Abstract PR5: Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors
Authors: Patsialou Antonia , Bravo-Cordero Jose Javier , Wang Yarong , Liu Huiping , Clarke Michael F. , Condeelis John S. 
Published: August 2014

Abstract
Metastasis is the main cause of death in breast cancer patients. Cell migration is an essential component of almost every step of the metastatic cascade, especially the early step of invasion inside the primary tumor. We have used intravital multiphoton microscopy to visualize the in vivo migration and invasion of human breast tumor cells in live primary tumors. We used two different tumors for this study: a. orthotopic xenografts of the highly metastatic breast cancer cell line MDA-MB-231, and b. low passage orthotopic xenografts of breast tumor cells isolated from a patient pleural effusion sample (TN1). The TN1 tumors were only passaged in vivo and never cultured. Both tumor cells were engineered to stably express GFP (green fluorescence protein) and were visualized in vivo based on their fluorescence emission. Collagen I matrix was visualized by second harmonic generation, while injected fluorescent dyes were used for visualization of blood vessels and stromal cells in the primary tumors. High-resolution 4D imaging of these two human tumors in vivo showed that they both shared common patterns of high-speed migration: a. cells moving as single entities irrespective of their neighboring cells, and b. multiple cells following each other in the same direction and in a single file but without cohesive cell junctions (i.e. multicellular streaming). We found that although the two tumors were derived from diverse genetic backgrounds, they exhibited almost identical proportions of either single or streaming cells, suggesting our observations may be generally applied to human breast cancer in vivo cell migration. Cells moving as multicellular streams were overall more frequent in both tumors, with approximately 60% of total motile cells moving as streams. Critically, we found that only multicellular streaming and not random migration of single cells was significantly associated with proximity to blood vessels. Macrophages were co-migrating with the tumor cells in the multicellular streams in both human tumors analyzed, and functional inhibition of macrophages in vivo by clodronate liposomes significantly decreased the frequency of multicellular streams, while increasing the frequency of single moving cells. Macrophage inhibition also significantly abrogated intravasation, as measured by the number of circulating tumor cells in the bloodstream of the treated mice. Since we found that macrophage inhibition reduced streaming migration while enhancing single cells migration, we deduce that multicellular streaming is the type of in vivo migration that most likely directly contributes to intravasation in human breast tumors. Finally, we analyzed the gene expression profile of the migratory tumor cells from both the MDA-MB-231 and the TN1 tumors and found that they both exhibited multiple gene expression changes in genes regulating cell motility. These gene expression changes were largely common in the two tumors and coordinated to regulate the basic actin motility machinery, leading to the same end phenotype of activated actin polymerization and enhanced migration. Our data is the first direct visualization of in vivo migration of live patient-derived breast tumor cells and the first assessment of the type of cell motility involved in tumor cell dissemination in a human tumor cell derived breast tumor.
This abstract is also presented as Poster A51.
Citation Format: Antonia Patsialou, Jose Javier Bravo-Cordero, Yarong Wang, Huiping Liu, Michael F. Clarke, John S. Condeelis. Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr PR5.