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Recent clinical trials have shown that the addition of immune checkpoint inhibitors (ICIs)–such as antibodies to PD-1 or to PD-L1–to platinum-based combination chemotherapy resulted in a significant extension of overall survival in patients with advanced non-small cell lung cancer (NSCLC). This synergistic effect is possibly attributable in part to the phenomenon of immunogenic cell death (ICD), which is characterized by the release of danger-associated molecular patterns (DAMPs) from dying tumor cells. Calreticulin (CRT) is one of the most potent DAMPs and its cell surface translocation (ecto-CRT) serves as an “eat me” signal to antigen-presenting cells (APCs), which plays a pivotal role in activating anti-tumor immunity. However, there is limited information which chemotherapeutic agents used in the treatment of NSCLC can induce the expression of ecto-CRT most robustly on the surface of dying lung cancer cells. In current study, to identify the most effective inducer of ecto-CRT, using flow cytometry, we compared the expression levels of ecto-CRT on different 3 NSCLC cell lines treated with 9 chemotherapeutic agents including oxaliplatin (as positive control), cisplatin, carboplatin, pemetrexed, gemcitabine, 5-fluorouracil, docetaxel, paclitaxel and vinorelbine under each value of IC50 (0.0023 μM~25.2 μM) determined by MTS assay after 72 hours exposure to each chemotherapeutic agent. We found that pemetrexed induced the highest expression of ecto-CRT compared with other chemotherapeutic agents in all NSCLC cell lines examined. There are significant positive correlations between positive rates of ecto-CRT and annexin V, commonly used to detect apoptotic cells, in all NSCLC cell lines (r=0.79~0.95, p=0.0001~0.0088). In addition, pemetrexed-inducible expression of ecto-CRT on A549 cells was remarkably reduced when pan-caspase inhibitor z-VAD-fmk was added, illustrating its dependency on the caspase activity associated with apoptosis. Furthermore, using a syngeneic mouse tumor model that is moderately sensitive to anti-PD-1 therapy, we confirmed that combination treatment with pemetrexed enhanced the in vivo antitumor effect compared with anti-PD-1 or pemetrexed monotherapy. Notably, in clinical settings, we found that the plasma concentrations of soluble CRT collected from 3 NSCLC patients treated with pemetrexed alone were increased during the first cycle of treatment in all patents examined. Collectively, our findings showed that pemetrexed is the most effective inducer of ecto-CRT in NSCLC cell lines among numerous chemotherapeutic agents, suggesting that the combinational use of pemetrexed on ICIs therapy might exert synergistic antitumor immunity by triggering abundant expression of ecto-CRT in parallel to apoptosis induction in local tumor tissues of patients with advanced NSCLC. Citation Format: Rie Furukawa, Hiroyuki Inoue, Hirono Tsutsumi, Ando Nobuhisa, Yuki Ikematsu, Keiichi Oota, Eiji Iwama, Yasuto Yoneshima, Kentaro Tanaka, Yoichi Nakanishi, Isamu Okamoto. Pemetrexed induces calreticulin expression most robustly in parallel with apoptosis among chemotherapeutic agents used in non-small cell lung cancer (NSCLC) treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2412.