Abstract
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 PDACin 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.
| Original language | American English |
|---|---|
| Pages (from-to) | 2023-2037 |
| Number of pages | 15 |
| Journal | Clinical Cancer Research |
| Volume | 27 |
| Issue number | 7 |
| DOIs | |
| State | Published - Apr 2021 |
| Externally published | Yes |
Bibliographical note
Funding Information:The results shown here are, in part, based on data generated by The Cancer Genome Atlas Research Network: https://www.cancer.gov/tcga. This project was supported by NIH/NCI grants R01CA151588, R01CA198074, and U01CA224145, and the American Cancer Society (to M. Pasca diMagliano). Thisworkwas supported by the University of Michigan Cancer Center support grant (P30CA046592), including an Administrative Supplement (to H.C. Crawford and M. Pasca diMagliano). This workwas supported by the NIH (R01 DC014428, R01 CA198074, and R01 118751 to B.L. Allen). Research reported in this article was supported by the University of Michigan Cancer Center Support grant (P30 CA046592) by the use of the following Cancer Center Shared Resource: Cell and Tissue Imaging. F. Bednar was funded by the Association of Academic Surgery Joel Roslyn Award. T.L. Frankel was funded by K08CA201581. Research reported in this article was supported by the NCI award number P30CA046592 by the use of the following Rogel Cancer Center Shared Resource: Transgenic Animal Models (to A.A. Dlugosz). This project was supported by NIH/NCI grants R01 CA118875 (to L. Syu), and P01 DK062041 and 5P30 CA046592 (to A.A. Dlugosz). S.B. Kemp was supported by T32 GM113900. N.G. Steele was supported by T32 CA009676 and is a recipient of the American Cancer Society Postdoctoral award PF-19-096-01 and the Michigan Institute for Clinical and Healthy Research Postdoctoral Translational Scholar Program fellowship award. E.S. Carpenter was supported by the American College of Gastroenterology Clinical Research award and by T32 DK094775. A. Rao and S. The were supported by institutional startup funds from the University of Michigan, a gift from Agilent Technologies, NCI grant R37 CA214955, and a Research Scholar grant from the American Cancer Society (RSG-16-005-01). C.J. Halbrook was supported by K99 CA241357 and P30 DK034933 NIH awards. The Tuveson laboratory was supported by the NIH Cancer Center Support grant P30 CA045508 and the Lustgarten Foundation, where D.A. Tuveson is a distinguished scholar and Director of the Lustgarten Foundation-designated Laboratory of Pancreatic Cancer Research. D.A. Tuveson was also supported by the Thompson Foundation, the Cold Spring Harbor Laboratory and Northwell Health Affiliation, the Northwell Health Tissue Donation Program, the Cold Spring Harbor Laboratory Association, and the NIH (NIH P30 CA45508, U01 CA210240, R01 CA229699, U01 CA224013, R01 CA188134, and R01 CA190092). This work was supported by a gift from the Simons Foundation (552716 to D.A. Tuveson). G. Biffi was a fellow of the Human Frontiers Science Program (LT000195/2015-L) and EMBO (ALTF 1203-2014) and was supported by a Cancer Research UK core funding (A27463) and by the Pancreatic Cancer and CMB Programmes of the Cancer Research UK Cambridge Centre. E. Elyada was a fellow of the Human Frontiers Science Program (LT000403/2014-L). J.B. Preall was supported by the Cold Spring Harbor Laboratory and Northwell Health Affiliation. Y. Park was supported by R50 CA211506. The authors would like to thank Tricia Tamsen and Judy Opp from the University of Michigan Advanced Genomics Core, and the Tissue Procurement Center at the University of Michigan. We would also like to thank Matthew Cochran and Terry Wightman at the University at the Rochester Medical Center Flow Cytometry Shared Resource Laboratory. We would like to thank Dr. Christ Wright for sharing the iKC (Ptf1a-CreERT; KrasLSL-G12D/+)mouse model. This work was performed with assistance from the University of Michigan Shared Resources: Biostatistics, Analytics & Bioinformatics; Flow Cytometry; Transgenic Animal Models; Tissue and Molecular Pathology; Structure & Drug Screening; Cell & Tissue Imaging; Experimental Irradiation; Preclinical Imaging & Computational Analysis; Health Communications; Immune Monitoring; and Pharmacokinetics. This work was performed with assistance from the Cold Spring Harbor Laboratory shared resources, which are supported by the NIH Cancer Center Support Grant P30 CA045508: Bioinformatics, Flow Cytometry, Animal, and Cell and Tissue Imaging Shared Resources.
