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.
Highlights
The incidence of gastric cancer is declining but remains a major cause of cancer-related death worldwide
As continuous human peritoneal mesothelial cell (HPMC) monolayers act as a barrier against peritoneal metastasis, once the diapedesis of exfoliated gastric cancer cells through mesothelial cell monolayers occurs, the abundant blood supply in the matrix beneath the mesothelium will offer a comfortable environment for metastatic gastric cancer cells and promote their colonization
Diapedesis was quantified as the number of tumor cells that were in contact with the mesothelium and classified into three stages according to their position relative to the mesothelium: 1) rounded - tumor cells with a spherical shape on the apical surface of the mesothelium; 2) migrating - tumor cells had penetrated through the mesothelial cell monolayers at mesothelial cell junctions with a portion of the cell body above the mesothelium and a portion of the cell body spread on the Matrigel beneath the mesothelial cell F-actin stress fibers; and 3) underneath - the entire tumor cell body was below the plane of the mesothelial cell stress fibers
Summary
The incidence of gastric cancer is declining but remains a major cause of cancer-related death worldwide. During the process of peritoneal metastasis, gastric cancer cells interact intercellularly with multiple cell types, and the interaction of the intra-abdominal exfoliated gastric cancer cells and the peritoneal mesothelial cells is of particular importance. Diapedesis of exfoliated gastric cancer cells through the mesothelial cell monolayer is an essential step during peritoneal metastasis. The interaction between these cells is accompanied by intercellular communication (IC), which is predominantly mediated by cellcell gap junctions [2]. Whether the Cx43 mediated gap junction plays an important role in diapedesis of gastric cancer cells through the peritoneal mesothelial barrier remains unclear. To address the hypothesis we examined the expression of Cx43 in primary gastric cancer tissues, exfoliated gastric cancer cells, and peritoneal metastatic tissues. In this study we show that Cx43 expression upregulates tumor cell diapedesis via a GJICdependent mechanism
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