The GLP-1R agonist semaglutide reshapes pancreatic cancer associated fibroblasts reducing collagen proline hydroxylation and favoring T lymphocyte infiltration

Abstract

Background: Metabolic syndrome represents a pancreatic ductal adenocarcinoma (PDAC) risk factor. Metabolic alterations favor PDAC onset, which occurs early upon dysmetabolism. Pancreatic neoplastic lesions evolve within a dense desmoplastic stroma, consisting in abundant extracellular matrix settled by cancer associated fibroblasts (CAFs). Hereby, dysmetabolism and PDAC association was analyzed focusing on CAF functions.

Methods: PDAC development upon dysmetabolic conditions was investigated in: 1) high fat diet fed wild type immunocompetent syngeneic mice by orthotopic transplantation of pancreatic intraepithelial neoplasia (PanIN) organoids; and 2) primary pancreatic CAFs isolated from chemotherapy naïve PDAC patients with/without an history of metabolic syndrome.

Results: The dysmetabolic-associated higher PDAC aggressiveness was paralleled by collagen fibril enrichment due to prolyl 4-hydroxylase subunit alpha 1 (P4HA1) increased function. Upon dysmetabolism, P4HA1 boosts collagen proline hydroxylation, intensifies collagen contraction strength, precluding PDAC infiltration. Noteworthy, semaglutide, an incretin agonist, prevents the higher dysmetabolism-dependent PDAC stromal deposition and allows T lymphocyte infiltration, reducing tumor development.

Conclusions: These results shed light on novel therapeutic options for PDAC patients with metabolic syndrome aimed at PDAC stroma reshape.

Keywords: Cancer associated fibroblasts; Collagen deposition; Diabetes; Obesity; Pancreatic ductal adenocarcinoma.

Fig. 1

HFD mice showed a higher volume of pre-neoplastic pancreatic lesions paralleled by a higher collagen deposition, a reduced oxidative phosphorylation and decreased tumor infiltration. A Left panels: Representative endoscopic ultrasound images of LFD and HFD mice at T1 (30 days) and T2 (90 days) from KPC organoid injection. T1 original scale bar, 1.5 mm; T2 original scale bar, 1.8 mm; n = 10 for each group. Right panels: Tumor size measurement at time point T1 (left panel; n = 12 for each group; ***p < 0.001) and T2 (right panel; n = 11 for each group; ***p < 0.001) in LFD (black circles) and HFD (black squares) mice bearing pre-neoplastic lesions. B Heatmap showing the 50 most differentially regulated genes in LFD and HFD mice at time point T1 identified by total RNA sequencing analysis (n = 3 for LFD group and n = 4 for HFD group). Red and blue represent over- and under-expressed genes, respectively. C Gene ontology analysis of differentially regulated transcripts between LFD and HFD mice at time point T1. Up-regulated genes depicted in blue bars and down-regulated genes in green bars. D Heatmap showing the 50 most differentially regulated genes in LFD and HFD mice at time point T2 identified by total RNA sequencing analysis (n = 4 for each group). Red and blue represent over- and under-expressed genes, respectively. E Gene ontology analysis of differentially regulated transcripts between LFD and HFD mice at time point T2. Up-regulated genes depicted in blue bars and down-regulated genes in green bars. F Left panels: Representative picrosirius red staining images of pancreatic lesions at time point T2 in LFD (upper panels) and HFD (lower panel) mice bearing pre-neoplastic lesions. Right panel: Quantitative assessment of percentage of picrosirius red stained positive area in LFD (black circles) and HFD (black squares) mice at time point T2 (n = 5 for each group; **p < 0.01). G Col1A1, Col4A1 and Col6A1 mRNA analysis in LFD (black circles) or HFD (black squares) mice. Data expressed as fold increase of average Ct of LFD after subtraction of the housekeeping gene p0 signal (n = 3; *p < 0.05 HFD vs. LFD mice). H Left panels: Representative confocal microscopy images depicting LFD (upper panel) and HFD (lower panel) mouse cryo-sections at time point T2 probed by an anti-CD3 antibody (green, left panels), an anti-E-Cadherin (white, middle left panels), and an anti-pan cytokeratin (red, middle right panels). Nuclei were counterstained with DAPI (blue, right panels). On the right, merged images. Original scale bar, 50 μm; n = 5. Right panels: quantification of CD3 positive cell MFI on area in LFD (black circles) and HFD (black squares) mice (n = 25; ***p < 0.001). Data expressed as average ± SEM. Data analyzed by Kolmogorov–Smirnov test

