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. 2021 Apr 1;27(7):2023-2037.
doi: 10.1158/1078-0432.CCR-20-3715. Epub 2021 Jan 25.

Inhibition of Hedgehog Signaling Alters Fibroblast Composition in Pancreatic Cancer

Nina G Steele #  1 , Giulia Biffi #  2   3   4 , Samantha B Kemp  5 , Yaqing Zhang  6 , Donovan Drouillard  6 , LiJyun Syu  7 , Yuan Hao  3   8 , Tobiloba E Oni  3   4 , Erin Brosnan  3   4 , Ela Elyada  3   4 , Abhishek Doshi  3   4 , Christa Hansma  1 , Carlos Espinoza  6 , Ahmed Abbas  1 , Stephanie The  9 , Valerie Irizarry-Negron  6 , Christopher J Halbrook  10 , Nicole E Franks  1 , Megan T Hoffman  10 , Kristee Brown  6 , Eileen S Carpenter  11 , Zeribe C Nwosu  10 , Craig Johnson  1 , Fatima Lima  6 , Michelle A Anderson  11 , Youngkyu Park  3   4 , Howard C Crawford  5   10   12 , Costas A Lyssiotis  5   10   12 , Timothy L Frankel  6 , Arvind Rao  9   10   13   14 , Filip Bednar  6 , Andrzej A Dlugosz  7   12 , Jonathan B Preall  3 , David A Tuveson  15   4 , Benjamin L Allen  16   12 , Marina Pasca di Magliano  16   5   6   12
Affiliations

Inhibition of Hedgehog Signaling Alters Fibroblast Composition in Pancreatic Cancer

Nina G Steele et al. Clin Cancer Res. .

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 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.

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Conflict of interest statement

Conflict of interest statement: D.A.T. discloses membership in the scientific advisory board for Leap Therapeutics, Surface Oncology, Cyngnal Therapeutics, Mestag Therapeutics, stock in Leap Therapeutics and Surface Oncology, Co-Founder of Mestag Therapeutics, Honorarium from Merck and sponsored research with ONO, Fibrogen, and Mestag Therapeutics. The other authors declare no conflict of interests.

