As a consequence of altered glucose metabolism, cancer cell intake is increased, producing large amounts of lactate which is pumped out the cytosol by monocarboxylate transporters (MCTs). MCT 1 and MCT4 are frequently overexpressed in tumors, and recently, MCT inhibition has been reported to exert antineoplastic effects. In the present study, MCT1 and MCT4 levels were assessed in esophageal adenocarcinoma (EAC) cells and the effects of the MCT-1 selective inhibitor AZD3965, hypoxia, and a glucose overload were evaluated in vitro. Two EAC cell lines (OE33 and OACM5.1C) were treated with AZD3965 (10–100 nM) under different conditions (normoxia/hypoxia) and also different glucose concentrations, and parameters of cytotoxicity, oxidative stress, intracellular pH (pHi), and lactate levels were evaluated. MCT1 was present in both cell lines whereas MCT4 was expressed in OE33 cells and only in a small proportion of OACM5.1C cells. Glucose addition did not have any effect on apoptosis nor cell proliferation. AZD3965 increased apoptosis and reduced proliferation of OACM5.1C cells, effects which were abrogated when cells were growing in hypoxia. MCT1 inhibition increased intracellular lactate levels in all the cells evaluated, but this increase was higher in cells expressing only MCT1 and did not affect oxidative stress. AZD3965 induced a decrease in pHi of cells displaying low levels of MCT4 and also increased the sodium/hydrogen exchanger 1 (NHE-1) expression on these cells. These data provide in vitro evidence supporting the potential of MCT inhibitors as novel antineoplastic drugs for EAC and highlight the importance of achieving a complete MCT inhibition.

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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.