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Pancreatic ductal adenocarcinoma (PDA) is the third most common cause of cancer death in the US with a 11% 5-year survival rate. A key contributor of this dismal prognosis is early and frequent metastatic spread with limited treatment options for patients. Metastasis in PDA is facilitated by a robust fibroinflammatory response in the primary tumor site, a large portion of which is composed of infiltrating immune cells, and survival during transit to establish in the secondary site. In the primary tumor reactive stroma, we found increased eosinophils in both human and mouse PDA compared to the normal pancreas. Eosinophils are an innate immune cell best known for their ability to control parasite responses and have been implicated in either pro- or anti-cancer roles in various solid tumors. We used the ΔdblGata mice, which lack eosinophils, to study the role of eosinophils during metastatic spread using a resectable model of PDA metastasis. This model of metastasis was developed to facilitate consistent seeding of lung metastasis in approximately 50 percent of mice from a transient subcutaneous tumor which undergoes subsequent surgical removal and harvest to evaluate metastasis at 4 weeks. We found that mice lacking eosinophils have increased metastasis to the lung in our resectable model of PDA metastasis but no difference in establishment of IV injected PDA tumor cells, suggesting eosinophil control at the primary tumor site. Single cell sequencing of the subcutaneous tumors finds differences in the angiogenic subtypes, tip and stalk cells, in mice lacking eosinophils. Sequencing of eosinophil populations in humans and mice find an increase in angiogenic signaling suggesting a regulatory role in tumor cell access to the circulation. We conclude that eosinophils in PDA inhibit the metastatic process through control of angiogenic signaling. Citation Format: Megan T. Hoffman, Patrick J. Lenehan, Jennifer Wang, Lestat Ali, Li Qiang, Amiko Uchida, Sara Vayrynen, Andressa Dias Costa, Michael Dougan, Max Heckler, Jonathan Nowak, Brian W. Wolpin, Stephanie K. Dougan. Eosinophils alter metastatic spread in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B018.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by an extensive fibroinflammatory stroma, which includes abundant cancer-associated fibroblast (CAF) populations. PDAC CAFs are heterogeneous, but the nature of this heterogeneity is incompletely understood. The Hedgehog pathway functions in PDAC in a paracrine manner, with ligands secreted by cancer cells signaling to stromal cells in the microenvironment. Previous reports investigating the role of Hedgehog signaling in PDAC have been contradictory, with Hedgehog signaling alternately proposed to promote or restrict tumor growth. In light of the newly discovered CAF heterogeneity, we investigated how Hedgehog pathway inhibition reprograms the PDAC microenvironment. Experimental Design: We used a combination of pharmacologic inhibition, gain- and loss-of-function genetic experiments, cytometry by time-of-flight, and single-cell RNA sequencing to study the roles of Hedgehog signaling in PDAC. Results: We found that Hedgehog signaling is uniquely activated in fibroblasts and differentially elevated in myofibroblastic CAFs (myCAF) compared with inflammatory CAFs (iCAF). Sonic Hedgehog overexpression promotes tumor growth, while Hedgehog pathway inhibition with the smoothened antagonist, LDE225, impairs tumor growth. Furthermore, Hedgehog pathway inhibition reduces myCAF numbers and increases iCAF numbers, which correlates with a decrease in cytotoxic T cells and an expansion in regulatory T cells, consistent with increased immunosuppression. Conclusions: Hedgehog pathway inhibition alters fibroblast composition and immune infiltration in the pancreatic cancer microenvironment.

