Abstract 6128: Establishment of spatial transcriptomics assay to study the mechanism of immune therapy against tumors
Authors: Wang Panpan , He Huacheng , Zhang Qiyao , Wang Jingjing , Lu Dan , Duan Fei , Fan Qiangqiang , Gu Qingyang , Ji Qunsheng 
Published: June 2022

Abstract
Background: Effective cancer immunotherapy is significantly affected by intratumoral heterogeneity, not only in different cell types and functions but also in the spatial pattern of immune cells within the tumor tissue. While most single-cell RNA sequencing technologies could enable the exploration of gene expression heterogeneity at the single-cell level, they cannot reveal spatial information within the tumor for us to map the whole transcriptome with morphological context. Spatial transcriptomics is an in situ capturing technique, which profiles gene expression at the RNA level, whilst preserving the spatial information of histological tissue sections. Thus we try to develop our spatial transcriptomics assay through a serial of validation experiments and to explore the mechanism of immune therapy against tumors.
Material and Method: Firstly, the tumor was frozen and sections were cryosectioned at 10 μm thickness. Sections processing including H&E staining, tissue optimization, permeabilization, reverse transcription, and cDNA library preparation was carried out following the user guide of Visium Spatial Gene Expression Reagent Kits (10X Genomics). And the final cDNA libraries were sequenced using the Illumina Novaseq instrument. Using gene expression matrix and aligned H&E image processed by Cell Ranger and Loupe Browser with default parameters, we performed data QC, normalization, dimensionality reduction, and clustering by Seurat and cell components identification in tumor microenvironment based on a multistep approach. Differential gene expression and spatial heterogeneity were analyzed in each cluster.
Results: Here we established a reliable system for spatial transcriptomics assay to elucidate the mechanism of anti-PD1 treatment in MC38 syngeneic colorectal tumor. Using this system, we constructed the gene expression spatial patterns of tumor cells and immune cells, including periphery and center of tumor cell, immune-cell enrichment area, and tumor cell surrounding the immune cell. Furthermore, we explored the cell-cell interactions in which the tumor cells directly interact with adjacent non-tumor cells, for example, macrophages. Using this system, we tried to investigate the mechanism of anti-PD-1 therapy against the tumors by comparing the spatial transcriptome profiles after the treatment with anti-PD-1 or isotype control in colorectal cancer models. Interestingly, changes in both gene-level and spatial levels were observed, and it will help reveal how the immune therapy may shake up the complex ecosystem.
Summary: We established a reliable system for spatial transcriptomics analysis and explored the mechanism of anti-PD-1 treatment against the colorectal cancer by using this system.
Citation Format: Panpan Wang, Huacheng He, Qiyao Zhang, Jingjing Wang, Dan Lu, Fei Duan, Qiangqiang Fan, Qingyang Gu, Qunsheng Ji. Establishment of spatial transcriptomics assay to study the mechanism of immune therapy against tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6128.