Fig 2: SHP2 inhibition promotes cytotoxic T-cell cytokine/granule production in CT-26 xenograft BALB/c mice. (A) H & E staining of tumor sections. (B) TUNEL assay for apoptotic cells in tumor tissue. (C) IHC for detecting IFN-? and GZMB in the tumor sections. (D) qPCR for Ifn-?, Prf and GzmB expression in tumors. Data are presented as mean ± SEM of 6 mice per group. (E) and (F) Intracellular staining for IFN-? and GZMB production in tumor infiltrating CD8+ T cells. Data are presented as mean ± SEM of 3 mice per group. *P < 0.05, **P < 0.01 vs. PBS. Prf, perforin; GzmB, granzyme B. Scale bar, 10 µm.

Fig 3: SHP2 inhibition synergizes with PD-1 blockade in MC-38 xenograft mice. The mice were subcutaneously inoculated with 1×106 MC-38 colon cancer cells, and treated with anti-PD-1 (5 mg/kg i.p. once every three days) and/or SHP099 (5 mg/kg i.p. every day) from day 3 onwards. All results presented are of day 13 post-inoculation. (A) Representative images of tumors. (B) Mean tumor volumes of different groups. (C) Mean tumor weights of different groups. (D) Tumor volume of individual mice. (E) Mean body weight of mice. (F) H & E staining of tumor sections. (G) TUNEL assay for apoptotic cells in tumor sections. (H) qPCR assay for the Ifn-γ, GzmB, Prf and FasL mRNA levels in tumor tissues. Data in (B), (C), (E), and (H) are presented as mean ± SEM of 6 mice per group. (I) IHC for IFN-γ and GZMB in tumor sections. (J) Intracellular staining for IFN-γ production in tumor infiltrating CD8+ T cells. Data in (J) are presented as mean ± SEM of 3 mice per group. (K) MTT viability assay for MC-38 colon cancer cells. Data are presented as mean ± SEM of three different experiments. *P < 0.05, **P < 0.01 vs. PBS. †P < 0.05 vs. SHP099+anti-PD-1. Prf, perforin; GzmB, granzyme B. Scale bar, 10 μm.

Fig 4: In vivo characterization of the antitumor efficacy of ANKET-IL-2v(A–C and E) Raji B cell lymphoma cells were subcutaneously engrafted in CB17 SCID mice.(A) Efficacy of 70 µg IL-2v/aNKp46/Fc/aCD20 or 1,500 µg Obinutuzumab injected i.v. once, 9 days after tumor engraftment.(B) Efficacy of 25 µg IL-2v/aNKp46/Fc/aCD20 or 25 µg IL-2v/aNKp46/Fc/IC injected i.v. once, 9 days after tumor engraftment.(C) Efficacy of two weekly i.v. injections of 25 µg IL-2v/aNKp46/Fc/aCD20, IL-2v/IC/Fc null/aCD20, or IL-2v/IC/Fc/aCD20 molecules.(D) Immunostaining for human NKp46 and Gzmb on sections of Raji tumors grown subcutaneously in RAG1ko huNKp46Tg mice, 3 days after treatment with 25 µg IL-2v/aNKp46/Fc/aCD20 or vehicle. The images shown are representative of n = 5 tumors.(E) Efficacy of a single i.v. injection of 25 µg IL-2v/aNKp46/Fc/aCD20 in the context of NK cell depletion with anti-Asialo-GM1. Dotted vertical lines represent the day of treatment. Each curve represents data from a single mouse. The data shown are representative of at least two independent experiments. The statistical analysis is described in the STAR methods, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig 5: SHP099 enhances the antitumor role of celastrol by promoting T-cell infiltration. (A) Infiltration of CD8+, CD4+, and GzmB+ cells in CRC xenografts treated with SHP099 alone, celastrol alone, or their combination using IHC examination. (B) Quantification of CD8+, CD4+, and GzmB+ cells in tumors treated with SHP099 alone, celastrol alone, or their combination. *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar: 20 µM.