The hen model of human ovarian cancer develops anti-mesothelin autoantibodies in response to mesothelin expressing tumors

<p>Abstract</p> <p>Objective</p> <p>Study of the hen immune system led to seminal contributions to basic immunological principles. Recent studies of spontaneous ovarian cancer in the laying hen show strikingly similar tumor types and antigen expression compared to human ovarian cancer, suggesting hens would be valuable for studies of tumor immunology and pre-clinical vaccine development. Circulating mesothelin is a relatively specific marker for human ovarian cancer and autoantibodies to mesothelin were reported. We hypothesized that hen tumors express mesothelin and that circulating anti-mesothelin antibodies occur in response to tumors.</p> <p>Methods</p> <p>Mesothelin mRNA expression was analyzed by RT-PCR in hen ovarian tumors and normal ovaries. Mesothelin protein expression was evaluated by immunohistochemistry (IHC) and two-dimensional SDS-PAGE Western blots. Anti-mesothelin antibodies were assessed by immunoassay of sera from hens with normal ovaries and with ovarian tumors.</p> <p>Results</p> <p>Significant mesothelin mRNA expression was observed in 57% (12/21) of hen ovarian tumors but not in normal ovaries and was found predominantly in serous tumors as in humans. Mesothelin protein was detected in tumors with mesothelin mRNA by IHC and 2D Western blots, but not in normal ovaries or tumors without mesothelin mRNA. Circulating anti-mesothelin antibodies occurred in 44% (n = 4/9) of hens with ovarian tumors which express mesothelin mRNA and were not found in hens with tumors that did not express mesothelin (n = 0/5) or normal ovaries (n = 0/5).</p> <p>Conclusion</p> <p>The results support the utility of the hen as a novel model for preclinical studies of mesothelin as a biomarker and a target for immunotherapy.</p

Association of Immunosuppression with DR6 Expression during the Development and Progression of Spontaneous Ovarian Cancer in Laying Hen Model

Ovarian cancer (OVCA) mainly disseminates in the peritoneal cavity. Immune functions are important to prevent OVCA progression and recurrence. The mechanism of immunosuppression, a hallmark of tumor progression, is not well understood. The goal of this study was to determine the immune system’s responses and its suppression during OVCA development and progression in hens. Frequencies of CD8+ T cells and IgY-containing cells and expression of immunosuppressors including IRG1 and DR6 in OVCA at early and late stages in hens were examined. Frequencies of stromal but not the intratumoral CD+8 T cells and IgY-containing cells increased significantly (P<0.01) during OVCA development and progression. Tumor progression was associated with increased expression of IRG1 and DR6 and decreased infiltration of immune cells into the tumor. Frequency of stromal but not intratumoral immune cells increases during OVCA development and progression. Tumor-induced IRG1 and DR6 may prevent immune cells from invading the tumor

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

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

Comparison of the number of immune cells in ovarian follicles determined by morphometric analysis.

<p>(<b>A</b>) B (Bu1a+) cells were rarely found in follicles and therefore were not counted. The number of CD4+ T cells increased slightly (p = 0.052) and the number of CD8+ T cells decreased (p = 0.009) in late stage ovarian tumors compared to normal ovaries. At each stage there were more CD8+ compared to CD4+ T cells (normal ovary, p = 0.003; early stage tumor, p = 0.008; late stage tumor, p = 0.007). Follicles were defined as shown in (<b>B</b>) and cells within the designated area were counted and the average determined per 2×10⁵ µm² area of follicle. Three sections from each ovary for each hen were counted at a magnification of 20x; scale bar = 50 µm. Error bars represent mean ± SEM.</p

Immune cell distribution in ovaries with late stage ovarian tumors.

<p>Similar regions of three tumor types are shown in serial sections stained for Bu1a, CD4 and CD8. <i>Top Row:</i> An example of sections from a serous ovarian tumor showing few CD4+ cells within the tumor compared to Bu1a+ and CD8+ cells. <i>Middle Row:</i> An example from a mucinous ovarian tumor showing deposits of Bu1a+, CD4+ and CD8+ cells between glands and throughout the tumor. <i>Bottom Row:</i> An example of an endometrioid ovarian tumor showing Bu1a+, CD4+ and CD8+ cells. Original magnification, 10x; scale bar = 100 µm. Stained CD8+ and CD4+ lymphocytes in selected areas (dotted boxes) are shown in insets at higher magnification; scale bar = 10 µm.</p