Inhibition of SRC family kinases facilitates anti-CTLA4 immunotherapy in head and neck squamous cell carcinoma

Abstract

The immune system plays a critical role in the establishment, development, and progression of head and neck squamous cell carcinoma (HNSCC). As treatment with single-immune checkpoint agent results in a lower response rate in patients, it is important to investigate new strategies to maintain favorable anti-tumor immune response. Herein, the combination immunotherapeutic value of CTLA4 blockade and SFKs inhibition was assessed in transgenic HNSCC mouse model. Our present work showed that tumor growth was not entirely controlled when HNSCC model mice were administered anti-CTLA4 chemotherapeutic treatment. Moreover, it was observed that Src family kinases (SFKs) were hyper-activated and lack of anti-tumor immune responses following anti-CTLA4 chemotherapeutic treatment. We hypothesized that activation of SFKs is a mechanism of anti-CTLA4 immunotherapy resistance. We, therefore, carried out combined drug therapy using anti-CTLA4 mAbs and an SFKs' inhibitor, dasatinib. As expected, dasatinib and anti-CTLA4 synergistically inhibited tumor growth in Tgfbr1/Pten 2cKO mice. Furthermore, dasatinib and anti-CTLA4 combined to reduce the number of myeloid-derived suppressor cells and Tregs, increasing the CD8⁺ T cell-to-Tregs ratio. We also found that combining dasatinib with anti-CTLA4 therapy significantly attenuated the expression of p-STAT3^Y705 and Ki67 in tumoral environment. These results suggest that combination therapy with SFKs inhibitors may be a useful therapeutic approach to increase the efficacy of anti-CTLA4 immunotherapy in HNSCC.

Keywords: CTLA4; Dasatinib; HNSCC; Immunotherapy; MDSCs; Tregs.

Fig. 1

Therapeutic effects of chemopreventive and chemotherapeutic anti-CTLA4 treatments. a Chemopreventive therapy schematic illustration. Tgfbr1/Pten 2cKO mice were administered tamoxifen for 5 consecutive days. Anti-mice CTLA4 mAb or PBS were administered i.p. on days 12, 15, 18, 21, and 24 after tamoxifen gavage. Tumor growth was monitored for up to 5 weeks. b Tumor growth curves of anti-mice CTLA4 and PBS group. The data are represented as mean ± SD, n = 5 (***p < 0.001; *p < 0.05). c Chemotherapeutic treatment schematic illustration. Tgfbr1/Pten 2cKO mice were administered tamoxifen for 5 consecutive days. Anti-mice CTLA4 mAb or PBS were administered intraperitoneally on days 26, 33, 40, and 47 after tamoxifen gavage. Tumor growth was monitored for up to 6 weeks. d Tumor growth curves of anti-mice CTLA4 and PBS. The data are represented as mean ± SD, n = 5 (*p < 0.05)

Fig. 2

Influence of chemotherapeutic anti-CTLA4 on the tumor microenvironment. a Representative flow cytometry staining of CD11b⁺Gr-1⁺ MDSCs and CD8⁺ T cells. Also shown are the number of IFN-γ producing CD8⁺ T cells and CD4⁺FOXP3⁺ Treg in the tumor. b Percentages of CD11b⁺Gr-1⁺ MDSCs in the gated population from tumor. The data are represented as mean ± SD, n = 5 (**p < 0.01). c Percentages of number of IFN-γ producing CD8⁺ T cells in the tumor. The data are represented as mean ± SD, n = 5 (ns not statistically significant). d Ratio of CD8⁺ T cell-to-CD4⁺FOXP3⁺ Treg in the tumor. The data are represented as mean ± SD, n = 5 (ns not statistically significant). e Representative immunofluorescence image for CD4⁺ and CD8⁺ T cell in the tumor microenvironment after anti-mice CTLA4 or PBS treatment. Scale bar 50 µm. f Quantification of CD4⁺ cells in tumor from anti-mice CTLA4 or PBS treatment groups. The data are represented as mean ± SD, n = 5 (ns not statistically significant). g Quantification of CD8⁺ cells in tumor from anti-mice CTLA4 or PBS treatment groups. The data are represented as mean ± SD, n = 5 (ns not statistically significant)

