PO-243 Exosomes from plasma of HNSCC patients treated with photodynamic therapy are biomarkers for epithelial-mesenchymal transition

Abstract

IntroductionPhotodynamic therapy (PDT) is a palliative treatment option for patients with head and neck squamous cell carcinoma (HNSCC). We and others have shown that PDT induces a local inflammatory reaction with the potential to initiate antitumor immune responses. Recent studies indicate that cancer cells can change their morphology after PDT due to cytoskeleton alterations and decreased cell adhesion. To see whether the cargo of exosomes released by these cancer cells reflects cellular alterations after PDT, plasma was collected from PDT-treated patients prior to and at time points after therapy for exosome isolation and molecular characterisation.Material and methodsHNSCC patients (n=9) were treated with PDT in a palliative setting. All patients had previously undergone several oncologic treatment regimens. Blood samples were taken before and after PDT. Exosomes were isolated from plasma by mini size exclusion chromatography and were co-incubated with cancer cells. Following co-incubation, tumour cell migration, proliferation and chemotaxis were measured. Expression on tumour cells of the EMT markers: Vimentin, EpCAM (by flow cytometry, PCR, immunofluorescence), Snail, Twist, ZEB1, Slug (by PCR), E-Cadherin, N-Cadherin (by flow and PCR) was determined. Total plasma exosomes were separated by immune capture on beads into CD3 +and CD3neg fractions. The CD3neg exosomes enriched in tumor-derived vesicles were tested by on-bead flow cytometry for the presence of E-Cadherin- and N-Cadherin.Results and discussionsExosomes harvested pre- and 24 hour after PDT contained high levels on N-Cadherin. In contrast exosomes isolated on day 7 and 4–6 weeks after PDT contained high levels of E-Cadherin. Exosomes collected before and 24 hour post PDT and co-incubated with tumour cells altered cell morphology and induced mesenchymal features, including co-expression of Vimentin, N-Cadherin, snail, slug, twist and ZEB1. In functional experiments, exosomes collected pre- and 24 hour after PDT significantly enhanced migration, proliferation and chemotaxis of tumour cells relative to exosomes harvested at two later time points.ConclusionWe have shown for the first time that PDT can change the mesenchymal character of tumour cells converting it into an epithelial phenotype, and that exosomes in plasma of the PDT –treated patients were responsible for mediating the conversion. In addition, plasma-derived exosomes served as biomarkers of response to PDT, as their molecular cargo reflected the phenotypic changes occurring in the tumour cells during therapy.