In vivo tumor models

All animal experiments were done according to protocols approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania. Animal housing was conducted in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, with a 12 h light/dark cycle and a temperature range of 68–74 °F. Six- to eight-week-old female C57BL/6 mice were purchased from Charles River. B6.129S2-Cd8^tm1Mak (CD8 KO) and B6.SJL-Ptprc^a Pepc^b/BoyJ (CD45.1) mice were purchased from The Jackson Laboratory. Six- to seven-week-old male and female SWV FRβ KO or WT mice were breed in-house.

For the assessment of antitumor efficacy of mFRβ CAR⁺ T cells on a mFRβ-expressing tumor model, C57BL/6 mice were implanted i.p. with 5 × 10⁶ ID8.mFRβ RFP⁻fLuc tumor cells. On days 4, 10, 17, 31, and 38 following tumor injection, mice received 5 × 10⁶ hMeso CAR-T cells (as control) or mFRβ CAR⁺ T cells. For the rest of experiments, mice were inoculated i.p. with 5 × 10⁶ ID8 RFP-fLuc, ID8.hMeso RFP-fLuc or ID8-OVA cells. A total of 4 × 10⁶ or 8 × 10⁶ CAR⁺ T cells (as indicated in the figure legends) were injected i.p. in 200 µL of PBS 21 days after tumor inoculation. Cy (150 mg/kg) was injected i.p. one day before T cell transfer and hIL-2 (Proleukin) was provided at 15 μg/dose in 100 µL i.p. for 3 consecutive days following T cell transfer. Tumor growth was monitored by bioluminescent imaging and weight gain as a surrogate marker for ascites formation. Mice were euthanized at indicated time points for analysis or when they had gained >20% of initial body weight. Bioluminescence imaging was performed by using IVIS Spectrum Imaging System and quantified with the Living Image Software (PerkinElmer). Mice were given an i.p. injection of 150 mg/kg d-luciferin (Caliper Life Sciences, Hopkinton, MA) and imaged under isofluorane anesthesia at the peak of photon emission. Survival curves were graphed considering an intermediary endpoint of weight gain >10% of initial body weight. Peripheral blood was collected via retro-orbital blood collection under isoflurane anesthesia. Fifty microliters per sample were labeled for CD45, CD11b, Ly6G, Ly6C, CD3, CD4 and CD8 and cell counts/µL of blood were calculated using Trucount tubes (BD Biosciences).

For studies with subcutaneous solid tumor models, C57BL/6 mice were inoculated subcutaneously with 0.25 × 10⁶ B16 melanoma or 0.5 × 10⁶ MC38 colon adenocarcinoma cells. After 14 days, tumors were collected and mechanically dissociated using gentleMACs tubes and dissociator according to the manufacturer’s recommendations (Miltenyi Biotec). Single-cell suspensions were stained for FACS analysis as described. For antitumor efficacy experiments, 10 days following tumor inoculation, mice were randomized in groups and treated with a single intravenous dose of 8 × 10⁶ mouse CAR⁺ T cells, accompanied with Cy + IL-2 conditioning as indicated above. Tumors were measured twice a week with caliper, and volumes were calculated as V = 1/2 × length (L) × width (W) × W. Mice were euthanized when tumor diameter was ≥2 cm.