Small putative NANOG, SOX2, and SSEA-4-positive stem cells resembling very small embryonic-like stem cells in sections of ovarian tissue in patients with ovarian cancer

Abstract

Background: In previous studies it has been found that in cell cultures of human adult ovaries there is a population of small stem cells with diameters of 2-4 μm, which are present mainly in the ovarian surface epithelium and are comparable to very small embryonic-like stem cells (VSELs) from bone marrow. These cells are not observed by histopathologists in the ovarian tissue due to their small size and unknown clinical significance. Because these cells express a degree of pluripotency, they might be involved in the manifestation of ovarian cancer. Therefore we studied the ovarian tissue sections in women with borderline ovarian cancer and serous ovarian carcinoma to perhaps identify the small putative stem cells in situ.

Methods: In 27 women with borderline ovarian cancer and 20 women with high-grade serous ovarian carcinoma the ovarian tissue sections were stained, per standard practice, with eosin and hematoxylin staining and on NANOG, SSEA-4 and SOX2 markers, related to pluripotency, using immunohistochemistry. We focused on the presence and localization of small putative stem cells with diameters of up to 5 μm and with the nuclei spread over nearly the full cell volume.

Results: In ovarian sections of both borderline ovarian cancer and serous ovarian carcinoma patients we were able to identify the presence of small round cells complying with the above criteria. Some of these small cells were NANOG-positive, were located among epithelial cells in the ovarian surface epithelium and as a single cell or groups of cells/clusters in typical "chambers", were found only in the presence of ovarian cancer and not in healthy ovaries and are comparable to those in fetal ovaries. We envision that these small cells could be related to NANOG-positive tumor-like structures and oocyte-like cells in similar "chambers" found in sections of cancerous ovaries, which could support their stemness and pluripotency. Further immunohistochemistry revealed a similar population of SSEA-4 and SOX2-positive cells.

Conclusions: We may conclude that putative small stem cells expressing markers, related to pluripotency, are present in the ovarian tissue sections of women with borderline ovarian cancer and high-grade serous ovarian carcinoma thus indicating their potential involvement in ovarian cancer.

Fig. 1

NANOG-positive cells (arrows) in the ovarian surface epithelium. They appeared among epithelial cells (a-d, g, h) without any special order, or just below them (e and f) in women with borderline ovarian cancer and high-grade serous ovarian carcinoma. (Light microscope: a-c, f, magnification 1000x; inverted microscope: d, e, g, h, magnifications 100x or 200x). Legend: brown-NANOG-positivity, blue-nuclei after HE staining. Red Bar: 100 μm for a-c, f, 50 μm for d and 10 μm for e, g, h

Fig. 2

Special “chambers” containing small, round cells (arrows) or groups/clusters (asterisks) of these cells. The diameter of these small, round cells was up to 5 μm with nuclei filling almost the whole cell volumes. They were located among epithelial cells or just below them in ovarian sections of women with borderline ovarian cancer after hematoxylin-eosin (HE) staining (a-h). In the vicinity of these “chambers” the ovarian surface epithelium was drastically changed with several layers of proliferated epithelial cells, epithelial cells of atypical shapes (e.g., extended) and formation of papillae. (Light microscope, magnification 1000x). Red Bar: 100 μm

Fig. 3

Small NANOG-positive cells (arrows) with diameters of up to 5 μm in “chambers”. These “chambers” containing small, round, NANOG-positive cells were located among epithelial cells in ovarian tissue sections of women with high-grade serous ovarian carcinoma (a-f). The surrounding epithelial cells also became NANOG-positive in some places (d-f), as possibly influenced by small cells in those “chambers”. Controls were provided: negative control (g) and positive control-testicular embryonal carcinoma with strongly stained cells in seminiferous tubules (h) (Light microscope: a, magnification 1000x; inverted microscope: b-h, magnifications 40x, 100x and 200x). Legend: brown-NANOG-positivity, blue-nuclei after HE staining. Red Bar: 10 μm for b-d, 50 μm for f, h and 100 μm for a, e, g

Fig. 4

Highly abundant “chambers” containing small, round cells (arrows) or clusters of cells (asterisks) in ovarian tissue. They were present in borderline ovarian cancer (a-c) and high-grade serous ovarian carcinoma (d-h) tissue. In a proportion of “chambers” the small, round cells were NANOG-positive and were stained brown. (Light microscope: a-c, magnifications 200x or 1000X; inverted microscope: d-h, magnifications 40x and 100x). Legend: brown-NANOG-positivity, blue-nuclei after HE staining. Red Bar: 100 μm for a-d and 50 μm for e-h

Fig. 5

Ovarian tissue sections with “chambers” of different shapes containing groups of small, round cells (arrows). These small cells had diameters of up to 5 μm. Their nuclei filled almost the whole cell volumes in two patients with high-grade serous ovarian carcinoma after HE staining (a-f). A proportion of these cells were NANOG-positive (g and h). “Chambers” were surrounded by epithelial/cancer cells. Figures a, b, c, e and f show the same structures at different magnifications to get better insight. (Light microscope: a-g, magnifications 100x, 200x and 1000x; inverted microscope: h, magnification 200x). Legend: brown-NANOG-positivity, blue-nuclei after HE staining. Red Bars: 10 μm for h and 100 μm for a-g

Fig. 6

Tumor-like structures (arrows) developed in “chambers” present in ovarian tissue (a-g). They were present in ovaries of women with borderline ovarian cancer and high-grade serous ovarian carcinoma. The tumor-like structures were strongly NANOG-positive (c-g). A small proportion of tumor-like structures in borderline ovarian cancer was mineralized and with no visible nuclei (b). Some tumor-like structures were composed of small cells with clearly visible nuclei (g). (Light microscope: a-d, g, magnification 100x and 1000x; inverted microscope: e-f, magnification 200x). Legend: brown-NANOG-positivity, blue-nuclei after HE staining. Red Bars: 100 μm for a-d, g and 10 μm for e, f

Fig. 7

Primitive oocyte-like cells in “chambers” present in ovarian tissue. They were present in borderline ovarian cancer (a) and high-grade serous ovarian carcinoma tissue (b-e). The primitive oocyte-like cells were slightly NANOG-positive (b-e) and small, round, NANOG-positive cells (arrows) were attached to them. The oocyte-like cells appeared from these small cells inside the ovarian tumor. The structure of cytoplasm in these cells was quite comparable to human oocytes. Figures b-e show the same structures at different magnifications to get better insight. (Light microscope: a, magnification 1000x; inverted microscope: b-e, magnifications 200x). Legend: O-oocyte-like cell. Legend: brown-NANOG-positivity, blue-nuclei after HE staining. Red Bars: 100 μm for (a), 50 μm for (b) and 10 μm for (c-e)

Fig. 8

Small putative stem cells (arrows) expressing SSEA-4 and SOX2-positivity after immunohistochemistry and blue nuclei after DAPI staining of ovarian sections. Small SSEA-4 and SOX2-positive cells were proliferated in ovarian surface epithelium or were found in typical “chambers” with atrophic and autofluorescent (af) tissue nearby. Legend: green-SSEA-4-positivity, red-SOX2-positivity, ose-ovarian surface epithelium, af-autofluorescence, er-erythrocytes (without nuclei). Red bars: 10 μm