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PDF file, 19KB, TUNEL staining of BTC cell lines untreated (NT) and treated (SAR) with Saracatinib at the concentration of 5 �M for 24 hours. A statistical significant increase of apoptosis was seen in all the cell lines ( p-value < 0.05) except in HuH28.

PDF file, 21KB, Ki-67 staining of tumor tissue sections derived from vehicle (A) and Saracatinib treated group (B). A statistical significant decrease of proliferation was seen in treated mice (C) (p-value = 0.0003). NT: vehicle-treated mice.

PDF file, 28KB, Statistical analysis of reduction of cell proliferation by saracatinib in BTC cell lines. A statistical significant inhibition was revealed up to 1.25 �M in all the cell lines, except for TGBC1, in which no significance was found. In x-axis, doses of Saracatinib. In y-axis, absorbance.

PDF file, 9KB, Basal activated Src expression in BTC cell lines.

PDF file, 548KB, Differentially expressed genes obtained in in vitro and in vivo experiments; Saracatinib-deregulated genes both in vitro and in vivo models of BTC.

PDF file, 9KB, Inhibition of cellular foci formation: Saracatinib had the ability to reduce the formation of cellular foci in all the cell lines at Dm of 1 μM after 72 hours of treatment.

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PDF file, 61KB, Clinical pathological characteristics of BTC patients.

Biliary tract carcinoma (BTC) has a poor prognosis due to limited treatment options. There is, therefore, an urgent need to identify new targets and to design innovative therapeutic approaches. Among potential candidate molecules, we evaluated the nonreceptor tyrosine kinase Src, observing promising antitumor effects of its small-molecule inhibitor saracatinib in BTC preclinical models. The presence of an active Src protein was investigated by immunohistochemistry in 19 surgical samples from patients with BTC. Upon saracatinib treatment, the phosphorylation of Src and of its downstream transducers was evaluated in the BTC cell lines TFK-1, EGI-1, HuH28, and TGBC1-TKB. The effect of saracatinib on proliferation and migration was analyzed in these same cell lines, and its antitumor activity was essayed in EGI-1 mouse xenografts. Saracatinib-modulated transcriptome was profiled in EGI-1 cells and in tumor samples of the xenograft model. Src was activated in about 80% of the human BTC samples. In cultured BTC cell lines, low-dose saracatinib counteracted the activation of Src and of its downstream effectors, increased the fraction of cells in G0–G1 phase, and inhibited cell migration. At high concentrations (median dose from 2.26–6.99 μmol/L), saracatinib was also capable of inhibiting BTC cell proliferation. In vivo, saracatinib treatment resulted in delayed tumor growth, associated with an impaired vascular network. Here, we provide a demonstration that the targeted inhibition of Src kinase by saracatinib is of therapeutic benefit in preclinical models of BTC. We propose our results as a basis for the design of saracatinib-based clinical applications. Mol Cancer Ther; 11(7); 1528–38. ©2012 AACR.

PDF file, 11KB, Representative images of p-Src expression in BTC case series. A: Negative control. B-C-D: p-Src expression scored 1+, 2+ and 3+ respectively.