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Inhibition of Hedgehog Signaling Alters Fibroblast Composition in Pancreatic Cancer a C Nina G

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Inhibition of Hedgehog Signaling Alters Fibroblast Composition in Pancreatic Cancer a C Nina G Published OnlineFirst January 25, 2021; DOI: 10.1158/1078-0432.CCR-20-3715 CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY Inhibition of Hedgehog Signaling Alters Fibroblast Composition in Pancreatic Cancer A C Nina G. Steele1, Giulia Biffi2,3,4, Samantha B. Kemp5, Yaqing Zhang6, Donovan Drouillard6, LiJyun Syu7, Yuan Hao3,8, Tobiloba E. Oni3,4, Erin Brosnan3,4, Ela Elyada3,4, Abhishek Doshi3,4, Christa Hansma1, Carlos Espinoza6, Ahmed Abbas1, Stephanie The9, Valerie Irizarry-Negron6, Christopher J. Halbrook10, Nicole E. Franks1, Megan T. Hoffman10, Kristee Brown6, Eileen S. Carpenter11, Zeribe C. Nwosu10, Craig Johnson1, Fatima Lima6, Michelle A. Anderson11, Youngkyu Park3,4, Howard C. Crawford5,10,12, Costas A. Lyssiotis5,10,12, Timothy L. Frankel6, Arvind Rao9,10,13,14, Filip Bednar6, Andrzej A. Dlugosz7,12, Jonathan B. Preall3, David A. Tuveson3,4, Benjamin L. Allen1,12, and Marina Pasca di Magliano1,5,6,12 ABSTRACT ◥ Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a deadly metry by time-of-flight, and single-cell RNA sequencing to study disease characterized by an extensive fibroinflammatory stroma, the roles of Hedgehog signaling in PDAC. which includes abundant cancer-associated fibroblast (CAF) popula- Results: We found that Hedgehog signaling is uniquely activated tions. PDAC CAFs are heterogeneous, but the nature of this hetero- in fibroblasts and differentially elevated in myofibroblastic CAFs geneity is incompletely understood. The Hedgehog pathway func- (myCAF) compared with inflammatory CAFs (iCAF). Sonic tions in PDAC in a paracrine manner, with ligands secreted by cancer Hedgehog overexpression promotes tumor growth, while Hedge- cells signaling to stromal cells in the microenvironment. Previous hog pathway inhibition with the smoothened antagonist, LDE225, reports investigating the role of Hedgehog signaling in PDAC have impairs tumor growth. Furthermore, Hedgehog pathway inhibition been contradictory, with Hedgehog signaling alternately proposed to reduces myCAF numbers and increases iCAF numbers, which promote or restrict tumor growth. In light of the newly discovered correlates with a decrease in cytotoxic T cells and an expansion in CAF heterogeneity, we investigated how Hedgehog pathway inhibi- regulatory T cells, consistent with increased immunosuppression. tion reprograms the PDAC microenvironment. Conclusions: Hedgehog pathway inhibition alters fibroblast Experimental Design: We used a combination of pharmacologic composition and immune infiltration in the pancreatic cancer inhibition, gain- and loss-of-function genetic experiments, cyto- microenvironment. Introduction 1 Department of Cell and Developmental Biology, University of Michigan, Ann Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy Arbor, Michigan. 2Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, England, United Kingdom. 3Cold Spring Harbor with a 5-year survival rate of approximately 10% (1). The PDAC Laboratory, Cold Spring Harbor, New York. 4Lustgarten Foundation Pancreatic microenvironment is characterized by an extensive stroma, comprised Cancer Research Laboratory, Cold Spring Harbor, New York. 5Molecular and of nonneoplastic cells and extracellular matrix (ECM) components, Cellular Pathology Graduate Program, University of Michigan, Ann Arbor, including cancer-associated fibroblasts (CAF). Within the stroma, 6 Michigan. Department of Surgery, University of Michigan, Ann Arbor, Michigan. CAFs secrete growth factors, immunomodulatory ligands, and ECM 7 Department of Dermatology, University of Michigan, Ann Arbor, Michigan. proteins (2–4). The Hedgehog signaling pathway is activated in PDAC 8Applied Bioinformatics Laboratories, NYU Grossman School of Medicine, New York, New York. 9Department of Computational Medicine and Bioinformatics, CAFs and has been associated with ECM deposition and pancreatic – University of Michigan, Ann Arbor, Michigan. 10Department of Molecular and tumorigenesis (5 10). In PDAC, Hedgehog signaling acts via a para- Integrative Physiology, University of Michigan, Ann Arbor, Michigan. 11Division of crine mechanism, whereby tumor-derived Hedgehog ligands activate Gastroenterology, Department of Internal Medicine, University of Michigan, Ann downstream signaling in CAFs through interactions with the canonical 12 Arbor, Michigan. Rogel Cancer Center, University of Michigan, Ann Arbor, Hedgehog receptor, patched (PTCH1), and the coreceptors, GAS1, 13 Michigan. Michigan Institute of Data Science (MIDAS), University of Michigan, CDON, and BOC (11–15). These interactions initiate a downstream Ann Arbor, Michigan. 14Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan. signaling cascade mediated by smoothened (SMO), which results in cellular responses driven by GLI proteins, the transcriptional effectors Note: Supplementary data for this article are available at Clinical Cancer of the Hedgehog pathway (16). However, reports investigating the role Research Online (http://clincancerres.aacrjournals.org/). of Hedgehog signaling in pancreatic cancer progression have been N.G. Steele and G. Biffi contributed equally to this article. contradictory. Initial studies indicated that Hedgehog pathway inhi- Corresponding Authors: Marina Pasca di Magliano, Department of Surgery, bitionintransplantationmodelsimpairsPDACgrowth(5,6,12,17,18). University of Michigan–Ann Arbor, 1500 E Medical Center Dr, Ann Arbor, MI Moreover, acute Hedgehog pathway inhibition in a genetically engi- 48019. Phone: 734-276-7104; E-mail: [email protected]; David A. Tuveson, neered mouse model (GEMM) of PDAC depleted the ECM, increased Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724. Phone: 516-367- vascular perfusion, and sensitized tumors to chemotherapy, providing 5246; E-mail: [email protected]; and Benjamin L. Allen, Phone: 734-615-7512, fi E-mail: [email protected] a modest survival advantage (19). However, these ndings failed to translate in clinical trials, which were largely unsuccessful or even Clin Cancer Res 2021;27:2023–37 detrimental to patient health, leading to early termination (20, 21). doi: 10.1158/1078-0432.CCR-20-3715 Subsequently, genetic inactivation of the ligand Sonic Hedgehog Ó2021 American Association for Cancer Research. (SHH) in the context of an oncogenic Kras-driven mouse model of AACRJournals.org | 2023 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2021 American Association for Cancer Research. Published OnlineFirst January 25, 2021; DOI: 10.1158/1078-0432.CCR-20-3715 Steele et al. Animal experiments Translational Relevance Orthotopic transplantation models A better understanding of the roles of signaling pathways C57BL/6J (BL/6J) mice (stock number 000664) were purchased differentially activated in distinct fibroblast populations will shed from The Jackson Laboratory. Injections of organoid cultures for the light on the complexity of fibroblast heterogeneity in pancreatic generation of orthotopically grafted organoid (OGO) tumors were cancer. Here, we show that Hedgehog pathway inhibition alters conducted as described previously (29). Typically, 1–2.5 Â 105 single fibroblast composition toward a more inflammatory microenvi- cells prepared from organoid cultures were resuspended as a 45 mL ronment, opening the possibility for future combination therapies. suspension of 50% Matrigel in PBS and injected into the pancreas. Tumors were imaged using the Vevo 3100 Ultrasound at two or three different orientations with respect to the transducer. Tumor volumes were measured at two angles, if possible, using the Vevo LAB Software pancreatic cancer led to cachexia and to poorly differentiated and program (version 2.2.0). To establish the orthotopic pancreatic cancer highly vascularized tumors, findings that were at least in part reca- models with the 7940b PDAC cell line (31), 5 Â 104 7940b cells (BL/6J þ pitulated by prolonged pharmacologic Hedgehog pathway inhibi- strain) were injected into the pancreas of BL/6J or Gli1lacZ/ hetero- tion (22, 23). These disparate outcomes, with Hedgehog signaling zygous mice derived from crossing Gli1lacZ/lacZ animals maintained on seemingly both promoting and restricting pancreatic cancer progres- a mixed genetic background (129S6/SvEvTac; C57BL/6J; Swiss Web- sion, indicate potentially pleiotropic roles for this pathway that have ster) with BL/6J animals (32). For Hedgehog pathway inhibition, mice yet to be fully elucidated. were subjected to once daily oral gavage for 12 days, starting 6 days The healthy pancreas is home to distinct fibroblast popula- after surgical implantation of tumor cells, with either 20 mg/kg of tions (24, 25), which have differing potential to expand during LDE225 (Sonidegib, ChemieTek, #NVP-LDE225) resuspended in carcinogenesis (26). We and others recently demonstrated that PDAC PEG400/5% dextrose solution (LDE treated) or the same volume of CAFs are also a heterogeneous population comprised of myofibro- PEG400/5% dextrose solution alone (vehicle treated). blastic CAFs (myCAF), inflammatory CAFs (iCAF), and antigen- presenting CAFs (apCAF; refs. 24, 25, 27–30). Given the different GEMMs þ functional roles proposed for these CAF subtypes, changes in the ratio BL/6J background (>20 backcrosses) KPC (KrasLSL-G12D/ ; þ of these populations may lead to distinct outcomes in PDAC pro- Trp53LSL-R172H/ ; Pdx1-Cre) mice used for the LDE225 study were gression. Therefore, we investigated whether Hedgehog modulation described previously (33). KPC mice were monitored by
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