Differential expression of aldehyde dehydrogenase 1a1 (ALDH1) in normal ovary and serous ovarian tumors

Abstract Background We showed there are specific ALDH1 autoantibodies in ovarian autoimmune disease and ovarian cancer, suggesting a role for ALDH1 in ovarian pathology. However, there is little information on the ovarian expression of ALDH1. Therefore, we compared ALDH1 expression in normal ovary and benign and malignant ovarian tumors to determine if ALDH1 expression is altered in ovarian cancer. Since there is also recent interest in ALDH1 as a cancer stem cell (CSC) marker, we assessed co-expression of ALDH1 with CSC markers in order to determine if ALDH1 is a potential CSC marker in ovarian cancer. Methods mRNA and protein expression were compared in normal human ovary and serous ovarian tumors using quantitative Reverse-Transcriptase PCR, Western blot (WB) and semi-quantitative immunohistochemistry (IHC). ALDH1 enzyme activity was confirmed in primary ovarian cells by flow cytometry (FC) using ALDEFLUOR assay. Results ALDH1 mRNA expression was significantly reduced (p Conclusions Total ALDH1 expression is significantly reduced in malignant ovarian tumors while it is relatively unchanged in benign tumors compared to normal ovary. Thus, ALDH1 expression in the ovary does not appear to be similar to breast, lung or colon cancer suggesting possible functional differences in these cancers. Significance These observations suggest that reduced ALDH1 expression is associated with malignant transformation in ovarian cancer and provides a basis for further study of the mechanism of ALDH1 in this process.</p

Localization of immune cells in early stage ovarian tumors.

<p>Bu1a+, CD4+ and CD8+ cells are shown in similar regions of serial sections that contain tumor foci. More CD8+ T cells and B cells are present in tumor foci than CD4+ T cells. (<b>A</b>) Early-stage tumor showing CD4+ cells in a tumor with poorly differentiated (PD) structure, and CD8+ and B cells in an adjacent well differentiated (WD) area. (<b>B</b>) A small lesion (dotted circle) containing CD8+ T cells and Bu1a+ cells, but not CD4+ cells. (<b>C</b>) An area with neoplastic cells containing CD8+ and Bu1a+ cells, but not CD4+ cells. Original magnification, 10x; scale bar = 100 µm. Stained CD8+ lymphocytes in a selected area (dotted box) is shown in an inset at higher magnification; scale bar = 10 µm.</p

Localization of immune cells in normal hen ovaries.

<p>Bu1a+, CD4+ and CD8+ cells (arrows) detected by immunohistochemistry in normal ovaries in the ovarian stroma (S), adjacent to follicles (f), in the thecal layer of follicles (double arrow) and were abundant in the cell layer under the ovarian surface epithelium (SE). Original magnification, 10x; scale bar = 100 µm. Selected areas (dotted boxes) are shown in an inset at higher magnification for Bu1a and CD8 staining; inset scale bars = 10 µm.</p

The number of Bu1a+ B cells and CD4+ and CD8+ T cells in normal ovaries and ovarian tumors determined by morphometric analysis.

<p>As shown in panels <b>A-C for normal (N), early (E) and late stage (L) ovarian cancer</b>, Bu1a+ cells and CD8+ T cells were more numerous overall than CD4+ T cells, particularly in late stage tumors. In panel <b>D</b>, the B and T cells counts were added. There was an overall increase in total (non-follicular) immune cells from normal ovary to late stage tumors. Cells were counted in multiple fields in three sections from each ovary for each hen at 20x magnification. The average number of B (Bu1a+) cells and CD4+ and CD8+ T cells was estimated from 11 hens with normal ovaries (number of fields counted was Bu1a = 524, CD4 = 425 and CD8 = 360), 8 hens with early stage ovarian cancer (number of fields counted was Bu1a = 228, CD4 = 190 and CD8 = 225) and 7 hens with late stage ovarian cancer (number of fields counted was Bu1a = 180, CD4 = 190 and CD8 = 200). Since the areas evaluated varied in size, all counts were normalized to 8×10⁴ µm².</p

Selection of hens with normal ovaries or ovarian tumors using (A–C) color Doppler ultrasound and (D–F) gross morphology of the ovaries.

<p>A normal ovary (<b>A and D</b>) with maturing ovarian follicles (F1, F2) and stromal blood vessels (arrows) with normal gross morphology showing maturing (F1, F2) ovarian follicles. An example of an abnormal ovary (<b>B and E</b>) that contains more blood vessels (arrows) than in the normal ovary, no detectable large mature follicles and a small solid tissue mass (circle) in the ovary. An example of late stage ovarian cancer (<b>C</b>) characterized by a large solid mass with increased blood flow (arrows) and profuse ascites (*).The gross morphology (<b>F</b>) confirmed the presence of multiple solid masses and tumor metastasis to other organs. Abbreviations: CT = cecal tonsil; F1–F2 = large preovulatory follicles; GI = gastrointestinal tract; OV = ovary; S = ovarian stroma; SM = solid tissue mass in the ovary; SP = spleen; UOD = upper oviduct; UT = uterus.</p

The number of ovarian CD4+, CD8+ and Bu1a+ cells estimated by flow cytometry.

<p>(<b>A</b>): Representative forward vs. side scatter plot for cells from a whole ovary showing the gate (R1) used for lymphocytes. (<b>B</b>) Cells were first gated using the gate in (A) and then stained with either Bu1a-APC, CD4-FITC or CD8-PE. The percent of labeled cells within the gate is shown in the lower right quadrant for each stain. (<b>C</b>) The total number of Bu1a-APC, CD4-FITC and CD8-PE labeled cells from normal and tumor hen ovaries (n = 3/group) are shown with the median indicated by the horizontal line.</p