Evaluating CAR T Cell Efficacy in Tumor-Bearing Mice

Immunodeficient NSG mice (Jackson Laboratory, ME) were implanted subcutaneously with MDA-MB-231 cells and injected intravenously with CAR T cells and then FITC-folate (as indicated) when tumors reached ~100 mm³ in size. Mice were maintained on folic acid-deficient diet (TD.95247, Envigo) in order to reduce the level of folic acid in mice to a physiological levels found in humans^{45 (link),46 (link)}. Tumors were measured every other day with calipers, and tumor volume was calculated according to equation: Tumor volume = ½(L × W²) where L is the longest axis of the tumor and W is the axis perpendicular to L. Mouse blood was also collected to measure cytokine levels (e.g., IL-2, IL-6, IFNγ, and TNFα) using LEGENDplex bead-based immunoassay (Biolegend, CA) and systemic toxicity was monitored by measuring bodyweight loss. All animal care and use followed by National Institutes of Health (NIH) guidelines and all experimental protocols were approved by the Purdue Animal Care and Use Committee.