Briefly, the tumor bearing colons and location-matched normal colons were cleaned, cut into 0.5-cm sections, and shaken twice in a Ca and Mg-free (CMF) solution containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 25 mM NaHCO3, 1 mM dithiothreitol (DTT), and 1 mM ethylenediaminetetraacetic acid (EDTA) (all from Sigma-Aldrich, St. Louis, MO), and 2% fetal bovine serum (FBS) (Gibco, Portland, OR, USA) at 37 ℃. Subsequently, the cell suspension was filtered using a 70-mm nylon cell strainer (Corning, NY, USA), and the cells were collected. The residual colon pieces were digested for 60 min in Roswell Park Memorial Institute 1640 Medium (RPMI-1640 medium) containing 10% FBS, 1 mg/mL of collagenase D (Roche Applied Science, Upper, Bavaria, Germany), and 0.08 mg/mL of DNase I (Sigma-Aldrich, St. Louis, MO) at 37℃, and the cells were collected. All the collected cells were mixed, and all the immune cells from were purified using 40% and 70% discontinuous percoll gradients. RNA libraries were generated by 10x Genomics Single Cell 3' V3 Library Gel Bead Kit and prepared for sequencing using standard Illumina protocols.