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. 2018 Oct 10;13(10):e0205494.
doi: 10.1371/journal.pone.0205494. eCollection 2018.

Ovarian stromal cells as a source of cancer-associated fibroblasts in human epithelial ovarian cancer: A histopathological study

Masayoshi Fujisawa  1 , Aye Moh-Moh-Aung  1 , Zheng Zeng  1 , Teizo Yoshimura  1 , Yoji Wani  2 , Akihiro Matsukawa  1
Affiliations

Ovarian stromal cells as a source of cancer-associated fibroblasts in human epithelial ovarian cancer: A histopathological study

Masayoshi Fujisawa et al. PLoS One. .

Abstract

Fibroblasts are a major component of cancer tissue and known to contribute to cancer progression. However, it remains unknown whether they are derived from local fibroblasts or of other origin. This study was designed to identify the contribution of local stromal cells to cancer stroma in human epithelial ovarian cancer. Seventy-six cases of surgically resected primary ovarian carcinoma (48 cases confined to the ovaries and 28 cases with distant metastases) and 17 cases of secondary ovarian tumor (e.g. colon cancer metastasized to the ovary) were enrolled in this study. The tissues were immunostained for forkhead box protein L2 (FOXL2), a transcription factor crucial for ovarian development and function, and markers for cancer-associated fibroblasts (CAFs) and inflammatory cells. Under normal condition, FOXL2 expression was restricted to ovarian stromal cells and some other types of cells in female genital tracts and never found in other sites of the body. FOXL2-positive cells were found in all primary and secondary tumors in the ovary, and were the dominant stromal cells in most cases. In contrast, only a few FOXL2-positive cells were found in peritoneal seeding sites of four serous carcinoma cases, and all the other tumors at extraovarian sites had no FOXL2-positive cells. FOXL2-positive cells in the ovarian lesion variably expressed CAFs markers, such as alpha-smooth muscle actin and fibroblast activating protein, as determined by double immunostaining. Background inflammation, but not histological subtype or origin of the neoplasm seemed to correlate with the proportion of FOXL2-positive cells. These results suggest that ovarian stromal cells are the main source of cancer stroma in the ovary but do not seem to move to distant sites via circulation together with tumor cells. Our results also support the hypothesis that cancer-associated fibroblasts may originate locally, which was previously demonstrated using animal models.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Distribution of FOXL2-positive stromal cells in the ovary.
(A and B) Normal ovarian cortex. Almost all stromal cells as well as granulosa cells (arrow head) show nuclear positivity for FOXL2. By contrast, none of the cells that constitute blood vessels (asterisk) have nuclear FOXL2. (C and D) Primary mucinous carcinoma. (E and F) Primary serous carcinoma. (G and H) Secondary ovarian tumor (ovarian metastasis of rectal adenocarcinoma). Most stromal spindle cells show nuclear positivity for FOXL2. (I and J) Primary endometrioid carcinoma, a rare example of primary ovarian cancer containing only a few FOXL2-postive cells. H&E (left panels) and FOLX2 immunostaining of the corresponding area (right panels; only nuclear staining is considered positive). Arrows indicate FOXL2-positive cells. The bar in (A) indicates 50μm, and the magnification is identical for all the pictures.
Fig 2
Fig 2. Distribution of FOXL2-positive stromal cells in extraovarian lesions.
(A and B) A metastatic site of mucinous ovarian carcinoma in the lung (the same case as Fig 1C and 1D). There are no FOXL2-positive stromal cells. (C and D) A peritoneal seeding site of serous ovarian carcinoma (the same case as Fig 1E and 1F). There are few FOXL2-positive cells. (E and F) The primary site of rectal cancer that caused secondary ovarian tumor (the same case with Fig 1G and 1H). There are no FOXL2-positive stromal cells. H&E (left panels) and FOLX2 immunostaining of the corresponding area (right panels). Arrows indicate FOXL2-positive cells. The bar in (A) indicates 50μm, and the magnification is identical for all the pictures.
Fig 3
Fig 3. The proportion of FOXL2-positive cells in cancer stroma.
The proportion of FOXL2-positive cells on each tissue section was scored using 5-tired scale and the results were plotted. High percentages of cancer stromal cells were FOXL2-positive in the ovary, whereas there were almost no FOXL2-positive cells outside the ovary (Ext-O). Asterisks indicate a significant difference between the groups by Mann-Whitney test (p < 0.05).
Fig 4
Fig 4. Expression of ASMA and FAP by FOXL2-positive ovarian cells.
(A-C) Tumor tissues were double stained for FOXL2 and ASMA or FAP. Nuclear FOXL2 was stained brown whereas cytoplasmic ASMA and FAP were stained red in B and C, respectively. Some FOXL2-positive cells were also stained positive for ASMA or FAP (arrows), but others were negative (arrow heads). (D and E) The frequency of ASMA and FAP positive cells in FOXL2-positive cells. ASMA and FAP were variously but at least focally expressed by FOXL2-positive cells of all cases. Asterisks indicate a significant difference between two groups (Kruskal-Wallis test followed by multiple comparison).
Fig 5
Fig 5. Correlation between chronic inflammation and FOXL2-positive cells in ovarian cancer stroma.
(A-E) An example of endometrioid carcinoma where FOXL2-positive stromal cells were scant (FOXL2-poor). (A) Low magnification of H&E. The area above the dotted line is cancer tissue and the area below the line is a pre-existing endometrial cyst. (B and C) CD138 immunostaining. Tumor area (B: red square in A) contained no plasma cells and nontumor area (C: blue square in A) had a few plasma cells (arrows). (D and E) CD163 immunostaining. Both tumor area (D) and nontumor area (E) had some macrophages (arrows). (F-I) The number of CD138-positive plasma cells (F and G) or CD163-positive macrophages (H and I) in one high power field was plotted, comparing between cases with FOXL2-poor stroma (score1 and 2) and those with FOXL2-rich stroma (score 3 and 4). The presence of plasma cells apart from tumor cells correlated with FOXL2-positive cell population, whereas the presence of plasma cells near tumor cells or macrophages in any place did not. The asterisk indicates a significant difference between the groups by Mann-Whitney test (p < 0.05).
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