Normal fibroblasts induce E-cadherin loss and increase lymph node metastasis in gastric cancer

Abstract

Background: A tumor is considered a heterogeneous complex in a three-dimensional environment that is flush with pathophysiological and biomechanical signals. Cell-stroma interactions guide the development and generation of tumors. Here, we evaluate the contributions of normal fibroblasts to gastric cancer.

Methodology/principal findings: By coculturing normal fibroblasts in monolayers of BGC-823 gastric cancer cells, tumor cells sporadically developed short, spindle-like morphological characteristics and demonstrated enhanced proliferation and invasive potential. Furthermore, the transformed tumor cells demonstrated decreased tumor formation and increased lymphomatic and intestinal metastatic potential. Non-transformed BGC-823 cells, in contrast, demonstrated primary tumor formation and delayed intestinal and lymph node invasion. We also observed E-cadherin loss and the upregulation of vimentin expression in the transformed tumor cells, which suggested that the increase in metastasis was induced by epithelial-to-mesenchymal transition.

Conclusion: Collectively, our data indicated that normal fibroblasts sufficiently induce epithelial-to-mesenchymal transition in cancer cells, thereby leading to metastasis.

Figure 1. Morphological changes in BGC-823 cells were associated with the epithelial-mesenchymal-transition.

Figure 1.1. Schematic of the experimental protocol. Figure 1 .2. Origin of the supernatant cells. (A) GFP-labeled BGC-823 cells (green). (B) BGC-823 cells that were cocultured with fibroblasts (red). (C) BGC-823 cells grew into clumps and demonstrated round shapes in suspension. (D) TBGCs were induced by coculturing. Magnification 100×. Figure 1 .3. Transformation from BGC-823 cells to TBGCs under a phase-contrast microscope. (A–D) BGC-823 cells in a dense culture system can form clusters and shed off cells into the suspension. (E–H) Passage of suspended tumor cells. Figure 1 .4. Immunofluorescent staining of pan-CK (red), E-cadherin (red), vimentin (red) and N-cadherin (red). BGC-823 cells (above) and TBGCs (below) were labeled with GFP (green). The nucleus was stained with DAPI (blue). Figure 1 .5. Heat plot of gene expression profiles analyzed using fluorescence quantitative RT-PCR. The depth of the color indicates relative gene expression. Figure 1 .6. Western blots of proteins. Figure 1 .7. Bar plot of protein expression determined using Western blot analysis. Bars denote the relative expression levels measured using IOD. Vertical lines denote standard differences. **p<0.01.

Figure 2. Proliferation, invasion, and mobility of TBGCs.

Figure 2.1.Line plot of the proliferation assay. Vertical lines denote standard differences. **p<0.01. Figure 2 .2. Line plot of the scratching assay. Vertical lines denote standard differences. **p<0.01. Figure 2 .3. Scratching assay of BGC-823 cells (above) and TBGCs (below) under a phase-contrast microscope. (A–D, E–H) Time from initial to 72 hours. Figure 2 .4. Box plot of the invasive and migration assays. Differences between BGC-823 cells and TGBCs are significant (p<0.01). Figure 2 .5. The top 2 photos show the results of the invasive modified transwell assay with Matrigel. Representative images of the transwell invasion assays for (A) BGC and (B) TBGC. Cells invading the underside of the transwell insert are shown. The bottom 2 photos are representative images of the transwell migration assays for (C) BGC and (D) TBGC. Cells migrating to the underside of the transwell insert are shown.

Figure 3. TBGCs exhibited cisplatin resistance.

Figure 3 .1. Twenty-four-hour line plot of the cisplatin-inhibition test. **p<0.01. Figure 3 .2. Twenty-four-hour line plot of the 5-FU inhibition test. Figure 3 .3. Flow cytometry was used to assess apoptosis in BGC and TBGC cells after exposure to different concentrations of cisplatin (20, 40 µM) for 24 hours. Cells were stained with Annexin V-FITC (marker of apoptosis) and propidium iodide (PI) (marker of dead cells). Figure 3 .4. Bar plot of cisplatin-induced apoptosis in BGC-823 cells and TBGCs. Bar graph showing the percentage of apoptotic cells according to flow cytometry. **p<0.01 vs cells in the dimethyl sulfoxide (DMSO) control wells.

Figure 4. TBGCs exhibit in vivo lymph node propensity in the vein injection group.

Figure 4 .1. Cumulative risk of lymph nodes metastasis in different groups that received tail vein injections. Figure 4 .2. HE staining (at 10 weeks) of organs in the group that received vein injections. Positivity was observed in the intestines of both groups and in the lungs of the BGC groups. No evident liver or kidney metastases were found. Figure 4 .3. Fluorescent tracing of tumor cells at week 2 in the groups that received tail vein injections. Tumor cells were labeled with green fluorescence as indicated by the yellow arrowheads. The nucleus was stained with DAPI (blue). Magnification 100×. Figure 4 .4. Immunohistochemical staining for E-cadherin and β-catenin in the lymph nodes of the groups that received tail vein injections at each time point. Magnification 200×. Figure 4 .5. Bar plot of the relative expression levels of E-cadherin and β-catenin in the lymph nodes of the groups that received tail vein injections at each time point. **p<0.01; *p<0.05.

Figure 5. TBGCs demonstrated low tumor formation but high lymph node metastasis capacity in our subcutaneous tumorigenesis model.

Figure 5.1.General tumorigenesis observations in the BGC and TBGC groups at each time point. Figure 5 .2. Cumulative risks of lymph nodes metastasis in the different tumorigenesis groups. Figure 5 .3. Fluorescent tracing of tumor cells at week 2 in the tumorigenesis groups. Tumor cells are labeled with green fluorescence and indicated by the yellow arrowheads. The nucleus was stained with DAPI (blue). Figure 5 .4. Immunohistochemical staining for E-cadherin and β-catenin in the lymph nodes of the tumorigenesis groups at each time point. Magnification 200×. Figure 5 .5. Bar plot of the relative expression of E-cadherin and β-catenin in the lymph nodes of the tumorigenesis groups at each time point. **p<0.01; *p<0.05. NA: no metastatic lymph nodes were observed in the BGC group.