5. GENERATION OF HUMAN INTESTINAL ORGANOIDS FROM INTESTINAL INFLAMED TISSUES.

MARÍA BELÉN POLO ; EMANUEL BARBIERA ROMERO; JULIÁN VACCARO; MANUELA ILID; LORENA MENENDEZ; PAULA BOROBIA; CECILIA ZUBIRI; ANABELLA ZOSI; LUCIANA GUZMAN; VIVIANA BERNEDO; EUGENIA M. ALTAMIRANO; GUSTAVO J. CORREA; MARTÍN YANTORNO; ALICIA SAMBUELLI; ANDRÉS ROCCA; BELÉN SARACHO; KARINA COLLIA; MARÍA VIRGINIA GENTILINI; GABRIEL GONDOLESI; RENATA CURCIARELLO; GUILLERMO H. DOCENA; CECILIA I. MUGLIA

Otro; LXXI REUNIÓN CIENTÍFICA ANUAL DE LA SOCIEDAD ARGENTINA DE INMUNOLOGÍA (SAI).; 2023

Fibroblast activation protein (FAP) is a transmembrane endopeptidase present in cancer associated fibroblasts, which contributes to extracellular matrix remodeling and mesenchymal cell activation, favoring tumoral growth. FAP overexpression has been reported in lung and breast cancer, under microRNA21 (miR-21) regulation. Nevertheless, FAP role is not completely described in colorectal cancer (CRC). We previously showed miR-21 overexpression in inflammatory bowel disease (IBD) patients´ mucosa and intestinal fibroblasts. IBD is a predisposing condition for CRC development; we aim to investigate FAP expression in the colonic mucosa of patients with chronic intestinal inflammation.Colonic biopsies and surgical samples were taken from macroscopically inflamed and uninflamed mucosa from patients with IBD, CRC, polyps, or healthy controls (HC). Mucosal pieces were fixed and staining for FAP and αSMA was performed by indirect immunofluorescence (IF). Total RNA was extracted from mucosal samples, and retro-transcribed. qPCR was performed on cDNA using specific primers for miR-21, fap, α-sma and HPRT. Relative expression was normalized to U6 for miRNA or to HPRT for the rest, and data were analyzed using the comparative threshold method (2−ΔCt). Fibroblast primary cultures were established after isolating lamina propria cells by mechanical and enzymatic digestion of biopsies or tissue sections. Cells were cultured in DMEM Glutamax 20% FBS with antibiotics. Fibroblast culture supernatants were collected and ultracentrifuged at 100.000xg for 2 h for exosome enrichment, which were visualized by atomic force microscopy. MiR21 expression was evaluated by qPCR in cells and exosome fractions. In vitroinduction of FAP was evaluated on fibroblasts by IF, after stimulation with TGFβ (1 and 10 ng/ml) or exosomal fraccion for 24 h. Images were acquired with Leica SP5 confocal microscope. FAP and α-SMA merge images were analyzed with Image J and QuPath softwares.We observed an overexpression of miR-21 and its target genes in the intestinal mucosa and fibroblasts from IBD and CRC patients, compared to HC (p