Fig. 2

HFD mice showed a higher expression of P4HA1, a PDAC unfavorable prognostic marker, and consequent higher hydroxyproline levels. A Representative WB analysis of P4HA1 in LFD or HFD mice at time point T2. Loading control: vinculin (n = 4 for each group). B Estimation of prognostic P4HA1 in PDAC patients by Kaplan–Meier survival curves according to P4HA1 gene expression levels (low expression Vs high expression) derived from transcriptomics data deposited in TCGA repository. C P4HA1 levels in different PDAC stratification groups. Upper panel: transcript levels of P4HA1 (expressed as z-score) in PDAC patient data extracted from the CPTAC portal displaying significant enrichment in stromal activated signature. Lower panel: P4HA1 transcript levels (expressed as z-score) in PDAC patient data extracted from the CPTAC portal displaying significant enrichment in hCAF infiltration signature. D Left panels: Representative confocal microscopy images depicting HFD mouse cryosections at time point T2 probed by an anti-P4HA1 antibody (green), an anti-Pancytokeratin (PanCK; red), and an anti-E-cadherin (E-Cad, white). Nuclei were counterstained with DAPI (blue). Right panels: Representative confocal microscopy images depicting HFD mouse cryosections at time point T2 probed by an anti-P4HA1 antibody (green) and an anti-alpha smooth muscle actin (aSMA; red). Nuclei were counterstained with DAPI (blue). Merged fluorescence images are shown (right panels). Original scale bar, 50 μm; n = 5. E Left panels: Representative confocal microscopy images depicting LFD (upper panels) and HFD (lower panels) mouse cryosections at time point T2 probed by an anti-E-cadherin antibody (E-Cad; white, left) and anti-hydroxyproline antibody (green, middle left). Nuclei were counterstained with DAPI (blue, middle right). Merged fluorescence images are shown (right; merge). Original scale bar, 100 μm; n = 5 (***p < 0.001). Right panel: Right panels: quantification of HydroxyP MFI on area in LFD (black circles) and HFD (black squares) mice (n = 25; ***p < 0.001). F Representative immunohistochemistry images depicting ND- (upper panel) and D- (lower panels) human PDAC specimens probed by an anti-P4HA1 antibody. Nuclei were counterstained with hematoxylin. “S” stands for stroma enriched region; “T” stand for tumor area. Original scale bar, 100 μm; n = 3. Data expressed as average ± SEM. Data analyzed by Kolmogorov–Smirnov test

Fig. 3

hpCAFs isolated from D PDAC patients showed higher P4HA1 protein expression paralleled by increased hydroxyproline levels and higher collagen mRNA expression contributing to higher contraction strength and immune exclusion. A Representative WB analysis of P4HA1 protein levels in hpCAFs isolated from ND or D PDAC patients. Loading control: vinculin (n = 5 for each group). B Col1A1, Col3A1, and Col5A1 mRNA analysis in hpCAFs isolated from ND (white circles) or D (black squares) PDAC patients. Data expressed as fold increase of average Ct of ND after subtraction of the housekeeping gene p0 signal (n = 4; *p < 0.05 D vs. ND patients). C Quantification of hydroxyproline levels in hpCAFs isolated from ND (white circles) and D (black squares) PDAC patients (n = 4 for each group; *p < 0.05 D vs. ND patients). D Upper panel: Representative images of contraction assay performed in hpCAFs derived from ND- or D- PDAC patients. Original scale bar, 1 cm. Lower panel: Quantification of contraction performed on hpCAFs derived from ND- (white circles) and D- (black squares) PDAC patients in cell-populated collagen hydrogel (n = 9 for each group; ***p < 0.001 D vs. ND patients). E Representative immunohistochemistry images depicting ND- (left panel) and D- (right panels) hpCAF-populated collagen hydrogel after 24 h contraction probed by an anti-hydroxyproline antibody. Nuclei were counterstained with hematoxylin. Original scale bar, 100 μm; n = 5. F Correlation plot between P4HA1 and T cell signature gene expression in PDAC patients according TCGA data showing a negative correlation coefficient (R = −0.24) with statistical significance (p = 0.001). G Left panels: representative confocal microscopy images depicting ND- (upper panels) and D- (lower panels) hpCAF-populated collagen hydrogel after 24 h contraction probed by an anti-CD3 antibody (green, left). Nuclei were counterstained with DAPI (blue, middle). Merged fluorescence images are shown (right; merge). Original scale bar, 100 μm; n = 5. Right panel: quantification of CD3 signal out (external white rectangle) or into (internal black rectangle) the disk. Data expressed as average ± SEM. Data analyzed by Kolmogorov–Smirnov test

