Abstract A21: Stromal-derived TGFβ facilitates pancreatic cancer development via repression of T-cell mediated cytotoxicity
Authors: DeCant Brian , Principe Daniel , Wayne Elizabeth , Mascarinas Emman , Munshi Hidayatullah , Jung Barbara , Grippo Paul 
Published: October 2015

Abstract
The roles of TGFβ in tumor development are seemingly paradoxical. Despite evidence suggesting TGFβ functions as an early stage tumor suppressor, TGFβ promotes the progression of many advanced cancers. For this reason, the effects of TGFβ across non-epithelial cell compartments were evaluated in the greater context of pancreatic cancer. Murine models expressing the human mutant KRASG12D allele (EL-KRAS) were crossed to either mice with epithelial expression of a truncated TGFBR2 (MT-Tgfbr2-DNR), or those with a global deficiency of TGFBR1 (Tgfbr1+/-). Epithelial TGFβ signaling was examined via western blotting and immunoprecipitation of protein lysates from human and mouse cell lines. Co-cultures of human pancreatic epithelial and stellate cells were established to assess paracrine TGFβ signaling with western blotting and ELISA analyses. Additionally, mouse tissue was collected and stained via immunohistochemistry or immunofluorescence. To examine the effects of TGFβ on the lymphoid compartment, mouse mesenteric lymph node was collected and subjected to flow cytometry. Pancreas specific immune activity was subsequently confirmed by immunostaining. In addition, 80 human pancreatic cancer patient samples were subject to immunohistochemical staining of SMAD4, p21, and GranzymeB and analyzed by two-way ANOVA. Despite similar disruption of epithelial TGFβ signaling in both models, the EL-KRAS/Tgfbr1+/- cohort exhibited protection against Kras-induced neoplasia, contrasting advanced disease in EL-KRAS/MT-Tgfbr2-DNR mice. These results suggest TGFBR-deficiency in a non-epithelial cell compartment attenuates the loss of tumor suppressive signaling in the epithelium. Co-cultures of pancreatic epithelial and stellate cells were established, indicating stromal overexpression of TGFβ1 in the presence of cancer epithelia. This stromal-derived TGFβ reduced lesion-targeted, cytotoxic CD8+ T-cell responses mediated by GranzymeB activation of caspase-3. The interruption of this mechanism in EL-KRAS/Tgfbr1+/- restores functional cytotoxicity, overpowering the tumor permissive phenotype of TGFBR1 reduction in pancreatic epithelium. In preparation for translation to the clinic, pancreatic cancer patients were examined for biomarkers of epithelial and tumor microenvironment TGFβ signaling. Statistical analysis revealed no significant correlation between the tumor suppressive SMAD/p21 axis and GranzymeB deposition, indicating cytotoxic responses may be restored following TGFβ inhibition in patients with SMAD4 loss in the epithelia. In summary, global deficiency of TGFBR1 restores a cytotoxic T-lymphocyte response by interrupting stromal overexpression of TGFβ, thereby protecting against mutant Kras-induced neoplasia. Given this protection, patients with simultaneous SMAD4 and GranzymeB deficiencies may be optimal candidates for inhibition strategies targeting the TGFβ pathway.
Citation Format: Brian DeCant, Daniel Principe, Elizabeth Wayne, Emman Mascarinas, Hidayatullah Munshi, Barbara Jung, Paul Grippo. Stromal-derived TGFβ facilitates pancreatic cancer development via repression of T-cell mediated cytotoxicity. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A21.