Tumour-site-dependent expression profile of angiogenic factors in tumour-associated stroma of primary colorectal cancer and metastases

Abstract

Background: Tumour-associated stroma has a critical role in tumour proliferation. Our aim was to determine a specific protein expression profile of stromal angiogenic cytokines and matrix metalloproteinases (MMPs) to identify potential biomarkers or new therapy targets.

Methods: Frozen tissue of primary colorectal cancer (n=25), liver (n=25) and lung metastases (n=23) was laser-microdissected to obtain tumour epithelial cells and adjacent tumour-associated stroma. Protein expression of nine angiogenic cytokines and eight MMPs was analysed using a multiplex-based protein assay.

Results: We found a differential expression of several MMPs and angiogenic cytokines in tumour cells compared with adjacent tumour stroma. Cluster analysis displayed a tumour-site-dependent stromal expression of MMPs and angiogenic cytokines. Univariate analysis identified stromal MMP-2 and MMP-3 in primary colorectal cancer, stromal MMP-1, -2, -3 and Angiopoietin-2 in lung metastases and stromal MMP-12 and VEGF in liver metastases as prognostic markers (P>0.05, respectively). Furthermore, stroma-derived Angiopoietin-2 proved to be an independent prognostic marker in colorectal lung metastases.

Conclusion: Expression of MMPs and angiogenic cytokines in tumour cells and adjacent tumour stroma is dependent on the tumour site. Stroma-derived MMPs and angiogenic cytokines may be useful prognostic biomarkers. These data can be helpful to identify new agents for a targeted therapy in patients with colorectal cancer.

Figure 1

Twenty-five samples of primary colorectal cancer (CRC), 25 samples of colorectal liver metastases (liver) and 23 samples of colorectal lung metastases (lung) were subjected to laser microdissection to obtain separately tumour and stroma tissues. After lysis, protein expression was determined using the new technology of a multiplex-based angiogenic cytokine and MMP assay. Expression analysis included angiopoietin-2, follistatin, G-CSF, HGF, IL-8, leptin, PDGF-BB, PECAM-1, VEGF, MMP-1, -2, -3, -7, -9, -10, -12 and -13 in tumour epithelial cells (red) and tumour-associated stroma (green). Unit: pg ml⁻¹. Each dot represents a single analysis of one tumour/stroma sample. P-values were adjusted for multiple testing using Hochberg's method. *P<0.05.

Figure 2

Tumour-site-dependent expression of MMPs and angiogenetic factors. Cluster analysis including all tumorous and stromal MMPs and angiogenic cytokines reveals a distinct expression pattern discriminating between primary colorectal cancer and lung metastases vs colorectal liver metastases. The expression pattern of colorectal liver metastases is characterised by a higher abundance of angiogenic cytokines and a lower expression of MMPs compared with primary colorectal cancer and lung metastases.

Figure 3

Kaplan–Meier curves display cancer-specific survival in correlation with (A) stromal expression of MMP-2 in primary colorectal cancer, (B) stromal expression of MMP-3 in patients with primary colorectal cancer, (C) stromal expression of MMP-12 in patients with colorectal liver metastases, (D) stromal expression of VEGF in patients with colorectal liver metastases, (E) stromal expression of MMP-1 in patients with lung metastases, (F) stromal expression of MMP-2 in patients with lung metastases, (G) stromal expression of MMP-3 in patients with lung metastases, (H) stromal expression of angiopoietin-2 in patients with lung metastases.