Biomedical Engineering Reference
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FIGURE 3.4: (See color insert.) Human peritoneal mesothelial cells
(HPMCs) were labeled with PKH26GL (red), seeded on fibronectin-coated
coverslips and grown until confluence at 37
C (as described in Figure 3.3).
Spheroids were generated using a modification of the hanging droplet method
[206]. Briey, cells were stained in green and then placed (50 cells/15 L of
RPMI with 15% FCS) on the cover of a tissue culture dish, and the cover
was placed over a dish containing PBS to prevent dehydration of the hanging
droplets. After 4 days, spheroids were seeded on HPMCs layer and allowed to
adhere for 3 h before imaging. Images show the 3D reconstruction of the same
field visualized from the upper, transverse, and bottom sides at time zero (A),
24 h (B), 48 h (C), and 72 h (D).
To experimentally test the ability of a cancer multicellular spheroid to dis-
seminate and to characterize its metastatic potential, the Laboratory of Im-
munogenetics of the Azienda Ospedaliero-Universitaria S.Giovanni Battista-
Molinette di Torino provides specific transmesothelial assays. Indeed, neoplas-
tic spheroids are generated, and seeded onto a mesothelial monolayer, gener-
ally anchored to an ECM-substrate; see Figure 3.4. The spheroid dissemina-
tion, defined as the tumor mass spreading on top of the monolayer without
forming invasive foci, is shown to require the interaction of 1-integrins with
the ECM proteins secreted by the mesothelial cells, with the contribution
of other adhesion molecules. The quantified overall adhesion levels of ascites
spheroids are somewhat lower than those reported for isolated carcinoma cells,
which possibly reflects a change in cell adhesive ability upon acquisition of the
spheroid morphology.
The process of invasion, determined by the establishment of proliferating
foci of ovarian tumor cells within the same focal plane as the layer, starts
with the retraction of some mesothelial cells (Figure 3.4(A)). Suddenly, the
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