FIBROBLAST ACTIVATION PROTEIN IS OVEREXPRESSED IN INFLAMED INTESTINAL MUCOSA AND MAY BE MODULATED BY MIR-21

ANAHÍ RENDÓN SORIA; EMANUEL BARBIERA ROMERO; MALENA FERREYRA COMPAGNUCCI; MARÍA BELEN POLO; MANUELA ILID; ANDRÉS ROCCA; ALICIA SAMBUELLI; JUAN ARRIOLA; MARÍA BELÉN SARACHO; KARINA COLLIA; MARÍA VIRGINIA GENTILINI; GABRIEL GONDOLESSI; GUSTAVO J. CORREA; MARTÍN YANTORNO; MARÍA VICTORIA MERCADER; JULIO DE MARÍA; MARTÍN RUMBO; GUILLERMO HORACIO DOCENA; CECILIA ISABEL MUGLIA; RENATA CURCIARELLO

San Luis

Congreso; LXXI Reunión Anual de la Sociedad Argentina de Inmunología; 2023

Sociedad Argentina de Inmunología

Fibroblast activation protein (FAP) is a transmembraneendopeptidase present in cancer associated fibroblasts, which contributes toextracellular matrix remodeling and mesenchymal cell activation, favoringtumoral growth. FAP overexpression has been reported in lung and breast cancer,under microRNA-21 (miR-21) regulation. Nevertheless, FAP role is not completelydescribed in colorectal cancer (CRC). We previously showed miR-21overexpression in inflammatory bowel disease (IBD) patients´ mucosa and intestinalfibroblasts. IBD is a predisposing condition for CRC development; we aim toinvestigate FAP expression in the colonic mucosa of patients with chronicintestinal inflammation. Colonic biopsies and surgical samples were taken frommacroscopically inflamed and uninflamed mucosa from patients with IBD, CRC,polyps, or healthy controls (HC). Mucosal pieces were fixed and staining forFAP and α-SMA was performed by indirect immunofluorescence (IF) . Total RNA wasextracted from mucosal samples, and retro-transcribed. qPCR was performed oncDNA using specific primers for miR-21,  fap, α-smaand HPRT.Relative expression wasnormalized to U6 for miRNA or to HPRT for the rest, and data were analyzedusing the comparative threshold method (2−ΔCt). Fibroblast primary cultures wereestablished after isolating lamina propria cells by mechanical and enzymaticdigestion of biopsies or tissue sections. Cells were cultured in DMEM Glutamax20% FBS with antibiotics. Fibroblast culture supernatants were collected andultracentrifuged at 100.000xg for 2 h for exosome enrichment, which werevisualized by atomic force microscopy. Mi-R21 expression was evaluated by qPCRin cells and exosome fractions. In vitroinduction of FAP was evaluated on fibroblasts by IF, after stimulation withTGF-β (1 and 10 ng/ml) or exosomal fraccion for 24 h. Images were acquired withLeica SP5 confocal microscope. FAP and α-SMA merge images were analyzed withImage J and QuPath softwares. We observed an overexpression of miR-21 and its targetgenes in the intestinal mucosa and fibroblasts from IBD and CRC patients,compared to HC (p<0,05). IF staining of FAP and α-SMA in colonic biopsiesshowed higher expression of these proteins in the stroma of inflamed tissue compared with HC (p<0,05),and both signals colocalized in inflamed areas. Incubation of fibroblasts withTGF-β 10 ng/ml or miR-21 rich exosomal fractions induced FAP expression, 30%and 91% FAP+ cells,respectively vs. 3%FAP+ cells in unstimulated conditions.   We conclude that miRNA-21 overexpression in stromalcells from chronic inflamed mucosa may participate in FAP activation pathways,implying the remodeling signature and increasing the risk of CRC development in IBD patients.