Fig 2: MSLN mediated the effects of Metformin on inhibiting the IL-6/STAT3 signaling. Western blot results showed the expression of MSLN, IL-6, p-STAT3 and STAT3 in untreated and 5 mM metformin-treated group. Quantitative analysis of the effects of metformin on IL-6 and p-STAT3 expression in control and 5 mM metformin-treated SKOV3 cells, data were showed in mean ± SEM, N = 5, ****P < .0001. All data were normalized to GAPDH and fold change was expressed in relation to the untreated group. Western blot results showed the expression of MSLN, IL-6, p-STAT3 and STAT3 in control (NC) and MSLN knock-out (KO) cells treated with 0 or 5 mM metformin. Quantitative analysis of the effects of metformin on IL-6 and p-STAT3 expression in control (NC) and MSLN knock-out (KO) SKOV3 cells treated with 0 or 5 mM metformin, data were showed in mean ± SEM, N = 5, ****P < .0001. All data were normalized to GAPDH and fold change was expressed in relation to the untreated group. Western blot results showed the expression of STAT3 in STAT3 deletion SKOV3 cells with or without metformin administration. SKOV3 cell viability was determined by CCK-8 at the indicated time point.

Fig 3: Characterization of 3rd-generation MSLN-CAR-T cells targeting MSLN(A) Schematic diagram of the MSLN-CAR-T transgene. (B) Proportion of M284bz infected primary human T cells determined by flow cytometry. (C) Histogram of the MSLN-CAR-T rate of M28z10 CAR infected cells and its control cells in three repeats. (D) The exogenous CD3ζ level detected by western blotting (mean ± SEM; ns, not significant; 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 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).