Selenium-Binding Protein 1 (SBP1) autoantibodies in ovarian disorders and ovarian cancer.

Infertility is a risk factor for ovarian cancer (OvCa). The goal was to determine if antibodies to selenium-binding protein 1 (SBP1), an autoantibody we identified in patients with premature ovarian failure (POF), occurs in both infertility and OvCa patients, and thus could be associated with preneoplasia. Anti-SBP1 was measured by immunoassay against recombinant SBP1, in sera from OvCa (n = 41), infertility (n = 92) and control (n = 87) patients. Infertility causes were POF, unexplained, irregular ovulation or endometriosis. The percent of anti-SBP1-positive sera was higher in POF (P = 0.02), irregular ovulation (P = 0.001), unexplained causes (P = 0.02), late (III-IV)-stage OvCa (P = 0.02) but was not significant in endometriosis, benign ovarian tumors/cysts, early stage (I-II) OvCa or uterine cancer compared to healthy controls. Anti-SBP1 was significantly higher in women with serous (P = 0.04) but not non-serous (P = 0.33) OvCa compared to controls. Also, we determined if anti-SBP1 was associated with CA125 or anti-TP53, markers often studied in OvCa. Anti-TP53 and CA125 were measured by established immunoassays. The ability of anti-SBP1 alone to discriminate infertility or OvCa from controls or when combined with anti-TP53 and CA125, to identify OvCa was evaluated by comparing the area under the curve (AUC) in ROC analysis. Anti-SBP1 alone discriminated infertility (AUC = 0.7; P = 0.001) or OvCa (AUC = 0.67; P = 0.03) from controls. The sensitivity and specificity of OvCa identification was increased by combining CA125, anti-TP53 and anti-SBP1 (AUC = 0.96). Therefore, anti-SBP1 occurs in infertile women with POF, ovulatory disturbances or unexplained infertility and in serous OvCa. This suggests an autoimmune process is associated with the development of serous OvCa.

Enhancement of Ovarian Tumor Detection by DR6-Targeted Ultrasound Imaging Agents in Laying Hen Model of Spontaneous Ovarian Cancer.

OBJECTIVE: The lack of an effective early detection test leads to high case to death ratio of women with ovarian cancer (OVCA). To improve early detection, tumor-associated imaging targets need to be established and imaging agents to image these targets need to be developed. Targeted imaging agents offer potential for improvement of signal intensities from their targets. Expression of death receptor 6 (DR6) by ovarian malignant cells and tumor-associated microvessels increases during OVCA development and represents a novel target for ultrasound imaging. The goal of this study was to examine the feasibility of newly developed DR6-targeted ultrasound imaging agents in enhancing early detection of ovarian tumors in laying hen model of spontaneous OVCA. MATERIALS AND METHODS: The study was conducted in an exploratory cross-sectional design using 4-year-old laying hens (n = 130). DR6-targeted imaging agents were developed by conjugating microbubbles with rabbit anti-chicken DR6 antibodies. Changes in signal intensity of ultrasound imaging were determined before and after injection of targeted imaging agents in hens with or without spontaneous OVCA. Following targeted imaging, normal or tumor ovaries were processed for histopathological and immunohistochemical studies. RESULTS: DR6-targeted imaging agents bound with their targets expressed by malignant cells and tumor-associated microvessels in the ovary. Compared with pretargeted imaging, targeted imaging is enhanced by approximately 40% ultrasound echo signal intensity (P < 0.001) from early- and late-stage OVCA. Differences in signal enhancement were not observed among different histological subtypes of OVCA at early or late stages. Higher imaging signal intensities were associated with enhancement in DR6 expression by ovarian malignant cells and increase in the frequency of DR6-expressing microvessels during OVCA development. CONCLUSIONS: The results of this study suggest that DR6-targeted imaging agents enhance the visualization of ovarian tumors and tumor-associated microvessels in hens with early-stage OVCA and will form a foundation for clinical studies.