Figures

Figure 1.
Figure 1.
HH pathway activation is higher in myCAFs compared to iCAFs in PDAC. (A) Uniform Manifold Approximation and Projection (UMAP) visualization of cell populations from single-cell RNA sequencing (scRNA-seq) of human adjacent normal/normal pancreas (n=3) and PDAC (n=16) tissues. The normal samples (n=3) were isolated from patients undergoing surgery for duodenal adenoma, ampullary carcinoma or PDAC, where an uninvolved portion of the pancreas was included in the resection. Different cell type clusters are color-coded. Data are from Steele et al. [35]. (B) Dot blot visualization of HH pathway gene expression level (color intensity) and frequency (size of dot) in different cell populations of human adjacent normal/normal pancreas (blue, n=3) and PDAC (red, n=16) samples from (A). Boxes highlight HH ligands (SHH, IHH, DHH) and HH targets (GLI1, PTCH1, PTCH2, HHIP). (C) UMAP visualization of scRNA-seq of the fibroblast clusters in pooled human adjacent normal pancreas (n=2) and PDAC (n=6) samples. Different CAF subtype clusters are color-coded. Data are from Elyada et al. [30]. (D) UMAP visualization of myCAF (ACTA2 coding for αSMA, TAGLN) and iCAF (IL6, CLEC3B) marker expression in the fibroblast clusters in human PDAC samples from (C). (E) UMAP visualization of HH target (GLI1, PTCH1) expression in the fibroblast clusters in human PDAC samples from (C). (F) Heatmaps of normalized expression of HH targets (GLI1, PTCH1, PTCH2, HHIP) and of HH receptor (SMO) and co-receptors (GAS1, CDON, BOC) in each fibroblast cluster in human PDAC samples from (C). Colors indicate log-scale gene counts. (G) Heatmap of scaled expression of HH ligands (Shh, Ihh, Dhh), HH targets (Gli1, Ptch1, Ptch2, Hhip), HH receptor (Smo) and co-receptors (Gas1, Cdon, Boc) in different cell populations of pancreatic tumors of the KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx1-Cre) mouse model of PDAC (n=4). Data are scaled such that the cluster with the lowest average expression = 0 and the highest = 1 for each gene. Data are from Elyada et al. [30]. (H) Representative RNA in situ hybridization (ISH) of Gli1 (white) and co-immunofluorescence (co-IF) of podoplanin (PDPN, green) in a KPC tumor. Counterstain, DAPI (blue). Scale bar, 20 μm. (I) Quantitation of Gli1 stain in PDPN+ (CAFs) and PDPN- (non-CAFs) cells in KPC tumors. Results show mean ± standard error of the mean (SEM) of 7 biological replicates. *** P < 0.001, unpaired Student t test. (J) UMAP visualization of scRNA-seq of the fibroblast clusters in KPC tumors (n=4) from Elyada et al. [30]. Different CAF subtype clusters are color-coded. (K) Heatmaps of normalized expression of HH targets (Gli1, Ptch1, Ptch2, Hhip), and of HH receptor (Smo) and co-receptors (Gas1, Cdon, Boc) in each fibroblast cluster from (J). Colors indicate log-scale gene counts. (L) Representative RNA ISH of Gli1 (white) and co-IF of PDPN (green) and alpha smooth muscle actin (αSMA, red) in a KPC tumor. Counterstain, DAPI (blue). Scale bar, 20 μm. The arrowhead points at a PDPN+αSMA+ cell with lower Gli1 expression; solid arrow points at a PDPN+αSMA+ cell with higher Gli1 expression. (M) Quantitation of Gli1 stain in αSMA+ PDPN+ (myCAFs) and αSMA- PDPN+ (non-myCAFs) cells in KPC tumors. Results show mean ± SEM of 7 biological replicates. ** P < 0.01, unpaired Student t test.
Figure 2.
Figure 2.
