Upregulated Matrisomal Proteins and Extracellular Matrix Mechanosignaling Underlie Obesity-Associated Promotion of Pancreatic Ductal Adenocarcinoma

Abstract

Diet-induced obesity (DIO) promotes pancreatic ductal adenocarcinoma (PDAC) in mice expressing KRasG12D in the pancreas (KC mice), but the precise mechanisms remain unclear. Here, we performed multiplex quantitative proteomic and phosphoproteomic analysis by liquid chromatography-tandem mass spectrometry and further bioinformatic and spatial analysis of pancreas tissues from control-fed versus DIO KC mice after 3, 6, and 9 months. Normal pancreatic parenchyma and associated proteins were steadily eliminated and the novel proteins, phosphoproteins, and signaling pathways associated with PDAC tumorigenesis increased until 6 months, when most males exhibited cancer, but females did not. Differentially expressed proteins and phosphoproteins induced by DIO revealed the crucial functional role of matrisomal proteins, which implies the roles of upstream regulation by TGFβ, extracellular matrix-receptor signaling to downstream PI3K-Akt-mTOR-, MAPK-, and Yap/Taz activation, and crucial effects in the tumor microenvironment such as metabolic alterations and signaling crosstalk between immune cells, cancer-associated fibroblasts (CAFs), and tumor cells. Staining tissues from KC mice localized the expression of several prognostic PDAC biomarkers and elucidated tumorigenic features, such as robust macrophage infiltration, acinar-ductal metaplasia, mucinous PanIN, distinct nonmucinous atypical flat lesions (AFLs) surrounded by smooth muscle actin-positive CAFs, invasive tumors with epithelial-mesenchymal transition arising close to AFLs, and expanding deserted areas by 9 months. We next used Nanostring GeoMX to characterize the early spatial distribution of specific immune cell subtypes in distinct normal, stromal, and PanIN areas. Taken together, these data richly contextualize DIO promotion of Kras-driven PDAC tumorigenesis and provide many novel insights into the signaling pathways and processes involved.

Keywords: KC mice; atypical flat lesions; diet-induced obesity; extracellular matrix; matrisomal proteins; pancreatic ductal adenocarcinoma; prognostic biomarkers; quantitative proteomic analysis; tandem mass tags; tumor microenvironment.

Figure 1

DIO-induced PDAC development. Representative pancreas histology (H&E) of PDAC development in female (upper panels) and male (lower panels) KC mice on a control diet (CD) versus a high-fat, high-calorie diet (HFCD) at 3, 6, and 9 months (mo) of age reveals extensive loss of acinar tissue concomitant with increased areas of fibrotic stroma, ADM, PanIN, and PDAC in HFCD-fed mice. Tumor tissues identified by a pathologist were selected to represent the effect of DIO in females at 9 mo and in males at 6 and 9 mo. Scale bar: 200 μm.

Figure 2

(A), representative Alcian Blue (AB) staining detects classical PanIN in the pancreas of KC mice fed a CD and HFCD. (B), quantitation of QuPath-classified AB+ cells in male and female pancreas tissues at different time points. (C), representative images of Trichrome staining at 6 mo show increases in collagen-stained areas in CD-fed and HFCD-fed KC mice. (D), stromal areas are also filled with ECM proteins in mice on a CD at 9 mo, and DIO induces smaller increases in mice exhibiting invasive pancreas/liver tumors. A dotted line demarcates a frontier between a tumor and an invaded liver area. (E), quantitation of a tissue area containing collagen stained blue with Masson’s Trichrome in KC mice on a CD versus an HFCD. The graphs in (B,E) show mean ± SEM. *, p < 0.05 vs. CD; #, p < 0.05 vs. 3 mo; ANOVA + post hoc Fisher’s LSD test. Scale bars, (A), 100 nm; (C,D), 200 nm.