Fig. 4

Glycemic control achieved by Semaglutide administration decreases PDAC stroma deposition favoring immune infiltration. A Left panel: Representative images of contraction assay performed in hpCAFs derived from D-PDAC patients untreated (SOL) or treated with SEMA. Original scale bar, 1 cm. Right panel: Quantification of contraction performed on hpCAFs derived from D-PDAC patients untreated (black cicrcles) or treated (blue squares) with SEMA in cell-populated collagen hydrogel (n = 7 for each group; ***p < 0.001 SEMA vs. SOL D-hpCAFs). B Oral glucose tolerance test (OGTT) in HFD (black circles) and HFD treated with semaglutide (SEMA; blue squares) mice (n = 3 for each group; ***p < 0.001 SEMA vs. HFD). C In vivo tumor size measurement at 60 days from treatment start in HFD (black squares; n = 12) and SEMA (blue rumbles; n = 12) mice bearing pre-neoplastic lesions (***p < 0.001 SEMA vs. HFD). D Representative hematoxilin/eosin images of pancreatic neoplasia at T2 time point in HFD (left panel) and SEMA (right panel) mice bearing pre-neoplastic lesions. Original scale bar, 100 μm; n = 5. E Heatmap showing the 50 most differentially regulated genes in HFD and SEMA mice at time point T2 identified by total RNA sequencing analysis (n = 4 for HFD group and n = 3 for SEMA group). Red and blue represent over- and under-expressed genes, respectively. F Gene ontology analysis of differentially regulated transcripts between HFD and SEMA mice at time point T2. Up-regulated genes depicted in blue bars and down-regulated genes in green bars. G Heatmap showing P4HA1 signature according GSEA analysis in T2 and SEMA mice identified by bioinformatics RNA sequencing analysis (n = 3 for SEMA group; n = 4 for LFD and HFD T2). Red and blue represent over- and under-categories, respectively. H Left panels: Representative picrosirius red staining images of pancreatic lesions at time point T2 in HFD (left panel) and SEMA (right panel) mice bearing pre-neoplastic lesions. Right panel: Quantitative assessment of percentage of picrosirius red stained positive area in HFD (black squares) and SEMA (blue rumbles) mice at time point T2 (n = 4 for each group; ***p < 0.001 SEMA vs. HFD). I Left panels: Representative immunofluorescence images depicting HFD (left panel) and SEMA (right panel) mouse cryosections at time point T2 probed by an anti-hydroxyproline antibody (green). Nuclei were counterstained with DAPI (blue). Merged images depicted. Original scale bar, 100 μm; n = 5. Right panels: quantification of hydroxyproline positive cells in HFD (black squares) and SEMA (blue rumbles) mice (n = 15). J Upper left panels: Representative confocal microscopy images depicting HFD (upper panels) and SEMA (lower panels) mouse cryosections at time point T2 probed by an anti-CD3 antibody (green), an anti-E-Cadherin (white), and an anti-pan cytokeratin (red). Nuclei were counterstained with DAPI (blue). Merged pictures were depicted on the right. Original scale bar, 50 μm; n = 5. Upper right panels: Representative confocal microscopy images depicting HFD (upper panels) and SEMA (lower panels) mouse cryosections at time point T2 probed by an anti-CD8 antibody (green), an anti-E-Cadherin (white), and an anti-pan cytokeratin (red). Nuclei were counterstained with DAPI (blue). Merged pictures were depicted on the right; n = 5. Left lower panel: quantification of CD3 positive cells in HFD (black squares) and SEMA (blue rumbles) mice. Right lower panels: quantification of CD8 positive cells in HFD (black squares) and SEMA (blue rumbles) mice (n = 25 for each group; ***p < 0.001 SEMA vs. HFD). Data expressed as average ± SEM. Data analyzed by Kolmogorov–Smirnov test (A, C, H, I, J) or 2-way-ANOVA (B)