Cotransplantation with specific populations of spina bifida bone marrow stem/progenitor cells enhances urinary bladder regeneration.

Spina bifida (SB) patients afflicted with myelomeningocele typically possess a neurogenic urinary bladder and exhibit varying degrees of bladder dysfunction. Although surgical intervention in the form of enterocystoplasty is the current standard of care in which to remedy the neurogenic bladder, it is still a stop-gap measure and is associated with many complications due to the use of bowel as a source of replacement tissue. Contemporary bladder tissue engineering strategies lack the ability to reform bladder smooth muscle, vasculature, and promote peripheral nerve tissue growth when using autologous populations of cells. Within the context of this study, we demonstrate the role of two specific populations of bone marrow (BM) stem/progenitor cells used in combination with a synthetic elastomeric scaffold that provides a unique and alternative means to current bladder regeneration approaches. In vitro differentiation, gene expression, and proliferation are similar among donor mesenchymal stem cells (MSCs), whereas poly(1,8-octanediol-cocitrate) scaffolds seeded with SB BM MSCs perform analogously to control counterparts with regard to bladder smooth muscle wall formation in vivo. SB CD34(+) hematopoietic stem/progenitor cells cotransplanted with donor-matched MSCs cause a dramatic increase in tissue vascularization as well as an induction of peripheral nerve growth in grafted areas compared with samples not seeded with hematopoietic stem/progenitor cells. Finally, MSC/CD34(+) grafts provided the impetus for rapid urothelium regeneration. Data suggest that autologous BM stem/progenitor cells may be used as alternate, nonpathogenic cell sources for SB patient-specific bladder tissue regeneration in lieu of current enterocystoplasty procedures and have implications for other bladder regenerative therapies.

Proteomic profiling of regenerated urinary bladder tissue in a non-human primate augmentation model.

Urinary bladder dysfunction can be caused by environmental, genetic, and developmental insults. Depending upon insult severity, the bladder may lose its ability to maintain volumetric capacity and intravesical pressure resulting in renal deterioration. Bladder augmentation enterocystoplasty (BAE) is utilized to increase bladder capacity to preserve renal function using autologous bowel tissue as a "patch." To avoid the clinical complications associated with this procedure, we have engineered composite grafts comprised of autologous bone marrow mesenchymal stem cells (MSCs) co-seeded with CD34+ hematopoietic stem/progenitor cells (HSPCs) onto a pliable synthetic scaffold [poly(1,8-octamethylene-citrate-co-octanol)(POCO)] or a biological scaffold (SIS; small intestinal submucosa) to regenerate bladder tissue in our baboon bladder augmentation model. We set out to determine the global protein expression profile of bladder tissue that has undergone regeneration with the aforementioned stem cell seeded scaffolds along with baboons that underwent BAE. Data demonstrate that POCO and SIS grafted animals share high protein homogeneity between native and regenerated tissues while BAE animals displayed heterogeneous protein expression between the tissues following long-term engraftment. We posit that stem cell-seeded scaffolds can recapitulate tissue that is nearly indistinguishable from native tissue at the protein level and may be used in lieu of procedures such as BAE.

Multipotent bone marrow cell-seeded polymeric composites drive long-term, definitive urinary bladder tissue regeneration.