Figure 3

Matrisomal proteins support DIO-induced PDAC development. (A), log₂-transformed HFCD/CD expression ratios. (B), volcano plot of HFCD/CD values with prominent down- (left) and upregulated (right) proteins labeled. (C), Gene Set Enrichment Analysis of HFCD- versus CD-fed mouse proteins within Naba Core Matrisome (see ref. [11]) gene set. (D), Ingenuity Pathway Analysis (IPA)-derived signaling network linking proteins upregulated by DIO with transcriptional regulators Yap1/Taz and discretely expressed matrisomal intercellular mediators such as Ccn2/Ctgf, Postn, and Bmp. (E), negative (blue box) and positive (orange box) upstream regulators of DIO-induced protein changes predicted by IPA. (F), unsupervised hierarchical clustering of proteins differentially elevated (red) or suppressed (green) in female (left) and male (right) KC mice by DIO at 6 mo. Protein names are shown on left. Legend indicates DIO-induced log2 fold change.

Figure 4

Motif analysis of 25 top (A), Proline-directed and (B), non-Proline-directed phosphopeptides that increased during DIO-induced PDAC induction in KC mice, using PhosphoSitePlus Tools. Each sequence logo depicts the preferred amino acid residues most found around the targeted Ser residues, with the preferred (larger) residues at each position stacked on top of lower-scoring ones. Kinases targeting the main sequences were also calculated and are shown.

Figure 5

(A), differential immunohistochemistry (IHC) localization of distinct PDAC markers of classical PanIN in KC mice. Representative staining for Alcian Blue (AB), Maspin, Muc4, and Agr2 marked the cells lining PanIN, and nuclear p-Erk and Yap/Taz marked both PanIN and stromal cells in CD-fed KC mice (upper panels). In more advanced tumor areas (lower panels; HFCD-fed obese mice), some glands exhibited dramatically diminished AB, Maspin, and Muc4 and patchy Agr2 staining. Also, p-Erk and Yap/Taz staining remained in a PanIN/glandular epithelial and stromal pattern and was intensified by DIO. (B), areas with thick bands of Masson’s Trichrome-stained (TRI) collagen corresponded closely to F4/80+ macrophage staining, and only to a lesser extent with α-Sma+ CAFs. (C), appearance of PanIN and nearby atypical flat lesions (AFLs) viewed adjacent to PanIN in a CD-fed KC mouse at 9 mo. Epithelial cells lining PanIN were AB+, Maspin+ and associated with a limited amount of α-Sma+ CAFs. In contrast, the disorganized epithelial cells of AFLs were AB/PAS-, Maspin-, and surrounded by α-Sma+ CAFs. Scale bars: (A), 50 μm; (B), 500 μm; (C), 100 μm.

Figure 6

NanoString GeoMx^® Digital Spatial Profiling of DIO-induced immune cell subtype enrichment in stromal and PanIN areas in KC mice. We analyzed pancreas tissues from CD- and HFCD-fed KC mice at 3 mo and 6 mo (n = 3 mice/group). We collected 8 ROIs per slide, total of 20 normal, 37 stromal, and 39 PanIN data points, and applied GeoMx^® Housekeeping and controls, immune cell profiling, immune cell typing, and immune activation status antibody panels. (A), representative H&E staining at 6 mo at 100 μm scale, to illustrate selected normal (red circle), PanIN (green circle), and stromal (yellow irregular) ROIs. (B), IF staining for epithelial cell pan-cytokeratin- (green), immune cell CD45- (yellow), and cell nuclei (blue)-guided ROI selection of normal, PanIN, or stromal areas as in (A). Scale bar, 250 μm. (C,D), heat maps of significant DIO-induced changes at (C), 3 mo; (D), 6 mo. Diet drove many important shifts in immune distribution at 3 mo. (E), volcano plot of PanIN (Pan) vs. stromal (St) regions. Stromal areas attracted immune cells (CD45) including myeloid cells (F4/80) and myofibroblasts (α-Sma). In contrast, PanIN area preferentially attracted immunosuppressive M2 macrophages (CD163). Dashed lines indicate cutoffs for significance (green horizontal, p = 0.05) and fold change (red vertical, plus or minus 1.5-fold) levels.