Fig. 3

Difference in SRC family protein levels after chemopreventive and chemotherapeutic anti-mice CTLA4 treatments. a Western blot analysis of p-SRC^Y416 expression in the tumor after chemopreventive anti-CTLA4 treatment. GAPDH was used as the loading control. b Western blot analysis p-SRC^Y416 expression in the tumor after chemotherapeutic anti-CTLA4 treatment. GAPDH was used as the loading control. c Representative tumor immunohistochemistry images from the chemotherapeutic treatment or control groups for p-SRC^Y416 and Ki67 on the day 25 and day 49 after tamoxifen inducing. Scale bar 50 µm. d Quantification of p-SRC^Y416 staining in tumor tissues of anti-mice CTLA4 or PBS treatment groups on the day 25 and day 49 after tamoxifen inducing. The data are represented as mean ± SD, n = 5 (***p < 0.001; ns not statistically significant). e Quantification of Ki67 staining in tumor tissues of anti-mice CTLA4 or PBS treatment groups on the day 25 and day 49 after tamoxifen inducing. The data are represented as mean ± SD, n = 5 (ns not statistically significant)

Fig. 4

Combination therapeutic effect of dasatinib and anti-mice CTLA4 mAb in Tgfbr1/Pten 2cKO HNSCC mouse model. a Schema of treatment. The mice received gavage of tamoxifen for 5 consecutive days. Anti-CTLA4 mAb were administered intraperitoneally on days 26, 28, 35, 42, and 49 after tamoxifen gavage. Dasatinib was administered i.p. on days 29–33, 36–40, 43–47, and 50–54 after tamoxifen gavage. b Representative images of a tumor from Tgfbr1/Pten 2cKO HNSCC mice treated with PBS, alone anti-mice CTLA4 mAb, alone dasatinib, combining anti-CTLA4 mAb and dasatinib treatment at days 21 and days 48 after tamoxifen gavage. c Tumor volume of mice in the four different groups (n = 5 each group, The data are represented as mean ± SD, ***p < 0.001). d Plots represent the tumor volume of individual mice for the control and combination treatment groups

Fig. 5

Patterns of intratumoral immune cell populations after combination therapy with dasatinib and anti-CTLA4 mAb. a Single-cell suspensions from tumors were stained for CD11b⁺Gr-1⁺ MDSCs; CD8⁺ T cell after single or combination treatment. In addition, the numbers of IFN-γ producing CD8⁺ T cells and CD4⁺FOXP3⁺ Tregs were determined. b The percentages of CD11b⁺Gr-1⁺ MDSCs in the gated population from tumor (left); the percentages of number of IFN-γ producing CD8⁺ T cells in the tumor (middle); and the ratio of CD8⁺ T-cell-to-CD4⁺FOXP3⁺ Tregs in the tumor (right) (the data are represented as mean ± SEM, n = 5. *p < 0.05; **p < 0.01; ***p < 0.001). c Representative immunofluorescence image of CD4⁺ and CD8⁺ T cells in tumor microenvironment after PBS, alone anti-CTLA4 mAb, alone dasatinib, or combination treatment. Scale bar 50 µm. d Quantification of CD4⁺ cells in tumor from PBS, alone anti-CTLA4 mAb, alone dasatinib, or combination treatment groups. The data are represented as mean ± SD, n = 5 (**p < 0.01; ***p < 0.001). e Quantification of CD8⁺ cells in tumor from PBS, alone anti-CTLA4 mAb, alone dasatinib, or combination treatment groups. The data are represented as mean ± SD, n = 5 (*p < 0.05; ***p < 0.001)

Fig. 6

Combination dasatinib with anti-mice CTLA4 therapy effectively decreased SRC family protein levels and inhibited STAT3 signalling pathway activation in Tgfbr1/Pten 2cKO HNSCC mouse model. a Western blot analysis of SRC family proteins: SRC, p-SRC^Y416, FAK, p-FAK^Y576/577, STAT3, and p-STAT3^Y705 in the tumor of mice treated with PBS, alone anti-mice CTLA4 mAb, alone dasatinib, or combination treatment. GAPDH was used as the loading control. b Quantification of the relative protein levels for SRC, p-SRC^Y416, FAK, p-FAK^Y576/577, STAT3, and p-STAT3Y⁷⁰⁵ in each group (the data are represented as mean ± SD; ***p < 0.001). c Representative tumor immunohistochemistry images from each group for p-SRC^Y416, p-STAT3^Y705, and Ki67. Scale bar 50 µm. d Quantification of p-SRC^Y416, p-STAT3^Y705, and Ki67 staining in tumor from PBS, alone anti-CTLA4 mAb, alone dasatinib, or combination treatment groups. The data are represented as mean ± SD, n = 5 (***p < 0.001)