Ct26 cells

Animal experiments were approved by a local ethics committee and performed under a United Kingdom Home Office license, in compliance with UK National Cancer Research Institute guidelines for the welfare of animals in cancer research,14 and with the ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines.15 All experiments were performed with a syngeneic mouse model, where CT26 murine colon carcinoma cells were implanted in an immunocompetent BALB/c mouse host. Mice were obtained from Harlan (Bicester, UK), and were housed under specific pathogen‐free conditions in individually ventilated cages holding a maximum of 6 animals, with appropriate bedding, nesting material, and a cardboard tunnel. Mice were housed on a 12 h/12 h light/dark cycle and were given filtered water and fed an appropriate rodent diet. CT26 cells (ATCC) were maintained in Dulbecco's modified eagle medium (DMEM), supplemented with 10% fetal calf serum (FCS) and 1% L‐glutamine (Invitrogen), and cultured to limited passage for 1‐2 weeks prior to implantation, with regular re‐screening for mycoplasma contamination. Mice were inoculated subcutaneously in the supraspinal position with
1×106 CT26 cells in 100 μL of phosphate‐buffered saline, and were treated when tumors were 250‐300 
mm3, as measured with callipers.

CT26 cells (murine CRC cells), HCT116 cells (human colorectal cancer cells), DC2.4 cells (mouse dendritic cell line ), and HUVEC cells (human umbilical vein endothelial cells) were acquired from ATCC. These cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium at 37 °C in a humidified atmosphere with 5% CO₂. The RPMI‐1640 medium was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin.
For the purpose of in vivo studies, BALB/C mice (female, 6–8 weeks old) were purchased from the Shanghai Wushi Experimental Animal Center (Shanghai, China). All animal studies were approved by the Institutional Animal Care and Use Committee at Fujian Medical University.

All mice were kept in IVC cages under SPF conditions. Six to eight weeks old female BALB/c mice (Janvier) were inoculated subcutaneously with 100 μl 5×10⁵ CT26 cells (ATCC CRL-2638) or 1.5×10⁶ RenCa cells (ATCC CRL-2947) in PBS into the flank and allowed to establish over 8-10 days. When reaching a size of 100-150 mm³ the mice were regrouped according to mean tumor volume, and subjected to infection. Regular caliper measurements in two dimensions allowed for volume calculations using the formula V = π/6 x (h x w²), wherein “h” = height and “w” = width. “Tumor size” denotes a measure of tumor development standardized to tumor volume at 0 dpi. Rag1^−/− mice were bred in house and females at six to ten weeks of age were deployed. To achieve neutropenic conditions, we adhered to the protocol of Westphal et al. [48 (link)] In brief: mice received repeated doses of 25 μg monoclonal rat anti-Gr-1 (RB6-8C5) or 25 μg monoclonal rat anti-Ly6G (1A8) i.p. Depletion was confirmed by flowcytometry.

Lan-1 cells, A204 and SupT1s were purchased from the ECACC. Lan-1 and A204 cells were cultivated in IMDM supplemented with 10% FCS and L-Glutamine. CT26 cells were purchased from ATCC and were transduced with a gammaretroviral cassette co-expressing GD2 and GD3 synthases to drive the biosynthesis of GD2. A single cell clone (clone 7) with stable and high GD2 expression was selected for in vivo studies.