Abstract
Metastasic breast cancer is the leading cause of death by malignancy in women worldwide. Tumor metastasis is a multistep process encompassing local invasion of cancer cells at primary tumor site, intravasation into the blood vessel, survival in systemic circulation, and extravasation across the endothelium to metastasize at a secondary site. However, only a small percentage of circulating cancer cells initiate metastatic colonies. This fact, together with the inaccessibility and structural complexity of target tissues has hampered the study of the later steps in cancer metastasis. In addition, most data are derived from in vivo models where critical steps such as intravasation/extravasation of human cancer cells are mediated by murine endothelial cells. Here, we developed a new mouse model to study the molecular and cellular mechanisms underlying late steps of the metastatic cascade. We have shown that a network of functional human blood vessels can be formed by co-implantation of human endothelial cells and mesenchymal cells, embedded within a reconstituted basement membrane-like matrix and inoculated subcutaneously into immunodeficient mice. The ability of circulating cancer cells to colonize these human vascularized organoids was next assessed in an orthotopic model of human breast cancer by bioluminescent imaging, molecular techniques and immunohistological analysis. We demonstrate that disseminated human breast cancer cells efficiently colonize organoids containing a functional microvessel network composed of human endothelial cells, connected to the mouse circulatory system. Human breast cancer cells could be clearly detected at different stages of the metastatic process: initial arrest in the human microvasculature, extravasation, and growth into avascular micrometastases. This new mouse model may help us to map the extravasation process with unprecedented detail, opening the way for the identification of relevant targets for therapeutic intervention.
Highlights
Metastatic disease is the main cause of death in breast cancer patients [1]
Confirming our previous work [6,10] we found that the coimplantation of genetically modified human umbilical vein endothelial cells (HUVEC) harboring the firefly luciferase (FLuc) gene (HUVECFLuc) and human bone marrow derived mesenchymal cells (MSC) (Figure S1-S4) embedded within a reconstituted basement membrane extract (BME, known as Matrigel) supplemented with VEGF, bFGF and heparin and inoculated subcutaneously into nude mice (Figure 1a), results in the formation of mature, stable human blood vessels
These vessels are functional, meaning that they are able to anastomose with preexisting mouse vessels, and can be assessed non-invasively and quantitatively by in vivo bioluminescent imaging (BLI) (Figure 1b)
Summary
Metastatic disease is the main cause of death in breast cancer patients [1]. Hematogenous cancer metastasis is a multistep process encompassing local invasion of cancer cells at primary tumors, intravasation into the blood vessel, survival in systemic circulation, extravasation, colonization and proliferation at the distant site, and ending with establishment of growing metastatic lesions [2]. Whole body bioluminescence imaging enables the dissemination of cancer cells through tissues to be monitored longitudinally in the same animal (but with low spatial resolution), whereas intravital microscopy (IVM), through confocal or multiphoton imaging of tumors expressing fluorescent proteins, enables spreading of a tumor to be observed with subcellular resolution. Such IVM studies usually rely on dorsal skin chambers or reversible skins flaps to increase sensitivity of detection and are limited to the primary tumor, providing only information over initial steps of metastasis. This technique is not suitable for following tumor progression, and internal metastasis in a live intact animal due to the invasiveness of the procedure [5]
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