Background: Immune checkpoint inhibition (ICI) has made significant breakthroughs in several tumor types including gastric cancer (GC) in recent years. However, single-agent pembrolizumab demonstrated remarkable and durable response in MSI and EBV GC because they are immune-enriched types. Recently, we reported on-treatment remodeling of the TME during chemotherapy in MSS AGC (Cancer Discov. 2021). We defined altered features including decreased tumor-associated macrophages (TAM), M1- macrophages repolarization and recruitment of NK cells, effector T cells. Methods: This phase 2, open-label, single-arm study was designed to assess the safety and efficacy of the CapeOx-pembrolizumab combination in advanced gastric cancer patients with HER2-negative. 38 patients were enrolled onto our CapeOx plus Pembrolizumab trial (NCT #04249739). All patients had MSS status with EBV-negative. Endoscopic biopsies were collected at baseline, after 1 cycle of chemotherapy alone and after 4 to 5 cycles of CapeOx + pembrolizumab to understand the impact of treatment to tumor TME. We performed coupling whole exome sequencing and whole transcriptome sequencing. Molecular features were extracted by exonic analysis. TME subtype was estimated by gene expression analysis. Results: Between 2020 and 2021, 38 patients were enrolled. There were 2 CR, 25 PR, 11 SD as best response.First, we found a number of somatic mutations (median 157.5, range 7-25,719) in pre-and on-treatment samples. Despite the creation and destruction of somatic mutation, we observed that ancestor mutations including TP53, MUC6 and APC previously reported in TCGA GC cohort, were preserved. At baseline (before chemotherapy), patients were classified to 5 immune-enriched (IE) subtye, 21 immune-depleted (D) subtype, ETC. Second, we observed a dynamic change following one cycle of CapeOx before the addition of pembrolizumab to chemotherapy. Nine (23.68%) out of thirty eight patients with immune-depleted or fibrotic pre-treatment TME became immune-favorable during treatment. Interestingly, seventeen (58.62%) out of twenty nine patients with immune-unfavorable pre-treatment TME became immune-favorable following CapeOx plus Pembrolizumab chemotherapy. Conclusions: This is the first study to demonstrate TME change during CapeOx plus pembrolizumab at molecular levels and will have clinically meaningful performance in patients with metastatic GC. Clinical trial information: NCT#04249739.