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

Abstract

BackgroundHuman mucoepidermoid carcinoma (MEC) is regarded as the most common primary salivary malignancy. High-grade MEC has a high risk of recurrence and poor prognosis. Tumor angiogenesis, induced by poorly differentiated cancer cells of high-grade MEC, contributes to tumor growth and metastasis. Therefore, elucidating molecular mechanisms underlying the pro-angiogenic ability of poorly differentiated MEC cells is critical for the understanding of high-grade MEC progression. It is well known that three-dimensional (3D) cell culture, in contrast with conventional two-dimensional (2D) culture, provides a better approach to in vitro recapitulation of in vivo characteristics of cancer cells and their surrounding microenvironment. The purpose of this study was to model a 3D environment for in vitro gene expression profiling of key molecules in poorly differentiated MEC cells for cancer neovascularization and compared them with traditional 2D cell culture.MethodsLow-passage poorly differentiated MEC cells, derived from human patient samples of high-grade MEC, were microencapsulated in sodium alginate gel microcapsules (3D culture) and compared with cells grown in 2D culture. Cancer cell proliferation was determined by MTT assays for 1 week, and gene expression of VEGF-A, bFGF and TSP-1 was analyzed by western blotting or ELISA. The hypoxic environment in 3D versus 2D culture were assessed by western blotting or immunofluorescence for HIF1α, and the effect of hypoxia on VEGF-A gene expression in 3D cultured cancer cells was assessed by western blotting with the use of the HIF1α inhibitor, 2-methoxyestradiol (2-MeOE2).ResultsWhen encapsulated in alginate gel microcapsules, low-passage poorly differentiated human MEC cells grew in blocks and demonstrated stronger and relatively unlimited proliferation activities. Moreover, significant differences were found in gene expression, with 3D-grown cancer cells a significant increment of VEGF-A and bFGF and a drastic reduction of TSP-1. Consistently, 3D-grown cancer cells secreted significantly more VEGF-A than 2D culture cancer cells. Furthermore, 3D-grown cancer cells showed significantly higher expression of HIF1α, a molecular indicator of hypoxia; the increased expression of VEGF-A in 3D cultured cancer cells was shown to be dependent on the HIF1α activities.ConclusionsThe present work shows the effects of 3D culture model by alginate microencapsulation on the proangiogenic potentials of low-passage poorly differentiated human MEC cells. Cancer cells in this 3D system demonstrate significant intensification of key molecular processes for tumor angiogenesis. This is due to a better modeling of the hypoxic tumor microenvironment during 3D culture.

The Role of Biomimetic Hypoxia on Cancer Cell Behaviour in 3D Models: A Systematic Review

“…3D tumour models are considered to better recapitulate the in vivo tumour microenvironment compared to standard 2D cultures. In this review we have identified a total of 37 publications [ 34 , 50 , 57 , 60 , 62 , 64 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 ] where 3D culture significantly altered cancer cell behaviour towards a more biomimetic response. Eighteen of these studies used 3D scaffold-based cultures and 18 studies used non scaffold-based, cell-generated extra-cellular matrix (ECM) cultures, with a single study using both approaches [ 73 ] ( Figure 3 ).…”

Design of spherically structured 3D in vitro tumor models -Advances and prospects

“…Such ability to isolate specific cells further increases the capacity of studying genetic and phenotypic alterations in specific sub-sets of the cultured cells. In a recent study by Yang and co-workers [217], spherical alginate-based microcapsules were used to cultivate low passage human mucoepidermoid cells, and also to isolate angiogenesis-related molecules released from these cancer cells. The analysis of 3D cultures phenotypes and genotypes revealed a higher expression of pro-angiogenic genes and hypoxia associated factors in comparison to those obtained in standard 2D cultures [217].…”

Customizable biomaterials as tools for advanced anti-angiogenic drug discovery

“…These systems demonstrated perfusable vascular networks growing within and around the tumor spheroids [139], as well as cancer cells migrating to and infiltrating vascular networks [140]. In many cases, cancer cells were partitioned inside alginate-based microcapsules to grow in their own isolated environments [141–144]. Alginate capsules are resistant to cell attachment but are also permeable to growth factors such as MCF-7 breast cancer cell-secreted VEGF and HIF1α [141].…”

“…In many cases, cancer cells were partitioned inside alginate-based microcapsules to grow in their own isolated environments [141–144]. Alginate capsules are resistant to cell attachment but are also permeable to growth factors such as MCF-7 breast cancer cell-secreted VEGF and HIF1α [141]. The vascularized microtumor model was further developed through the incorporation of alginate capsules containing MCF-7 cells into vascularized Collagen-I hydrogels.…”