Overexpression of matrix metalloproteinase 9 in tumor epithelial cells correlates with colorectal cancer metastasis

Abstract

Colorectal cancer mortality largely reflects metastasis, the spread of the disease to distant organs. Matrix metalloproteinase 9 (MMP-9) is a key regulator of metastasis and a target for anticancer strategies in colon cancer. Here, the overexpression of MMP-9 in pure tumor epithelial, but nor stromal, cell populations was associated with metastatic progression of colorectal cancer, as defined by reverse transcriptase-polymerase chain reaction (qRT-PCR) and confirmed by immunostaining. Thus, cancer cell MMP-9 represents a novel, selective prognostic and predictive factor that may be exploited for more effective disease stage stratification and therapeutic regimen selection in patients with colorectal cancer.

Figure 1

Isolation of pure epithelial and stromal cell populations from primary tumors and matched normal mucosa of patients with colorectal cancer. Following collagenase digestion and MACS sorting, the epithelial (MACS elution) or stromal (MACS flow‐through) cells of normal adjacent tissue (NAT) and colorectal tumors (Tumor) isolated from patients were subjected to FACS sorting. Pure intestinal epithelial (red box for PE‐conjugated anti‐CD104) or stromal (green box for FITC‐conjugated anti‐CD45) cell populations were collected for further analysis.

Figure 2

Relative MMP‐9 expression levels in colorectal tumor cell populations exhibit prognostic significance. (A) Stromal and epithelial cells from primary colorectal tumors (Tumor) and normal adjacent tissue (NAT) isolated by MACS and FACS sorting were subjected to RT‐PCR. Sample levels of MMP‐9 mRNA were normalized to respective β‐actin mRNA using the formula 2^{[(MMP‐9 Ct ) −(β‐actin Ct )]}, where Ct is the sample threshold cycle number. The lines connect data from matched specimens from the same patient. The box plots denote median and values from the 25th to 75th percentile; the whiskers denote range of values. (B) Lymph nodes involvement in the different subgroups of patients as defined by the relative expression of tumor cell MMP‐9 compared to the matched normal mucosa cell MMP‐9. ↑MMP‐9 = MMP‐9 overexpression; ↓MMP‐9 = MMP‐9 downregulation; E = tumor epithelial cells; S = tumor stromal cells.

Figure 3

MMP‐9 overexpression in tumor epithelial cells is a biological marker of colorectal cancer metastasis. Association between MMP‐9 overexpression in tumor cell populations and lymph node involvement (left panels). Compared to those with tumor MMP‐9 downregulation, patients with MMP‐9 overexpression in tumor epithelial, but not stromal, cells exhibit advanced disease stage (middle panels) and increased lymph node tumor burden (right panels). N categories and disease stage scores are specified in Table 2 and are based on the AJCC/TNM system. ¹⁹ ↑MMP‐9 = MMP‐9 overexpression; ↓MMP‐9 = MMP‐9 downregulation. The values in the middle and right panels are means ± SEM. *p <0.05 by the Wilcoxon two‐sample test.

Figure 4

Overexpression of tumor epithelial cell MMP‐9 in metastatic colorectal cancer is detected by immunostaining. IHC (left panels) and immunofluorescence (right panels) analyses of primary tumors (Tumor) and matched normal adjacent tissues (NAT) from colorectal cancer patients with metastatic disease progression. For IHC (magnification 40×), the tissues were stained with specific goat polyclonal anti‐MMP‐9 (brown) and hematoxylin (blue, nuclei). For immunofluorescence, the tissues were stained with DAPI (blue, nuclei) and specific antibodies against MMP‐9 (red) and the epithelial‐specific marker β‐catenin (green) and subjected to confocal microscopy (magnification 100×).