To date, there are no efficacious translational solutions for end-stage urinary bladder dysfunction. Current surgical strategies, including urinary diversion and bladder augmentation enterocystoplasty (BAE), utilize autologous intestinal segments (e.g. ileum) to increase bladder capacity to protect renal function. Considered the standard of care, BAE is fraught with numerous short- and long-term clinical complications. Previous clinical trials employing tissue engineering approaches for bladder tissue regeneration have also been unable to translate bench-top findings into clinical practice. Major obstacles still persist that need to be overcome in order to advance tissue-engineered products into the clinical arena. These include scaffold/bladder incongruencies, the acquisition and utility of appropriate cells for anatomic and physiologic tissue recapitulation, and the choice of an appropriate animal model for testing. In this study, we demonstrate that the elastomeric, bladder biomechanocompatible poly(1,8-octamethylene-citrate-co-octanol) (PRS; synthetic) scaffold coseeded with autologous bone marrow-derived mesenchymal stem cells and CD34+ hematopoietic stem/progenitor cells support robust long-term, functional bladder tissue regeneration within the context of a clinically relevant baboon bladder augmentation model simulating bladder trauma. Partially cystectomized baboons were independently augmented with either autologous ileum or stem-cell-seeded small-intestinal submucosa (SIS; a commercially available biological scaffold) or PRS grafts. Stem-cell synergism promoted functional trilayer bladder tissue regeneration, including whole-graft neurovascularization, in both cell-seeded grafts. However, PRS-augmented animals demonstrated fewer clinical complications and more advantageous tissue characterization metrics compared to ileum and SIS-augmented animals. Two-year study data demonstrate that PRS/stem-cell-seeded grafts drive bladder tissue regeneration and are a suitable alternative to BAE.

Proteomic profiling of regenerated urinary bladder tissue with stem cell seeded scaffold composites in a non-human primate bladder augmentation model.

Urinary bladder insult can be caused by environmental, genetic, and developmental factors. Depending upon insult severity, the bladder may lose its ability to maintain capacity and intravesical pressures resulting in renal deterioration. Bladder augmentation enterocystoplasty (BAE) is employed to increase bladder capacity to preserve renal function using autologous bowel tissue as a "patch." To avoid the clinical complications associated with this procedure, we have engineered composite grafts comprised of autologous bone marrow mesenchymal stem cells (MSCs) with CD34+ hematopoietic stem/progenitor cells (HSPCs) co-seeded onto a pliable synthetic scaffold [POCO; poly(1,8-octamethylene-citrate-co-octanol)] or a biological scaffold (SIS; small intestinal submucosa) to regenerate bladder tissue in a baboon bladder augmentation model. We set out to determine the protein expression profile of bladder tissue that has undergone regeneration with the aforementioned stem cell seeded scaffolds along with baboons that underwent BAE. Data demonstrate that POCO and SIS grafted animals share high protein homogeneity between native and regenerated tissues while BAE animals displayed heterogenous protein expression between the tissues following long-term engraftment. We posit that stem cell seeded scaffolds can recapitulate tissue that is almost indistinguishable from native tissue at the protein level and may be used in lieu of procedures such as BAE.

Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis.

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain unclear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degradation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs in vitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs in vitro and in a C. elegans model in vivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demonstrate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.

Association of interleukin 16 with the development of ovarian tumor and tumor-associated neoangiogenesis in laying hen model of spontaneous ovarian cancer.

OBJECTIVE: Tumor-associated neoangiogenesis (TAN) is an early event in ovarian tumor development. Interleukin 16 (IL-16) is a proangiogenic cytokine that stimulates production of neoangiogenic factors. The goal of this study was to determine the association of IL-16 with tumor development and ovarian TAN in laying hens, an animal model of spontaneous ovarian cancer (OVCA). METHODS: Sera and ovarian tissues from 3-year-old laying hens were collected and processed for histopathologic, immunoassay, immunohistochemistry, immunoblotting, and molecular biological studies to determine the tissue expression and serum levels of IL-16. Samples were divided into 3 groups based on the diagnosis of the histopathologic ovarian tissue examination, namely, normal (healthy control, n = 81), early (n = 23 including 11 with microscopic OVCA), and late stages (n = 16) of OVCA. RESULTS: Serum levels of IL-16 were significantly higher in hens with microscopic, early, and late stages of OVCA than normal hens (P < 0.0001). The frequencies of IL-16 cells in tumor-bearing ovaries were significantly higher than normal hens (P < 0.05). The expression of IL-16 protein and mRNA were stronger in tumor-bearing ovaries than normal ovaries. In addition to ovarian stroma, IL-16 was also expressed by the epithelial cells of the tumor in OVCA hens. Differences in serum levels and ovarian IL-16 expression were not significant among different histological subtypes of OVCA including serous, endometrioid, and mucinous. Similar to the serum levels and ovarian expression of IL-16, the densities of neoangiogenic microvessels were significantly higher in hens with tumor-bearing ovaries than normal hens. CONCLUSIONS: The results of the study suggest that changes in serum levels of IL-16 are associated with tumor development and TAN. Thus, serum IL-16 levels may be an indicator of ovarian TAN at the early stage of OVCA.

