Isolation and Culture of Colon Cancer Cells

The LoVo, SW620, HCT116, SW48, and SW480 human colon cancer cell lines were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China). The XhCRC cells were established as previously described 28 (link). Xenograft tumors were minced and incubated in DMEM/F12 (Thermo Fisher Scientific; Waltham, MA, USA) containing 1.5 mg/mL collagenase IV (Thermo Fisher Scientific), 20 µg/mL hyaluronidase (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), and 1% penicillin/streptomycin (Thermo Fisher Scientific) at 37 °C for 1 h. To remove red blood cells, the cells were treated with red blood cell lysis buffer (Thermo Fisher Scientific) on ice for 10 min, then washed twice with PBS. Isolated single cells were stained with EpCAM and subjected to fluorescence-activated cell sorting (FACSAria II, BD Biosciences; San Jose, CA, USA) to purify EpCAM-positive tumor cells. All cell lines were cultured in DMEM (Thermo Fisher Scientific) with 10% FBS (Thermo Fisher Scientific) and incubated at 37 °C with 5% CO₂ in a cell culture incubator. Mycoplasma was routinely tested using a mycoplasma detection kit (Thermo Fisher Scientific).