Innovative Immunohistochemistry Identifies Mmp-9 Expressing Macrophages At The Invasive Front Of Murine Hcc

AIM: To investigate the proteolytic contribution of tumor-associated macrophages (TAM) in tumor invasion, we analyzed whether TAM at the invasive front of small HCC in Abcb4(-/-)-mice show an enhanced expression of MMP-9.METHODS: Liver cryosections of the hepatocellular carcinoma (HCC) invasive front from 12 mo old Abcb4(-/-)-mice were stained for collagen type. and MMP-9 using Alexa488 and Alexa568 labeled secondary antibodies. Afterwards, the Alexa568 dye was bleached and the macrophage marker F4/80 was visualized using Alexa568 labeled secondary antibodies. Finally, photographs of the invasive tumor front were digitally overlaid and analyzed.RESULTS: After complete bleaching of the primary dye, specific fluorescence staining of a third antigen, here F4/80, was successfully performed on the same histological section. With this method, we were able to identify conglomerates of matrix metalloproteinase (MMP-9) expressing macro-phages within the tumor capsule of HCC.CONCLUSION: MMP-9 expressing macrophages are involved in matrix remodelling at the invasive tumor front of HCC. The described staining protocol provides a simple yet powerful extension of conventional immuno- histochemistry, facilitating visualization of at least three different antigens plus nuclei in one single histological section. (C) 2010 Baishideng. All rights reserved.