Isolation of CRC Liver Metastatic Cells

Human or mouse CRC liver metastatic tissues were harvested aseptically from CRCLM patients or tumor-bearing mice and then digested with a tumor dissociation kit (Miltenyi Biotec). A single-cell suspension of tissues was obtained using a GentleMAC tissue processor (Miltenyi Biotec) according to the manufacturer’s instructions. Tumor cells were isolated using anti-EpCAM beads (Miltenyi Biotec) and characterized as EpCAM⁺ (Biolegend) with purity greater than 95% by flow cytometry. LSECs were isolated using anti-CD146 beads (Miltenyi Biotec) and characterized as CD146⁺ (Miltenyi Biotec) with purity greater than 95% by flow cytometry as described previously (64 (link)). MDSCs were characterized as negative for HLA-DR (Biolegend) and Lin (Biolegend) but positive for CD33 (Biolegend) and CD11b (Biolegend), which were sorted by flow cytometry as described previously (65 (link)).