Proteomic profiling identifies markers for inflammation-related tumor-fibroblast interaction

Abstract

Background: Cancer associated fibroblasts are activated in the tumor microenvironment and contribute to tumor progression, angiogenesis, extracellular matrix remodeling, and inflammation.

Methods: To identify proteins characteristic for fibroblasts in colorectal cancer we used liquid chromatography-tandem mass spectrometry to derive protein abundance from whole-tissue homogenates of human colorectal cancer/normal mucosa pairs. Alterations of protein levels were determined by two-sided t test with greater than threefold difference and an FDR of < 0.05. Public available datasets were used to predict proteins of stromal origin and link protein with mRNA regulation. Immunohistochemistry confirmed the localization of selected proteins.

Results: We identified a set of 24 proteins associated with inflammation, matrix organization, TGFβ receptor signaling and angiogenesis mainly originating from the stroma. Most prominent were increased abundance of SerpinB5 in the parenchyme and latent transforming growth factor β-binding protein, thrombospondin-B2, and secreted protein acidic-and-cysteine-rich in the stroma. Extracellular matrix remodeling involved collagens type VIII, XII, XIV, and VI as well as lysyl-oxidase-2. In silico analysis of mRNA levels demonstrated altered expression in the tumor and the adjacent normal tissue as compared to mucosa of healthy individuals indicating that inflammatory activation affected the surrounding tissue. Immunohistochemistry of 26 tumor specimen confirmed upregulation of SerpinB5, thrombospondin B2 and secreted protein acidic-and-cysteine-rich.

Conclusions: This study demonstrates the feasibility of detecting tumor- and compartment-specific protein-signatures that are functionally meaningful by proteomic profiling of whole-tissue extracts together with mining of RNA expression datasets. The results provide the basis for further exploration of inflammation-related stromal markers in larger patient cohorts and experimental models.

Keywords: Cancer associated fibroblasts; Colorectal cancer; Extracellular matrix organization; Inflammation signature; Proteomic profiling; SPARC; THBS2.

Fig. 1

Proteom analysis of CRC. a Protein abundances in tumor versus normal tissue samples. Volcano plot representing differences in LFQ values (log2FC, fold-changes in logarithmic scale to the base of two) of proteins including corresponding p values (logarithmic scale). Red labeled proteins are higher abundant in the tumor tissue whereas green labeled proteins are higher abundant in the normal tissue samples. b Stromal contribution. Proteomics derived data of significantly regulated proteins were plotted against stromal contribution of their respective mRNA [41]. Red indicates upregulated proteins of stromal origin. Blue indicates the 2 stromal proteins THBS2 and SPARC plus the parenchymal SerpinB5 that were chosen for IHC staining

Fig. 2

Proteomic alterations associated with inflammation, ECM organization, TGFβ receptor signaling pathway and angiogenesis. Schematic presentation of identified regulated proteins and their functional annotation according to literature. Most proteins were confirmed and grouped by GO-term enrichment analysis of biological processes using the Cytoscape plugin Cluego. Significant associations were found with ECM-organization (14 proteins, p > 0.0001), angiogenesis (8 proteins, p < 0.0001) and TGFβ receptor signaling pathways (3 proteins, p = 0.0017). Inflammation was indirectly covered by terms like “response to corticosteroid” (3 proteins, p < 0.0001), “blood vessel development” (8 proteins, p < 0.0001) and “endothelial cell migration” (4 proteins, p < 0.0001). Blue represent proteins with lower abundance in the tumor, while red indicates upregulation compared to adjacent normal mucosa

Fig. 3

mRNA expression of tissues derived from healthy volunteers and CRC patients [70]. For analyzing gene expression of stage II CRC tumors, we obtained the dataset GSE44076 consisting of tissue expression data of 50 healthy volunteers (healthy) as well as 98 CRC samples (tumor) and paired normal adjacent mucosa (normal). a–f upregulated stromal markers; g: SerpinB5. Overall, expression increased from healthy to normal and again from normal to tumor tissue; h, i for mast cell and macrophage markers, expression was highest in the normal tissue compared to both healthy and tumor tissue, with the tumor showing the lowest expression. Statistical analysis for differential gene expression was perfomed by using empirical Bayesian statistics with FDR detection according to the Benjamini–Hochberg method. *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant

Fig. 4

Consensus Molecular Subtype Classification of upregulated stromal markers and SerpinB5 [34]. For comparing gene expression between the subtypes and selected markers, raw data of published data sets were obtained as used in the consensus subtype classification of CRC [34], processed, analyzed and differences plotted as log2-foldchange (log2-FC). a–e expression of 5 selected stromal markers that were upregulated on protein level in our data set. f results for SerpinB5, a parenchymal derived marker. Statistical analysis of log2-foldchanges were performed by using empirical Bayesian statistics. Adjusted p values were < 0.001 except for those marked non-significant (ns)

Fig. 5

Tissue localization of selected proteins. Serial sections of normal mucosa and tumor tissue were stained using antibodies directed against SerpinB5 (a, b), THBS2 (e, f), SPARC (i, j), and αSMA (m, n). a, e, i, m representative area of normal intestinal mucosa. b, f, j, n representative area of tumor tissue. Scale bars correspond to 500 µm. A selected area was magnified ×20 and is shown in the inserts. Staining intensity for all images was quantified using Definiens software. The diagrams depict the pooled quantification obtained from 26 tumor samples and corresponding normal tissue with regard to overall tumor and normal tissue (c) or tumor and normal epithelium for SerpinB5 (d). For THBS2, quantification results are depicted for total tumor and normal areas (g) or tumor and normal stromal compartments (h). For SPARC, quantification is presented for tumor and normal stromal compartments (k) and specifically for low/high staining intensities (l). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: not significant, according to Mann–Whitney U test