxaliplatin and then irinotecan (HCT116-OXA/R and HCT116/OXA/SN38/R), UBE2R also markedly increased compared to the control. The level of expression of UBE2R2 showed a linear relationship with the number of resistant drugs.

Conclusion: Our results suggest a novel mechanism of chemotherapy associated with UBE2R2. We are further analyzing exosomes using resistant cell lines obtained by sequentially exposing them to anticancer drugs and exploring additional mechanisms of resistance related to UBE2R2.

Citation Format: Chi Hoon Maeng, Minji Choi, Kwang Pyo Kim, Hyeong Min Lee. Mass spectrometry-based proteome profiling of extracellular vesicles and their potential roles in drug resistance of gastrointestinal cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2516.