Abstract
Metastasis is a multistep process, during which circulating tumor cells traffic through diverse anatomical locations. Stable inducible marking of tumor cells in a manner that is tightly spatially and temporally controlled would allow tracking the contribution of cells passing through specific locations to metastatic dissemination. For example, tumor cells enter the lymphatic system and can form metastases in regional lymph nodes, but the relative contribution of tumor cells that traffic through the lymphatic system to the formation of distant metastases remains controversial. Here, we developed a novel genetic switch based on mild transient warming (TW) that allows cells to be marked in a defined spatiotemporal manner in vivo. Prior to warming, cells express only EGFP. Upon TW, the EGFP gene is excised and expression of mCherry is permanently turned on. We employed this system in an experimental pancreatic cancer model and used localized TW to induce the genetic switch in tumor cells trafficking through tumor-draining lymph nodes. Thereby we found that tumor cells disseminating via the lymphatics make a major contribution to the seeding of lung metastases. The inducible genetic marking system we have developed is a powerful tool for the tracking of metastasizing cells in vivo.
Highlights
Cancer cells disseminating from primary tumors pass through different conduits and anatomical sites, which can affect the outcome of the metastatic process.[1]
Development of a green–red fluorescent protein genetic switch triggered by transient warming (TW) To develop an inducible system for marking disseminating tumor cells in a spatiotemporally controlled manner, we first created a promoter element that can be activated by TW
Here we report the establishment of a tightly spatiotemporally controlled inducible genetic switch system, which allowed us to assess the contribution of tumor cell trafficking through the lymphatic system to the seeding of distant metastases
Summary
Cancer cells disseminating from primary tumors pass through different conduits and anatomical sites, which can affect the outcome of the metastatic process.[1] Dissecting the contribution of cells that traffic through distinct locations at specific points in time requires their labeling in a precise spatiotemporal manner, ideally using an inducible genetic switch that can permanently mark the disseminating cells. Current inducible gene expression systems typically employ soluble inducers, such as tetracycline/doxycycline or (4-hydroxy)tamoxifen.[2] While such treatments allow temporal control of gene expression, spatial restriction of the induction is difficult to achieve, due to diffusion and systemic distribution of the inducer. Leakiness due to basal promoter activity is often a problem. High spatiotemporal precision of cell labeling can be achieved using phototransformation of fluorescent proteins
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