Alginate-embedded HuH-7 cells increase MMP-9 and reduce OCLN expression in vitro
The use of 3D models for cell culture has many advantages over monolayer cell culture. Many different 3D models were developed for different approaches [10] and one of them is the use of alginate capsules [19]. Sodium alginate is one of the materials most commonly chosen because it form hydrogels with desirable characteristics such as lightweight, flexibility and mechanical stability [20]. Alginate encapsulation was reported to increment cell division, aid in extracellular matrix assembly and exacerbate tumor characteristics [21–23].
Our results show that HuH-7 cells change their behavior when undergo 3D culture environment, as described for other tumor cells [24]. Moreover, these changes occur in important genes for tumor progression, such as proteases and adhesion molecules.
Proteases are important to trigger cell proliferation, differentiation, matrix remodeling, vascularization and migration. All these events take place during normal development but also during malignant progression. MMP-9 is a protease widely studied because it is important to promote the whole process of cell colonization, hence proliferation, tumor invasion and metastasis [25]. According to our results, cell culture in capsules promotes a high increment in MMP–9 expression. It is reasonable to think that the activation of this kind of molecule is triggered by the surrounding matrix formed by sodium alginate.
Although the three-dimensional cell culture aggregates do not always lead to the formation of a complex 3D structure, they introduce biomechanical and biophysical stimuli that promote a controlled environment promoting the assembly of “tumors tissue type” [26]. As the cells in 2D are exposed to a uniform environment with sufficient availability of nutrients and oxygen, they don’t experience the three-dimensional solid tumor environment with a gradient of chemical and critical biological signals that can benefit or harm cell growth and tumor development [8]. Hypoxia is one of the triggers of EMT, characterized by decreasing cellular junctions for the subsequent infiltration of surrounding tissues. This study observed a decrease in the expression of OCLN, which participates in the tight junctions between cells. This result may not due to lack of oxygen, but simply because during the process of encapsulation cells are isolated from one another, surrounded by the alginate matrix, thus unable to establish such cell contacts. However, the possibility that cells inside the capsule receive less oxygen cannot be discarded. Hypoxia-related genes and oxygen measurements must be subsequently analyzed.
However, other analyzed EMT and metastasis markers (p65, VEGF and I–CAM) did not differ after 4 days in 3D culture. These molecules are frequently overexpressed in 3D cultures and in cancer cells in vivo [16, 27–29]. The reasons why we failed to detect a difference in these molecules in the present study are not clear. It may be related to the length of time cells were kept in culture. An interesting analysis would be to compare results at different times, plotting temporal changes in gene expression [21]. Studies have already shown the viability of encapsulated cells for longer culture periods, up to 45 days [30]. In order to improve the model, an increment in time culture could be carried out to asses if a longer period induces different expression of other markers. It would also be important to asses different alginate concentrations to investigate the effect of this variable on EMT and metastatic progression markers in HuH-7 cells, according to studies performed for viral infections [19].
On the other hand, some limitations should be considered. Because of the small size for handling and the modifications suffering during the fixing process, it is difficult to obtain slides for histological analysis of good quality from 3D cultures. Also, as can be seen in Fig. 3, not all the encapsulated cells could be marked because of the lack of permeability of the alginate. This could hamper cytological analysis using antibodies. It would be very interesting to confirm gene expression data with protein analysis by western blot. Unfortunately, in our experience, it is very difficult to obtain good quality protein fraction from encapsulated cells, as alginate usually contaminates cell extracts, even after dissolving the beads. Nevertheless, the use of capsules as a 3D model has the advantage of decreasing the use of animals for research. Moreover, it is a cost-effective method, thus providing a useful platform for the study of cancer cells [21].