Abstract A52: Modulation of Hedgehog signaling alters immune infiltration in pancreatic cancer

Abstract

Abstract Background: Pancreatic ductal adenocarcinoma (PDA) has a dismal 5-year survival rate of 9%, making this disease one of the deadliest human malignancies. Primary barriers to the treatment of pancreatic cancer include extensive stromal interactions and sustained immune suppression. Aberrant Hedgehog (HH) pathway activity is a hallmark of pancreatic tumorigenesis. Tumor-derived HH ligands signal in a paracrine fashion to the surrounding stroma to influence tumor growth. Expression of HH ligands increases during PDA progression, and previous work has shown that genetic deletion of Sonic HH from the epithelium of mice with pancreatic tumors results in increased Indian HH (Ihh) expression. Methods: Ihh was deleted in tumor cells lines (IhhKO) derived from a genetically engineered mouse model of pancreatic cancer (KrasG12D;Trp;P48-Cre), using CRISPR/Cas-9 gene editing to assess the role of Ihh in the tumor microenvironment. The level of HH signaling was determined using tumor cell co-cultures with Gli1lacZ fibroblasts (derived from mice with a lacZ reporter allele knocked into the Gli1 locus), in which beta galactosidase activity serves as a readout for HH signaling. WT and IhhKO tumor cells were orthotopically transplanted into the pancreas of syngeneic C57BL/6 mice. Human pancreas samples were obtained from surgical resection of pancreatic adenocarcinoma or fine-needle biopsy procedure (FNB). Immune profiling of mouse and human pancreatic tumors was performed using Cytometry Time-of-Flight analysis, and tumor composition was analyzed by single-cell RNA sequencing. In vitro cultures with pancreatic fibroblasts treated with either WT or IhhKO tumor cell conditioned media (CM) were cultured to assess tumor crosstalk. Results: Tumor cells lacking Ihh were generated through CRISPR/Cas-9 deletion, and this was confirmed by qRT-PCR. Co-culture of IhhKO tumor cells with Gli1lacZ fibroblasts results in decreased Gli1 expression both in vitro and in vivo. Immune profiling revealed that tumors lacking Ihh have significantly fewer macrophages (CD11b+/F4/80+), resulting in decreased presence of immunosuppressive factors such as arginase 1 and PDL1. Immune profiling of human PDA revealed similar populations of immunosuppressive myeloid cells present in tumors. In vitro co-cultures demonstrated CCL2 expression was reduced in pancreatic fibroblasts cultured with IhhKO-CM, providing mechanistic insight into the in vivo phenotype observed. Further, scRNA seq analysis suggests that modulation of HH signaling in the tumor microenvironment alters chemokine and immunomodulatory signaling pathways driven by fibroblasts in the pancreatic tumor microenvironment. Significance of Impact: HH signaling in pancreatic fibroblasts contributes to the establishment of an immune-suppressive environment in pancreatic cancer. Combining methods to target HH signaling and immune checkpoint therapy has translational potential in treating pancreatic cancer patients. Citation Format: Nina Steele, Samantha Kemp, Valerie Irizarry-Negron, Veerin Sirihorachai, Ahmed Abbas, Eileen Carpenter, Christopher Halbrook, Carlos Espinoza, Costas Lyssiotis, Howard Crawford, Timothy Frankel, Filip Bednar, Ben Allen, Marina Pasca di Magliano. Modulation of Hedgehog signaling alters immune infiltration in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A52.