Tumor heterogeneity has impacts on tumor biopsy strategy, characterization of actionable targets and drug resistance. At lung cancer, recent study demonstrated that the discordance rates of EGFR mutation implying tumor heterogeneity in metachronous and synchronous settings were 14.3% and 9.1% respectively. And other study further showed intratumoral EGFR mutational heterogeneity was correlated with the response and prognosis of EGFR-TKIs. Extracellular vesicles (EVs) such as exosomes serve as the transporter of bioactive molecules between cells and become one of the major mechanisms contributing intratumoral heterogeneity via intercellular transference of genetic information. Since most patients harboring EGFR mutation showed excellent response, we hypothesized that EVs such as exosome mediating the crosstalk between EGFR mutant cell and EGFR wild type cell contributing the change of sensitivity of EGFR wild type cell to EGFR-TKI in heterogeneous NSCLC. We used ultrafiltration (UF) method to isolate the exosomes. The conditioned media were centrifuged and then the supernatants were passed through 0.22 μm filters and then Vivaspin nanomembrane concentrators step by step. We first incubated EGFR wild type lung cancer cell (CL1-5) with medium containing PKH26 labeled exosomes derived from PC9 cell (EGFR Del 19) for 24 h. The uptake of exosomes from PC9 in CL1-5 cell can be observed under immunofluorescence microscope and recorded by time-lapse microscope. And the EGFR Del19 DNA and specific protein can be detected in recipient CL1-5 cells by digital PCR and Western blotting respectively. Then EGFR wild type lung cancer cell (CL1-0, CL1-5 and H1299) were first treated with exosome from themselves or PC9 cell respectively for 48h and then subjected to gefitinib (0.1 μM) for 72h. We found only PC9 cell derived EV sensitized EGFR wild type cell to gefitinib. To mimic tumor heterogeneity, we next tested the significance of exosome on EGFR-TKI sensitivity in co-culture system with pretreatment with or without GW4869, which blocks exosome secretion by inhibiting neutral sphingomyelinase. We first verified that treating lung cancer cell with GW4869 at concentrations of 2.5, 5 and 10μM inhibited exosome secretion without cytotoxicity. We demonstrated that EGFR wild type lung cancer cell became sensitive to EGFR-TKI after co-culture with PC9 cell for 48h and then subjected to gefitinib for 72h. However, the pretreatment with GW4869 for 48 hours reversed the sensitivity to EGFR-TKI in co-culture system with PC9. We further compared the exosomal miRNAs from PC9 to those from CL1-5 and identified a panel of discriminative miRNAs. Some miRNA among the panel such as miR-200 family have been identified associated with resistance to EGFR-TKI. Our study, at least in part, suggests that exosome release from EGFR mutant cell might be a possible mechanism for excellent EGFR-TKI sensitivity even in heterogeneous NSCLC.

Citation Format: Chien-Chung Lin, Wu-Chou Su, Chin-You Wu. The role of exosomes and exosomal miRNA in mediating EGFR-TKI sensitivity in heterogeneous NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 217.