Exploring the characteristics of immortalized human ovarian surface epithelial cell lines

Abstract

The origins of epithelial ovarian cancer (EOC) have long been debated, with proposed sources including ovarian surface epithelial (OSE) cells, secondary Müllerian tract structures, or fallopian tube epithelium. Despite being the second most common gynecological cancer and a leading cause of death in the United States, in vitro cell models mimicking normal ovarian epithelial cells and their malignant counterparts are lacking. To address this gap, we established immortalized human OSE (IHOSE) cell lines that demonstrate stable in vitro growth without malignant properties. IHOSE cell lines were unique cell lines by analyzing short tandem repeat (STR) profiling. In addition, the epithelial characteristics were confirmed by cytokeratin 7 and cytokeratin 18 marker expression. IHOSE cell lines were subjected to Opal multiplex immunohistochemistry (IHC) analysis, which established four distinct subtypes based on marker dominance. These studies offer the most basic but essential cellular characterization information for IHOSE cell lines, providing critical data that can guide the selection of cells when inducing normal controls or disease models.

Keywords: Epithelial ovarian cancer; Immortalized human OSE (IHOSE) cell lines; Opal multiplex-IHC; Single-cell heterogeneity.

Fig. 1

The novel six IHOSE cell lines were established via SV40 T or HPV E6/E7. (A) Representative images show the morphology of aged cells (indicated by arrows). Scale bar: 200 μm, 200× magnification. (B) After the immortalization process, the left images show that epithelial cells are cultured. The right image is epithelial cells with fibroblast, and they isolate only epithelial cells excluding fibroblast using a Cloning ring. (C) The expression levels of SV40 T antigen and HPV E6/E7 in IHOSE cells were detected by RT-PCR. β-actin was used as a loading control. (D) The expression levels of indicated protein were measured by immunoblotting in the IHOSE and cancer cell lines. ɑ-actinin was used as a loading control. (E) Cytoskeleton was detected by Immunofluorescence using an anti-ɑ-tubulin (green) antibody in six IHOSE cell lines. Nuclei are stained with Hoechst (blue). Scale bar: 100 μm, 400× magnification.

Fig. 2

Maintained cell morphology of IHOSE for ten passages (A) Growth curves and doubling time of cells were determined by counting the cells every 2 days for 8 days. Error bars represent mean ± SD. (B). Representative images showed high and low-density cell morphology from six IHOSE cell lines. The upper panel shows the early passage of cells; the lower panel shows the late passage of cells. Scale bar: 200 μm, 200 × magnification. (C). The expression levels of indicated protein were measured by immunoblotting in the IHOSE cell lines of early and late passages. ɑ-actinin was used as a loading control.

Fig. 3

Non-malignancy characteristics of IHOSE cell lines. (A) The expression levels of indicated protein were measured by immunoblotting in the IHOSE and cancer cell lines. ɑ-actinin was used as a loading control. (B) The expression levels of CA-125 were confirmed via immunohistochemistry staining in the IHOSE and cancer cell lines. Scale bar: 50 μm. (C) Anchorage-independent soft agar assay performed in IHOSE and TOV112D cell lines. Colonies were stained by MTT solution. Representative images of colonies captured with 40x and 100x magnification. Scale bar: 10 μm and 5 μm, respectively.

Fig. 4

7-Color Multiplex IHC to visualize IHOSE and ovarian cancer cell lines in FFPE sections of the cell block. (A) The cell lines were analyzed in 7-marker panels in one FFPE section, showing the colocalized image of 7-markers. Biomarkers and colors are shown on the right. Scale bar: 50 μm. (B) Two cell lines represented the colocalized images of the 7-marker and corresponding single-color images; 50 μm and 20 μm, respectively.

Fig. 5

Single-cell populations in IHOSE and ovarian cancer cell lines. Violin (A) and ridge (B) plots depict the expression levels of each marker. (C) UMAP plot showing single-cell populations of IHOSE and ovarian cancer cell lines.

Fig. 6

The pie chart displays the proportions of phenotype in each cell line. Pie-chart showing the phenotypes of IHOSE (A) and ovarian cancer cell lines(B). (C) Among the 57 phenotypes, only those accounting for >2 % are indicated.