Background: Immunotherapy, especially anti-programmed cell death protein 1 (PD-1) antibodies, has yielded significant and durable tumor response in melanoma, renal cell carcinoma, and other cancer types. In contrast, immunotherapy applied major breakthrough achievement only on patients with microsatellite instability high (MSI-H) metastatic colorectal cancer (mCRC). However, MSI-H mCRC comprised only 1.8-4% of total mCRC patients. It is crucial to investigate immune pathways to develop a new strategy for immunotherapy in treatment of microsatellite stable (MSS) mCRC. Recently, some evidences indicate that interferon (IFN)-γ pathway is critical for anti-PD-1 therapy. In this study, we emphasized on evaluating the response of MSS CRC cell lines to IFN-γ. Methods: We characterized two MSS CRC cell lines, namely SW480 (KRAS G12V mutation, BRAF wild type) and COLO320 (KRAS wild type, BRAF wild type) for our studies. The impacts of interferon-γ (IFN-γ) on cell surface expressions of different isotypes of major histocompatibility complex (MHC) class I and MHC class 1 related molecule A/B (MICA/B, the NK cell ligand) were explored. Different concentration of IFN-γ with different incubation time were also examined. Results: Both cell lines demonstrated low baseline human leukocyte antigen (HLA)-ABC and HLA-BC expression and the expression significantly increased in response to IFN-γ stimulation. We further focused on the SW480 cell line. The SW480 demonstrated low expression of all MHC class I isotypes, including HLA-ABC, HLA-BC, HLA-A, HLA-C, HLA-E, HLA-F, HLA-G and NK cell ligand MICA/B. The IFN-γ specifically stimulated the expression of HLA-A, but all other isotypes were not responsive to IFN-γ stimulation. In summary, IFN-γ significantly stimulates the expression of HLA-ABC, HLA-BC and HLA-A without stimulating HLA-C. The NK cell ligand MIC A/B was also not responsive to IFN-γ stimulation. The stimulatory effect of HLA-ABC and HLA-A in response to IFN-γ stimulation was positively correlated with IFN-γ dosage, which demonstrated highest expression level in response to 800U/ml IFN-γ stimulation. The stimulatory effect in response to IFN-γ stimulation was also positively correlated with increased incubation time with IFN-γ. Conclusion: These findings reveal that regardless of their KRAS mutation status, both MSS CRC cell lines possess a phenotype that may be not responsive to anti-PD-1 therapy due to low expression of MHC class I molecules. The IFN-γ specifically elicits the expression of stimulatory isotype, HLA-A and HLA-B, without eliciting all other inhibitory MHC class I isotypes. These results indicated that IFN-γ demonstrated pure adaptive immune stimulatory effect on CRC cell lines. Analysis of T- and NK cell-responsive immune markers along with IFN-γ signaling pathway may help us to survey possible combination therapy with anti-PD-1 treatment in CRC. Citation Format: Yi-Hsin Liang, Kuan-Yu Chan, Chang-Cheng Lee, Te-Jung Chen, Ann-Lii Cheng, Kun-Huei Yeh. IFN-γ elicits stimulatory MHC class I isotypes in human colorectal carcinoma cell lines with genetic features of microsatellite stable [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 608.