TGFβ Signaling in the Pancreatic Tumor Microenvironment Promotes Fibrosis and Immune Evasion to Facilitate Tumorigenesis

Abstract

In early pancreatic carcinogenesis, TGFβ acts as a tumor suppressor due to its growth-inhibitory effects in epithelial cells. However, in advanced disease, TGFβ appears to promote tumor progression. Therefore, to better understand the contributions of TGFβ signaling to pancreatic carcinogenesis, we generated mouse models of pancreatic cancer with either epithelial or systemic TGFBR deficiency. We found that epithelial suppression of TGFβ signals facilitated pancreatic tumorigenesis, whereas global loss of TGFβ signaling protected against tumor development via inhibition of tumor-associated fibrosis, stromal TGFβ1 production, and the resultant restoration of antitumor immune function. Similarly, TGFBR-deficient T cells resisted TGFβ-induced inactivation ex vivo, and adoptive transfer of TGFBR-deficient CD8(+) T cells led to enhanced infiltration and granzyme B-mediated destruction of developing tumors. These findings paralleled our observations in human patients, where TGFβ expression correlated with increased fibrosis and associated negatively with expression of granzyme B. Collectively, our findings suggest that, despite opposing the proliferation of some epithelial cells, TGFβ may promote pancreatic cancer development by affecting stromal and hematopoietic cell function. Therefore, the use of TGFBR inhibition to target components of the tumor microenvironment warrants consideration as a potential therapy for pancreatic cancer, particularly in patients who have already lost tumor-suppressive TGFβ signals in the epithelium. Cancer Res; 76(9); 2525-39. ©2016 AACR.

Conflict of interest statement

Disclosures: The authors have no conflicts to disclose.

©2016 American Association for Cancer Research.

Figures

Figure 1. Global TGFBR Deficiency Protects Against Pancreatic Tumorigenesis Despites Epithelial Deficits in Downstream TGFβ Signaling

(a) El-Kras (KRAS) mice with mutant KRASG12D expression is restricted to the pancreas acinar compartment via a rat elastase promoter were employed as a model of early pancreatic tumorigenesis. (b) KRAS mice were crossed to mice conditionally expressing a dominant negative TGFBR2 in epithelial tissues (TE) to form KTE mice. (c) KRAS mice were next crossed to mice with heterozygous deletion of Tgfbr1 (TG) to form KTG. (d,e) At one year, 100% of KRAS animals present with cystic papillary neoplasia (yellow arrows), though gland architecture is predominantly intact. The KTE cohort has noticeable increases in lesion size (yellow arrows), frequency, and severity, contrasted by KTG mice where the majority of the gland is normal (N=15). (f,g) Tissues were stained with anti-CK19, and pancreatic acini in KRAS and KTE begin to take on a more ductal morphology demonstrating acinar-to-ductal metaplasia. In KTG, a majority of animals express CK19 solely in normal ducts. (h,i) Tissues were next stained with an antibody specific to mutant RASG12D, the expression of which was restricted to the E-Cadherin positive epithelial cells in all three models. (j-k) Proliferation was assessed by IHC staining for PCNA. Proliferation was at an intermediate level in KRAS mice compared to higher levels in KTE mice, and overall lower levels in KTG animals. (l-o) Tissue sections from were next stained with anti-pSMAD2 and anti-p21, two downstream targets of TGFβ. These results confirm the deficiency of these signals in the neoplastic epithelia of KRAS and KTE, as well as in the normal epithelia of KTG. (*, P < 0.05. N=5 mice per group unless otherwise specified).

Figure 2. Conditional and Global TGFBR Deficiency Have Opposing Effects on Tumor Associated Fibrosis

(a,b) Fibrosis was assessed via trichrome staining, and KRAS mice have an intermediate phenotype with respect to KTE, where fibrosis is increased, and KTG, where fibrosis is decreased. N=4 per group. (c,d) Tissue sections were next dual stained for Collagen IA (green) and the epithelial marker E-Cadherin, indicating increased matrix deposition in KTE mice, and reduced matrix in KTG mice. (e,f) Tissue sections were next dual stained for αSMA, a marker of pancreas stellate cells. The stroma of KRAS mice had modest αSMA expression, though KTE mice had strong αSMA staining and KTG mice had very little αSMA expression, even near neoplastic lesions (*, P < 0.05. N=5 per group). (g,h) Tissue sections were next restrained for pSMAD2 and the stroma evaluated, showing high pSMAD2 staining in the stroma of KRAS and KTE mice, yet little to no staining in KTG. (i) hPSCs were incubated with recombinant TGFβ1 and lysed after 24 hours. After TGFβ1 incubation, these cells had an increase in collagen IA deposition as well as a reduction in p21, consistent with increased fibrosis and stellate cell proliferation, respectively. (j,k) PANC1 cells were co-cultured with hPSCs in transwell inserts. The co-culture was pulsed with TGFβ1. After 24 hours, these cells had a dramatic increase in downstream TGFβ-signaling, namely increased pSMAD2 and p21.

