Fig 2: Evaluation of the anti-tumor capacity of MSLN-CAR-T cells in vitro(A) The surface expression of MSLN receptor level on solid tumor cell lines (OVCAR3, SKOV3, HeLa, ES2, AGS, HCT116) detected by flow cytometry. (B) The cytotoxicity of MSLN-CAR-T cells against solid tumor cell lines was analyzed by RTCA assay. The red arrow represents the time point of effector cell addition. (C) Levels of IFN-γ and TNF-α released by MSLN-CAR-T cells analyzed by ELISA after incubated with cells for 24 h. (D) A549 and HeLa weren’t shown, mean ± SEM; p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Fig 3: Anti-tumor efficacy of MSLN-CAR -T cells against colorectal cancer in vivo(A) Schema of the experimental events and nodes. In this process, model mice were injected with 5 × 105 CRC cells HCT116. 10 days after injection, 12 mice were randomly divided into 3 groups (n = 4): 5 × 106 MSLN-CAR-T cells-treated group, control-T cells-treated group, and PBS of the same volume-treated group. After only one injection, we started observing the mice. (B and C) The tumor size (B) and mice weight (C) variated with MSLN-CAR-T cells, control T cells, or PBS injection among the 28 days. (D) Size of tumor mass differed in MSLN-CAR-T cells, control T cells, or PBS-treated mice. (E) Weight of tumor mass differed in MSLN-CAR-T cells, control T cells, or PBS-treated mice. On day 20, the mice were sacrificed; the CD3+ T cell amount in the PB of HCT116 CDX model is as shown (F). The level of IFN-γ in mouse serum was evaluated by ELISA as shown (G). (H) Immunohistochemistry (IHC) analysis of granzyme B in tumor sections (n = 4, mean ± SEM; p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Fig 4: High expression of MSLN in CRC and OC samples(A and B) MSLN expression was detected in CRC, CPC, OC, and OB tissues. CRC, colorectal cancer; CPC, colorectal para-cancerous tissue; OC, ovarian cancer tissue sample; OB, ovarian benign tissue. Scale bar, 100 μm. (C and D) Comparison of MSLN expression level between CRC and CPC or OC and OB groups. Stacked bar graph was derived from the Tables 1 and 2. High positive means positive target expression >50%, medium positive means positive target expression 20%~50%, and low positive means positive target expression <20%.

Fig 5: Anti-tumor efficacy of MSLN-CAR-T cells against ovarian cancer in vivo(A) Schema of the experimental events and nodes. In this process, model mice were injected with 1 × 106 OC cells SKOV3. 7 days after injection, 8 mice were randomly divided into 2 groups (n = 4): 2.5 × 106 MSLN-CAR-T cells-treated group and control-T cells-treated group. (B and C) The tumor size (B) and mice weight (C) variation with MSLN-CAR-T cells, control T cells, or PBS injection among the 35 days.(D) Size of SKOV3 CDX tumor mass differed in MSLN-CAR-T cells, control T cells or PBS treated mice. (n = 4, mean ± SEM; p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).