Establishment of five immortalized human ovarian surface epithelial cell lines via SV40 T antigen or HPV E6/E7 expression

Abstract

Background: Human ovarian surface epithelial (HOSE) cells are a critical cell source for ovarian cancer research; however, they are difficult to obtain and maintain under standard laboratory conditions in large quantities. The aim of this study was to generate immortalized HOSE (IHOSE) cells with maintained properties to the original cell source, thereby guaranteeing a sufficiently large cell quantity for ovarian cancer research.

Methods: HOSE cells isolated from four non-cancer patients and five IHOSE cell lines were established by induction of HPV-E6/E7 expression or SV40 large T antigen using a lenti-viral system. Each of IHOSE cells was confirmed to be distinct by STR profiling. RNA-sequencing was used to compare gene expression profiles in HOSE, IHOSE and ovarian cancer cells.

Results: RNA-sequencing results revealed a stronger linear correlation in gene expression between IHOSE and HOSE cells (R2 = 0.9288) than between IHOSE or HOSE cells and ovarian cancer cells (R2 = 0.8562 and R2 = 0.7982, respectively). The gene expression pattern of 319 differentially expressed genes revealed minimal differences between HOSE and IHOSE cells, while a strong difference between ovarian cancer cells and HOSE or IHOSE cells was observed. Furthermore, the five IHOSE cell lines displayed morphological characteristics typical of epithelial cells but showed a lower level of EpCAM, CD133 and E-cadherin, as cancer stem marker, than ovarian cancer cells. Moreover, unlike cancer cells, IHOSE cells could not form colonies in the anchorage-independent soft agar growth assay.

Conclusion: These findings demonstrate that five newly established IHOSE cell lines have characteristics of progenitor HOSE cells while exhibiting continuous growth, and thus, should be highly useful as control cells for ovarian cancer research.

Fig 1. Characterization of five immortalized human ovary surface epithelial (IHOSE) cell lines.

(A) Representative images showing the morphology of cells from five IHOSE cell lines. SE (Upper panel = Low density of cells; Bottom panel = High density of cells). Scale bar: 400 μm, 100× magnification. (B) Mycoplasm contamination testing was performed using 100 ng genomic DNA by RT-PCR. (C) The expression levels of SV40 T antigen and HPV E6/E7 protein in IHOSE cells were detected by RT-PCR. β-actin and GAPDH were used as loading controls. (D) The expression levels of indicated protein were determined by western blot analysis in the five IHOSE and cancer cell lines. β-actin and GAPDH were used as loading controls. (E) Cells (4 × 10⁴) were seeded in a 6-well plate and counted every 2 days up to 8 days. Cell proliferation was determined using an automated cell counter. Results indicate cell number ± SD.

Fig 2. Analysis of differentially expressed genes (DEGs) between IHOSE, HOSE, and ovarian cancer (Cancer) cells using RNA sequencing.

(A) Scatter plot revealing a linear correlation of gene expression pattern according to comparative analysis in HOSE versus IHOSE, IHOSE versus Cancer, and HOSE versus Cancer. (B) Venn diagram showing a common denominator in 319 DEGs based on sorting conditions of 2-fold change, a read-count threshold > 0.1, and p < 0.05. (C) Heatmap of 319 DEGs in three analyzed groups. (D) Heat map of downregulated genes (Cluster 1) or upregulated genes (Cluster 2) in ovarian cancer compared to HOSE and IHOSE. (E) Gene ontology related to biological process ordered according to p-value (–log10). Functional annotation clustering was performed using the DAVID algorithm (Upper panel = Cluster 1; Lower panel = Cluster 2). (F) Dendrogram for the clustering of each cell with Euclidean distance metric. (G) Validation of RNA-sequencing gene expression levels by real-time PCR for four common DEGs in the three groups. Data are represented as the mean fold change ± SE (Upper panel = RNA sequencing; Lower panel = Real-time PCR).

Fig 3. Immortalized human ovary surface epithelial (IHOSE) cell lines have no malignant characteristics.

(A) The expression levels of indicated protein for the cancer stem markers EpCAM, CD133, and E-cadherin were measured by western blot analysis (Star = non-specific band). (B) Soft agar colony formation of 5 IHOSE and OVCAR3 cell lines stained by 5 mg/mL MTT solution. Representative images of wells and colonies (Colonies = 100× magnification).