Three‐dimensional culture of melanoma cells profoundly affects gene expression profile: A high density oligonucleotide array study

Abstract

Growth in three-dimensional (3D) architectures has been suggested to play an important role in tumor expansion and in the resistance of cancers to treatment with drugs or cytokines or irradiation. To obtain an insight into underlying molecular mechanisms, we addressed gene expression profiles of NA8 melanoma cells cultured in bidimensional monolayers (2D) or in 3D multicellular tumor spheroids (MCTS). MCTS containing 10-30,000 cells were generated upon overnight culture in poly-Hydroxyethylmethacrylate (polyHEMA) coated plates. Kinetics of cell proliferation in MCTS was significantly slower than in monolayer cultures. Following long-term culture (>10 days), however, MCTS showed highly compact and organised cell growth in outer layers, with necrotic cores. Oligonucleotide microarray analysis of the expression of over 20,000 genes was performed on cells cultured in standard 2D, in the presence of collagen as model of extracellular matrix (ECM), or in MCTS. Gene expression profiles of cells cultured in 2D in the presence or absence of ECM were highly similar, with >/=threefold differences limited to five genes. In contrast, culture in MCTS resulted in the significant, >/=threefold, upregulation of the expression of >100 transcripts while 73 were >/=threefold downregulated. In particular, genes encoding CXCL1, 2, and 3 (GRO-alpha, -beta, and gamma), IL-8, CCL20 (MIP-3alpha), and Angiopoietin-like 4 were significantly upregulated, whereas basic FGF and CD49d encoding genes were significantly downregulated. Oligonucleotide chip data were validated at the gene and protein level by quantitative real-time PCR, ELISA, and cell surface staining assays. Taken together, our data indicate that structural modifications of the architecture of tumor cell cultures result in a significant upregulation of the expression of a number of genes previously shown to play a role in melanoma progression and metastatic process.

A three-dimensional microenvironment alters protein expression and chemosensitivity of epithelial ovarian cancer cells in vitro

“…49 MIB1 staining revealed that cells cultured in 3D were less proliferative than cells cultured in 2D, which is similar to previous reports for other cell types. 9,11 We also observed a reduction in p53 expression, which may also reflect the lower proliferation rates of 3D cultured cells. MIB1/p53-positive cells tended to be located in a proliferative zone on the outer surface of the spheroids.…”

“…9,11 Cell lines were epithelial and had been established either from primary tumors, ascites, or metastases and represented the main histological subtypes of EOC, namely serous, endometrioid, clear cell, and mucinous ovarian cancer. All cell lines, with the exception of OVCA429 and OVCA433, formed MCA structures under static culture conditions.…”

“…can be achieved by culturing primary tissues or established cell lines within extracellular matrix gels, synthetic scaffolds, rotary cell culture systems, or on low/non-adherent culture plastics; [6][7][8][9][10][11] these 3D cell culture techniques all aim to restore the 3D architecture that characterizes normal tissues and solid tumors alike. Studies have shown that gene expression profiles of 3D-cultured cells are dramatically different to monolayer cultures and more closely resemble the profiles of the tissues of origin than their 2D counterparts.…”

“…Studies have shown that gene expression profiles of 3D-cultured cells are dramatically different to monolayer cultures and more closely resemble the profiles of the tissues of origin than their 2D counterparts. [11][12][13][14] 3D multicellular aggregate (MCA) structures also contain many features absent in 2D cultures that are present in primary tissues. For example, in MCAs of tumor cells hypoxic and necrotic areas can develop, regions that are commonly found in primary tumors and known to be associated with chemoresistance.…”

Epithelial-To-Mesenchymal Transition in Pancreatic Ductal Adenocarcinoma: Fiction or Fact?

“…In particular, in melanoma cells it was described that cell cultures in a 3D system, allowing structural modifications of the architecture of tumor cell cultures in monolayer and the maintenance of cell-cell interactions, exhibited a significant up-regulation of the expression of a number of genes previously shown to play a role in melanoma progression and metastatic process. 21 Our experimental findings in 3D-spheroids can contribute to describe the phenotype of PDAC cells in relation to EMT, suggesting a concomitant expression of "epithelial" and "mesenchymal" markers in these cells. This suggests an EMT-related phenotype for PDAC cells and strengthens the general assumption that an inverse correlation between E-cadherin expression and invasive potential of carcinoma cells is not absolute.…”