Proteomic analyses of ECM during pancreatic ductal adenocarcinoma progression reveal different contributions by tumor and stromal cells

Abstract

Pancreatic ductal adenocarcinoma (PDAC) has prominent extracellular matrix (ECM) that compromises treatments yet cannot be nonselectively disrupted without adverse consequences. ECM of PDAC, despite the recognition of its importance, has not been comprehensively studied in patients. In this study, we used quantitative mass spectrometry (MS)-based proteomics to characterize ECM proteins in normal pancreas and pancreatic intraepithelial neoplasia (PanIN)- and PDAC-bearing pancreas from both human patients and mouse genetic models, as well as chronic pancreatitis patient samples. We describe detailed changes in both abundance and complexity of matrisome proteins in the course of PDAC progression. We reveal an early up-regulated group of matrisome proteins in PanIN, which are further up-regulated in PDAC, and we uncover notable similarities in matrix changes between pancreatitis and PDAC. We further assigned cellular origins to matrisome proteins by performing MS on multiple lines of human-to-mouse xenograft tumors. We found that, although stromal cells produce over 90% of the ECM mass, elevated levels of ECM proteins derived from the tumor cells, but not those produced exclusively by stromal cells, tend to correlate with poor patient survival. Furthermore, distinct pathways were implicated in regulating expression of matrisome proteins in cancer cells and stromal cells. We suggest that, rather than global suppression of ECM production, more precise ECM manipulations, such as targeting tumor-promoting ECM proteins and their regulators in cancer cells, could be more effective therapeutically.

Keywords: ECM; PDAC; PanIN; pancreatitis.

Fig. 1.

Proteomic analysis of enriched ECM and ECM-associated proteins in human patient samples and genetically engineered mouse models. (A) Simplified schematic of the ECM enrichment and liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based quantification of normal and diseased pancreas samples. See Materials and Methods for details. The arrow points to ECM pellet. (B) Western blot showing stepwise removal of cytosolic (1), nuclear (2), membrane (3), and cytoskeletal (4) fractions of proteins and final enrichment of ECM. Example shown is an extraction from a mouse KPC PDAC tumor. (C) Total abundance from summed precursor ion peak areas for all peptides (ion counts) and protein numbers from different groups of ECM and non-ECM proteins. Data from the human PDAC TMT set A (hA) are shown. (D) ECM from all stages has abundant collagens (>90%). An increase in the abundance of different groups of proteins relative to collagens I and III was observed in PanIN, PDAC, and CP ECM compared to normal pancreas. Abundance for each sample was calculated by splitting the combined precursor ion peak areas (MS1-based) in proportion to the individual reporter ion intensities (MS/MS-based) for each sample. Data from hA set are shown.

Fig. 2.

Comparisons among diseased pancreas matrices and normal pancreas matrix. (A–C) Volcano plots comparing normal pancreas to PanIN (A), PDAC (B), and pancreatitis (C) of human samples using ECM protein TMT reporter-ion ratios to show the increase of many ECM proteins. Proteins with Benjamini–Hochberg 2-tailed t test adjusted P values less than 0.1 (Materials and Methods) and fold changes more than 1.5 are highlighted. Note that many proteins up-regulated in PanIN are further up-regulated in PDAC. (D) Venn diagram showing the overlap of significantly overrepresented proteins (P_adj < 0.1, fold change > 1.5) in each stage (Dataset S1D). The identities of the proteins in each field are shown; proteins in each matrisome category are ordered from the most abundant (Top) to the least abundant (Bottom), as decided by the mean abundance of the hA and hB sets. When there are too many proteins to list here, see Dataset S1D for complete information.

Fig. 3.

Comparisons of global changes in ECM and ECM-associated proteins in the course of PDAC progression in human and mouse. Hierarchical clustering of the protein-level TMT ratios by Pearson correlation using all identified matrisome proteins grouped almost all PDAC samples separately from normal and PanIN samples in both human (A) and mouse (B). Over 200 ECM and ECM-associated proteins were identified from both human and mouse sets (C), with a majority being significantly overrepresented (P_adj < 0.1) in PDAC compared to normal. Only proteins identified in both pairs of TMT MS (hA and hB for human; mA and mB for mouse) were included. Note that collagens I and III are the most prevalent proteins overall. (D) Pearson correlation analysis showed that core matrisome proteins (collagens, glycoproteins, and proteoglycans) and ECM regulators have the best correlations between mouse and human in normal, PanIN, and PDAC stages. Input was the abundance of all individual proteins identified in both sets of TMT MS for human and mouse. (E and F) Comparisons of protein abundance in normal pancreas and PDAC from human (E) and mouse (F). Proteins above the diagonal line are selectively overrepresented in PDAC. The gray regions are enlarged and plotted on a log scale to show all proteins. The dotted lines parallel to the diagonal line on the log-scale plots indicate 1.5-fold more (Top line) or less (Bottom line) than normal samples. Note the good agreement between human and mouse data with respect to up-regulated proteins.

Fig. 4.

Definition of matrisome proteins derived from cancer cells and stromal cells and their characterization. (A) Schematic overview of the xenograft ECM MS experiment. Venn diagram shows cell-of-origin assignments for a large fraction (180/294) of ECM proteins identified from human patients. “Human patient MS” group includes proteins that are identified in both A and B sets of TMT MS analyses (SI Appendix, Fig. S1C). “Xenograft MS” group includes proteins that are detected in at least 2 out of 4 xenograft lines, and by at least 2 species-specific peptides. On the Right, the numbers of matrisome proteins are broken down into their cellular origins, and their matrisome categories are also shown. (B) Stromal cells (mouse) contribute more ECM mass than do cancer cells (human), even more so in organoid lines (HuO1, HuO2) as compared with 2D lines (AsPC1, BxPC3). Abundances calculated from summed precursor ion chromatographic peak area of LC-MS/MS peptides. (C and D) Cancer-cell–derived ECM includes proteins from all ECM categories (C), while stromal cells primarily make collagens and glycoproteins (D).

Fig. 5.

Survival and pathway analyses on matrisome proteins derived from cancer cells and stromal cells. (A and B) ECM proteins significantly overrepresented in human PDAC TMT MS comparing PDAC to normal with P_adj < 0.02 were tested against both TCGA (A) and QCMG (B) human PDAC datasets. Cancer-cell–derived proteins (red) and both-derived proteins (gray) correlated with small Cox regression P values and large hazard ratios (HRs), indicative of short survival. In contrast, many stroma-derived proteins (blue) tend to correlate with good survival (low hazard ratio). Proteins that do not significantly correlate with survival (Cox regression P > 0.05) are grayed out in Lower part of figure. (C) The identity of the proteins that significantly correlate with survival (Cox regression P < 0.05). Refer to color legends in the figure for the meaning of the color coding of each column. (D) Ingenuity Pathway Analysis (IPA) was performed on proteins that are overrepresented in PDAC and made by cancer cells and/or stromal cells (Materials and Methods). The top 8 predicted regulators of the significantly overrepresented matrisome proteins exclusively in cancer cell and stromal cells, as well as in both compartments, are shown. Refer to Dataset S4 for the complete lists of regulators. The bolded genes are predicted direct regulators.