LAUSR

Introduction: In recent years, single-cell RNA sequencing (scRNA-seq) and high-dimensional spatial transcriptomics (ST-seq) have contributed significantly to our understanding of the tumor microenvironment (TME) in many cancers. This is essential for understanding the tumor-stromal crosstalk. Thus, integrating scRNA-seq data with ST-seq data will facilitate understanding intercellular communication in TMEs. Material and Method: We performed and combined ST-seq in CRC with public scRNA-seq. And, immunohistochemistry (IHC) (HLA-G, CD68, and SPP1) was performed on 20 CRC surgical specimens collected in our hospital. To verify this, we knocked out H2-M3 (HLA-G in humans) in mouse CRC cell lines (MC-38 cells) using the CRISPR/Cas9 system and examined H2-M3 KO tumor volume in xenograft mouse models. Results: We identified co-localized cells with CRC cells at the invasive front and those in the center based on the spatial distribution estimated by an analytical pipeline, Cell2location. At the invasive front, CRC cells co-localized more frequently with SPP1+ macrophages than other cells. To dissect the molecular machinery and cell types inducing SPP1+ macrophages, we found ligand activity by another pipeline, NicheNet. The prominent signal to SPP1+ macrophages was mediated by several ligand-receptor pairs including HLA-G- immunoglobulin-like transcript (ILT)2/ILT4. Next, we disclosed the effect on CD8+ cells, and SPP1+ macrophages secrete IL-10 suppressing the immune activity of CD8+ T cells. And the ratio of HLA-G-positive cancer cells was predominantly higher in areas with more SPP1+ macrophages by IHC in CRC. In vivo analysis, H2-M3 KO reduced CRC tumor volume and tumor tissue from H2-M3 KO cells exhibited higher SPP1 and CD68 staining compared with wild type cells. Conclusion: SPP1+ macrophages and cancer cells secreting high levels of HLA-G may represent targets to improve the prognosis of patients with CRC. Further analysis is currently underway with a focus on early-stage cancer in carcinoma in adenoma. Citation Format: Masahiro Hashimoto, Yuki Ozato, Yusuke Nakano, Tadashi Abe, Yuichi Hisamastu, Takeo Toshima, Yusuke Yonemura, Mamoru Uemura, Takaaki Masuda, Hirofumi Yamamoto, Masaki Mori, Hidetoshi Eguchi, Yuichiro Doki, Koshi Mimori. Spatial transcriptomics deciphers the cellular society of advanced colorectal cancer [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 5855.