Lexin Wang ^1,2 Xue Zhou ^3* Haisheng Yan ^1,2* Lexin Wang ⁴ Binbin Wang ^5* Yuheng Gu ³ Yaping Miao ^1,2 Weining Fan ^1,2* Ke Xu ^6* Shangke Huang ^3* Jie Liu ^7*

• ¹ Ningxia Medical University, Yinchuan, China
• ² General Hospital of Ningxia Medical University, Yinchuan, Ningxia Hui Region, China
• ³ Department of Oncology, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan Province, China
• ⁴ The Affiliated Hospital of Shaoxing University, Shaoxing, Zhejiang Province, China
• ⁵ Intensive Care Unit, Gansu Provincial People's Hospital, Lanzhou, Gansu Province, China
• ⁶ Department of Ophthalmology, Chongqing General Hospital, Chongqing, China
• ⁷ Department of General Surgery, Dazhou Central Hospital, DaZhou, China

Background: Tryptophan Metabolism-associated Genes (TMGs), such as ECHS1 and ALDH2, are crucial in cancer progression through immunosuppressive mechanisms, particularly in Gastric Cancer (GC). This study explores their effects on the Tumor Microenvironment (TME). Additionally, it examines their potential as novel immunotherapy targets.We utilized single-cell and bulk transcriptomic technologies to analyze the heterogeneity of GC. Non-negative Matrix Factorization (NMF) clustering identified key TMGs, and extensive RNA-seq analyses were performed to pinpoint prognostic genes and potential immunotherapy targets.Furthermore, through PCR analyses we found that ECHS1 and ALDH2 gene expression plays a regulatory role in the migration, invasion and inflammatory factor in AGS and SNU-1 cell lines. The interference effect of si-ECHS1 and ad-ALDH2 was validated using cell scratch assay in AGS and SNU-1 cell line.2 Results: We observed a statistically significant correlation between ECHS1 and ALDH2 expression and increased TME heterogeneity.Our findings also revealed that ECHS1 down-regulation and ALDH2 up-regulation contribute to reduced TME heterogeneity, decreased inflammation, and inhibited AGS and SNU-1 tumor cells migration and proliferation. GSVA enrichment analysis highlighted the NF-kappa B(NF-κB)signaling pathway as specifically regulated by TMGs.Furthermore,ECHS1 and ALDH2 modulated CD8+ and CD4+ T cell activities, impacting GC progression.In vitro experiments further solidified our conclusions by showcasing the inhibitory effects of Si-ECHS1 and ad-ALDH2 on the invasive and proliferative capabilities of AGS and SNU-1 cells.Moreover, Si-ECHS1 and ad-ALDH2 gene expression effectively reduced the expression of inflammatory factors IL-10,IL-7,CXCL8 and IL-6, leading to a remarkable alleviation of chronic inflammation and the heterogeneous nature of the TME.This research highlights the importance of ECHS1 and ALDH2 in GC progression and immune modulation, suggesting that targeted therapies focusing on these genes offer promising avenues for personalized immunotherapy in GC. These findings hold potential for improving patient survival and quality of life. Future studies on the NF-κB signaling pathway's role in this context are warranted to further elucidate the mechanisms underlying TMG-mediated immune modulation in GC.