JHIBRG:Abstract Dec 14 2006

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Metastasis, Apoptosis and MMP-9 (Intact Organ Video Microscopic Imaging for Visualizing Metastasis) by Jungwhan Kim
The process of hematogenous metastasis is initiated when tumor cells enter the circulation. These cells then arrest in distant organs. After arrest, survival, proliferation and angiogenesis are needed to result in a macroscopic metastasis. These processes are rarely separated in the study of metastasis as most of the assays use colony formation as an endpoint. The development of assays for pulmonary arrest and for subsequent tumor cell proliferation and survival in vivo now allow the delineation of particular steps of metastasis. While many genes have been shown to affect metastasis in general, the specific points in the metastatic process where most act have not been characterized.

We have developed new methods to evaluate the fate of circulating tumor cells in the lung measuring both pulmonary vascular attachment and apoptosis in vivo. We had previously proposed that a propensity for apoptosis might distinguish metastatic cells from non-metastatic cells, but we did not distinguish between the ability of the tumor cells to attach to the pulmonary endothelium and the induction of apoptosis. Indeed if non-metastatic cells failed to attach, this might account for increased apoptosis. To observe attachment and apoptosis in vivo during the steps of pulmonary metastasis, we have developed imaging methods allowing us to examine the initial (1 hour after infusion) attachment of individual tumor cells to the pulmonary vasculature and to visualize subsequent apoptosis by those attached tumor cells. These methods revealed that similar numbers of metastatic and non-metastatic tumor cell lines were fixed to the pulmonary vasculature one hour after intravenous infusion indicating that initial arrest was not a rate limiting factor in the lung metastatic cascade. By 7 days however, most of the non-metastatic cells had been cleared from the lung yet the metastatic cells had formed small colonies suggesting that the non-metastatic cells might be more susceptible to apoptosis in vivo. Translocation of cytoplasmic BAD-GFP fusion proteins to the mitochondria during apoptosis leads to a punctate fluorescent pattern that allowed us to mark apoptotic events in vivo and provides an assay for apoptosis in living cells. Microscopic observation of the tumor cells transfected with BAD-GFP after intravenous injection into nu/nu mice revealed translocation of BAD-GFP in many more of the attached, non-metastatic melanoma or transformed rat embryo cells over 4- 24h. Metastatic melanoma cells appear to be better able to negotiate the barrier of survival in the circulation after attachment than non-metastatic cells.

Matrix metalloproteinase–9 (MMP-9) has been shown to augment tumor progression and metastasis. However since many metastasis assays do not isolate different steps in the metastatic cascade, the points at which MMP-9 affected metastasis were not known. We show that MMP-9 affects metastasis through its enzymatic activity on the surface of prostate cancer cells leading to enhanced pulmonary arrest. Prostatic carcinoma cells with downregulated MMP-9 arrest less well than cells with MMP-9. Pulmonary metastasis from mice with TRAMP induced tumors is less in MMP-9 deficient than in wild-type mice. Arrest in lungs by prostatic carcinoma cells from TRAMP tumors is enhanced by exposure to MMP-9. MMP-9 supplied either by the host or by the tumor cell acts to enhance arrest of the tumor cells in the lungs.