Transforming Growth Factor β Signaling in the Pancreatic Tumor Microenvironment
Creators: name: Principe, Daniel R
Publisher: name: University of Illinois at Chicago
Year: 2024

description: Despite considerable progress for several difficult-to-treat cancer histologies, pancreatic ductal adenocarcinoma (PDAC) remains remarkably lethal with a 5-year survival rate of approximately 10%. The universally poor clinical outcomes associated with PDAC can be attributed primarily to the late stage of presentation, as well as widespread resistance to conventional therapy. Additionally, PDAC tumors are largely non-immunogenic, and most patients have failed to demonstrate therapeutic response to immunotherapy. To this end, Transforming Growth Factor β (TGFβ) is of particular importance to the pathobiology of PDAC, though the underlying mechanisms through which TGFβ contributes to pancreatic tumorigenesis are poorly understood and often contradictory. 
We have previously demonstrated that loss of TGFβ signaling in epithelial cells is associated with highly aggressive disease forms. However, selective blockade of TGFβ signals in T-cells leads to immune-mediated regression of pancreatic neoplasms (Principe et al., 2016b). Though interesting, it is important to note that pharmacologic inhibition of the TGFβ pathway has shown little therapeutic efficacy in clinical trials. Similarly, systemic blockade of TGFβ signals does not lead to anti-tumor immune responses in vivo. In this work, we first describe the current treatment paradigm for PDAC in the clinic followed by comprehensive discussions of immunotherapy for PDAC and the known roles of TGFβ signaling in PDAC pathobiology. We then explore the immunologic consequences of TGFβ signal inhibition in transgenic 
 We next describe the immunologic consequences of systemic TGFβ inhibition in transgenic models of PDAC, and demonstrate that administration of TGFβ-inhibitors led to the accumulation of Programmed Death Ligand 1 (PD-L1) on the tumor surface. PD-L1 is a co-inhibitory molecule that is often upregulated in the pancreatic tumor microenvironment. Expression of PD-L1 on the epithelial cell surface is critical in the evasion of T-cell mediated cytotoxicity, and its inhibition is being explored in the management of PDAC (Dong et al., 2002; Iwai et al., 2002). Using genetic models of advanced PDAC, we then determined that concomitant inhibition of both TGFβ and PD-L1 receptors led to a substantial reduction in the neoplastic phenotype, improving survival and reducing disease-associated morbidity in vivo.
 In the subsequent section, we explore the immune modifying capacity of the standard of care chemotherapy medication Gemcitabine. Using a combination of mouse models, primary cell line-derived xenografts, as well as tumor cell lines, we first evaluated chemotherapy-induced alterations in the tumor secretome and immune surface proteins by high throughput proteomic arrays. In addition to enhancing antigen presentation and immune checkpoint expression, Gemcitabine consistently increased the synthesis of CCL/CXCL chemokines and TGFβ-associated signals. These secreted factors altered the composition of the tumor stroma, conferring Gemcitabine-resistance to cancer-associated fibroblasts in vitro and further enhancing TGFβ1 biosynthesis. We therefore combined Gemcitabine with anti-PD-1 in transgenic models of murine PDAC, which failed to alter disease course unless mice also underwent genetic or pharmacologic ablation of TGFβ signaling. In the setting of TGFβ signal deficiency, Gemcitabine and anti-PD-1 led to a robust CD8+ T-cell response and decrease in tumor burden, markedly enhancing overall survival. These results suggest that Gemcitabine may prime PDAC tumors for immune checkpoint inhibition by enhancing antigen presentation, but requires the inhibition of an immunosuppressive cytokine barrier. Given the current lack of third line treatment options, this approach warrants consideration in the clinical management of Gemcitabine-refractory PDAC. 
 Finally, we explore the immune consequences of mutations to TGFβ target SMAD4 in PDAC. Using a combination of genomic databases and PDAC specimens, we found that tumors with loss of SMAD4 have a comparatively poor T-cell infiltrate. SMAD4 loss was also associated with a reduction in several chemokines with known roles in T-cell recruitment, which was recapitulated using knockdown of SMAD4 in PDAC cell lines. While exogenous TGFβ modestly reduced PD-L1 expression in SMAD4-intact cell lines, SMAD4 and PD-L1 positively correlated in human PDAC samples. PD-L1 status was closely related to tumor-infiltrating lymphocytes, particularly IFNγ-producing T-cells, which were more abundant in SMAD4-expressing tumors. Low concentrations of IFNγ upregulated PD-L1 in tumor cells in vitro, even when administered alongside high concentrations of TGFβ. Hence, while SMAD4 may have a modest inhibitory effect on PD-L1 in tumor cells, SMAD4 indirectly promotes PD-L1 expression in the pancreatic tumor microenvironment by enhancing T-cell infiltration and IFNγ biosynthesis. These data suggest that pancreatic cancers with loss of SMAD4 represent a poorly immunogenic disease subtype, and SMAD4 status warrants further exploration as a predictive biomarker for cancer immunotherapy, including the regimens described above.