Aberrant expression of Cx43 is associated with the peritoneal metastasis of gastric cancer and Cx43-mediated gap junction enhances gastric cancer cell diapedesis from peritoneal mesothelium

Bo Tang, Zhi-hong Peng, Pei-wu Yu, Ge Yu, Feng Qian, Dong-zhu Zeng, Yong-liang Zhao, Yan Shi, Ying-xue Hao, Hua-xing Luo, Bo Tang, Zhi-hong Peng, Pei-wu Yu, Ge Yu, Feng Qian, Dong-zhu Zeng, Yong-liang Zhao, Yan Shi, Ying-xue Hao, Hua-xing Luo

Abstract

The process of peritoneal metastasis involves the diapedesis of intra-abdominal exfoliated gastric cancer cells through the mesothelial cell monolayers; however, the related molecular mechanisms for this process are still unclear. Heterocellular gap-junctional intercellular communication (GJIC) between gastric cancer cells and mesothelial cells may play an active role during diapedesis. In this study we detected the expression of connexin 43 (Cx43) in primary gastric cancer tissues, intra-abdominal exfoliated cancer cells, and matched metastatic peritoneal tissues. We found that the expression of Cx43 in primary gastric cancer tissues was significantly decreased; the intra-abdominal exfoliated cancer cells and matched metastatic peritoneal tissues exhibited increasing expression compared with primary gastric cancer tissues. BGC-823 and SGC-7901 human gastric cancer cells were engineered to express Cx43 or Cx43T154A (a mutant protein that only couples gap junctions but provides no intercellular communication) and were co-cultured with human peritoneal mesothelial cells (HPMCs). Heterocellular GJIC and diapedesis through HPMC monolayers on matrigel-coated coverslips were investigated. We found that BGC-823 and SGC-7901 gastric cancer cells expressing Cx43 formed functional heterocellular gap junctions with HPMC monolayers within one hour. A significant increase in diapedesis was observed in engineered Cx43-expressing cells compared with Cx43T154A and control group cells, which suggested that the observed upregulation of diapedesis in Cx43-expressing cells required heterocellular GJIC. Further study revealed that the gastric cancer cells transmigrated through the intercellular space between the mesothelial cells via a paracellular route. Our results suggest that the abnormal expression of Cx43 plays an essential role in peritoneal metastasis and that Cx43-mediated heterocellular GJIC between gastric cancer cells and mesothelial cells may be an important regulatory step during metastasis. Finally, we observed that the diapedesis of exfoliated gastric cancer cells through mesothelial barriers is a viable route of paracellular migration.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Cx43 expression in normal gastric…
Figure 1. Cx43 expression in normal gastric tissue specimens, primary gastric carcinoma tissues, matched metastatic peritoneal tissues and intra-abdominal exfoliated gastric cancer cells.
(A) Cx43 was expressed in normal epithelia with typical membranous and cytoplasmic staining. (B) Cx43 expression in well-differentiated gastric cancer tissues, cancer cells showed a positive membrane and cytoplasmic staining pattern. (C) Cx43 showed a major positive staining in cytoplasm in moderately differentiated gastric cancer tissues (D) Cx43 expression in poorly differentiated gastric cancer tissues, tumour cells show a negative staining pattern. (E) Cx43 expression in metastatic peritoneal tissues showed a positive staining pattern. (F) Intra-abdominal exfoliated gastric cancer cells were determined by Hematoxylin and eosin (H and E) staining. (G, H, I) The expression of Cx43 in intra-abdominal exfoliated gastric cancer cells was accessed by immunofluorescence. Original magnification: (A-F, ×400).
Figure 2. Effects of Cx43 on in…
Figure 2. Effects of Cx43 on in vitro adhesion of BGC-823 and SGC-7901gastric cancer cells to mesothelial cells after transfection.
(A) Cx43 and Cx43T154A expression of BGC-823 and SGC-7901 gastric cancer cells after transfection. (B, C) Effects of Cx43 on in vitro adhesion of BGC-823 and SGC-7901 gastric cancer cells to mesothelial cells. Adherent tumor cells were counted (magnification: ×200), the results showed that adhesion of cells transfected with Cx43 or Cx43T154A was significantly increased compared to that of cells transfected with empty vector (*P

Figure 3. Fluorescence recovery images after photobleaching.