HH pathway inhibition impairs PDAC growth. (A) Schematic of orthotopic transplants of 7940b KPC PDAC cells with Ihh wild-type (WT) or knockout (KO) in C57BL/6J (BL/6J) or Gli1lacZ/+ mice. (B) Tumor weights at day 18 post-transplantation of the experiment from (A). Results show mean ± SEM of 6 biological replicates. ** P < 0.01, one-way ANOVA. (C) Representative RNA ISH images of Gli1 in Ihh WT or KO tumors in BL/6J mice (left panels), and of X-GAL stain and co-IF of αSMA (magenta), beta-galactosidase (β−GAL) (green) and DAPI (blue) in Ihh WT or KO tumors in Gli1lacZ/+ mice (middle and right panels). Inserts, magnifications. Scale bars, 50 μm (left panels), 100 μm (middle panels), 25 μm (right panels). (D) Quantitation of Gli1 RNA ISH in Ihh WT or KO tumors in BL/6J mice. Results show mean ± SEM on 6 biological replicates. *** P < 0.001, unpaired Student t test. (E) Quantitation of X-GAL stain in Ihh WT or KO tumors in Gli1lacZ/+ mice. Results show mean ± SEM on 6 biological replicates. ** P < 0.01, unpaired Student t test. (F) Schematic of orthotopic transplants of 7940b KPC PDAC cells into BL/6J or Gli1lacZ/+ mice, followed by a 12-day treatment with 20 mg/kg smoothened (SMO) inhibitor LDE225 or vehicle by daily oral gavage. (G) Tumor weights at day 18 post-transplantation of the experiment from (F). Results show mean ± SEM of 4–6 biological replicates. *** P < 0.001, one-way ANOVA. (H) Representative RNA ISH images of Gli1 in vehicle- or LDE225- treated tumors in BL/6J mice (left panels), and of X-GAL stain and co-IF of αSMA (red), βGal (green) and DAPI (blue) in vehicle- or LDE225- treated tumors in Gli1lacZ/+ mice (middle and right panels). Inserts, magnifications. Scale bars, 50 μm (left panels), 100 μm (middle panels), 25 μm (right panels). (I) Quantitation of Gli1 RNA ISH in LDE225- or vehicle- treated tumors in BL/6J mice. Results show mean ± SEM on 4 biological replicates. *** P < 0.001, unpaired Student t test. (J) Quantitation of X-GAL stain in LDE225- or vehicle- treated tumors in Gli1lacZ/+ mice. Results show mean ± SEM on 4 biological replicates. *** P < 0.001, unpaired Student t test. (K) Schematic of 2-week treatment of tumor-bearing KPC mice with 20 mg/kg LDE225 or vehicle by daily oral gavage. U/S, ultrasound. (L) Tumor volume as measured by U/S of vehicle- (n=9) and LDE225- (n=8) treated KPC tumors from (K). Results show mean ± SEM. * P < 0.05, unpaired Student t test. (M) Representative RNA ISH of Gli1 in 2-week vehicle- (n=11) and LDE225- (n=10) treated KPC tumors. Scale bar, 100 μm. (N) Quantitation of Gli1 stain in vehicle- (n=11) and LDE225- (n=10) treated KPC tumors. Results show mean ± SEM. *** P < 0.001, unpaired Student t test.
Figure 3.
Figure 3.
Two-week HH pathway inhibition alters the fibroblast compartment in PDAC. (A) Representative immunohistochemistry (IHC) of PDPN in vehicle- (n=10) and LDE225- (n=11) treated KPC tumors. Scale bar, 50 μm. (B) Quantitation of PDPN stain in vehicle- (n=10) and LDE225- (n=11) treated KPC tumors. Results show mean ± SEM. ** P < 0.01, unpaired Student t test. (C) Schematic of fluorescence-activated cell sorting (FACS) strategy for bulk RNA-seq of fibroblasts from vehicle- and LDE225- treated KPC tumors. (D) Gene set enrichment analysis (GSEA) of cell cycle signature in FACS-sorted CAFs from LDE225-treated KPC tumors (n=2) compared to FACS-sorted CAFs from vehicle-treated controls (n=3). (E) RNA-seq expression of proliferation markers (Mki67, Ccnb2 and Cks2) in FACS-sorted CAFs from vehicle- (n=3) and LDE225- (n=2) treated KPC tumors. Results show mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, unpaired Student t test. (F) Co-IF of αSMA (red) and PDPN (green) in vehicle- (n=7) and LDE225- (n=4) treated KPC tumors. Counterstain, DAPI. Scale bar, 20 μm. (G) Quantitation of myofibroblastic CAFs (αSMA+ PDPN+ DAPI+) in vehicle- (n=7) and LDE225- (n=4) treated KPC tumors. Results show mean ± SEM. P = 0.05, unpaired Student t test. (H) Quantitation of non-myofibroblastic CAFs (αSMA- PDPN+ DAPI+) in vehicle- (n=7) and LDE225- (n=4) treated KPC tumors. Results show mean ± SEM. * P < 0.05, unpaired Student t test.
Figure 4.
Figure 4.