Funding Information:
The results shown here are, in part, based on data generated by The Cancer Genome Atlas Research Network: https://www.cancer.gov/tcga. This project was supported by NIH/NCI grants R01CA151588, R01CA198074, and U01CA224145, and the American Cancer Society (to M. Pasca di Magliano). This work was supported by the University of Michigan Cancer Center support grant (P30CA046592), including an Administrative Supplement (to H.C. Crawford and M. Pasca di Magliano). This work was supported by the NIH (R01 DC014428, R01 CA198074, and R01 118751 to B.L. Allen). Research reported in this article was supported by the University of Michigan Cancer Center Support grant (P30 CA046592) by the use of the following Cancer Center Shared Resource: Cell and Tissue Imaging. F. Bednar was funded by the Association of Academic Surgery Joel Roslyn Award. T.L. Frankel was funded by K08CA201581. Research reported in this article was supported by the NCI award number P30CA046592 by the use of the following Rogel Cancer Center Shared Resource: Transgenic Animal Models (to A.A. Dlugosz). This project was supported by NIH/NCI grants R01 CA118875 (to L. Syu), and P01 DK062041 and 5P30 CA046592 (to A.A. Dlugosz). S.B. Kemp was supported by T32 GM113900. N.G. Steele was supported by T32 CA009676 and is a recipient of the American Cancer Society Postdoctoral award PF-19-096-01 and the Michigan Institute for Clinical and Healthy Research Postdoctoral Translational Scholar Program fellowship award. E.S. Carpenter was supported by the American College of Gastroenterology Clinical Research award and by T32 DK094775. A. Rao and S. The were supported by institutional startup funds from the University of Michigan, a gift from Agilent Technologies, NCI grant R37 CA214955, and a Research Scholar grant from the American Cancer Society (RSG-16-005-01). C.J. Halbrook was supported by K99 CA241357 and P30 DK034933 NIH awards. The Tuveson laboratory was supported by the NIH Cancer Center Support grant P30 CA045508 and the Lustgarten Foundation, where D.A. Tuveson is a distinguished scholar and Director of the Lustgarten Foundation–designated Laboratory of Pancreatic Cancer Research. D.A. Tuveson was also supported by the Thompson Foundation, the Cold Spring Harbor Laboratory and Northwell Health Affiliation, the Northwell Health Tissue Donation Program, the Cold Spring Harbor Laboratory Association, and the NIH (NIH P30 CA45508, U01 CA210240, R01 CA229699, U01 CA224013, R01 CA188134, and R01 CA190092). This work was supported by a gift from the Simons Foundation (552716 to D.A. Tuveson). G. Biffi was a fellow of the Human Frontiers Science Program (LT000195/2015-L) and EMBO (ALTF 1203-2014) and was supported by a Cancer Research UK core funding (A27463) and by the Pancreatic Cancer and CMB Programmes of the Cancer Research UK Cambridge Centre. E. Elyada was a fellow of the Human Frontiers Science Program (LT000403/2014-L). J.B. Preall was supported by the Cold Spring Harbor Laboratory and Northwell Health Affiliation. Y. Park was supported by R50 CA211506. The authors would like to thank Tricia Tamsen and Judy Opp from the University of Michigan Advanced Genomics Core, and the Tissue Procurement Center at the University of Michigan. We would also like to thank Matthew Cochran and Terry Wightman at the University at the Rochester Medical Center Flow Cytometry Shared Resource Laboratory. We would like to thank Dr. Christ Wright for sharing the iKC (Ptf1a-CreERT; KrasLSL-G12D/+) mouse model. This work was performed with assistance from the University of Michigan Shared Resources: Biostatistics, Analytics & Bioinformatics; Flow Cytometry; Transgenic Animal Models; Tissue and Molecular Pathology; Structure & Drug Screening; Cell & Tissue Imaging; Experimental Irradiation; Preclinical Imaging & Computational Analysis; Health Communications; Immune Monitoring; and Pharmacokinetics. This work was performed with assistance from the Cold Spring Harbor Laboratory shared resources, which are supported by the NIH Cancer Center Support Grant P30 CA045508: Bioinformatics, Flow Cytometry, Animal, and Cell and Tissue Imaging Shared Resources.
Funding Information:
G. Biffi reports grants from Cancer Research UK and Human Frontier Science Program during the conduct of the study. Y. Zhang reports grants from NIH during the conduct of the study and outside the submitted work. M.A. Anderson reports other from GlaxoSmithKline, Boehringer Ingelheim, Boston Scientific, and Olympus outside the submitted work. A. Rao reports other from Voxel Analytics, LLC, Agilent Technologies, Genophyll, LLC, and Texas A&M IBT and grants from NIH and ACS outside the submitted work. D.A. Tuveson reports grants from NIH NCI, Lustgarten Foundation, and Simons Foundation during the conduct of the study. Dr. Tuveson also reports grants from ONO, Fibrogen, and Merck; other from Leap Therapeutics, Surface Oncology, and Cygnal Therapeutics; and grants and other from Mestag Therapeutics outside the submitted work, as well as has a patent as coinventor, “Hedgehog pathway inhibitors” filed by CRT/CRUK January 23, 2009 as US61/205,837 (Olive and colleagues, 2009) and PCT US2010/021816 filed January 22, 2010 issued to CRUK/CRT. B.L. Allen reports grants from NIH and University of Michigan during the conduct of the study. No disclosures were reported by the other authors.
Publisher Copyright:
© 2021 American Association for Cancer Research.
