Tumor Regression Induced by CAR T Cells and Glycolysis

Ovalbumin (OVA) and human CD19-expressing MC38 colon adenocarcinoma (MC38^OVA and MC38^hCD19) cells were grown at 37 °C and 5% CO₂ in standard DMEM medium supplemented with 10% FBS (Sigma), 2 mM glutamine, 1 mM sodium pyruvate, 1% penicillin/streptomycin, 2 mM Hepes and 0.1 mM non-essential amino acids (NEAA, all Gibco). 5 x 10⁵ MC38^OVA cells were subcutaneously (s.c.) injected into the flanks of 8-12 weeks old C57BL/6 mice. Six days after tumor inoculation, mice were irradiated sublethally (6 Gy). The following day, mice were injected intravenously (i.v.) with 2 x 10⁶ OT-I CD8⁺ T cells that were isolated from the spleen and peripheral lymph nodes of OT-1 mice and stimulated with 0.5 μg/ml anti-CD3 (clone 145-2C1), 1 μg/ml anti-CD28 antibodies (clone 37.51, both Bio X Cell) and 10 ng/ml rhIL-2 (Peprotech) for 72 h with or without 1 mM 2-desoxyglucose (2-DG, Sigma). Tumor size was assessed every day using a caliper. For some experiments, tumor tissue was harvested and digested with 1 mg/ml collagenase type I and 100 μg/ml DNase I (both Roche) and T-cell infiltration analyzed by flow cytometry. Alternatively, Rag1^–/– mice were s.c. injected with 1 × 10⁶ MC38^hCD19. After 7 days, 2 x 10⁶ anti-hCD19 CAR T cells^{33 (link)} treated with and without 2 mM 2-DG for 2 days were adoptively transferred i.v.