Biology Reference
In-Depth Information
5-7 days
Fig. 7.5 Tumor cell invasion and intravasation and inflammatory cell influx in the CAM sponta-
neous metastasis assay. The schematic on the
left
depicts tissue composition of the CAM at the time
of tumor cell application on day ten of chick embryo incubation. From 0.5 to 2
10
6
tumor cells
(
green
) are applied in a volume of 20-25
m
l to the top of dropped/lowered CAM. The dropped CAM
is not subsequently injured and remains covered with the ectoderm (the layer of
blue cells
), which is
underlined with the basement membrane (
grey
) and ectoderm capillary plexus (
red irregular
circles
filled with nucleated erythrocytes). Larger terminal capillaries (
bulb-like structures
) lay
right beneath ectoderm plexus and protrude to the mesoderm filled with the ECM fibrils, stromal
fibroblasts, and arterial and venous blood vessels containing erythrocytes, monocytes, and neu-
trophils. The mesoderm is underlined by the thin endoderm, which separates CAM from the
allantoic cavity. Within 5-7 days (
right
), the applied tumor cells can form a large tumor mass,
which compresses the CAM and makes it much thinner than the original membrane. The staining
for blood vessels allows for visualization of the compressed ectoderm plexus at the tumor border
and also for larger vessels in the mesoderm and angiogenic vessels in the tumor mass. Tumor cells
with high potential of dissemination and metastasis appear to escape from the primary by breaching
the ectoderm layer, the basement membrane, and ectoderm plexus. The escaped tumor cells invade
the mesoderm and appear to congregate and wrap around blood vessels and capillaries and sometimes
are visualized intravascularly within the blood vessels. Although traversed with numerous angiogenic
blood vessels, the density of tumor cells usually does not allow for unambiguous identification of
intravascular tumor cells within the primary tumor. Importantly, tumor development is associated
with the influx of inflammatory, MMP-delivering neutrophils (MMP-9) and monocytes/macrophages
(MMP-13), which can be identified both within the primary tumor and at the tumor/CAM border by a
correspondingMMP staining. Most of the intravasated tumor cells are captured at the capillary plexus
throughout the CAM serving as the lung analog of the adult animal, but can also be identified as
spontaneously metastasized to different internal organs, including liver, lungs, and brain
Therefore, intravasation of tumor cells measured as an overall outcome of cell
dissemination from primary tumor cells placed atop the CAM cannot be unequivo-
cally attributed to the MMP-dependent proteolysis of BMs underlying the CAM
vessels.
That intravasation could be significantly dampened by general MMP inhibition,
but not by specific targeting of tumor-derived MMPs, provides strong evidence for
the regulation of tumor cell intravasation by stromal, most probably inflammatory
MMPs. Data in favor of this notion in mammalian model systems are scarce, but
some elegant studies demonstrate that macrophages often assist tumor cell entry
