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.