Abstract C113: Multiple modalities of single cell analyses to evaluate the tumor microenvironment in clinical specimens
Authors: Kashima Yukie , Fukuoka Shota , Togashi Yosuke , Kamada Takahiro , Suzuki Ayako , Nakamura Yoshiaki , Shitara Kohei , Ohashi Akihiro , Yoshida Taku , Taoka Naofumi , Kawase Tatsuya , Wada Teiji , Inaki Kocihiro , Chihara Masataka , Suzuki Yutaka , Tsuchihara Katsuya , Kobayashi Susumu S , Nishikawa Hiroyoshi , Doi Toshihiko 
Published: March 2020

Abstract
Background: Tumor tissues are composed of heterogeneous populations, including cancer cells, immune cells, and stromal cells. These cells are spatiotemporally interacted and play critical roles in tumor development. To understand the complexity of these tumor microenvironment more comprehensively, single-cell analysis is being at the forefront as a novel technology. In this study, we investigated immune-profiling of peripheral blood mononuclear cells (PBMCs) and clinical specimens from gastrointestinal cancer patients using single-cell analyses. Furthermore, we technically characterized two distinct methodologies of the single-cell analysis. Methods: We employed Chromium v2-based single cell RNA sequence (scRNA-seq) analysis and Helios-based CyTOF analysis. PBMCs and the endoscopically- or surgically-resected tumors from gastrointestinal cancer patients were used. PBMCs were divided to two samples, applying to scRNA-seq and CyTOF. The tumor tissues from each patient were divided to three samples, applying to scRNA-seq, CyTOF, and IHC. For scRNA-seq and CyTOF, samples were treated with the specific enzyme at 37 °C for 30 minutes using GentleMACS to isolate cells at a single-cell levels. The unsorted single-cells were subjected to the scRNA-seq and CyTOF analyses. Results: In PBMCs, scRNA-seq identified subpopulations into T cell and NK cells, B cells, and other immune-related cells. Due to similarity of gene expression and low depth of data, it is difficult to distinguish NK cell and T cell in scRNA-seq. In contrast, CyTOF appears to identify the differences between them more clearly. In tumor samples, scRNA-seq classified cells into epithelial, fibroblast/endothelial, CD45+ immune cells, and CD45-CD19-CD138+ plasma cells. CyTOF didn’t detect plasma cells due to the lack of markers. These plasma cells were also confirmed by IHC in tumor tissues. When FOXP3+CD4+ regulatory T (Treg) cells, which play immunosuppressive roles in antitumor immunity, were focused, scRNA-seq analysis classified Treg cells in multiple clusters, indicating that Treg cells have heterogeneity and can play various roles in the tumor microenvironment. Conclusions: We demonstrated using the two-distinct methodologies that single-cell analysis is a powerful tool to classify the cell types in clinical specimens. Although deeper analysis is required, our results may help us to identify various cell population, which could not be identified by other modalities, leading to novel insights in the tumor microenvironment. Taken with the technical advantages for each methodology, the single-cell analysis would be more powerful tools to understand the complexity of the tumor microenvironment.
Citation Format: Yukie Kashima, Shota Fukuoka, Yosuke Togashi, Takahiro Kamada, Ayako Suzuki, Yoshiaki Nakamura, Kohei Shitara, Akihiro Ohashi, Taku Yoshida, Naofumi Taoka, Tatsuya Kawase, Teiji Wada, Kocihiro Inaki, Masataka Chihara, Yutaka Suzuki, Katsuya Tsuchihara, Susumu S Kobayashi, Hiroyoshi Nishikawa, Toshihiko Doi. Multiple modalities of single cell analyses to evaluate the tumor microenvironment in clinical specimens [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C113. doi:10.1158/1535-7163.TARG-19-C113