Pancreatic ductal adenocarcinoma (PDA) is unique as it is marked by a dramatic increase in the collagen deposition tumor microenvironment. This microenvironment in pancreatic tumors is composed of immune cells and reactive fibroblasts, with the tumor cells themselves comprising only a small proportion of the overall mass. Nearby normal epithelial tissue is also reactive, undergoing a transdifferentiation event known as acinar-to-ductal metaplasia (ADM), where normal acinar cells are replaced by metaplastic duct-like structures. Collectively neoplastic and metaplastic epithelium orchestrate the immune and fibrotic response, and understanding how the pancreatic epithelia communicate to the microenvironment may lead to more effective clinical treatments. Metaplastic tuft cells (MTCs) are a specialized subset of the epithelium that has the potential to drive tumor progression through communication with the microenvironment and modulate PDA progression. Also known as solitary chemosensory cells, tuft cells were first discovered in rodent luminal surfaces more than 60 years ago. They are characterized by the “tuft” of microvilli reaching into the lumen and, only recently, studies have started to determine the role of normal tuft cells in different organs. These studies determined that tuft cells have different roles depending on the organ in which they reside. Studies have shown that tuft cells are not present in a normal pancreas. MTCs are only present in the pancreas in PanINs during PDA progression in both humans and mice. Furthermore, the population of MTCs in the pancreas disappears as PDA progresses into invasive carcinoma. MTCs also express other markers outside of the taste sensing proteins, including POU2f3/SKN-1alpha, and DCLK1. We know little about the role of MTCs in the pancreas, but prior studies have suggested their role as a progenitor cell during PDA. However, these studies do not exclusively mark MTCs during their genesis in a progressive model of PDA due to a lack of mouse model as well as the complexity of culturing them ex vivo. We have generated a unique mouse model to drive lineage tracing of MTCs during PDA, and I have preliminary data to suggest that tuft cells can transdifferentiate into neuroendocrine-like cells during PDA progression. Neuroendocrine (NE) cells are found in aggressive cancers such as prostate and small cell lung cancer. They are characterized by markers such as Synaptophysin (SYP), as well as an upregulation of MYC which has been found to be more aggressive in different cancers. Rosalie Sears, at the Oregon Health and Science University, a specialist of MYC function in PDA, has shown that NE cells in the pancreas not only originate from acinar cells, where MTCs originate but whose presence is associated with poor survival and resistance to treatment in PDA. She has also shown that MYC plays a key role in the regulation of NE cells in PDA. Through my dissertation research, I will investigate tuft to NE-like transdifferentiation and its role in PDA progression. Citation Format: Daniel J. Salas-Escabillas, Megan Hoffman, Howard C. Crawford. Understanding the lineage of metaplastic tuft cells in the progression of PDA [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-059.

Pancreatic ductal adenocarcinoma (PDA) is the third most common cause of cancer death in the US with a 9% 5-year survival rate. PDA is characterized by a robust stromal reaction, a large portion of which is composed of infiltrating immune cells and supported by fibroblasts. This fibroinflammatory response promotes both the initiation and progression of PDA. Key to this progression is the crosstalk between the tumor epithelium and immune compartment. Metaplastic tuft cells (MTCs) are a subset of the neoplastic epithelium derived by the transdifferentiation of acinar cells. Tuft cells have been characterized to have a “sense-and-respond” role in luminal surfaces throughout the body, using the taste chemosensory system to promote an inflammatory response in infected and damaged tissues. Among the pancreatic epithelium, MTCs uniquely express components of the gustation sensory system including taste receptors, TRPM5 and αGustducin, as well as an array of inflammatory cytokines, suggesting they are capable of promoting an immune response to extracellular challenges. To explore this hypothesis, we ablated αGustducin, a key protein in the chemosensory cascade, in a model of pancreatitis-associated tumorigenesis. We found that neoplasia in the GNAT3-/-; KrasG12D; PTF1aCreERT mice initiates and progresses more rapidly, a difference associated with alteration in immune cell subtypes. To further quantitate changes in the immune response, we performed mass cytometry (CyTOF), as a way to explore changes in cell number and type, and single-cell sequencing, to more deeply probe the unique gene signatures of cells within the tumor stroma. These large-scale analyses suggest substantial changes in the number of T-cell and myeloid cell populations, as well as alterations of fibroblasts and myeloid cell polarity. We conclude that MTCs use chemosensory signaling to promote the formation of a tumor-suppressive microenvironment. Citation Format: Megan T. Hoffman, Stephanie The, Daniel J. Salas-Escabillas, Kathleen E. DelGiorno, Filip Bednar, Robert F. Margolskee, Arvind Rao, Howard C. Crawford. The role of metaplastic tuft cell chemosensory signaling 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 A19.

