Src-signaling interference impairs the dissemination of blood-borne tumor cells

Abstract

Although solid tumors continuously shed cells, only a small fraction of the neoplastic cells that enter the blood stream are capable of establishing metastases. In order to be successful, these cells must attach, extravasate, proliferate and induce angiogenesis. Preclinical studies have shown that small-molecule ATP-competitive Src kinase inhibitors can effectively impair metastasis-associated tumor cell functions in vitro. However, the impact of these agents on the metastatic cascade in vivo is less well understood. In the present studies, we have examined the ability of saracatinib, a dual-specific, orally available inhibitor of Src and Abl protein tyrosine kinases, to interfere with the establishment of lung metastases in mice by tumor cells introduced into the blood stream. The results demonstrate that Src inhibition most effectively interferes with the establishment of secondary tumor deposits when treatments are administered while tumor cells are in the initial phases of dissemination.

Figures

Fig. 1

Median number (a) and sizes (b) of neoplastic cell foci in the lungs of mice determined 21 days after iv injection of KHT sarcoma cells. Beginning the day after cell injection, the mice were treated daily with either the drug carrier or saracatinib (10 or 25 mg/kg). Bars and hatched areas = 90% and 75% confidence intervals, respectively; stars = statistical significance of p < 0.05 (Wilcoxon’s rank-sum test) compared with controls.

Fig. 2

Median number of neoplastic cell foci in the lungs of mice determined 21 days after iv injection of KHT sarcoma cells. In (a) 5×103 (squares, triangles) and 2×103 (circles) KHT cells were injected. In (b) 2×103 KHT cells were used. Treatment in (a) was daily 10 mg/kg doses of saracatinib commencing on day 1; treatment in (b) was 10 mg/kg administered daily on days 3–7 and 10–14. Bars and hatched areas = 90% and 75% confidence intervals, respectively; stars = statistical significance of p < 0.05 (Wilcoxon’s rank-sum test) compared with controls.

Fig. 3

Median number of lung colonies assessed 21 days post tumor cell injection in mice treated with daily doses of 10 mg/kg saracatinib either on days 3–7 or 10–14 (a) or days 3–7 or 17–21 (b). Bars and hatched areas = 90% and 75% confidence intervals, respectively; stars = statistical significance of p < 0.05 (Wilcoxon’s rank-sum test) compared with controls.

Fig. 4

Median number of KHT sarcoma lung colonies determined 21 days after injecting 5×103 (circles) or 2×103 (squares, triangles) tumor cells. Saracatinib treatment(s) consisted of a single 10 mg/kg dose administered on day 0, four doses of 10 mg/kg given on days 0–3, or daily doses of 10 mg/kg administered through the course of the experiments. Bars and hatched areas = 90% and 75% confidence intervals, respectively; stars = statistical significance of p < 0.05 (Wilcoxon’s rank-sum test) compared with controls.

Fig. 5

Number of non-adherent KHT sarcoma cells remaining in the supernatant 2 h after 1×106 tumor cells were added to a monolayer of endothelial cells. Tumor cells were pretreated with 0.1% DMSO (control) or saracatinib (0.5, 2.5 or 5 μM) for 24 h prior to their addition to the endothelial cells. After 2 h, the number of tumor cells remaining unattached in the supernatant was counted. Data are the mean number of cells in 6-well plates (± SE for 3 experiments); stars = P<0.05 versus control.

Fig. 6

The effect of a 24 h exposure of saracatinib (1–10 μM) on HMVEC-L Src and Src phosphorylation (a), tube formation (b–e), and motility (f) are illustrated. Bars in b–e equal 100 μ. The level of VEGF determined in the media of KHT sarcoma cells after a 24 h saracatinib treatment is shown in (g). Data are the results from three independent experiments ± SE; stars = P<0.05 versus control.

Fig. 7

The effect of saracatinib on the ability of KHT sarcoma cells to induce angiogenesis was determined by either pretreating the tumor cells for 24 h with saracatinib (0–5 μM) prior to injection (a) or treating the mice with daily 10 mg/kg doses of saracatinib post tumor cell inoculation (b). Bars and hatched areas = 90% and 75% confidence intervals, respectively; stars = statistical significance of p < 0.05 (Wilcoxon’s rank-sum test) compared with controls.