(A) The indicated cell lines were treated (in independent experiments, performed in triplicate) with different doses of cabozantinib (µM), for the indicated times. (B) Percentage of dead cells was measured by exclusion of propidium iodide (PI). Cells were treated as in (A). (C) After 72 h of treatment, cells were plated at low density, and colonies were scored after at least 1 week of incubation (in the absence of the drug). A full-colour version of this figure can be found at https://doi.org/10.1530/ERC-23-0232.

(A) The indicated cell lines were treated (in independent experiments, performed in triplicate) with different doses of cabozantinib (µM), for the indicated times. Distribution of cells in the different phases of cell cycle was measured by propidium iodide. (B) BON1 cells were stained for DNA content: arrows point to cells arrested during mitosis in a polynucleated state. A full-colour version of this figure can be found at https://doi.org/10.1530/ERC-23-0232.

BON1 cells were treated with different doses of cabozantinib or sunitinib (µM), for the indicated times. (A) Analysis of MET phosphorylation. (B) Analysis of mTOR pathway. (C) Analysis of MCL-1 levels.

Engraftment of NEN cells in zebrafish embryos. Representative epifluorescence images of Tg(fli1a:EGFP)^y1 embryos injected with PBS (control; A) and implanted with red-stained BON-1 cells (B, B’ and C), NCI-H727 cells (D, D’ and E) and NCI-H720 cells (F, F’ and G). Embryos were imaged at 24 h post injection (hpi). The red channel was omitted in panels B, B’, D, D’, F and F’ to facilitate the observation of tumor-induced angiogenesis (green). NEN cells stimulated endothelial sprouting from the SIV plexus within 24 hpi. B’, D’ and F’ are the digital magnification of white-boxed regions. All images are oriented so that rostral is to the left and dorsal is at the top. Scale bar, 100 μm. The graph H showed the quantification of tumor-induced angiogenesis at 24 hpi. NCI-H720 value have been set to 1.0. The graph I showed the quantification of tumor-induced angiogenesis in embryos after 24 h of treatment with DMSO and CAB (0.25 and 2.5 µM). Control (DMSO) values have been set to 1.0. The values reported in the graphs represent the mean ± s.e.m. ^*P < 0.05;^***P < 0.001. A full-colour version of this figure can be found at https://doi.org/10.1530/ERC-23-0232.

Invasiveness of NEN cells in grafted zebrafish embryos. Overlay of representative fluorescent and bright field images of embryos grafted with red-stained BON-1 (A–C), NCI-H727 (D–F) and NCI-H720 (G–I) cells at 0 (A, D, G) and 48 hpi (B, C, E, F, H, I). For each injected cell line, the tail region was imaged at 48 hpi (C, F, I), showing the spread of NEN cells throughout the embryo body. All images are oriented so that rostral is to the left and dorsal is at the top. Scale bar, 100 μm. The graph J showed the quantification of NEN spread in the tail region at 48 hpi. NCI-H727 value has been set to 1.0. The graph K showed the quantification of NEN spread after 48 h of treatment with DMSO and CAB (0.25 and 2.5 µM). Control (DMSO) values have been set to 1.0. The values reported in the graphs represent the mean ± s.e.m. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001. A full-colour version of this figure can be found at https://doi.org/10.1530/ERC-23-0232.

BON1 cells were inoculated into nude mice, and treatment was performed as described in the main text. (A) Tumor size. (B) Representative hematoxylin–eosin staining of tumors at the end of treatment. A full-colour version of this figure can be found at https://doi.org/10.1530/ERC-23-0232.