Mitochondrial calcium uniporter deletion prevents painful diabetic neuropathy by restoring mitochondrial morphology and dynamics.

ABSTRACT: Painful diabetic neuropathy (PDN) is an intractable complication affecting 25% of diabetic patients. Painful diabetic neuropathy is characterized by neuropathic pain accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability, resulting in calcium overload, axonal degeneration, and loss of cutaneous innervation. The molecular pathways underlying these effects are unknown. Using high-throughput and deep-proteome profiling, we found that mitochondrial fission proteins were elevated in DRG neurons from mice with PDN induced by a high-fat diet (HFD). In vivo calcium imaging revealed increased calcium signaling in DRG nociceptors from mice with PDN. Furthermore, using electron microscopy, we showed that mitochondria in DRG nociceptors had fragmented morphology as early as 2 weeks after starting HFD, preceding the onset of mechanical allodynia and small-fiber degeneration. Moreover, preventing calcium entry into the mitochondria, by selectively deleting the mitochondrial calcium uniporter from these neurons, restored normal mitochondrial morphology, prevented axonal degeneration, and reversed mechanical allodynia in the HFD mouse model of PDN. These studies suggest a molecular cascade linking neuropathic pain to axonal degeneration in PDN. In particular, nociceptor hyperexcitability and the associated increased intracellular calcium concentrations could lead to excessive calcium entry into mitochondria mediated by the mitochondrial calcium uniporter, resulting in increased calcium-dependent mitochondrial fission and ultimately contributing to small-fiber degeneration and neuropathic pain in PDN. Hence, we propose that targeting calcium entry into nociceptor mitochondria may represent a promising effective and disease-modifying therapeutic approach for this currently intractable and widespread affliction. Moreover, these results are likely to inform studies of other neurodegenerative disease involving similar underlying events.

Long-lived mitochondrial cristae proteins in mouse heart and brain.

Long-lived proteins (LLPs) have recently emerged as vital components of intracellular structures whose function is coupled to long-term stability. Mitochondria are multifaceted organelles, and their function hinges on efficient proteome renewal and replacement. Here, using metabolic stable isotope labeling of mice combined with mass spectrometry (MS)-based proteomic analysis, we demonstrate remarkable longevity for a subset of the mitochondrial proteome. We discovered that mitochondrial LLPs (mt-LLPs) can persist for months in tissues harboring long-lived cells, such as brain and heart. Our analysis revealed enrichment of mt-LLPs within the inner mitochondrial membrane, specifically in the cristae subcompartment, and demonstrates that the mitochondrial proteome is not turned over in bulk. Pioneering cross-linking experiments revealed that mt-LLPs are spatially restricted and copreserved within protein OXPHOS complexes, with limited subunit exchange throughout their lifetimes. This study provides an explanation for the exceptional mitochondrial protein lifetimes and supports the concept that LLPs provide key structural stability to multiple large and dynamic intracellular structures.

BET1 variants establish impaired vesicular transport as a cause for muscular dystrophy with epilepsy.