Figure 3. Fluorescence recovery images after photobleaching.

1.photobleaching mesothelial cells; 2.adjacent gastric cancer cells. A,…

Figure 3. Fluorescence recovery images after photobleaching.
1.photobleaching mesothelial cells; 2.adjacent gastric cancer cells. A, before bleaching; B, C, D, E, F representing images in 0, 1, 2, 3, 4min after photobleaching, respectively.

Figure 4. Tumor cell diapedesis through mesothelial…

Figure 4. Tumor cell diapedesis through mesothelial cell monolayers.

Optical sections obtained by laser scanning…

Figure 4. Tumor cell diapedesis through mesothelial cell monolayers.
Optical sections obtained by laser scanning confocal microscopy at the focal levels indicated in (A, E, I) identified three major stages of diapedesis: round on top of (A–D), migrating through (E–H), or located underneath (I–L) the mesothelium. (B) Gastric cancer cells with a round shape had filopodial extensions presented in the cytoplasm (arrows). (H) Gastric cancer cells located in mesothelial cell-cell junctions had long spindle and thick bundles of F-actin presented close to cytoplasm (arrows). (K, L) Those that completed diapedesis showed prominent stress fibers distributed loosely in cytoplasm (arrows).

Figure 5. Connexin 43 mediated gap junctional…

Figure 5. Connexin 43 mediated gap junctional intercellular communication enhances tumor cell diapedesis.

Adherent cells…

Figure 5. Connexin 43 mediated gap junctional intercellular communication enhances tumor cell diapedesis.
Adherent cells in the process of diapedesis and cells that had completed diapedesis were scored together as having migrated cells successfully. (A) Percentage of adherent migrating cells in BGC-823 group cells(%), compared to BGC-823v (control), a significant increase in diapedesis of BGC-823Cx43 cells was observed at 1, 4 and 7 h (*p
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References
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This work was supported by grant No. 30801097 and No. 81272366 from the National Natural Science Foundation of China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Figure 3. Fluorescence recovery images after photobleaching.
Figure 3. Fluorescence recovery images after photobleaching.
1.photobleaching mesothelial cells; 2.adjacent gastric cancer cells. A, before bleaching; B, C, D, E, F representing images in 0, 1, 2, 3, 4min after photobleaching, respectively.
Figure 4. Tumor cell diapedesis through mesothelial…
Figure 4. Tumor cell diapedesis through mesothelial cell monolayers.
Optical sections obtained by laser scanning confocal microscopy at the focal levels indicated in (A, E, I) identified three major stages of diapedesis: round on top of (A–D), migrating through (E–H), or located underneath (I–L) the mesothelium. (B) Gastric cancer cells with a round shape had filopodial extensions presented in the cytoplasm (arrows). (H) Gastric cancer cells located in mesothelial cell-cell junctions had long spindle and thick bundles of F-actin presented close to cytoplasm (arrows). (K, L) Those that completed diapedesis showed prominent stress fibers distributed loosely in cytoplasm (arrows).
Figure 5. Connexin 43 mediated gap junctional…
Figure 5. Connexin 43 mediated gap junctional intercellular communication enhances tumor cell diapedesis.
Adherent cells in the process of diapedesis and cells that had completed diapedesis were scored together as having migrated cells successfully. (A) Percentage of adherent migrating cells in BGC-823 group cells(%), compared to BGC-823v (control), a significant increase in diapedesis of BGC-823Cx43 cells was observed at 1, 4 and 7 h (*p

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