Figure 6: Immunofluorescent staining of fixed spheroids
composed of HepG2s:HSCs (4:1), 20k cells initially per spheroid cultured
with different oxygenating MPs for 10 d stained with A) Anti-Vinculin
(for focal adhesions, yellow), B) E-Cadherin (for adherens, purple), and
Hoechst 33342 (for nuclei, blue) using confocal microscopy on central
plane. C) Quantitative analysis of images using ImageJ. Statistics via
one-way ANOVA and Tukey’s post hoc testing with A as the highest
mean and D with the lowest mean, p< 0.05. Mean ± SD, n =2
spheroids with S=3.
Figure 6A shows representative confocal microscope images of
liver spheroids cultured with various modified MPs and stained for
vinculin. We observed high levels of vinculin expression in the
laminin-511 group suggesting better integrin mediated focal adhesion
kinase (FAK) activation and focal adhesion formation. In connection to
our findings, the Mitaka group investigated the deposition of laminins
containing the α5 chain in the liver cancer environment and concluded
that laminin-511 and -521 are major isoforms in liver tumors. They
showed that cancer cells could specifically interact with these ligands
through integrin α3β1 and α6β4 which are widely expressed in
hepatocellular carcinoma.[48,49] Laminin-111
primary binding integrins are α6β1[50] and
α7β1[49] while fibronectins main integrin
receptors are α5β1, α8β1, αVβ1.[51] HepG2 cells
are known to express α2, α6, β1, and β4 integrin subunits[52] and very low levels of the fibronectin
receptors, presenting the possibility that they may respond to
laminin-511 and 521 but not fibronectin and laminin-111 simply because
they do not express the appropriate integrin receptors.
Equally important in the liver to integrin activation is cell-cell
adherins formation. Hepatocytes in the liver arrange with one surface in
direct contact with blood and the other surface forming tight junctions
with adjacent cells. For every eight hepatocytes, one HSC helps to
maintain the storage of vitamin A and protects hepatocytes in case of
injury.[53] Thus, we studied physical linkage
between cells to explore the effect of modified MPs on E-cadherin
expression levels. E-cadherin, a cell-cell adhesion molecule in
epithelial tissues, is localized on the surfaces of cells and allows
physical linkage between neighboring cells. [54]Figure 6B shows increased expression of E-cadherin in spheroids
cultured with laminin-511 and -521 modified MPs as compared with other
groups (p < 0.01). Importantly, we observed high
expression of E-cadherin at the edges, which is associated with more
heterotypic cell-cell contacts. The degree of E-cadherin contact has
been shown to affect proliferation and liver-specific
function.[55] Taken together, analysis of vinculin
and E-cadherin expression suggest that the phenotype of cells was
maintained better in groups cultured with laminin-511 and -521 and may
indeed play a role in modulating hepatocytes functions. Another
important aspect of adherens-type junctions is stabilization of the
interactions between adjacent cells, which is a crucial factor in
forming and maintaining 3D cellular aggregates. In our previous study
with liver spheroids,[16] we showed that HSCs play
a central role in compactness and formation of stable liver spheroids.
Improved E-cadherin expression prompted us to determine if we could use
MPs modified with laminin 511 or 521 instead of HSCs to induce the cells
form compact spheroids. Our data suggested that although these MPs lead
to smaller size spheroids and more cell-cell junctions compared to HepG2
only spheroids (Figure S3), they failed to achieve the
compactness caused by HSCs. This is likely because HSC secrete many
biomolecules, cytokines, and growth factors leading to better shape and
stability. In the liver, HSCs do not constitute direct cell adhesions
with liver sinusoidal endothelial cells (LSECs), whereas they establish
adherens junctions with hepatocytes through thorn-like microprojections
or spines [56] with E-cadherin as the main
adhesion molecule.