Figure 3. Pancreatic Stromal Cells Mediate TGFβ Overexpression in the TME

(a) Media from isolated PANC1 and hPSCs was compared to media from PANC1/hPSC co- cultures. In the co-culture media, the amount of TGFβ1 was significantly (P=0.0001. N=4) higher than the sum of that in isolated cell media. (b) hPSCs were treated with the TGFBR-inhibitor Galunisertib, and inhibitor efficacy was assessed by western blotting for pSMAD2. (c) hPSCs were treated with Galunisertib for 24 hours, indicating that TGFBR-inhibition reduces endogenous TGFβ1 production. (d-e) hPSCs were pretreated with Galunisertib prior to co-culture with untreated PANC1 cells. hPSC and PANC1 cells were lysed separately. In the co-culture, Galunisertib pretreated hPSCs had dramatically lower levels of endogenous TGFβ1. PANC1 cells cultured in the presence of TGFβ-insensitive stroma (Stromal-Gal) had a normal response to exogenous TGFβ1, with no change in the amount of endogenous TGFβ1 ligand. These results suggest that the stroma is responsible for the increase in TGFβ1 in the co-culture setting. (f,g) TGFβ1 expression was analyzed via IHC staining in KRAS, KTE, and KTG cohorts. KRAS and particularly KTE mice had localization of TGFβ1 to the stroma, where KTG had no detectable staining in the stroma, even near neoplastic lesions (yellow arrow). (h) T cells were localized to the TGFβ-rich stroma in KRAS and KTE, and the TGFβ-low stroma of KTG mice by dual staining for Vimentin and CD3.

Figure 4. Global TGFBR Deficiency Promotes a Cytotoxic Response Against Pancreatic Lesions

(a,b) Spleens from KRAS, KTE, and KTG mice were stained for pSMAD2, suggesting TGFβ signaling is intact in the hematopoietic cells of KRAS and KTE mice, but not in KTG (c,d) When compared to KRAS, T cell infiltration was increased near the lesions of both KTE and KTG mice. However, in KTE, these cells are largely FoxP3+, suggesting increased peripheral tolerance, consistent with a strong TGFβ presence. This was not observed in KTG mice, where nearly all CD3+ cells did not express FoxP3. (e-l) Functional cell-mediated cytotoxicity was evaluated by lesion-specific staining of CD8, Interferon gamma (IFNγ), GranzymeB, and Cleaved Caspase-3. Each of these signals was absent in neoplastic lesions of KRAS and KTE mice, yet stained very strongly in the few neoplastic and metaplastic lesions observed in KTG mice. N=4 mice per group.

Figure 5. TGFBR Deficient Cytotoxic T Cells Resist Inactivation ex Vivo

(a-b) The presence of live CD4 and CD8 cells in the mesenteric lymph nodes of KRAS and KTG mice was detected via flow cytometry, indicating an approximate 30-fold increase in the number of live CD8+ cells in KTG compared to KRAS mice. (*, P < 0.05. N=4 per group) (c) 1 million peripheral blood mononuclear cells (PBMCs) were cultured ex vivo from control and TG animals. Media was subject to GranzymeB ELISA, revealing a 3-fold increase in GranzymeB secretion by TG PBMCs consistent with increased cytotoxic activity (*, P < 0.05. N=3). (d) PBMC cultures from control and TG mice and maintained ex vivo and pulsed with recombinant TGFβ1. CD8 activity was assessed by CD8+CD69+ dual staining. TG PBMCs resisted TGFβ1-induced inactivation. Control PBMCs pre-treated with Galunisertib displayed similar resistance to TGFβ1-induced inactivation. (e)Ex vivo PBMC cultures of control mice were pulsed with recombinant TGFβ1. After 24 hours, CD4+ cells displayed an increase in CD25 and FoxP3 expression, consistent with an induced Treg phenotype. This experiment was repeated with cells from TG animals, which resisted TGFβ1-induced CD25 and FoxP3 expression. Additionally, nongenic cells pre-incubated with the TGFBR-inhibitor Galunisertib behaved like TG lymphocytes, and resulted an induced Treg phenotype. (*, P < 0.05. N=3).

Figure 6. Adoptive Transfer of TGFBR-Deficient CD8+ T Cells Promotes a Cytotoxic Response Against PanIN Lesions

(a) Ptf1a-Cre/LSL-KRAS (KC) mice were generated to provide a model of PanIN-disease. (b,c) CD8+ T cells were isolated by dyanabeads, and purity measured by flow cytometry. 2 million CD8+ cells were then transferred to two-month-old KC animals. (d) One month after adoptive transfer, KC animals with control CD8+ cells (KCAT) or TG CD8+ cells (KCAT-TG) were euthanized and subject to pathological analysis by H&E. Lesion frequency and leukocyte infiltration (yellow arrows) was quantified per high power field (*, P < 0.05). (f) T cell infiltration was measured by CD3/E-Cadherin dual staining, or staining with anti-CD8, revealing an increased cytotoxic presence in KCAT-TG. (g) Functional cell-mediated cytotoxicity was evaluated by lesion-specific staining of CD3/E-Cadherin, CD8, CK19/GranzymeB, and Cleaved Caspase-3. The increase in GranzymeB and Cleaved Caspase-3 in abnormal tissues and the surrounding stroma suggest an increase in anti-tumor cytotoxicity in KCAT-TG animals.

Figure 7. The Tumor Promoting Effects of TGFβ in the Tumor Microenvironment are Independent of Epithelial TGFβ Signaling

(a-f) Human pancreatic cancer sections were stained for TGFβ1, SMAD4, p21, GranzymeB, and Mason's Trichrome. Stains were then and independently scored by two blinded investigators from 0-3+ based on intensity. The number of patients in each scoring category is displayed via histogram. (g) Two-way ANOVA revealed a highly significant (P=0.0001) interaction between epithelial staining for SMAD4 and p21, yet none (p>0.05) between SMAD4 and GranzymeB. (h) Two-way ANOVA also revealed a highly significant (P=0.0006) inverse correlation between TGFβ1 and GranzymeB expression, and a significant (P=0.041), positive association between TGFβ1 and Trichrome staining.