Hyaluronan in vitro modulation of macrophages in colorectal adenocarcinoma microenvironment

SPINELLI FIORELLA; VITALE DAIANA; ALANIZ LAURA

Atenas

Conferencia; 2nd Matrix Biology Europe (MBE) Conference; 2016

Hyaluronan(HA), a glycosaminoglycan normally present in the extracellular matrix, acts as a modulator of immune and angiogenic responses(1). At homeostasis, high-molecular weight HA (HMW) is predominant, having hydrodynamic properties whereas the low-molecular weight (LMW) form is present mainly during inflammation(1). Macrophages are able to bind HA, previous studies showed that LMW HA polarize macrophages to M1 phenotype in vitro whereas HMW HA is associated with a M2 activation(2). HA present in tumor microenvironment serves as a signal for recruiting tumor associated macrophages(3), that are able to regulate angiogenesis releasing pro-angiogenic factors(1,4).Aim: To evaluate the effect of HA, in its HMW and LMW form, on colorectal adenocarcinoma tumor-pulsed human macrophages using the THP-1 cell line. Methods: The human monocytic cell line THP-1 was differentiated into active macrophages with PMA (25ng/ml). 72h later, were pulsed with tumor lysates(TL) (prepared with LoVo colorectal adenocarcinoma cell line) and treated with HA (20ug/ml) LMW (1,5-1,8x106Da) or HMW (1-3x105Da) for 24h. Macrophages were characterized with CD80 and MHC II by flow cytometry. Gene expression levels of CD44 and TLR4, were analyzed through RT-qPCR. IL10, IL12 and VEGF biosynthesis were evaluated by ELISA assay and MMP-2 by zymography. Results: When THP-1 macrophages were treated with TL alone and together with HA, HMW or LMW, an up-regulation of MHC II and CD80 was observed, levels similar to M1 control. When pulsed with TL plus HA HMW, the biosynthesis of IL-12, IL-10 and VEGF diminished. However, in the presence of TL alone and TL plus HA LMW, their synthesis was higher. We found increased levels of MMP-2 activity when pulsed with TL plus HA HMW and a lower increase when treated with TL plus HA LMW, in comparison to basal conditions. We found significate differences in TLR4 expression when treated with TL alone and together with HA LMW. Conclusion: HA is able to modulate macrophages phenotype in the tumor microenvironment as well as their angiogenic capacity, key aspects for tumor development. Our results provide the first evidence that in tumor context HA is able to modulate not only macrophages phenotype, but also their cytokines profile and involvement in tumor angiogenesis.Bibliography: (1)Spinelli FM et al. The immunological effect of hyaluronan in tumor angiogenesis. Clin & Trans Immunol 2015; 4:e52; (2)Rayahin JE et al. (2016) High and Low Molecular Weight Hyaluronic Acid Differentially Influence Macrophage Activation. ACS Biomaterials Science and Engineering 7:481?493; (3)Kobayashi N et al. Hyaluronan deficiency in tumour stroma impairs macrophage trafficking and tumour neovascularization. Cancer Res 2010; 70:7073?7083; (4)Murdoch C & Lewis CE. Macrophage migration and gene expression in response to tumour hypoxia. Int J Cancer 2005; 117:701?708.