Direct cancer-stromal interaction increases fibroblast proliferation and enhances invasive properties of scirrhous-type gastric carcinoma cells

Abstract

Background: Scirrhous-type gastric carcinoma (SGC) exhibits an extensive submucosal fibrosis and extremely poor patient prognosis. We investigated the importance of the cancer-stromal interaction in the histogenesis of SGC.

Methods: Gastric fibroblasts NF-25 and intestinal fibroblasts NF-j2 were co-cultured with SGC-derived (HSC-39) or non-SGC-derived (HSC-57 and HSC-64) cells. To identify genes that are up- or downregulated in NF-25, complementary DNA (cDNA) microarray analysis was performed. The antibody against vascular-cell adhesion molecule-1 (VCAM-1) was used for cell growth test and immunohistochemistry. Moreover, the impact of interaction with NF-25 fibroblasts on HSC-39 cells was investigated using western blot and reverse transcription-polymerase chain reaction.

Results: HSC-39 cells stimulated growth of NF-25 but not NF-j2 when co-cultured. Induction of VCAM-1 in NF-25 fibroblasts was identified, which was specific when co-cultured with HSC-39 but not with non-SGC-derived HSC-57 and HSC-64 cells. Neutralising antibody to VCAM-1 suppressed NF-25 growth in dose-dependent manners. In tissue samples, positive immunoreactivity of VCAM-1 in SGC-derived fibroblasts was significantly higher than that in non-SGC-derived fibroblasts. Furthermore, interaction with NF-25 fibroblasts not only induced the epithelial-mesenchymal transition-like change, but also expressions of matrix metalloproteinase- related genes in HSC-39 cells.

Conclusion: Direct interaction between SGC cells and gastric fibroblasts establishes the tumour microenvironment and reinforces the aggressiveness of SGC.

Figure 1

Cell–cell contact with SGC-derived HSC-39 cells upregulated NF-25 gastric fibroblasts’ growth. (A) Immunofluorescence of NF-25 fibroblasts co-cultured with HSC-39 cells. NF-25 fibroblasts and HSC-39 cells were stained with vimentin (green) and cytokeratin (red) ( × 200). (B) Growth curves of NF-25 gastric fibroblasts and NF-j2 intestine fibroblasts in the presence or absence of co-incubation with HSC-39 cells. Before counting of the numbers of NF-25 and NF-j2 fibroblasts, HSC-39 cells co-cultured were excluded by separation by a magnetic beads method. To examine the effect of soluble factors, NF-25 fibroblasts and HSC-39 cells were separately co-maintained using a 1-μm pore-sized Boyden Chamber inserts. ^*<0.01.

Figure 2

Identification of genes differentially expressed in NF-25 gastric fibroblasts in the presence or absence of cell–cell contact with HSC-39 cells. (A) Illustration of the strategy of cDNA microarray. NF-25 fibroblasts (1 × 10⁵ cells dish⁻¹) were maintained for 48 h in the presence or absence of HSC-39 cells (1 × 10⁵ cells dish⁻¹). HSC-39 cells were separated with magnetic beads epithelial cell enrichment. (B) Results of SAM plot analysis. Cy5 positive (light blue), genes upregulated in NF-25 fibroblasts co-cultured with HSC-39 cells; Cy3 positive (green), genes upregulated in NF-25 fibroblasts; positive (blue), genes equally expressed and negative (red), genes, which were not expressed.

Figure 3

Induction of VCAM-1 expression in NF-25 gastric fibroblasts is specifically induced by direct interaction with HSC-39 cells. NF-25 fibroblasts were co-incubated with HSC-39 (SGC-derived), HSC-57 (non-SGC-derived) and HSC-64 (non-SGC-derived) cells for 48 h. Cancer cells were separated with magnetic beads epithelial enrichment. (A) Results of RT–PCR analysis. The levels of β-actin expression were used as control. (B) Results of western blotting. The levels of β-actin expression were used as control. (C) Immunofluorescence and morphological changes of NF-25 fibroblasts in the presence or absence of co-culture with HSC-39 cells ( × 200). NF-25 fibroblasts were visualised with antibodies against vimentin (red) and VCAM-1 (green).

Figure 4

Effects of neutralising antibodies to VCAM-1 and integrin-α4 on NF-25 fibroblasts growth in the presence of co-culture with HSC-39 cells. NF-25 fibroblasts (1 × 10⁵ cells dish⁻¹) were co-maintained with HSC-39 cells (1 × 10⁵ cells dish⁻¹) and the various amounts of each antibody were added to the media. The numbers of NF-25 fibroblasts were counted 48 h after addition of these neutralising antibodies. (A) Effects of anti-VCAM-1 antibody (0–10 μg ml⁻¹). (B) Effects of anti-integrin-α4 antibody (0–10 μg ml⁻¹). Non-specific mouse IgG (5 μg ml⁻¹) was used as negative control. The experiments were performed thrice.

Figure 5

Immunohistochemistry of VCAM-1, Snail and E-cadherin expressions under VCAM-1-positive SGC (signet-ring cell carcinoma) condition and VCAM-1-negative non-SGC condition (moderately differentiated tubular adenocarcinoma). Histological examination was performed by haematoxylin and eosin (H&E) staining. Original maginification: × 200.

Figure 6

Effects of cell–cell contact on downregulation of cell adhesion molecules in NF-25 fibroblasts and induction of EMT-like change in HSC-39 cells. Results of western blot. Twenty-four hours after incubation of NF-25 and HSC-39 cells, the cells were separated by magnetic beads method. The levels of β-actin expression were used as control.

Figure 7

Effects of cell–cell contact on upregulation of MMPs in HSC-39 cells. Results of RT–PCR. Twenty-four hours after incubation of NF-25 fibroblasts and HSC-39 cells, the cells were separated by magnetic beads method. The levels of β-actin expression were used as control.