Stromal TGFβR2 signaling: a gateway to progression for pancreatic cancer.

The function of transforming growth factor β (TGFβ) in the progression of pancreatic ductal adenocarcinoma (PDA) is complex and therapeutic targeting of this pathway is challenging. We showed that antibody-mediated inhibition of stromal Tgfβr2 prevented or reversed epithelial plasticity resulting in a potent reduction of metastasis in xenograft models of PDA.

Pancreatic cancer is the fourth leading cause of cancer-related mortality in the United States, and has proved to be a formidable challenge with regard to treatment. An elevated level of transforming growth factor β (TGFβ) is a negative prognostic indicator for patients diagnosed with pancreatic ductal adenocarcinoma (PDA) and over 50% of human PDAs have mutations in the TGFβ pathway. This makes the TGFβ pathway an attractive therapeutic target. However, the function of TGFβ in the development and progression of PDA is complex and severely dysregulated in advanced tumors. As in other epithelial tumors, TGFβ functions as a tumor suppressor early in the development of PDA, but switches to a tumor promoter function late in the disease process. This switch, together with findings that direct neutralization of Tgfβ accelerates tumor growth in some mouse models of PDA, makes targeting the TGFβ pathway challenging. In this study, we found that selected neutralization of murine Tgfβr2 with a monoclonal antibody (2G8) led to a differentiated epithelial tumor cell phenotype and potent anti-metastatic effects. These effects were seen in multiple mouse models of PDA including 4 xenograft models and syngeneic and genetic (LSL-Kras ; Cdkn2a ; p48-Cre) models. In the xenograft setting in particular, these effects can be attributed directly to inhibition of stromal Tgfβr2 because 2G8 is a mouse-specific monoclonal antibody.

In each PDA model, Tgfβr2 neutralization significantly reduced metastasis and cell proliferation, while increasing apoptosis in the primary tumor. Interestingly, the effect of 2G8 on primary tumor size was not predictive of the effect on metastasis. In vitro data corroborated these findings, as 2G8 had no effect on tumor cell viability. However, 2G8 reduced a stimulatory effect of conditioned media from murine stromal cells on tumor cell migration and anchorage-independent growth. Together, the in vivo metastasis data and in vitro characterization studies strongly implicate stromal Tgfβr2 as a critical driver of PDA dissemination. These results are consistent with the characterization of primary tumor tissue in each model after treatment with 2G8. 2G8 consistently reduced the level of activated fibroblasts, collagen deposition, and microvessel density associated with primary PDA. These observations are concordant with Tgfβr2 as a regulator of ECM deposition and the fibroblast phenotype in the PDA microenvironment. Arguably though, the most interesting finding was the effect on tumor cell epithelial to mesenchymal transition (EMT); tumors treated with 2G8 displayed a more epithelial or differentiated phenotype than tumors exposed to control or gemcitabine alone. This is demonstrated convincingly in the genetic and syngeneic models of PDA (). In the genetic model, 2G8-treated mice had significantly more pancreatic intraepithelial neoplasia (PanIN) lesions than those treated with saline control or gemcitabine. Additionally, in the syngeneic model using Pan02 cells, a highly mesenchymal murine pancreatic tumor cell line, 2G8 induced prominent expression of epithelial cadherin (E-cadherin) and epithelial membrane expression of β-catenin together with decreased vimentin expression, whereas Pan02 tumors treated with control or gemcitabine showed a less differentiated phenotype. β-catenin is expressed on the membrane of epithelial cells and translocates into the nucleus during the process of EMT, and vimentin is a marker of mesenchymal cells and/or cells undergoing EMT. These dramatic phenotypic effects were dependent on the presence of stromal cells because treatment of PDA cells in vitro with 2G8 could not drive epithelial differentiation. TGFβ is a pleiotrophic cytokine that also promotes immune suppression. In this study, 2G8 reversed the immune suppressive effects of Tgfβ as demonstrated by an increase in the ratio of pro-inflammatory M1 to anti-inflammatory M2 macrophages (M1:M2) and natural killer (NK) cells, with a concomitant decrease in myeloid-derived suppressor cells (MDSCs) and T regulatory cells. Although we did not specifically evaluate effects on T- and B-cell immunity in this study, our findings were concordant in immunocompromised (xenograft) and immunocompetent models, demonstrating that blockade of stromal Tgfβr2 promotes a proinflammatory phenotype. However, the contribution of these changes to the promotion of a differentiated tumor cell phenotype and the reduction in metastasis remains to be elucidated. It is plausible that 2G8 induction of NK cell activity is critical for the antimetastatic activity observed given the results of Zhong et al., who observed that 2G8 induced an increase in NK cell infiltration and killing activity in syngeneic breast tumors.

Figure 1.

Transforming growth factor β receptor 2 (Tgfβr2) blockade promotes an epithelial phenotype in murine tumors in vivo. A-E, LSL-Kras; Cdkn2a; p48-Cre (KIC) mice establish tumors and precursor pancreatic intraepithelial neoplasia (PanIN) lesions by 4 weeks of age. At this time point the mice were randomized to receive saline (Control), gemcitabine (Gem), 2G8, or 2G8 + Gem for 4 weeks. (A) Analysis of tumor architecture by hematoxylin and eosin (H&E) staining (scale bar, 100 μm). 2G8-treated tumors had significantly more PanIN and epithelial lesions than mice treated with Gem or Control. (B) This was confirmed with Periodic Acid-Schiff (PAS)-Alcian Blue staining, which marks mucin-secreting PanIN lesions but not invasive lesions (PAS-Alcian Blue, purple; scale bar, 100 μm). Additionally, 2G8 tumors had significantly increased expression of epithelial cadherin (E-cadherin or ECAD) (C) and decreased expression of vimentin (D) by immunohistochemistry. (E) Representative images of these tumors demonstrate the predominant epithelial phenotype of 2G8-treated tumors (ECAD, red; vimentin, green; scale bar, 50 μm). (F, G) 2G8 induces a similar epithelial phenotype in murine pancreatic Pan02 tumors. Immunohistochemical analysis of β-catenin (F) and ECAD (G) in Pan02 tumors treated with control, Gem, 2G8, or 2G8+Gem. Bar graphs represent mean+/−SEM. **, P < 0.01; #, P < 0.0001 vs. control; ^^, P < 0.001; ^^^, P < 0.0001 vs. Gem. Reproduced with permission from Ostapoff et al.

Overall, this study further defines the importance of stromal TGFβ signaling in the development and progression of PDA. Pharmacologic blockade of Tgfβr2 resulted in a proinflammatory immune cell phenotype, a decrease in mature/activated fibroblasts, and a decrease in collagen deposition. These microenvironmental changes occurred in concert with tumor cell epithelial differentiation and a reduction in metastasis, leading us to conclude that Tgfβ signaling within stromal cells has a direct influence on pancreatic tumor cell phenotype and pancreatic cancer progression. Whether this impact is secondary to a distinct soluble factor released into the tumor microenvironment, or to effects on other cells in the microenvironment such as pancreatic stellate cells, mesenchymal stem cells, and endothelial cells, remains to be seen. We suspect that the complexity of TGFβ biology in cancer is intimately tied to the interplay between stromal cells and the tumor cells that recruit them. We propose that signaling through TGFβR2 on stromal cells is a requisite pathway for a subset of tumors that stimulates the release of prometastatic soluble factors from stromal cells. Identifying tumors that rely on this pathway and the factors that are produced by stromal cells in response to activation of TGFβR2 are remaining challenges.