The tumor microenvironment promotes cancer progression and cell migration

Abstract

The tumor microenvironment contributes to cancer progression, in part through interactions between tumor and normal stromal cells. This study analyzed morphological and molecular changes induced in co-cultured human fibroblasts (HFs) and the MG-63 osteosarcoma cell line. Co-cultured cell monolayers were morphologically analyzed using high resolution scanning electron microscopy (HR-SEM), and trans-well assays were performed to assess cell migration and invasion. Proteins involved in inflammatory responses, cancer cell invasion, and angiogenesis were assessed using western blotting. HR-SEM showed progressive spatial orientation loss by fibroblasts in contact with MG-63s, while MG-63s proliferated rapidly and invaded HF space. Trans-well assays showed enhanced MG-63 migration in the presence of HFs. IL-6 expression was increased in co-cultured HFs, possibly stimulated by TNF-α. HFs do not normally express YKL-40 but did so in co-culture. Band densitometric analyses showed that increasing YKL-40 expression was followed by VEGF overexpression, especially in MG-63s. Finally, our results confirmed fibroblasts as the main matrix metalloproteinase source in this tumor microenvironment. Our study sheds new light on how tumor-stroma interactions promote tumor development and progression, and may support identification of novel anti-cancer therapeutics.

Keywords: co-culture; human fibroblasts; osteosarcoma; tumor microenvironment; tumor stroma.

Figure 1. HR-SEM analyses

HF control at 24 h (Bar: 10 μm) A. Flat and elongated morphology (arrow). MG-63 control at 24 h (Bar: 10 μm) B. Polygonal morphology (star). HFs C. and MG-63s D. after 24 h of co-culture (Bar: 10 μm). HFs E. and MG-63s F. after 48 h of co-culture. Loss of fibroblast orientation (E., arrow); reduced MG-63 proliferation (Bar: 100 μm) and formation of clusters (F., stars). HFs G. and MG-63s H. after 72 h of co-culture (Bar: 100 μm). MG-63 invasion of HF space (H., stars). HFs I. and MG-63s J. after 96 h of co-culture (Bar: 100 μm). Overlap of MG-63s with HFs (I., arrow). Detail of MG-63s at 96 h of co-culture K. Pseudopod protrusions (stars) and pre-uropod region (arrow) (Bar: 10 μm). Detail of MG-63 control at 96 h (Bar: 10 μm) L.

Figure 2. Cell migration assay

MG-63 cell migration (%) A. * represents a difference between MG-63 cells cultured with and without HFs, P<0.05. Giemsa staining of migrating MG-63 cells, with or without HFs B.

Figure 3. Western blot for TNF-α

Densitometric analysis A. TNF-α expression in co-cultured HFs and MG-63 cells B. * represents a difference between co-cultured cells and HF or MG-63 cells alone. P<0.05.

Figure 4. Western blot for IL-6

Densitometric analysis A. IL-6 expression in co-cultured HFs and MG-63 cells B. * represents a difference between co-cultured cells and HF or MG-63 cells alone. P<0.05.

Figure 5. Western blot for YKL-40

Densitometric analysis A. YKL-40 expression in co-cultured HFs and MG-63 cells B. * represents a difference between co-cultured cells and HF or MG-63 cells alone. P<0.05.

Figure 6. Western blot for VEGF

Densitometric analysis A. VEGF expression in co-cultured HFs and MG-63 cells B. * represents a difference between co-cultured cells and HF or MG-63 cells alone. P<0.05.

Figure 7. Western blot for MMP-9

Densitometric analysis A. MMP-9 expression in co-cultured HFs and MG-63 cells B. * represents a difference between co-cultured cells and HF or MG-63 cells alone. P<0.05.