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its receptor is well correlated clinically with invasive carcinomas of the breast
and prostate (Sherwood and Lee, 1995; Klijn et al., 1994). Growth factors
including EGF are present in serum, macrophages, platelets and smooth
muscle cells near blood vessels (Calabro et al., 1997; Kume and Gimbrone,
1994). Release of growth factors from these cells or endothelial cells could
provide a gradient that would produce a chemotactic response toward blood
vessels. This is important since intravasation, the movement of cells into blood
vessels, is a major route for egress of carcinoma cells from tumours (Wyckoff
et al., 2000). In fact, we have shown a strong correlation between the density
of live carcinoma cells in the blood and the number of both single cells and
metastases in the lungs establishing intravasation as a key step for metastasis.
The behaviour behind this correlation at the single cell level was proposed to
be chemotaxis of carcinoma cells within the primary tumour toward growth
factors like EGF that are associated with blood vessels, and differences in the
ability of carcinoma cells to resist lysis upon intravasation (Wyckoff et al.,
2000).
We have investigated the interaction of carcinoma cells with blood vessels in
metastatic primary tumours in more detail and determined if carcinoma cells
have the intrinsic ability to locomote toward blood vessels and to intravasate
without fragmentation. To do this we used GFP expression by carcinoma cells
and time-lapse confocal microscopy to image the behaviour of single
carcinoma cells near blood vessels. Imaging was carried out in metastatic
primary tumours generated by injection of a metastatic mammary carcinoma
cell line, MTLn3, into the mammary fat pads of Fisher rats. As shown in
Figure 11.1, carcinoma cells are elongated and polarized towards blood
vessels suggesting that there is a vessel-mediated attraction for the cells. That
intravasation is an active process requiring each cell to cross an intact vessel
wall is suggested by the failure of intravenously (IV) injected low molecular
weight fluorescent dextran to escape from the vessels during intravasation.
Furthermore, time-lapse imaging of carcinoma cells in contact with blood
vessels demonstrates the ability of carcinoma cells actively to crawl into the
blood vessel as solitary cells using amoeboid locomotion (Farina et al., 1998).
These time-lapse microscopy results correlate directly with the large number
of GFP-expressing carcinoma cells observed in the circulation of the primary
tumours and their absence from the blood vessels of non-metastatic tumours
(Wyckoff et al., 2000).
These observations suggest mechanisms by which increased carcinoma cell
orientation and locomotion towards blood vessels could increase the e
ciency
with which they intravasate. The orientation could be induced by
chemoattractants diffusing from the blood vessel and its associated cell
layers. In culture, metastatic carcinoma cells (MTLn3) show dramatic
chemotaxis to growth factors while non-metastatic cells (MTC) do not
(Figure 11.2). MTLn3 cells express more EGF receptors than MTC cells
