PO-267 Notch-dependent tumourigenic mechanism associated with exosome signalling

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IntroductionTumour microenvironment (TME) plays a crucial role in tumours initiation, progression and response to therapy. It is becoming increasingly clear that one of the most important promoters of tumour growth and progression is a subpopulation of fibroblasts – cancer-associated fibroblasts (CAFs). There is emerging evidence that tumor-derived (TD) exosomes could stimulate TME fibroblasts to acquire an ‘activated’ phenotype that is usually characterised by α-smooth-muscle actin (α-SMA) expression and secretion of growth factors (TGF-β, etc.). Notch signalling pathway is involved in a variety of developmental processes and its alterations are known to play an oncogenic role in tumour progression. Notch activation can also lead to CAFs-mediated tumour growth stimulation, invasion and premetastatic niche formation. This study aims to explore Notch-dependent mechanism responsible for primary tumour cells remote stimulation of CAF phenotype formation during tumour progression.Material and methodsWestern-blot analysis was used for exosome markers evaluation, Notch signalling components and α-SMA expression levels in human colorectal adenocarcinoma cell line (HCT116) and primary human dermal fibroblasts (HF). The α-SMA+ HF proportion was estimated using immunofluorescence microscopy. Female BALB/c nude mice were used for all experiments in vivo. TGF-β levels in conditioned media were estimated by ELISA.Results and discussionsFibroblasts with full-length Notch1 surface exposure (HF Notch1+) were able to stimulate HCT116 xenograft growth when injected in mixture in contrast with HF Notch1-. Moreover, if HCT116 and HF Notch1+ were injected in different mouse sides tumour growth was also enchanced. Luciferase reporter gene system revealed Notch1 signalling transactivation in HF Notch1+ during HF and HCT116 cocultivation in vitro as well as separate HF Notch1+ and HCT116 injection in vivo correlated with increased proportion of α-SMA+ HF. Considering that Notch signalling is mediated via a cell-cell receptor-ligand interactions we proposed a possible exosome-mediated distant Notch1 signalling. TD exosomes analysis revealed Notch ligand Jagged1 enrichment in TD exosomes that could stimulate HF Notch1+ distant activation with subsequent TGF-β-mediated stimulation of tumour growth.ConclusionWe have shown a positive feedback loop between tumour and stromal cells based on exosome-dependent Notch signalling molecular mechanism driving tumour progression. The study was supported by Russian Science Foundation (RSCF), grant No. 14-15-00467.