Alginate-embedded HuH-7 cells increase MMP-9 and reduce OCLN expression in vitro

Abstract

BackgroundHepatocellular carcinoma is a common cancer, ranking third in cancer-associated deaths. An important cause of cancer patients’ mortality is metastasis. At the start of metastasis progression, there is an epithelial-mesenchymal transition, characterized by matrix degradation, junction reductions and vessels formation. HuH-7 is a cell line used in research as an in vitro model for hepatocellular carcinoma. It is known that two-dimensional growth reflects tumor characteristics poorly. In contrast, three-dimensional cultures provide a better approach to the study of tumorigenic potential. The purpose of this work was to mimic a three-dimensional environment in order to assess gene expression of some epithelial-mesenchymal transition and metastasis progression markers in HuH-7 cells and compare them with traditional two-dimensional culture model.MethodsHuH-7 cells were encapsulated in sodium alginate (three-dimensional model) to be compared with cells grown in two-dimensional flasks. After 4 days in culture, gene expression of Matrix metallopeptidase 9, Occludin, p65, Intercellular adhesion molecule 1 and Vascular endothelial growth factor A was analyzed by qPCR and cytoskeleton assessment was performed by rhodamine-phalloidin staining.ResultsDifferences were found in gene expression, with a high increment of Matrix metallopeptidase 9 and Occludin reduction. The cytoskeleton morphology also showed differences, with a cytoplasm restricted only near the nuclei in the three-dimensional model.ConclusionsThis work shows the effects of using sodium alginate capsules as a three-dimensional model to the study of HuH-7. Cells in this 3D system show key markers of epithelial-mesenchymal transition, such as Matrix metallopeptidase 9 overexpression and Occludin down-regulation.

A decellularized matrix enriched collagen microscaffold for a 3D in vitro liver model

“…To enhance the stability of the microscaffolds over a longer period of time (>1 week), 1% alginate was added to all the compositions as the crosslinking agent. 31 This biopolymer was used for its high biocompatibility and reversible crosslinking ability in the presence of calcium ions. Alginate-based systems provide the benefit of extracting the cells from these scaffolds for further analysis by disrupting the calcium crosslinks.…”

Microencapsulation of low-passage poorly-differentiated human mucoepidermoid carcinoma cells by alginate microcapsules: in vitro profiling of angiogenesis-related molecules

“…Originally developed for tissue engineering [ 16 , 17 ], cell microencapsulation with alginate hydrogels has been recently adopted as a suitable model for 3D in vitro culture in cancer researches [ 11 ]. Previous studies showed that alginate microencapsulation enhanced cancer cell proliferation and survival, modulated cancer cell migration and invasiveness and exacerbated tumor malignancies [ 14 , 15 , 18 – 21 ]. Our results demonstrated that alginate microencapsulation increased the proangiogenic potentials of low-passage poorly-differentiated human MEC cells.…”

“…However, multicellular tumor cell aggregates enabled cancer cells to experience a controlled 3D environment seen in solid tumors, where a gradient of chemical and critical biological signals, such as nutrients and oxygen, promote the assembly of “tumors tissue type” [ 15 ]. Therefore, cancer cells in 3D are under hypoxic conditions [ 15 , 21 ]. In contrast, low-passage poorly-differentiated human MEC cells in 2D monolayer culture are exposed to a uniform environment with sufficient availability of nutrients and oxygen [ 9 , 21 ].…”

“…Therefore, cancer cells in 3D are under hypoxic conditions [ 15 , 21 ]. In contrast, low-passage poorly-differentiated human MEC cells in 2D monolayer culture are exposed to a uniform environment with sufficient availability of nutrients and oxygen [ 9 , 21 ]. This is reflected by the differential expression of HIF1α for 2D versus 3D cultured cancer cells.…”