Two-week HH pathway inhibition alters the ratio of myCAFs and iCAFs in PDAC. (A) Schematic of flow cytometry strategy for myCAFs and iCAFs from 2-week vehicle- and LDE225- treated KPC tumors. (B) Representative flow plots showing the gating strategy for the analysis of DAPI- CD45- CD31- EpCAM- PDPN+ CAFs in 2-week vehicle- (n=7) and LDE225- (n=6) treated KPC tumors. (C) Flow cytometry analysis of myCAFs (calculated from DAPI+ singlets) in vehicle- (n=7) and LDE225- (n=6) treated KPC tumors. Results show mean ± SEM. Unpaired Student t test. (D) Flow cytometry analysis of iCAFs (calculated from DAPI+ singlets) in vehicle- (n=7) and LDE225- (n=6) treated KPC tumors. Results show mean ± SEM. * P < 0.05, unpaired Student t test. (E) Proportions of myCAF, iCAF and apCAF subtypes from the PDPN+ gate in vehicle- (n=7, top) and LDE225- (n=6, bottom) treated KPC tumors, as measured by flow cytometry analysis. Results show average % of biological replicates. (F) Flow cytometric analysis of the iCAF/myCAF ratio from the PDPN+ gate in vehicle- (n=7) and LDE225- (n=6) treated KPC tumors. Results show mean ± SEM. * P < 0.05, unpaired Student t test. (G) GSEA of the myCAF gene signature in FACS-sorted CAFs from 2-week LDE225-treated KPC tumors (n=2) compared to FACS-sorted CAFs from vehicle-treated controls (n=3). The myCAF gene signature was defined from the study by Öhlund et al. [27]. (H) RNA-seq expression of myCAF markers (Acta2, Thy1, Tagln, Tgfb1) in FACS-sorted CAFs from vehicle- (n=3) and LDE225- (n=2) treated KPC tumors. Results show mean ± SEM. No statistical difference was observed as calculated by unpaired Student t test. (I) GSEA of the iCAF gene signature in FACS-sorted CAFs from 2-week LDE225-treated KPC tumors (n=2) compared to FACS-sorted CAFs from vehicle-treated controls (n=3). The iCAF gene signature was defined from the study by Öhlund et al. [27]. (J) RNA-seq expression of iCAF markers (Dpt, Clec3b, C3, Cxcl12) in FACS-sorted CAFs from vehicle- (n=3) and LDE225- (n=2) treated KPC tumors. Results show mean ± SEM. * P < 0.05, unpaired Student’s t-test.
Figure 5.
Figure 5.
Two-week HH pathway inhibition alters the immune infiltration in pancreatic tumors. (A) Top: schematic of 2-week treatment of tumor-bearing KPC mice with 20 mg/kg LDE225 or vehicle prior to CyTOF (Cytometry by time of flight) analysis. U/S, ultrasound. Bottom: table of CyTOF panel including metal tag, antibody target, and cell type predominantly expressed on. See Table S1 for detailed antibody information. (B) Manual gating of CyTOF data for total myeloid cells (CD45+CD11b+), macrophages (CD11b+F4/80+), PDL1+ macrophages (F4/80+PD-L1+) and CD206+ macrophages (F4/80+CD206+) in 2-week vehicle- (n=7) and LDE225- (n=6) treated KPC tumors. Results show mean ± SEM. * P < 0.05, unpaired Student’s t-test. (C) Manual gating of CyTOF data for CD8+, CD4+ and CD4+CD25+ T cells as a percentage of total CD3+ T cells. Results show mean ± SEM. * P < 0.05, ** P < 0.01, unpaired Student’s t-test. (D) Representative IHC of CD8A in 2-week vehicle- and LDE225- treated KPC tumors. Inserts, magnifications. Scale bar, 50 μm. (E) Quantitation of CD8A stain in 2-week vehicle- (n=12) and LDE225- (n=11) treated KPC tumors. Results show mean ± SEM. * P < 0.05, unpaired Student’s t-test. (F) Representative IHC of FOXP3 in 2-week vehicle- and LDE225- treated KPC tumors. Inserts, magnifications. Scale bar, 50 μm. (G) Quantitation of FOXP3 stain in 2-week vehicle- (n=12) and LDE225- (n=11) treated KPC tumors. Results show mean ± SEM. ** P < 0.01, unpaired Student’s t-test. (H) Model explaining the role of HH signaling and the effects of HH inhibition in the PDAC microenvironment. Cancer-secreted HH ligands, such as SHH and IHH, activate HH signaling in surrounding fibroblasts (arrow), especially in myCAFs (left panel). HH inhibition leads to a reduction in myCAFs and an increase in iCAFs, and to decreased CD8+ T cells and more abundant regulatory T cells (right panel).
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