Role of Matrix Metalloproteinase-9 in LHBETATAG Retinal Tumor Growth

Abstract

Purpose: : Metalloproteinase-9 (MMP-9), a gelatinase, is essential for the extracellular matrix (ECM) remodeling required for tumor growth and angiogenesis. Macrophage secreted MMP-9 is essential for the growth of several tumors including ovarian and melanoma tumors. The purpose of this study is to analyze the role of MMP-9 in LH_BETAT_AG retinal tumor growth.

Methods: : This study was approved by the IACUC and follows ARVO guidelines. Eyes were enucleated from twenty-four LH_BETAT_AG mice at 4, 8, 12 and 16 weeks of age (n=6 for each age group). MMP-9 density was detected by immunohistochemistry. To analyze the role of MMP-9 on tumor growth, LH_BETAT_AG MMP-9 knock-out mice were created through two cross breedings: (1) MMP-9 knock-out mice with LH_BETAT_AG mice to produce an F1 generation, and (2) a dihybrid cross between F1 MMP-9 heterozygotes and LH_BETAT_AGMMP-9 heterozygotes to produce retinal tumors with the MMP-9 knock-out gene. Gelatinase MMP-9 activity was measured using in situ zymography and immunohistochemistry.

Results: : Immunohistochemistry of MMP-9 gelatinase activity showed that MMP-9 expression increases with tumor progression, being most prevalent in advanced disease at 16 weeks of age (p=0.0339), confirming in situ zymography results. Tumor burden significantly increased by 12-fold in the MMP-9 knock-out mice. Homozygous MMP-9-(+)(+) mice presented with small-size tumors (0.72 mm²), whereas heterozygous MMP-9-(+)(-) mice presented tumors that increased in size by 305% (2.2 mm²) and homozygous MMP-9-(-)(-) mice presented large tumors that increased in size by 1153% (8.3 mm²).

Conclusions: : We have previously shown that MMP-9 activity increases with tumor progression. We have further shown that intraocular treatment with anti-angiogenic therapy reduces MMP-9 protein levels and activity and reduces tumor burden. In this study we demonstrate that depletion of MMP-9 results in increased tumor burden. We hypothesize that these unexpected results may be explained by: 1) MMP-9 gelatinase activity may be involved in tumor suppression through MI polarized macrophages, which cause tissue destruction; and 2) lack of MMP-9 results in overexpression of other gelatinases such as MMP-2.

Keywords: retina • tumors • immunomodulation/immunoregulation