BET1 is required, together with its SNARE complex partners GOSR2, SEC22b, and Syntaxin-5 for fusion of endoplasmic reticulum-derived vesicles with the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi. Here, we report three individuals, from two families, with severe congenital muscular dystrophy (CMD) and biallelic variants in BET1 (P1 p.(Asp68His)/p.(Ala45Valfs*2); P2 and P3 homozygous p.(Ile51Ser)). Due to aberrant splicing and frameshifting, the variants in P1 result in low BET1 protein levels and impaired ER-to-Golgi transport. Since in silico modeling suggested that p.(Ile51Ser) interferes with binding to interaction partners other than SNARE complex subunits, we set off and identified novel BET1 interaction partners with low affinity for p.(Ile51Ser) BET1 protein compared to wild-type, among them ERGIC-53. The BET1/ERGIC-53 interaction was validated by endogenous co-immunoprecipitation with both proteins colocalizing to the ERGIC compartment. Mislocalization of ERGIC-53 was observed in P1 and P2's derived fibroblasts; while in the p.(Ile51Ser) P2 fibroblasts specifically, mutant BET1 was also mislocalized along with ERGIC-53. Thus, we establish BET1 as a novel CMD/epilepsy gene and confirm the emerging role of ER/Golgi SNAREs in CMD.

Inflammasome expression is higher in ovarian tumors than in normal ovary.

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 IL1ß 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, IL1ß, 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; IL1ß, 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, IL1ß, 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.

Do current bladder smooth muscle cell isolation procedures result in a homogeneous cell population? Implications for bladder tissue engineering.

PURPOSE: Conventional techniques used to harvest and culture bladder smooth muscle cells (SMCs) have been thought to yield homogeneous populations of SMCs. In order to delineate the cellular composition of tissue derived bladder cells, this study was conducted to determine whether current culturing techniques result in a uniform population of bladder SMCs that may be utilized for bladder tissue engineering. METHODS: Patient derived bladder muscle was isolated and manually minced followed by enzymatic digestion. Cells were cultured in D: -valine alpha-MEM with decreasing levels of fetal bovine serum then fixed and permeabilized for flow cytometric and immunofluorescent analyses. Antibody staining of cultured cells consisted of alpha-SMA, von Willebrand factor, pan-cytokeratin, CD31, and CD90. Cells were visualized using directly conjugated fluorescein isothiocyanate primary or IgG-Alexa-555 conjugated secondary antibodies. RESULTS: Flow cytometric analyses revealed mixed populations of cells expressing non-SMC epitopes as corroborated by immunofluorescent studies. High density oligonucleotide array analysis revealed expression levels of known bladder SMC genes and the expression of endothelial and fibroblast related markers (P < 0.005). CONCLUSIONS: Phenotypic analyses demonstrate cell heterogeneity when SMCs are acquired and cultured through conventional methods. Standardized criteria based upon objective experimentation need to be established in order to better characterize bladder SMCs that are to be utilized for bladder tissue engineering.

Prevalence of antitumor antibodies in laying hen model of human ovarian cancer.

Antitumor antibodies are associated with tumors in human cancers. There is relatively little information on the timing and progression of antibody response to tumors. The objective of the study was to determine if spontaneous ovarian cancer in the egg-laying hen is associated with antitumor antibodies. Antibodies were detected by immunoassay and immunoblotting using proteins from normal ovary and ovarian tumors. Candidate antigens were identified by mass spectrometry of immunoreactive spots cut from 2-dimensional gels and Western blot. Antitumor (serum reacting against tumor ovarian extract) and antiovarian (serum reacting against normal ovarian extract) antibodies were significantly associated with ovarian cancer (67%; P

Histopathology of ovarian tumors in laying hens: a preclinical model of human ovarian cancer.

