Desmin expression in colorectal cancer stroma correlates with advanced stage disease and marks angiogenic microvessels

Abstract

Introduction: Biomarkers that improve stratification of colorectal cancer patients for adjuvant therapy versus resection alone, or that are predictive of response to therapeutic agents, have the potential to greatly improve patient selection for such therapies. The aim was to determine proteins differentially expressed within the malignant epithelial glands and closely associated stromal elements compared to matched normal mucosa, and to characterise the over-expression of one such protein as a potential biomarker.

Methods: Protein from laser microdissected tumor and normal mucosa was analysed by two dimensional difference gel electrophoresis (2D DIGE) and mass spectrometry to determine differentially over expressed tumor proteins. Tumor over-expression of one such protein, desmin, was quantified using immunofluorescence staining in a larger cohort. Dual staining for desmin and vimentin, or desmin and von Willebrand factor, was performed to determine the cell type of interest.

Results: Desmin expression was significantly increased between stage I and III tumors, (P < 0.0001), and stage II and III tumors, (P < 0.0001). Strong focal desmin expression was found in stroma directly adjacent to carcinomatous glands and microvessels. These cells showed co-localisation of desmin and vimentin in close association with cells expressing VWF, indicating they were pericytes. Significantly higher levels of desmin-positive pericytes were observed in late stage tumors, consistent with increased angiogenesis.

Conclusion: Pericyte coverage of vasculature is a marker of vessel maturation, hence desmin expression may have use as a marker for microvessel maturation. Clinical trials will be needed to determine its use in identifying tumors that will be less responsive to anti-angiogenic therapy.

Figure 1

Scarce labelling 2D DIGE on protein from laser microdissected tissue. A, fixed and stained tumor tissue section (LHS), arrow points to malignant epithelial glands, unfixed and unstained section prior to LMD (centre), and after LMD (RHS). B, a scanned gel image of Cy5-labelled tumor protein with the circled proteins identified as significantly over-expressed in tumor versus matched normal mucosa across the 4 pairs (DeCyder™). Spot 1, identified by MS/MS as desmin shows an apparent molecular weight (MW) of 53 kDa, and isoelectric point (pI) of 5.21.

Figure 2

Desmin immunofluorescence staining. Representative examples of desmin staining with Alexa 488 (green) conjugated secondary antibody, and DAPI nuclei stain (blue) performed on colon tissues. A, tumor section showing stromal desmin staining. B, enlargement of white boxed area in A showing desmin positive cells surrounding a crypt. C, from the matched normal tissue section showing desmin-positive muscularis mucosa across the top of the section. Note the much reduced desmin expression in the normal epithelial tissue. Scale bar = 100 μm.

Figure 3

Scatter plot of % area of desmin staining according to tumor stage. Areas of muscularis mucosa and vasculature were excluded from analysis. Levels of staining were significantly increased between early stage (stage I and II) and stage III tumors, * P < 0.0001.

Figure 4

Desmin and vimentin co-localisation. Dual immunostaining staining performed on stage III tumor using Alexa 488 (green)- and Cy5 (red)- conjugated secondary antibodies for desmin and vimentin respectively, counterstained with DAPI (blue). Co-expression of desmin and vimentin by the same cell results in overlapping of the fluors, and appears yellow. Scale bar = 100 μm. Large amounts of vimentin staining appear between the epithelial crypts given vimentin is a fibroblast/mesenchymal marker and will stain stromal cells, endothelial cells and pericytes. The co-staining pattern of desmin and vimentin appears to be in cross and vertical sections of microvessels. Red arrows indicate areas of tissue stroma, white arrows malignant epithelial glands and yellow arrows cross-sectioned blood vessels showing co-localisation of desmin and vimentin.

Figure 5

Desmin and von Willebrand factor (VWF) immunostaining. Dual immunostaining performed on stage III tumor tissue. Desmin was visualised with an Alexa 488 (green) conjugated secondary and VWF with an Alexa 568 (red) conjugated secondary, counterstained with DAPI (blue), scale bar = 100 μm. Co-localisation of the proteins appears yellow. Red arrows indicate cells positive for VWF, white arrows cells positive for desmin and yellow arrows co-localisation for desmin and VWF. The tumor section shows a blood vessel outlined by cells showing co-localisation of desmin (pericytes) and VWF (endothelial cells) in juxtaposition.