Abstract
The progression of malignant tumors does not depend exclusively on the autonomous properties of cancer cells; it is also influenced by tumor stroma reactivity and is under strict microenvironmental control. By themselves, stromal cells are not malignant, and they maintain normal tissue structure and function. However, through intercellular interactions or by paracrine secretions from cancer cells, normal stromal cells acquire abnormal phenotypes that sustain cancer cell growth and tumor progression. In their dysfunctional state, fibroblast and immune cells produce chemokines and growth factors that stimulate cancer cell growth and invasion. In our previous work, we established an in vitro model based on a monolayer co-culture system of healthy human fibroblasts (HFs) and human osteosarcoma cells (the MG-63 cell line) that simulates the microenvironment of tumor cells and healthy cells. The coexistence between MG-63 cells and HFs allowed us to identify the YKL-40 protein as the main marker for verifying the influence of tumor cells grown in contact with healthy cells. In this study, we evaluated the interactions of HFs and MG-63 cells in a transwell co-culture system over 24 h, 48 h, 72 h, and 96 h. We analyzed the contributions of these populations to the tumor microenvironment during cancer progression, as measured by multiple markers. We examined the effect of siRNA knockdown of YKL-40 by tracking the subsequent changes in gene expression within the co-culture. We validated the expression of several genes, focusing on those involved in cancer cell invasion, inflammatory responses, and angiogenesis: TNF alpha, IL-6, MMP-1, MMP-9, and VEGF. We compared the results to those from a transwell co-culture without the YKL-40 knockdown. In a pro-inflammatory environment promoted by TNF alpha and IL-6, siRNA knockdown of YKL-40 caused a down-regulation of VEGF and MMP-1 expression in HFs. These findings demonstrated that the tumor microenvironment has an influence on the gene expression of healthy surrounding tissues and on the process of tumorigenicity and it is emerging as attractive targets for therapeutic strategies.
Highlights
The constituents of the tumor microenvironment can arrive from two major avenues: recruitment from local tissue and systemic recruitment from distant tissues via circulation
A similar trend was observed in co-cultured human fibroblasts (HFs) with Human cartilage glycoprotein–39 (YKL-40)-silenced MG-63 cells: an increased amount of TNF-α mRNA in HFs and a constant but low concentration in MG-63 cells (Figure 1)
We identified YKL-40 as the main marker for verifying the influence of tumor cells grown in contact with healthy cells, and we found that YKL-40 may have synergistic effects: in HFs, it can act to insulin-like growth factor-1, leading to an inflammatory condition and promoting an up-regulation of Matrix metalloproteinases (MMPs)-1 expression, remodeling the extracellular matrix (ECM)
Summary
The constituents of the tumor microenvironment can arrive from two major avenues: recruitment from local tissue and systemic recruitment from distant tissues via circulation. Tumor cells may alter the surrounding stroma through direct cell contact or by secreting paracrine-soluble factors, inducing cell modification and extracellular matrix (ECM) alterations [1] In this context, inflammation and infection have received special attention; many inflammatory mediators may influence www.impactjournals.com/oncotarget cell proliferation and tumor development, and they are responsible for invasion and immunosuppressive signaling through the production of angiogenic and growth factors, chemokines, cytokines, and matrix metalloproteinases [2]. Through intercellular interactions or by paracrine secretions from cancer cells, normal stromal cells acquire abnormal phenotypes that sustain cancer cell growth and tumor progression In their dysfunctional state, fibroblast and immune cells produce chemokines and growth factors that stimulate cancer cell growth and invasion. The coexistence between MG-63 cells and HFs allowed us to identify the YKL-40 protein as the main marker for verifying the influence of tumor cells grown in contact with healthy cells
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