The high mortality rate due to ovarian cancer (OVCA) is attributed to the lack of an effective early detection method. Because of the nonspecificity of symptoms at early stage, most of the OVCA cases are detected at late stages. This makes the access to women with early-stage disease problematic and presents a barrier to development and validation of tests for detection of early stage of OVCA in humans. Animal models are used to elucidate disease etiologies and pathogenesis that are difficult to study in humans. Laying hen is the only available animal that develops OVCA spontaneously; however, detailed information on ovarian tumor histology is not available. The goal of this study was to determine the histological features of malignant ovarian tumors in laying hens. A total of 155 young and old (1-5 years of age) laying hens (Gallus domesticus) were selected randomly and evaluated grossly and microscopically for the presence of ovarian tumors. Histological classification of tumors with their stages and grades was determined with reference to those for humans. Similar to humans, all 4 types including serous, endometrioid, mucinous, and clear cell or mixed carcinomas were observed in hen ovarian tumors. Some early neoplastic as well as putative ovarian lesions were also observed. Similarities in histology, metastasis, and stages of hen OVCA to those of humans demonstrate the feasibility of the hen model for additional delineation of the mechanism underlying ovarian carcinogenesis, preclinical testing of new agents for the prevention, and therapy of this disease.

Selenium-Binding Protein 1 expression in ovaries and ovarian tumors in the laying hen, a spontaneous model of human ovarian cancer.

OBJECTIVE: Reduced Selenium-Binding Protein 1 (SELENBP1) expression was recently shown in multiple cancers. There is little information on the expression and function of SELENBP1 in cancer progression. In order to develop a better understanding of the role of SELENBP1 in ovarian cancer, our objective was to determine if SELENBP1 is expressed in the normal ovaries and ovarian tumors in the egg-laying hen, a spontaneous model of human ovarian cancer. METHODS: SPB1 mRNA expression in normal ovary (n=20) and ovarian tumors (n=23) was evaluated by RT-PCR. Relative levels of mRNA were compared by quantitative RT-PCR (qRT-PCR) in selected samples. SELENBP1 protein expression was evaluated by 1D Western blot and immunohistochemistry with a commercial anti-human SELENBP1 antibody. RESULTS: SELENBP1 mRNA and protein was expressed in 100% of normal and ovarian tumors and qRT-PCR confirmed decreased mRNA expression in 80% of ovarian tumors. SELENBP1 was primarily localized in surface epithelial cells of normal ovaries. In ovaries containing early tumor lesions, SELENBP1 expression was reduced in the surface epithelium near the tumor and was expressed in tumor cells, while more distant regions with normal histology retained SELENBP1 expression in the surface epithelium. CONCLUSIONS: We have shown for the first time that SELENBP1 is expressed in both normal ovaries and ovarian tumors in the hen and that SELENBP1 expression is altered in the vicinity of the tumor. Furthermore, SELENBP1 expression in normal ovarian surface epithelium and in ovarian tumors parallels that previously reported for ovarian cancer in women.

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.

Immune cells in the normal ovary and spontaneous ovarian tumors in the laying hen (Gallus domesticus) model of human ovarian cancer.

BACKGROUND: Spontaneous ovarian cancer in chickens resembles human tumors both histologically and biochemically. The goal was to determine if there are differences in lymphocyte content between normal ovaries and ovarian tumors in chickens as a basis for further studies to understand the role of immunity in human ovarian cancer progression. METHODS: Hens were selected using grey scale and color Doppler ultrasound to determine if they had normal or tumor morphology. Cells were isolated from ovaries (n = 6 hens) and lymphocyte numbers were determined by flow cytometry using antibodies to avian CD4 and CD8 T and B (Bu1a) cells. Ovarian sections from another set of hens (n = 26) were assessed to verify tumor type and stage and to count CD4, CD8 and Bu1a immunostained cells by morphometric analysis. RESULTS: T and B cells were more numerous in ovarian tumors than in normal ovaries by flow cytometry and immunohistochemistry. There were less CD4+ cells than CD8+ and Bu1a+ cells in normal ovaries or ovarian tumors. CD8+ cells were the dominant T cell sub-type in both ovarian stroma and in ovarian follicles compared to CD4+ cells. Bu1a+ cells were consistently found in the stroma of normal ovaries and ovarian tumors but were not associated with follicles. The number of immune cells was highest in late stage serous tumors compared to endometrioid and mucinous tumors. CONCLUSIONS: The results suggest that similar to human ovarian cancer there are comparatively more immune cells in chicken ovarian tumors than in normal ovaries, and the highest immune cell content occurs in serous tumors. Thus, this study establishes a foundation for further study of tumor immune responses in a spontaneous model of ovarian cancer which will facilitate studies of the role of immunity in early ovarian cancer progression and use of the hen in pre-clinical vaccine trials.

