Inflammasome expression is higher in ovarian tumors than in normal ovary

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Luborsky, J., Barua, A., Edassery, S., Bahr, J. M., & Edassery, S. L. Inflammasome expression is higher in ovarian tumors than in normal ovary. Plos One, 15(1), (2020): e0227081, doi:10.1371/journal.pone.0227081.Chronic inflammation fundamentally influences cancer risk and development. A mechanism of chronic inflammation is the formation of inflammasome complexes which results in the sustained secretion of the pro-inflammatory cytokines IL1β and IL18. Inflammasome expression and actions vary among cancers. There is no information on inflammasome expression in ovarian cancer (OvCa). To determine if ovarian tumors express inflammasome components, mRNA and protein expression of NLRP3 (nucleotide-binding domain, leucine-rich repeat family, pyrin domain containing 3), caspase-1, IL1β, and IL18 expression in hen and human OvCa was assessed. Chicken (hen) OvCa a valid model of spontaneous human OvCa. Hens were selected into study groups with or without tumors using ultrasonography; tumors were confirmed by histology, increased cellular proliferation, and expression of immune cell marker mRNA. mRNA expression was higher for hallmarks of inflammasome activity (caspase-1, 5.9x increase, p = 0.04; IL1β, 4x increase, p = 0.04; and IL18, 7.8x increase, p = 0.0003) in hen OvCa compared to normal ovary. NLRP3, caspase-8 and caspase-11 mRNA did not differ significantly between tumor and non-tumor containing ovaries. Similar results occurred for human OvCa. Protein expression by immunohistochemistry paralleled mRNA expression and was qualitatively higher in tumors. Increased protein expression of caspase-1, IL1β, and IL18 occurred in surface epithelium, tumor cells, and immune cells. The aryl hydrocarbon receptor (AHR), a potential tumor suppressor and NLRP3 regulator, was higher in hen (2.4x increase, p = 0.002) and human tumors (1.8x increase, p = 0.038), suggesting a role in OvCa. Collectively, the results indicate that inflammasome expression is associated with hen and human OvCa, although the NLR sensor type remains to be determined.This research was made possible by NIH grant NCI R03CA182120 (JL), DOD grant W81XWH-08-1-0203 (JL) and Swim Across America (AB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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

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

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