Abstract
BackgroundThe role of the microenvironment during the initiation and progression of carcinogenesis is thought to be of critical importance, both for the enhanced understanding of fundamental cancer biology as well as for improving molecular diagnostics and therapeutics. The aim of this study was to establish an in vitro model based on a co-culture of healthy human fibroblasts (HFs) and human osteosarcoma cells (MG-63s) to simulate the microenvironment including tumor and healthy cells.MethodsThe HFs and MG-63s were in vitro co-cultured for a period of time ranging from 24 h to 7 days. Cell morphology and organization were studied using phase contrast microscopy while the expression of Human Cartilage Glycoprotein 39 (YKL-40), Vascular Endothelial Growth Factor (VEGF) and Matrix Metalloprotease 1 (MMP1) was investigated by Real Time PCR and Western Blotting.ResultsThe results showed a characteristic disposition of tumor and healthy co-cultured cells in columns which are not visible in tumor and healthy cells grown singularly. The expression of YKL-40, VEGF and MMP1 significantly changed in co-cultured cells compared to HFs and MG-63s separately cultured.ConclusionsWe concluded that the tumor microenvironment has an influence on the protein expression of the healthy surrounding tissues and the process of tumorigenicity.
Highlights
The role of the microenvironment during the initiation and progression of carcinogenesis is thought to be of critical importance, both for the enhanced understanding of fundamental cancer biology as well as for improving molecular diagnostics and therapeutics
The MG-63s had a polygonal morphology after 24 h of culture (Figure 1C) while the human fibroblasts (HFs) showed a fibroblastic morphology with an elongated cell body after 24 h of culture (Figure 1D)
QRT-PCR of Human cartilage glycoprotein–39 (YKL-40) and Vascular Endothelial Growth Factor (VEGF) in co-cultured HFs and MG-63 cells To examine changes in protein expression during the coexistence of HFs and MG-63 cells, the mRNA expression of YKL-40 and VEGF was evaluated using a quantitative real time polymerase chain reaction in cells grown in co-culture, and the results were compared to the same cells grown separately as controls
Summary
The role of the microenvironment during the initiation and progression of carcinogenesis is thought to be of critical importance, both for the enhanced understanding of fundamental cancer biology as well as for improving molecular diagnostics and therapeutics. The continual upregulation of enzymes, such as matrix metalloproteinases (MMPs) by stromal fibroblasts can disrupt the ECM; invading immune cells can overproduce factors which promote abnormal gene expression [1] These conditions are normally reversible but, when inflammation is sustained, the normal organization of cells might lead to gene expression instability within the healthy cells and the acquisition of tumorigenic potential [2]. A tumor microenviroment includes invasive cancer cells and stromal cells, consisting in cancer-associated non-malignant fibroblasts, which provide an essential communication network via secretion of growth factors and chemokines This condition leads to an altered ECM and provides additional oncogenic signals enhancing cancer-cell proliferation and invasion [4]. While the stromal cells are not malignant per se, their role in supporting cancer growth and development is vital to the survival of the tumor [5,6]
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