ENZYMES INVOLVED IN EXTRACELLULAR MATRIX PROTEOGLYCAN´S SYNTHESIS AS A POTENTIAL THERAPEUTIC TARGET FOR COLORECTAL CANCER STEM CELLS

ARIADNA BIROCCO; CURCIO SOFÍA; BLACHMAN AGUSTÍN; CALABRESE GRACIELA; ZLOTOLOW NICOLE

Congreso; Reunión Conjunta SAIC SAIB AAFE NANOMEDar; 2021

Cancer Stem Cells (CSC) are characterized by self-renewal, differentiation, chemoresistance and phenotypic reversibility, which is associated with worse prognosis in tumors. Interaction with the microenvironment is one of the factors related with stemness. The aim of the present work is to analyze the expression of extracellular matrix proteoglycans in CSC derived from cell lines. CSC were enriched by scaffold free 3D culture (colonospheres) of human colorectal cancer cell line HCT116 in the presence of bFGF and EGF, employing ultra-low attachment plates in serum-free culture conditions. After 14 days of culture, microscopy studies were performed to assess colonosphere formation. Stemness was addressed by the expression of master genes SOX2, Nanog and CD44 by RT-PCR. Decorin and biglycan proteoglycan´s core protein expression was also analyzed by RT-PCR. Moreover, glycosaminoglycan and protein quantification was addressed by ionic exchange chromatography of the culture medium followed by colorimetric determination. RT-PCR was performed for the study of glycosaminoglycan synthesis enzyme expression. Bright light microscopy showed colonospheres around 50-100um. The expression of master genes was heterogeneous among cultures correlated with an heterogeneous expression of decorin (number of experiments, n =3). On the other hand, no biglycan expression was detected among different colonospheres (n=3). No differences were registered in glycosaminoglycan/protein ratio among spheres (0,274 ± 0.127). Nevertheless, Chondroitin-4-O- Sulfotransferase (C4ST) expression was detected in colonospheres while no expression was observed for Dermatan-4-O-Sulfotransferase (D4ST). The heterogeneity presented by 3D cultures represents the heterogeneity reported for CSC within tumors and C4ST and D4ST pattern suggests differences in GAG chain´s quality. Therefore, colonospheres are a suitable model for the study of GAG enzymes as potential therapeutic targets.