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Cancer Research, vol. 60: 2504-2511, 2000

A Critical Step in Metastasis: In vivo Analysis of Intravasation at the Primary Tumor

Jeffrey B. Wyckoff^*, Joan G. Jones^#, John C. Condeelis^*, and Jeffrey E. Segall^*† 

Departments of ^*Anatomy and Structural Biology and ^#Pathology 

Abstract

Detailed evaluation of all steps in tumor cell metastasis is critical for evaluating the cell mechanisms controlling metastasis. Using GFP transfectants of metastatic (MTLn3) and nonmetastatic (MTC) cell lines derived from the rat mammary adenocarcinoma 13762 NF, we have measured tumor cell density in the blood, individual tumor cells in the lungs, and lung metastases. Correlation of blood burden with lung metastases indicates that entry into the circulation is a critical step for metastasis. To examine cell behavior during intravasation, we have used GFP technology to view these cells in time lapse images within a single optical section using a confocal microscope. In vivo imaging of the primary tumors of MTLn3 and MTC cells indicates that both metastatic and nonmetastatic cells are motile and show protrusive activity. However, metastatic cells show greater orientation towards blood vessels, and larger numbers of host cells within the primary tumor while nonmetastatic cells fragment when interacting with vessels. These results demonstrate that a major difference in intravasation between metastatic and nonmetastatic cells is detected in the primary tumor and illustrate the value of a direct visualization of cell properties in vivo for dissection of the metastatic process.

Figure 1:MTLn3-GFP cells orient towards blood vessels, while MTC-GFP cells do not. Orientation of MTLn3-GFP cells (A) and MTC-GFP cells (B) to blood vessels in the primary tumor. A) MTLn3-GFP cells (green) near the vessel (red) are seen to orient themselves toward the vessel in an elongated fashion (arrows) as opposed to those away from the vessel (arrowheads). B) MTC-GFP cells (green) randomly associate with the vessel (red), and remain elongated and oriented to each other away from the vessel. Scale bar = 25um.

Figure 2:MTC-GFP cells are able to move in a primary tumor by first sending out a thin leading edge protrusion through a small space. Protrusive movement of a whole MTC-GFP cell within the primary tumor viewed in a single optical section in situ. (A) Initial position of cell. (B) Initial extension of protrusion. (C) Further protrusion. Outline shows starting position. The cell moves at a speed of 3.7 um/min, images are 8 min apart. Scale bar = 25um

Figure 3:MTLn3-GFP cells exhibit linear translocation in situ. Movement of MTLn3-GFP cell in a single optical section of the primary tumor. The cell moves over a distance of 22 um. (A) The cell seen in its starting position (outlined, with arrow). (B) Cell has moved to a new position 6 min later (arrow). Its starting position is outlined. The cell moves at 3.6 um/min. Scale bar = 25 um.

Figure 4: MTC-GFP cells exhibit linear translocation in situ. Movement of a MTC-GFP cell in a single optical section of the primary tumor. (A) Cell is shown outlined in starting position (arrow) (B-C) Cell at 6 min intervals showing a progressively linear movement (arrows). Outline shows original position. The cell moves at a speed of 3.6 um/min, images are 4 min apart. Scale bar = 25 um.

Figure 5: The protrusions of MTC-GFP cells in situ are seen to break off from the cell body and move away rapidly. (A) A cell protrusion prior to being broken off (arrow). (B,C) Fragment broken off from the protrusion (arrows). Outline shows starting position. Speed of fragment after breaking away from protrusion suggests movement into vascular flow (large hash marks delineate vessel). Initial speed of protrusion is 2.8um/min. After release it increases to 11-15 um/min. Images are 1 min apart. Scale bar = 25 um

Figure 6:Host cells can be visualized as shadows on top of white fluorescent cells in the primary tumor. Host cell movement over MTLn3-GFP cells in a single optical section of the primary tumor viewed in situ. (A-C) Host cells are seen as black shapes (arrows) moving over fluorescent tumor cells (MTLn3-GFP). Outline shows starting position. Host cells move at 7.7 um/min, images are 7 min apart. Scale bar = 25 um.