Background: Tumors are complex ecosystems composed of different cell types with different phenotypes, status and gene profiles. Commonly used GEP tools like bulk RNA Sequencing could only display the gene expression profiles as a whole, and cannot reflect the heterogeneous tumor cell change or immune composition in a tumor. Single cell RNA Sequencing can be a good tool to implement it as single cell level. However, the method and experiment system should be optimized to make sure the result is reliable and interpretable. Thus we try to develop and optimize our scRNAseq assay through a series of validation step to make sure the reliability of the results, to find the mechanism of immune therapy against tumors.

Methods: Sample processing were validated for each step separately, including tumor dissociation, dead cell removal and blood cell lysis. Single-cell 3′ library generation was performed on the 10x Genomics Chromium Controller following the manufacturer's protocol for the Single Cell 3' Reagent Kits v3.1 (10x Genomics), and libraries were sequenced using Illumina Novaseq instrument. Using gene expression matrix processed by Cell Ranger with default parameters, we performed data QC, normalization, clustering by Seurat and cell type identification based on a multistep approaches. Differential gene expression were analyzed in each clusters.

Results: In this study, we established a reliable system for scRNASeq analysis to elucidate the mechanism of anti-PD-1 treatment, and the interaction between the tumor and immune cells. We optimized the system from the following 3 aspects: 1) sample processing method to make sure the cell yield, viability and no bias introduced. 2) Library size that sensitive enough for our sub-population analysis. And 3) whether to sort immune cells before the library construction. Using this system, we tried to find the mechanism of anti-PD-1 against the tumors by comparing the scRNASeq profiles changed after the treatment in two breast cancer models, EMT6 and 4T1, which are responsive and non-responsive to anti-PD-1 treatment, respectively. Both models were treated with anti-PD-1 or isotype control and tumors were collected for scRNASeq analysis. Interestingly, changes in both tumors cells and tumor micro-environment were observed, and it will help us to reveal how the immune therapy may shake up the complex ecosystem.

Summary: We optimized and established a reliable system for scRNASeq analysis to explore the mechanism of anti-PD-1 treatment against the breast cancer.

Citation Format: Qiyao Zhang, Jingjing Wang, Panpan Wang, Tianqi Tang, Yaqiong Pei, Wenli Zhang, Qingyang Gu, Qunsheng Ji. Establishment and Optimization of scRNASeq assay to find the mechanism of immune therapy against tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2721.