Animal study

To value the tumorigenesis of SCCs enriched in in vivo model, xenograft tumor were dissected and digested 30 days after inoculation. Tumor cells were collected by EpCAM⁺ FACS sorting from single cell suspensions and injected subcutaneously into the flank of Nude mice a gradient dose of 10000, 5000 and 1000 cells (n=8 for each group). The mice were examined visually every day up for 6 weeks. To determine growth potential, tumor cells from xenograft tumor were collected and inoculated to Nude mice in a high dose (5×10⁵ cells/mouse, 5 mice for each group). Tumor growth was monitored once every 5 days up to 30 days.

For adoptive transfer mice model, Nude mice bearing colorectal carcinomas (1 week after being subcutaneous inoculation with tumor cells) received intravenous transfusion of DC-CIK cells (1.5×10⁷ cells per mouse, once every three days for four times). Tumor growth was monitored every 5 days.

To value the tumorigenesis of SCCs enriched in in vitro with or without DC-CIK killing, tumor cells were cocultured with DC-CIK cells for 48 h in vitro. Then they were isolated by EpCAM+ sorting and injected into the flank of Nude mice at a gradient dose of 1000, 5000, 10000 or 50000 cells. The mice were examined visually every day up to 6 weeks.

For colorectal tumor metastasis model, LoVo cells were transduced with CMV-Fluc-IRES-GFP lentiviral expression vector (GeneChem, shanghai, China). GFP positive cells were enriched through FACS sorting and designated as LoVo-GFP cells. SCCs-GFP cells were enriched from LoVo-GFP cells as described above. Nude mice were inoculated with LoVo-GFP or SCCs-GFP cells by the injection of 1×10⁶ cells into spleen. On day 35 after inoculation, mice were sacrificed and tumor nodes on both spleen and liver were observed and photoed under the stereomicroscope. To assay tumor cell arrest in lung during blood flow, LoVo and SCCs cells were labeled with CFSE, and injected into mice via tail vein (2×10⁶ cells/mouse, n=5 for each group). Lungs of mice were harvested 5 h or 24 h after the injection. Frozen sections were prepared and analyzed by fluorescence microscopy.

All these experiments were repeated three times.