Abstract Tumor-infiltrating lymphocytes (TILs) play a major role in predicting response to immunotherapy in solid tumors. However, few studies have analyzed TIL distribution and associated genomic signatures based on proximity to the tumor-stromal border (TSB). Here, we describe TIL density, IFN-γ signature, and tumor mutational burden (TMB), according to set distances from the TSB, in The Cancer Genome Atlas (TCGA) pan-carcinoma dataset. For spatial TIL analysis, we used Lunit SCOPE IO, an AI-powered H&E whole-slide image analyzer, which identifies and quantifies tumor and lymphocyte cells within cancer or stroma area. In TCGA pan-carcinoma (n=7,468) dataset, the median values of cancer area and cancer stroma were 46.8 (interquartile range [IQR] 21.6-87.0) and 26.6 (IQR 10.9-54.5) mm2, respectively, and those of TIL density in each area were 72.1 (IQR 34.1-160.1) and 747.2 (IQR 330.4-1575.9)/mm2, showing 10-fold enrichment in stromal TIL (sTIL) compared to intratumoral TIL (iTIL). Subdividing the cancer area in 10 micron-wide steps from the TSB toward the tumor core, the median proportions of 0-10, 10-20, 20-30 (IT 0-10, IT 10-20, IT 20-30), and greater than 30 microns (IT 30-core) were 17.7%, 20.8%, 13.6%, and 47.9%, respectively. For subdividing the cancer stroma outwards from the TSB, the median proportions of 0-10, 10-20, 20-30 (ST 0-10, ST 10-20, ST 20-30), and greater than 30 microns (ST 30-outside) were 13.6%, 19.4%, 13.5%, and 53.5%, respectively. From the tumor core to TSB, median values of TIL density were 53.4 (IT 30-core), 59.3 (IT 20-30), 69.3 (IT 10-20), 127.4 (IT 0-10)/mm2, respectively. From the TSB to the remaining stroma, median values of TIL density were 98.7 (ST 0-10), 567.6 (ST 10-20), 708.8 (ST 20-30), and 896.1 (ST 30-outside)/mm2, respectively. Hence, the density of TIL generally increased from the tumor core to the outside of cancer stroma. Of note, IFN-γ signature showed the highest level of correlation with TIL density in IT 10-20 (Spearman’s rho [ρ] 0.5146) or IT 0-10 (ρ 0.5143), compared with other subdivided cancer areas (ρ 0.4567-0.4924), and cancer stromas (ρ 0.1454-0.4529). However, TMB was highly correlated with TIL density in ST 20-30 (ρ 0.3162) or ST 30-outside (ρ 0.3278), compared with other subdivided cancer stromas (ρ 0.0588-0.2843) and cancer areas (ρ 0.0784-0.1208). In conclusion, AI-powered analysis of the tumor micro-environment reveals that iTIL density increases from the tumor core to the outside of tumor-stromal border. IFN-γ signature is high in the area of the tumor-stromal border on the tumor side, while tumor mutational burden is associated with sTIL far from the tumor-stromal border. Citation Format: Sanghoon Song, Gahee Park, Sukjun Kim, Sangjoon Choi, Seokhwi Kim, Wonkyung Jung, Mingu Kang, Mohammad Mostafavi, Heon Song, Aaron Valero, Sérgio Pereira, Donggeun Yoo, Seulki Kim, Seunghwan Shin, Ken Nesmith, Chan-Young Ock. Micron-resolution spatial analysis near the tumor-stromal border reveals a distinct density distribution of tumor-infiltrating lymphocytes and related genomic features. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5389.