GFP Reporter Cell Line - ID8

Epithelial ovarian cancer (EOC) patients are often diagnosed with peritoneal metastasis and ascites, i.e., fluid accumulation in the peritoneal cavity containing non-malignant cells. However, the interaction mechanism between EOC and non-malignant cells before peritoneal metastasis remains unclear. To investigate this issue, researchers used multiphoton microscopy to observe intact EOC spheroids and assess their invasiveness. Through morphological evaluation, immunohistochemical/immunofluorescence staining, and single-cell RNA sequencing analysis, mesothelial cells were found to be the main component of ascites. Almost all EOC cells were spheroids, with 60% containing mesothelial cells. EOC cells can rapidly form aggregated spheroids with mesothelial cells, and these aggregated cancer-mesothelial spheroids (ACMS) can invade the collagen layer or mesothelial layer. The formation of ACMS by mesothelial cells initiates the invasion process. RNA sequencing analysis showed significant changes in RNA expression in mesothelial cells, while changes in ovarian cancer cells were smaller. Transforming growth factor-β1-stimulated mesothelial cells exhibited increased invasive pseudopodia formation and upregulated fascin-1 expression. These findings suggest that ovarian cancer cells alter mesothelial cells through ACMSs, thus explaining the rapid spread of ovarian cancer cells within the peritoneal cavity.

To test the hypothesis that mesothelial cells exist within cancer spheroids, researchers constructed a Wt1-CreERT2; tdTomato reporter gene mouse model. The Wt1-CreERT2 mouse strain, combined with a fluorescent reporter system, effectively tracks the lineage of mesothelial cells in various organs, including the peritoneum, heart, and kidneys. In this model, Wt1-positive cells expressed tdTomato fluorescent protein after tamoxifen induction (Figure 1K). In the peritoneal cavity, researchers confirmed that tdTomato was expressed only in the mesothelial layer four weeks after tamoxifen induction, and these cells simultaneously expressed WT1 and tdTomato on the surface of the peritoneum and greater omentum (Figure 1L). They injected green fluorescent protein (GFP)-labeled ID8 mouse ovarian cancer cells into the peritoneal cavity. Four weeks after injection, researchers isolated cancer spheroids from the malignant ascites. More than 70% of these spheroids consisted of GFP-positive ID8 and tdTomato-positive mesothelial cells (Figure 1M and N).

Figure 1. Mesothelial cells are present in ovarian cancer spheroids. (Uno K, et al., 2026)