RNA-sequencing of tumor-infiltrated T cells in colorectal tumors

The goal of this study is to identify differentially expressed genes in colorectal tumor samples enriched for exhausted T cells. CD38 expression was used as a representative marker for T cell exhaustion. The comparison was made between CD38high (top 33rd percentile of all samples) and CD38low (bottom 33rd percentile of all samples) samples. Tumor samples for the Immunoprofiler was transported from various cancer operating rooms (ORs) as well as from outpatient clinics. All patients consented by the UCSF IPI clinical coordinator group for tissue collection under a UCSF IRB approved protocol (UCSF IRB# 20-31740). Tumor or metastatic tissue was thoroughly chopped with surgical scissors and transferred to GentleMACs C Tubes (Miltenyi Biotec) containing 20 uL/mL Liberase TL (5 mg/ml, Roche) and 50 U/ml DNAse I (Roche) in RPMI 1640 per 0.3 g tissue. GentleMACs C Tubes were then installed onto the GentleMACs Octo Dissociator (Miltenyi Biotec) and incubated for 45min according to the manufacturer’s instructions. Samples were then quenched with 15 mL of sort buffer (PBS/2% FCS/2mM EDTA), filtered through 100 um filters and spun down. Red blood cell lysis was performed with 175 mM ammonium chloride if needed. Cells were then incubated with Human FcX (Biolegend) to prevent non-specific antibody binding. Cells were then washed in DPBS and incubated with Zombie Aqua Fixable Viability Dye (Thermo). Following viability dye, cells were washed with sort buffer and incubated with cell surface antibodies mix diluted in the BV stain buffer (BD Biosciences) following manufacturer instruction for 30 minutes on ice in the dark and subsequently fixed in either Fixation Buffer (BD Biosciences) or in Foxp3/Transcription Factor Staining Buffer Set (eBioscience) if intracellular staining was required.