TYROSINE KINASE INHIBITORS IN NEUROENDOCRINE TUMORS: FROM IN VITRO TO ZEBRAFISH MODEL

(1) Background: Neuroendocrine neoplasms (NENs) are a group of tumors that arise from neuroendocrine cells throughout the body, with the lungs and gastrointestinal tract being the most common sites of origin. In patients with NENs and distant metastases, surgery is generally not curative. Although well-differentiated and low-grade NENs, classified as neuroendocrine tumors (NETs), are usually less aggressive than poorly-differentiated NENs, they can develop distant metastases in about 15% of cases. These patients require chronic medical management. However, the clinical efficacy of these treatments is limited by the low objective response rate, due to the occurrence of tumor resistance and the high biological heterogeneity of these neoplasms. (2) Research problem: We addressed this study on two rare NETs: lung neuroendocrine tumors (LNETs) and medullary thyroid carcinoma (MTC). LNETs represent about 2% of lung tumors, while MTCs are rare thyroid tumors caused by mutations in the RET proto-oncogene. Both NETs are well-differentiated neoplasms and are known to be highly vascularized. Therefore, they represent a potential target for tyrosine kinase inhibitors (TKIs) selective for receptors involved in angiogenesis. The aim of this project was to evaluate the antitumor activity of several new TKIs both in vitro, using LNETs (NCI-H727, UMC-11 and NCI-H835) and MTC (TT and MZ-CRC-1) cell lines, and in vivo, adopting a novel zebrafish xenograft model to study angiogenesis. In LNETs we tested: sulfatinib, a small molecule that inhibits the Vascular Endothelial Growth Factor Receptor (VEGFR) 1, 2, and 3, and the Fibroblast Growth Factor Receptor type 1 (FGFR1); cabozantinib, a multi-target inhibitor selective for VEGFR2, c-Met, Kit, Axl and Flt3; and axitinib, a multi-target TKI of VEGFR1, 2, 3 and Platelet-Derived Growth Factor Receptor-beta (PDGFRβ). In MTC we tested: sulfatinib; SPP86, a RET-specific inhibitor; and SU5402, an inhibitor of the FGFR1 and VEGFR2. (3) Methodology: In LNETs and MTC cells the effects of selected TKIs have been evaluated in vitro through: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays, for assessing cell viability; flow-cytometer analysis, for the evaluation of cell cycle and apoptosis; and wound-healing assay, to study cell migration. In vivo we took advantage of the transgenic zebrafish line of Tg(fli1a:EGFP)y1. Through the xenotransplantation of NET cells in the subperidermal space near the subintestinal vein, we assessed the effects of TKIs on tumor-induced angiogenesis and cancer dissemination. (4) Key Results: In LNET cell lines we observed a dose-dependent decrease in cell viability after incubation with all TKIs. This effect seems to be related to the perturbation of the cell cycle and induction in apoptosis. In NCI-H727 wound healing assay showed a significant reduction in cell migration only after incubation with cabozantinib. In the zebrafish model, we found a significant reduction of the tumor-induced angiogenesis in implanted LNET cell lines after treatment with all TKIs. Cabozantinib and axitinib were more potent than sulfatinib in inhibition of angiogenesis, while cabozantinib was the most efficient in reducing cell migration from the transplantation site to the tail. In MTC cell lines, sulfatinib, SU5402 and SPP86 showed a decrease in cell viability, confirmed by the significant reduction in S phase cell population. Moreover, sulfatinib and SPP86 showed for both cell lines a significant induction of apoptosis. Sulfatinib and SPP86 inhibited the migration of TT and MZCRC-1 cells, evaluated through the wound healing assay, while SU5402 was able to inhibit migration only in TT cells. In vivo we observed a significant reduction of TT cells-induced angiogenesis in zebrafish embryos after treatment with sulfatinib and SPP86. (5) Conclusions: Despite sulfatinib resulted the most potent compound in terms of inhibition of LNET cell proliferation, cabozantinib showed in vivo the most effective impact in reducing tumor-induced angiogenesis. Cabozantinib was the only TKI able to inhibit in vivo the dissemination of implanted LNET cells. According to these data, cabozantinib could represent a potential candidate in the therapy of patients with highly vascularized LNET. In MTC cell lines, SPP86 and sulfatinib displayed a similar antitumor activity both in vitro and in vivo, suggesting a good efficacy of specific RET inhibitors (SPP86) with potentially less adverse effects than multitarget TKIs (sulfatinib). In addition, this study showed that the zebrafish model for NETs represents an innovative tool for drug screening with several advantages compared with rodent models: rapidity of procedure, animal immune suppression is not required, lower number of tumor cells for implant and the optical transparency provides a real-time monitoring of cell-stromal interactions and cancer progression in living animals.