Pma and ionomycin

Fresh tumor samples were obtained immediately after surgical resection, of which adipose or necrotic tissues were removed carefully. To collect single‐cell suspensions, tumors were minced and digested in RPMI‐1640 with collagenase I (Sangon; 250 U ml⁻¹) and DNase I (Sangon; 50 U ml⁻¹) for 3 hours at 37 C according to the instructions. Red blood cell lysis buffer was then used for removing red blood cells. After stimulation with cell stimulation cocktail (PMA and ionomycin, eBioscience) and Golgistop (BD Biosciences) for 4 hours, cells were stained with membrane markers in dark for 45 minutes at 4 C after applying Human Fc block (BD Biosciences). Cells were stained with intracellular markers after permeabilization by the BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences) or BD Pharmingen Transcription Factor Buffer Sets (BD Biosciences). Detailed information on antibodies for flow cytometry is listed in Table S1. Cells were collected and analyzed on the FACSCelesta (BD Biosciences) in 1 hour. In addition, part of the fresh tumor tissues was formalin fixed and paraffin embedded for MDK IHC staining to stratify the tumor into high/low intratumoral MDK expression groups.