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Problem: We hypothesize that activated peritoneal immune cells can be redirected to target ovarian tumors. Here, we obtain fundamental knowledge of the peritoneal immune environment through deep immunophenotyping of T cells, dendritic cells (DC), and innate lymphoid cells (ILC) of ovarian cancer patients. Method of study: T cells, DC, and ILC from ascites of ovarian cancer patients (n = 15) and peripheral blood of post‐menopausal healthy donors (n = 6) were immunophenotyped on a BD Fortessa cytometer using three panels—each composed of 16 antibodies. The data were analyzed manually and by t‐SNE/DensVM. CA125 levels were obtained from patient charts. Results: We observed decreased CD3+ T cells and a higher proportion of activated CD4+ and effector memory CD4+/CD8+ T cells, plasmacytoid DC, CD1c+ and CD141+ myeloid DC and CD56Hi NK cells in ascites. t‐SNE/DensVM identified eight T cell, 17 DC, and 17 ILC clusters that were unique in the ascites compared to controls. Hierarchical clustering of cell frequency distinctly segregated the T‐cell and ILC clusters from controls. Increased CA125 levels were associated with decreased CD8+/CD45RA+/CD45RO−/CCR7− T cells. Conclusion: The identified immune clusters serve as the basis for interrogation of the peritoneal immune environment and the development of novel immunologic modalities against ovarian cancer. [ABSTRACT FROM AUTHOR]

Over three decades have passed since the first report on the expression of CA125 by ovarian tumors. Since that time our understanding of ovarian cancer biology has changed significantly to the point that these tumors are now classified based on molecular phenotype and not purely on histological attributes. However, CA125 continues to be, with the recent exception of HE4, the only clinically reliable diagnostic marker for ovarian cancer. Many large-scale clinical trials have been conducted or are underway to determine potential use of serum CA125 levels as a screening modality or to distinguish between benign and malignant pelvic masses. CA125 is a peptide epitope of a 3-5 million Da mucin, MUC16. Here we provide an in-depth review of the literature to highlight the importance of CA125 as a prognostic and diagnostic marker for ovarian cancer. We focus on the increasing body of literature describing the biological role of MUC16 in the progression and metastasis of ovarian tumors. Finally, we consider previous and on-going efforts to develop therapeutic approaches to eradicate ovarian tumors by targeting MUC16. Even though CA125 is a crucial marker for ovarian cancer, the exact structural definition of this antigen continues to be elusive. The importance of MUC16/CA125 in the diagnosis, progression and therapy of ovarian cancer warrants the need for in-depth research on the biochemistry and biology of this mucin. A renewed focus on MUC16 is likely to culminate in novel and more efficient strategies for the detection and treatment of ovarian cancer. [ABSTRACT FROM AUTHOR]

Problem MUC16 ( CA125) released from ovarian tumors binds to NK cells and monocytes via the inhibitory receptor Siglec-9. Here, we investigate whether MUC16 also binds to circulating immune cells during pregnancy and in women with preeclampsia. Method of study MUC16 binding was monitored by flow cytometry and immunoprecipitation, and RT- PCR was used to monitor indigenous expression in immune cells. Serum CA125 levels were measured by a clinical assay. Results MUC16 was equally distributed on Siglec-9pos CD16pos/ CD56dim and CD16neg/ CD56br NK cells in the healthy pregnant and preeclampsia groups. While serum CA125 levels and number of NK and monocytes were similar, increased binding of MUC16 was observed on these immune cells in the preeclampsia cohort as compared to the healthy pregnant samples. Conclusion MUC16 binding to NK cells and monocytes likely contributes to tolerance of the fetal allograft from maternal responses and may also serve as a novel biomarker for preeclampsia. [ABSTRACT FROM AUTHOR]

The ovarian tumour marker MUC16 (CA125) inhibits the cytotoxic responses of human natural killer (NK) cells and down-regulates CD16. Here we show that approximately 10% of the peripheral blood NK cells (PBNK) from the epithelial ovarian cancer (EOC) patients are CD16– CD56br whereas 40% of the peritoneal fluid NK (PFNK) carry this phenotype, which is usually associated with NK cells from the lymph nodes or human decidua. PBNK from healthy donors exposed to PF show a significant increase in the CD16– CD56br population. This shift in phenotype is not caused by increased apoptosis of the CD16+ CD56dim cells or selective proliferation of the CD16– CD56br NK cells. Thus, the terminal differentiation of the CD16– CD56br NK cells to CD16+ CD56dim subset that occurs during normal NK cell development may actually be a reversible step. A majority of the NK cell receptors (NKp46, NKp44, NKG2D, CD244, CD226, CD158a, CD158b, and CD158e) studied were down-regulated in the PFNK. MUC16 binds selectively to 30–40% of CD16+ CD56dim NK cells in EOC patients indicating that phenotypic alterations in these cells are mediated by tumour-derived soluble factors. Similar to EOC, MUC16 in early pregnancy also binds to NK cells suggesting shared mechanisms of NK cell suppression in feto-maternal tolerance and immune evasion by ovarian cancers. [ABSTRACT FROM AUTHOR]

