Project name: scRNAseq of orthotopic mismatch repair deficient (dMMR) or chromosomally instable (CIN) colorectal cancer

Description: Colorectal cancers (CRCs) deficient in DNA mismatch repair (dMMR) contain abundant CD8+ tumor infiltrating lymphocytes (TILs) responding to the abundant neoantigens from their unstable genomes. Priming of such tumor-targeted TILs first requires recruitment of CD8+ T cells into the tumors, implying that this is an essential prerequisite of successful dMMR anti-tumor immunity. We have discovered that selective recruitment and activation of systemic CD8+ T cells into dMMR CRCs strictly depends on overexpression of CCL5 and CXCL10 due to endogenous activation of cGAS/STING and IFN signaling by damaged DNA. TIL infiltration into orthotopic dMMR CRCs is neoantigen-independent and followed by induction of a resident memory-like phenotype key to the anti-tumor response. CCL5 and CXCL10 could be upregulated by common chemotherapies in all CRCs, indicating that facilitating CD8+ T cell recruitment underlies their efficacy. Induction of CCL5 and CXCL10 thus represents a tractable therapeutic strategy to induce TIL recruitment into CRCs where local priming can be maximized even in neoantigen-poor CRCs.Overall design: Orthotopic CRC experiments were performed by injecting 1.5x105 dMMR or CIN MC38 CRC cells (containing targeted mutations in Mlh1 or Kras, respectively) in 50 µl PBS into the wall of the descending colon using a flexible needle inserted through the working channel of an endoscope and visualized via the ColoView imaging system. Orthotopic tumour growth was monitored by endoscopy and tumours were harvested after 14-21 days and dissociated by digestion in an enzyme cocktail (RPMI containing 0.5 mg/ml collagenase IV (SigmaAldrich), 10 µg/ml DNaseI (SigmanAldrich), 10% FBS, 1% penicillin-streptomycin and 1% HEPES buffer) for 30 minutes at 37˚C in a shaking incubator at 200 rpm. Dead cells were removed using the EasySep Dead Cell Removal Kit (StemCell). CD45+ cells were purified using magnetic beads (StemCell) and then mixed at a 5:1 ratio of CD45+ cells to tumor cells. Samples were submitted to the University of Alberta High Content Analysis Core for processing with a Single Cell Immune Profiling kit (10X Genomics) and sequenced by Novogene. The data were first processed using the Cell Ranger v5.0 pipeline (10X Genomics) and then further analyzed using the Seurat package for R (v3.0) as described previously (Stuart et al., 2019; Wang et al., 2019).

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: ModelOrganism

Last updated: 2021-06-22