Pancreatic ductal adenocarcinoma (PDA) is the second most common cause of cancer death in the US with a 10% 5-year survival rate and, as such, is one of the most common deadly cancers. PDA is characterized by a robust stromal reaction, a large portion of which is composed of infiltrating immune cells. This inflammatory response has been previously shown to promote both the initiation and progression of PDA. Current research has been to understand the interaction between tumor cells and infiltrating immune cells and the contribution to cancer progression. In this work we focus on a specific cell population present in the epithelium during pancreatic tumorigenesis called a tuft cell. Tuft cells have been found in luminal surfaces throughout the body, most notably characterized in the intestine where these cells have been shown to directly regulate the immune response to parasitic infection, but have also been found in the nasal, respiratory and bladder epithelium. These solitary chemosensory cells, as they are also known, use a common chemosensory pathway, previously characterized in type-II taste cells, in order to interact with the luminal environment. Interestingly, tuft cells in the metaplastic pancreas uniquely express TRPM5 and alpha-gustducin, both components of the chemosensory cascade, suggesting these cells have the potential to mediate a response to extracellular signals. Pancreatic tuft cells also make an array of cytokines and immunomodulatory factors that can directly alter the immune response in the tumor microenvironment. These data leads us to hypothesize that tuft cells utilize the chemosensory response pathway to remodel the tumor microenvironment in PDA. In order to answer this question, we ablated a key protein in the chemosensory cascade, alpha-gustducin, and found that this loss increases aggressiveness in a model of pancreatitis-associated tumorigenesis. We are currently exploring differences in the tumor microenvironment that may explain these results. Citation Format: Megan T. Hoffman, Howard Crawford. Tuft cell chemosensory signaling during pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1004.

The tuft cell is a chemosensory cell type that can interpret signals from luminal surfaces throughout the body in order to activate downstream effectors. One clear effector pathway of the tuft cell promotes a type-two immune response in the intestine after parasitic infection, suggesting that this cell population has the ability to communicate with the immune system. Furthermore, tuft cells have also been shown to activate sensory nerve fibers to lead to the production of a neuroinflammatory signal in the nasal epithelium. Therefore, tuft cells can promote effector inflammatory responses through direct immune cell signaling and through sensory nerve fibers. Pancreatic ductal adenocarcinoma (PDA) is characterized by a robust stromal reaction, a large component of which is composed of an immune response. It has been previously studied that this inflammatory response can function to promote both the initiation and progression of PDA. One mechanism by which inflammatory cells can be recruited to the pancreas is through neuroinflammatory pathways. Work with collaborators has shown that pancreatic sensory neurons are required for the progression of PDA, as ablation of this cell type significantly prolongs survival. Interestingly, tuft cells in the metaplastic pancreas have been shown to appear during transdifferentiation and progression to PDA. These tuft cells also possess key signaling elements that have the potential to mediate the response to extracellular signals, such as α-gustducin and TRPM5. Metaplastic tuft cells also produce the proteins necessary to make or release prostaglandins, acetylcholine and ATP, all of which can stimulate either a direct immunomodulatory signal or activate sensory nerve fibers. This leads us to hypothesize that tuft cells are a key mediator of signals to the stromal immune compartment and to sensory neurons in order to promote a robust immune response. In order to answer this question we are using chemosensory knockout animals to ablate critical signaling components of the chemosensory cascade to study the progression of PDA. Citation Format: Megan T. Hoffman, Shan Gao, Howard Crawford.{Authors}. Tuft cell signaling can induce pancreatic inflammation and promote pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B69.