Fig. 3. Systemic infusions of tumor-specific CAR-T cells produce only modest therapeutic benefits.

Ten days after 4T1-ROR1 tumor cells were transplanted into the mammary glands of BALB/cJ mice, the animals were intravenously injected with 10⁷ ROR1–28z CAR-transduced T cells or tumor-irrelevant (anti-GP75) control lymphocytes. The CAR-T cells co-expressed click beetle red luciferase (CBR-luc) reporter so we could measure the dynamics of programmed lymphocyte targeting. (A) Sequential bioluminescence imaging of the adoptively transferred T cells in three representative mice from each cohort (n = 8). (B) CBR-luc signal intensities from T cells at the tumor site examined via sequential bioluminescence imaging every two days after cell transfer. At the indicated time points, pairwise differences in photon counts between treatment groups were analyzed with the Wilcoxon rank-sum test. (C) Multicolor flow cytometry of cells recovered from tumors six days after CAR-T cell infusion. Adoptively transferred ROR1–28z CAR-transduced T cells were identified by positive labeling for CD3 and the c-myc tag incorporated in the CAR (see Fig. 2A). (D) Progression of 4T1-ROR1 tumor growth following therapy. Every line represents one animal and the dots indicate tumor sizes, as determined by caliper measurements. Shown are results from eight animals pooled from three independent experiments. (E) Kaplan-Meier survival curves for treated versus control mice. ms, median survival. Statistical analysis was performed using the log-rank test and P < 0.05 was considered significant.