Integrated analysis of genomic and transcriptomic profiles to examine CD73/NT5E as a driver of drug-resistance and immunosuppression in biliary cancer (BTC).
Authors: Carotenuto Pietro , Salati Massimiliano , Barbato Anna , Piscopo Fabiola , Evangelista Lorenzo , Gambardella Gennaro , Sarnataro Sergio , Petrillo Angelica , Daniele Bruno , Pisapia Pasquale , Iaccarino Antonino , Salatiello Maria , Spallanzani Andrea , Gelsomino Fabio , Malapelle Umberto , Iuliano Antonella , Troncone Giancarlo , Reggiani Bonetti Luca , Dominici Massimo , Franco Brunella 
Published: January 2023

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Background: Despite the improved outcome achieved by the addition of the anti-PD-L1 durvalumab to standard chemotherapy, the vast majority of BTC do not benefit from chemo-immunotherapy. Immunosuppressive microenvironment is a dominant feature of BTC, involved in tumour progression and drug resistance. Here, we investigated the biological role and treatment implications of the adenosine-producing enzyme CD73 in a clinically-annotated cohort of BTC. CD73, an ecto-5’-nucleotidase (NT5E), creates an immunosuppressive tumour-promoting microenvironment by converting ATP to adenosine. Methods: Immunohistochemistry for CD73, CD4/CD8 and FOXP3 as well as whole-exome and transcriptomic sequencing were performed on resected specimens of 80 BTC (Illumina Platform). Spatial Transcriptomics was performed by using Visium Spatial Gene Expression-10x Genomics). Silencing of CD73 was obtained by transient transfection od CD73-siRNA, while genetic Knock-out by using CRISPR-Cas system technology in two BTC cell lines. Tumor growth was assessed in 2D and in 3D cell culture by using MTS assay and spheroid growth analysis before and after treatment with selected drugs. Statistical and Survival, correlation analyses were performed. Results: High CD73 expression (CD73high) was associated with older age (>70 years, p=0.01), gallbladder subsite (p=0.03), and nodal involvement (p=0.04). CD73high tumours were significantly enriched in infiltrating FOXP3+ T lymphocytes (p<0.001). CD73high status was independent predictor of poorer prognosis at the multivariate analysis (p=0,03) together with ECOG PS ≥2 (p=0,001) and the pathological stage (p=0,025) and was associated with a remarkably shorter RFS in patients treated with adjuvant chemotherapy (p=0,011). At the transcriptomic level, CD73high tumours were significantly enriched in upregulated EMT, TNF-alfa/NFKB, hypoxia and G2/M checkpoint signaling pathways and p53, BMI1, MEL18, EGFR and K-RAS genes. In in vitro models, siRNA-mediated depletion and CRISPR-CAS9 gene KO of CD73 sensitized both BTC 2D and 3D culture to cisplatin/gemcitabine treatment. The pharmacological inhibition of CD73 by AMCP improved the sensitivity of BTC cell lines to cisplatin/gemcitabine treatment. Finally, Spatially resolved transcriptomics of CD73high revealed a critical role of CD73 in tumor immunity and therapeutic response. Conclusions: We showed that CD73high BTC display aggressive biological features, poorer prognosis and resistance to standard chemotherapy. The therapeutic targeting of this adenosinergic ectonucleotidase by clinically-available compounds has the potential to enhance the efficacy of conventional treatment in BTC.