Abstract: Objectives.: CA125 expresses specific oligosaccharides that can inhibit the cytotoxicity of human natural killer (NK) cells. The current study was undertaken to determine the ability of CA125 to modulate NK cell-mediated cytotoxicity. Methods.: CA125 was isolated from OVCAR-3 cells and its purity was determined by ELISA and ultra-sensitive mass spectrometric analysis. Peripheral blood-derived NK were treated with CA125 and standard cytotoxicity assays were performed using 51Cr-labeled K562 cells as targets. The expression of cell surface and intracellular markers on NK cells was determined by either flow cytometry or Western blot analysis. Results.: NK cells incubated with CA125 for 72 h exhibited a 50–70% decrease in the lysis of K562 targets. Incubation with CA125 for 4 h and 24 h had no effect on NK-mediated cytolysis. Inhibition of NK function was observed at CA125 concentrations (10,000–100,000 U/ml) that are expected to be significantly lower than those observed in the tumor microenvironment. Co-stimulation with IL-2 did not abrogate the NK inhibitory response of CA125. CA125 did not reduce proliferation or induce apoptosis of NK cells and alter the expression of p56lck, phospholipase Cγ1, ZAP70, or CD3ζ. CA125 did, however, induce major downregulation of CD16 and minor decrease in expression of CD94/NKG2A. Conclusions.: Our ongoing research and recent work performed by other laboratories highlights the potential physiologic role of this mucin. Based on the data presented here, it is likely that the tumor-derived CA125 acts as a suppressor of the immune response that is directed against the ovarian tumors. [Copyright &y& Elsevier]

Simple Summary: High-grade serous ovarian cancer is a fatal disease typically detected at an advanced stage when options for effective treatment are significantly limited. The lack of a screening modality to identify ovarian cancer in its early stage continues to be a major impediment in the management of this malignancy. The serum biomarker CA125, a repeating peptide epitope present in the sialomucin, MUC16, is unsuitable as a screening test. We have demonstrated that immune cells of ovarian cancer patients capture miniscule amounts of CA125 on their cell surface. Here, we report an automated, sensitive, alignment-free microscopy platform to qualitatively and quantitatively assess the low-abundance binding of CA125 to circulating leucocyte subsets. Through a comparison of the CA125 levels on immune cell subsets of ovarian cancer patients versus healthy donors, we demonstrate that our new technique can serve as a novel diagnostic platform for detection and monitoring of ovarian cancer. MUC16, a sialomucin that contains the ovarian cancer biomarker CA125, binds at low abundance to leucocytes via the immune receptor, Siglec-9. Conventional fluorescence-based imaging techniques lack the sensitivity to assess this low-abundance event, prompting us to develop a novel "digital" optical cytometry technique for qualitative and quantitative assessment of CA125 binding to peripheral blood mononuclear cells (PBMC). Plasmonic nanoparticle labeled detection antibody allows assessment of CA125 at the near-single molecule level when bound to specific immune cell lineages that are simultaneously identified using multiparameter fluorescence imaging. Image analysis and deep learning were used to quantify CA125 per each cell lineage. PBMC from treatment naïve ovarian cancer patients (N = 14) showed higher cell surface abundance of CA125 on the aggregate PBMC population as well as on NK (p = 0.013), T (p < 0.001) and B cells (p = 0.024) compared to circulating lymphocytes of healthy donors (N = 7). Differences in CA125 binding to monocytes or NK-T cells between the two cohorts were not significant. There was no correlation between the PBMC-bound and serum levels of CA125, suggesting that these two compartments are not in stoichiometric equilibrium. Understanding where and how subset-specific cell-bound surface CA125 takes place may provide guidance towards a new diagnostic biomarker in ovarian cancer. [ABSTRACT FROM AUTHOR]

Abstract Objective MUC16, the mucin that contains the CA125 epitopes, suppresses the cytolytic responses of human NK cells and inhibits the efficacy of therapeutic antibodies. Here, we provide further evidence of the regulatory role of MUC16 on human and murine NK cells and macrophages. Methods Target cell cytolysis and doublet formation assays were performed to assess effects of MUC16 on human NK cells. The effect of MUC16 on ovarian tumor growth was determined in a mouse model by monitoring survival and ascites formation. Innate immune cells from spleens and peritoneal cavities of mice were isolated and stimulated in vitro with anti-CD40 antibody, lipopolysaccharide and IFN-γ and their ability to cytolyse MUC16 expressing and non-expressing cells was determined. Results We confirm that MUC16 inhibits cytolysis by human NK cells as well as the formation of NK-tumor conjugates. Mice implanted with MUC16-knockdown OVCAR-3 show >2-fold increase in survival compared to controls. Murine NK cells and macrophages are more efficient at lysing MUC16-knockdown cells. In vitro cytotoxicity assays with NK cells and macrophages isolated from mice stimulated with anti-CD40 antibody showed 2–3-fold increased activity against the MUC16-knockdown cells as compared to matching target cells expressing this mucin. Finally, knockdown of MUC16 increased the susceptibility of cancer cells to ADCC by murine splenocytes. Conclusions For the first time, we demonstrate the immunoregulatory effects of MUC16 on murine NK cells and macrophages. Our study implies that the immunoregulatory role of MUC16 on murine NK cells and macrophages should be considered when examining the biology of MUC16 in mouse models. Highlights • The mucin MUC16 carries the peptide epitope that defines the biomarker CA125. • We provide data supporting the immunosuppressive effects of MUC16. • MUC16 prevents human immune cells from conjugating with ovarian cancer cells. • MUC16 also inhibits the cytolytic responses of murine NK and macrophages. • Studies on MUC16 in murine models should consider the suppressive effects of MUC16 on murine immune cells. [ABSTRACT FROM AUTHOR]