Targeting transforming growth factor-β2 by antisense oligodeoxynucleotide accelerates T cell-mediated tumor rejection in a humanized mouse model of triple-negative breast cancer

Abstract

Transforming growth factor-beta (TGF-β) pathway mediates suppression of antitumor immunity and is associated with poor prognosis in triple-negative breast cancer (TNBC). In this study, we generated a humanized animal model by transplanting human peripheral blood mononuclear cells into immunodeficient mice followed by inoculation of MDA-MB-231 cells and subsequently analyzed the role of TGF-β2 in the interaction between human T cells and human tumor cells. Following reconstitution of the human immune system, inhibition of TGF-β signaling by TGF-β2 antisense oligodeoxynucleotide (TASO) resulted in accelerated tumor growth inhibition. TGF-β2 inhibition also resulted in downregulation of peripheral Foxp3 + regulatory T cells (Treg), whereas no effect was seen in the expression of CD8 + cytotoxic T cells. Analysis of the TASO-treated mice serum revealed elevated levels of human IFN-γ and reduced levels of human IL-10 and TGF-β2. Moreover, TGF-β2 inhibition resulted in increased CD8 + T cell infiltration, whereas the reduced infiltration of Tregs into the tumor partly resulted from decreased expression of CCL22. Decreased intratumoral Tregs facilitated the activation of cytotoxic T cells, associated with increased granzyme B expression. These results indicate that TASO potentiated T cell-mediated antitumor immunity, and it is proposed that TGF-β2 may be a promising target in the immunotherapeutic strategy of TNBC.

Keywords: Antitumor immunity; Breast cancer; Humanized model; TGF-β2 antisense.

Fig. 1

Generation of huPBL NSG mice (Preliminary experiment). Human CD3 + T cell dominant humanized mouse model is established by engrafting human PBMCs into NSG mouse. Subpopulations of human immune cells were measured by FACS analysis at days 7 and 25 after transplantation of human PBMCs into NSG mice. a Flow cytometry plots of human CD45 + cells in total gated cells, human T cells (hCD3 + cells) and human cytotoxic T cells (hCD3 + CD8 + cells) in human CD45 + -gated population. Flow cytometry data show the percentage of b human CD45 + cells, and the percentage of c human CD3 + cells and d human CD3 + CD8 + cells of the human CD45 + cells in blood (n = 4)

Fig. 2

Effects of TGF-β2 inhibition by TASO on tumor growth in humanized mice. Tumor regression is accelerated by TASO administration in MDA-MB-231 xenografted huPBL NSG mice. a Experimental scheme for in vivo experiment. b Tumor volumes were measured by caliper every alternate day from day 4 and calculated using the formula (length) × (width)²/2. c Tumor weights and d corresponding images of tumor were measured at study termination (day 19 after tumor inoculation). The results are expressed as mean ± S.E.M. obtained from five mice. ^##p < 0.01 vs. Tumor only; *p < 0.05 and **p < 0.01 vs. PBMC + vehicle (Dunnett’s test)

Fig. 3

Effects of TASO on TGF-β2 production and TGF-β signaling in tumor. TGF-β2 synthesis and TGF-β downstream signaling in tumor are inhibited by TASO administration. Human TGF-β2 production in tumor was measured by IHC staining. a representative images and b its relative intensity were observed. TGF-β signaling was measured by western blot analysis of p-SMAD2/3 and SMAD2/3 in the tumor. c Representative band images of proteins and d the relative intensity of p-SMAD2/3 levels to SMAD 2/3 levels are presented. The results are expressed as mean ± S.E.M. **p < 0.01 vs. PBMC + vehicle (Dunnett’s test). Each image is representative of 4–5 mice per group. Bar = 100 μm

Fig. 4

Effects of TGF-β2 inhibition by TASO on reconstitution of human T cells in blood. The subpopulation of Tregs is regulated by TGF-β2 inhibition in blood. Subpopulations of human Tregs and CD8 + T cells in peripheral blood were measured by FACS analysis on days 17 and 19 after tumor inoculation, respectively. a The flow cytometry plots of human Tregs (hCD25 + Foxp3 + cells) in human CD4 + -gated population, and human cytotoxic T cells (hCD3 + CD8 + cells) in human CD45 + -gated population are presented. Flow cytometry data showing the percentage of b human Tregs (hCD25 + Foxp3 + cells) among human CD4 + cells, and c human cytotoxic T cells (hCD3 + CD8 + cells) among the human CD45 + cells in blood were measured. The results are expressed as mean ± S.E.M. obtained from five mice. **p < 0.01 vs. PBMC + vehicle (Dunnett’s test)

Fig. 5

Effects of TGF-β2 inhibition by TASO on recruitment of human T cells in spleen. Subpopulation of Tregs is regulated by TGF-β2 inhibition in spleen. a Recruitment of human T cells in spleen was measured by IHC staining against human Foxp3 and human CD8. The human Foxp3 positive cells b and human CD8 + cells c were counted, and the density of cells (no. of 0.1 mm²) for phenotype of Tregs and cytotoxic T cells was determined, respectively. The results are expressed as mean ± S.E.M. obtained from five mice. **p < 0.01 vs. PBMC + vehicle (Dunnett’s test). Bar = 100 μm

Fig. 6

Effects of TGF-β2 inhibition by TASO on changes in human cytokine levels in huPBL NSG mice. Human cytokine levels are altered by TGF-β2 inhibition in huPBL NSG mice. Mouse sera were collected and assayed for the presence of a human TGF-β2, b human IL-10, and c human IFN-γ. The results are expressed as mean ± S.E.M. obtained from five mice. ^##p < 0.01 vs. Tumor only. *p < 0.05 and **p < 0.01 vs. PBMC + vehicle (Dunnett’s test)

Fig. 7

Effects of TGF-β2 inhibition by TASO on infiltration of human T cells into tumors and their activation. Infiltration and activation of human T cells in tumors are regulated by TGF-β2 inhibition. Infiltration and activation of human T cells in the tumor were measured by IHC staining against human CCL22, human Foxp3, human CD8, and granzyme B. a Each image is representative of 4–5 mice per group. The human Foxp3 positive cells c and human CD8 + cells d in tumor were counted, and the density of cells (no. of 0.1 mm²) for phenotype of Tregs and cytotoxic T cells was determined, respectively. Relative intensity of b human CCL22 and e granzyme B were measured. ^#p < 0.05 and ^##p < 0.01 vs. Tumor only. *p < 0.05 and **p < 0.01 vs. PBMC + vehicle (Dunnett’s test). Bar = 100 μm