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Abstract Background: Gastric cancer (GC) is the fifth most prevalent malignant tumor worldwide, and the fourth most common cause of mortality. c-MET has been reported to be associated with poor prognosis in GC. Over the decades, treatment approaches targeting HGF/c-MET have been developed including small molecule tyrosine kinase inhibitors, anti-c-MET and anti-HGF monoclonal antibodies. Unfortunately, the results of previous study support the development of acquired resistance to HGF/c-Met inhibitors. They suggest a possible resistance mechanism involving poor MET-status detection, crosstalk between c-MET and compensatory signaling pathways, the occurrence of acquired a new mutation, however, knowledge regarding the mechanism of c-MET inhibitor is limited. 2. Purpose: We identify new markers and mechanisms that induce drug resistance when c-MET-targeted inhibitors are treated in c-MET-amplified GC cell lines in 3D culture, and discover new therapeutic targets for GC via single cell-RNA sequencing analysis. 3. Material and Methods: Single cell-RNA sequencing is performed on c-MET amplified GC cell line in the 2D and 3D culture methods according to the presence or absence of c-MET inhibitor treatment. Through DEG and clustering analysis, the candidate genes estimated to impart resistance are selected and verified by qPCR. Through over-expression, induction of resistance for c-MET inhibitor treatment with target gene is evaluated in c-MET amplified GC cell line. c-MET inhibitor selected gene is constructed as a xenograft model to reveal the mechanism of drug resistance. Retrospective analysis confirms the relationship between target gene expression level and the resistance for c-MET inhibitor or other TKI treated patients with GC. 4. Results: Treatment of c-MET-amplified GC cell lines with c-MET inhibitors under the conditions of 2D and 3D cultures confirmed that resistance was induced only in the 3D. We performed a high-precision single cell RNA-Seq analysis of 18056 single cells in total from c-MET amplified GC cell line MKN-45 in 2D/3D culture control as well as corresponding samples with volitinib treatment investigate their gene expression signatures. Fifteen selected candidate genes presumed to induce drug resistance were screened and validated by qPCR, with a total of six genes consistent with the sequencing results. After over-expression of six genes to the c-MET amplified gastric cancer cell line, c-MET inhibitors were treated and cell viability was performed to check for resistance to cell viability. Finally, ERO1A and VEGFA were over-expressed to confirm the resistant for the c-MET inhibitor treatment in vitro and in vivo. 5. Discussion: We plan to build a xenograft mouse model with candidate genes such as ERO1a and VEGFA to prove whether the candidate gene overcomes drug resistance when they are on or off. Citation Format: Hajung Kim, Jinhyeon Kim, Seung-Jae Myung, Charny Park, Sun young Kim. Unraveling c-MET targeting resistance mechanisms in gastric cancer via single-cell transcriptome analysis; Novel therapeutic target discovery of ERO1A and VEGFA abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 5848.
Kim et al. (Fri,) studied this question.