Expression of death receptor 6 by ovarian tumors in laying hens, a preclinical model of spontaneous ovarian cancer.

Tumor-associated neoangiogenesis and suppression of antitumor immunity are hallmarks of tumor development and progression. Death receptor 6 (DR6) has been reported to be associated with suppression of antitumor immunity and tumor progression in several malignancies. However, expression of DR6 by malignant ovarian epithelial tumors at an early stage is unknown. The goals of this study were to determine whether DR6 is expressed by malignant ovarian epithelial tumors at an early stage and to examine whether DR6 expression is associated with ovarian cancer (OVCA) progression in a laying hen model of spontaneous OVCA. Expression of DR6 was examined in normal and malignant ovaries, normal ovarian surface epithelial (OSE) cells, or malignant epithelial cells and in serum of 3-year-old hens. The population of microvessels expressing DR6 was significantly higher in hens with early-stage OVCA than hens with normal ovaries (P < .01) and increased further in late-stage OVCA. The results of this study showed that, in addition to microvessels, tumor cells in the ovary also express DR6 with a significantly higher intensity than normal OSE cells. Similar patterns of DR6 expression were also observed by immunoblot analysis and gene expression studies. Furthermore, DR6 was also detected in the serum of hens. In conclusion, DR6 expression is associated with OVCA development and progression in laying hens. This study may be helpful to examine the feasibility of DR6 as a useful surrogate marker of OVCA, a target for antitumor immunotherapy and molecular imaging and thus provide a foundation for clinical studies.

Expression of Leukocyte Inhibitory Immunoglobulin-like Transcript 3 Receptors by Ovarian Tumors in Laying Hen Model of Spontaneous Ovarian Cancer.

Attempts to enhance a patient's immune response and ameliorate the poor prognosis of ovarian cancer (OVCA) have largely been unsuccessful owing to the suppressive tumor microenvironment. Leukocyte immunoglobulin-like transcript 3 (ILT3) inhibitory receptors have been implicated in immunosuppression in several malignancies. The expression and role of ILT3 in the progression of ovarian tumors are unknown. This study examined the expression and association of ILT3 in ovarian tumors in laying hens, a spontaneous preclinical model of human OVCA. White Leghorn laying hens were selected by transvaginal ultrasound scanning. Serum and normal ovaries or ovarian tumors were collected. The presence of tumors and the expression of ILT3 were examined by routine histology, immunohistochemistry, Western blot analysis, and reverse transcription-polymerase chain reaction. In addition to stromal immune cell-like cells, the epithelium of the ovarian tumors also expressed ILT3 with significantly high intensity than normal ovaries. Among different subtypes of ovarian carcinomas, serous OVCA showed the highest ILT3 staining intensity, whereas endometrioid OVCA had the lowest intensity. Similar to humans, an immunoreactive protein band of approximately 55 kDa for ILT3 was detected in the ovarian tumors in hens. The patterns of ILT3 protein and messenger RNA expression by ovarian tumors in different subtypes and stages were similar to those of immunohistochemical staining. The results of this study suggest that laying hens may be useful to generate information on ILT3-associated immunosuppression in OVCA. This animal model also offers the opportunity to develop and test anti-ILT3 immunotherapy to enhance antitumor immunity against OVCA in humans.