Matrix metalloproteinase (MMP)-9 in cancer-associated fibroblasts (CAFs) is suppressed by omega-3 polyunsaturated fatty acids in vitro and in vivo

Abstract

Cancer associated fibroblasts (CAFs) are responsible for tumor growth, angiogenesis, invasion, and metastasis. Matrix metalloproteinase (MMP)-9 secreted from cancer stroma populated by CAFs is a prerequisite for cancer angiogenesis and metastasis. Omega-3 polyunsaturated fatty acids (omega-3 PUFA) have been reported to have anti-tumor effects on diverse types of malignancies. Fat-1 mice, which can convert omega-6 to omega-3 PUFA independent of diet, are useful to investigate the functions of endogenous omega-3 PUFA. To examine the effect of omega-3 PUFA on tumorigenesis, TC-1 cells, a murine epithelial cell line immortalized by human papillomavirus (HPV) oncogenes, were injected subcutaneously into fat-1 or wild type mice. Tumor growth and angiogenesis of the TC-1 tumor were significantly suppressed in fat-1 compared to wild type mice. cDNA microarray of the tumors derived from fat-1 and wild type mice revealed that MMP-9 is downregulated in fat-1 mice. Immunohistochemical study demonstrated immunoreactivity for MMP-9 in the tumor stromal fibroblasts was diffusely positive in wild type whereas focal in fat-1 mice. MMP-9 was expressed in primary cultured fibroblasts isolated from fat-1 and wild type mice but was not expressed in TC-1 cells. Co-culture of fibroblasts with TC-1 cells enhanced the expression and the proteinase activity of MMP-9, although the protease activity of MMP-9 in fat-1-derived fibroblasts was lower than that in wild type fibroblasts. Our data suggests that omega-3 PUFAs suppress MMP-9 induction and tumor angiogenesis. These findings may provide insight into mechanisms by which omega-3 PUFAs exert anti-tumor effects by modulating tumor microenvironment.

Figure 1. Tumor growth rates in fat-1 and wild type (WT) mice.

5×10⁶ murine TC-1 cells suspended in 100 µl of DMEM were injected s.c. into each of 10 fat-1 and wild type mice. Tumor volume, based on caliper measurements, was calculated at 7 and 14 days after injection according to the following formula: (tumor volume) = 1/2×(the shortest diameter) ²×(the largest diameter). Mean values with standard deviations are presented. Asterisks indicate those comparisons (fat-1 vs. wild type mice) with statistical significance (p<0.05).

Figure 2. Omega-3 PUFAs suppress tumor vasculogenesis.

CD31 immunostaining of the TC-1 tumor derived from wild type (WT) mice (A) and fat-1 (B). Bars indicate 200 µm. (C) Microvessel densities in TC-1 tumors are expressed as the representative number of labeled vessels in 4 fields (n = 5). Mean values with standard deviations are presented. Asterisks indicate those comparisons (fat-1 vs. wild type mice) with statistical significance (p<0.05).

Figure 3. MMP-9 expression is downregulated in TC-1 tumors from fat-1 mice.

Total RNA was extracted from TC-1 tumors, followed by reverse transcription. MMP-9 mRNA levels were measured by qRT-PCR. Expression levels of MMP-9 were normalized to β-actin as an internal standard (n = 4 in each group). Asterisks indicate those comparisons (fat-1 vs. wild type (WT) mice) with statistical significance (p<0.05). MMP-9 immunostaining of TC-1 tumors derived from wild type (WT) and fat-1 mice. Bars indicate 200 µm in low-power fields, 50 µm in high-power fields.

Figure 4. MMP-9 expression and enzymatic activity in primary-cultured fibroblasts.

(A) Primary fibroblasts isolated from murine lungs were cultured. Total RNA from fibroblasts was reverse transcribed and MMP-9 mRNA levels were measured by qRT-PCR. Expression levels of MMP-9 were normalized to β-actin as an internal standard. Data are the representative of three independent experiments. The data were analyzed by using the Student's t-test. Asterisks indicate those comparisons (fat-1 vs. wild type (WT) mice) with statistical significance (p<0.05). (B) Gelatin zymography: Supernatant from primary fibroblast cultures were collected and separated by electrophoresis. Gelatinase activities were visualized by standard staining techniques.

Figure 5. The increased MMP-9 expression and activity in TC-1/fibroblast co-cultures is inhibited in fat-1 mice.

(A) Isolated fibroblasts were co-cultured with TC-1 cells for 24 hours and expression levels of MMP-9 in the fibroblasts were measured by RT-qPCR. Expression levels of MMP-9 were normalized to β-actin as an internal standard. The data are representative of three independent experiments. The data were analyzed using the Student's t-test. Asterisks indicate those comparisons (fat-1 vs. wild type (WT) mice) with statistical significance (p<0.05). “N.D.” indicates ‘not detected’. (B) Gelatin zymography: Supernatants from fibroblast homotypic cultures and fibroblast/TC-1 co-cultures were collected and separated by electrophoresis. Gelatinase activities were visualized by standard staining techniques. (C, D) For semi-quantitative analyses, gelatin zymography bands were analyzed using image analysis software. Results are represented as mean ±SEM of three independent experiments. The data were analyzed using the Student's t-test. Asterisks indicate those comparisons (fat-1 vs. wild type (WT) mice) with statistical significance (p<0.05).