apyrase and Ovarian-Neoplasms

Hydrolysis of extracellular ATP to adenosine (eADO) is an important immune checkpoint in cancer immunology. We here investigated the impact of the eADO pathway in high-grade serous ovarian cancer (HGSC) using multiparametric platforms.. We performed a transcriptomic meta-analysis of eADO-producing CD39 and CD73, an eADO signaling gene signature, immune gene signatures and clinical outcomes in approximately 1200 patients with HGSC. Protein expression, localization and prognostic impact of CD39, CD73 and CD8 were then performed on approximately 1000 cases on tissue microarray, and tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry and single-cell RNA sequencing on a subset of patients.. Concomitant CD39 and CD73 gene expression, as well as high levels of an eADO gene signature, were associated with worse prognosis in patients with HGSC, notably in the immunoregulatory molecular subtype, characterized by an immune-active microenvironment. CD39 was further associated with primary chemorefractory and chemoresistant human HGSC and platinum-based chemotherapy of murine HGSC was significantly more effective in CD39-deficient mice. At protein level, CD39 and CD73 were predominantly expressed by cancer-associated fibroblasts, and CD39 was expressed on severely exhausted, clonally expanded and putative tissue-resident memory TILs.. Our study revealed the clinical, immunological, subtype-specific impacts of eADO signaling in HGSC, unveiled the chemoprotective effect of CD39 and supports the evaluation of eADO-targeting agents in patients with ovarian cancer.

Topics: 5'-Nucleotidase; Adenosine; Adenosine Triphosphate; Animals; Antigens, CD; Antineoplastic Agents; Apyrase; Biomarkers, Tumor; Cell Line, Tumor; Databases, Genetic; Female; Gene Expression Profiling; GPI-Linked Proteins; Humans; Hydrolysis; Mice, Knockout; Middle Aged; Neoplasm Grading; Neoplasms, Cystic, Mucinous, and Serous; Ovarian Neoplasms; RNA-Seq; Signal Transduction; Single-Cell Analysis; Transcriptome; Xenograft Model Antitumor Assays

The detection of tumor-specific T cells in solid tumors is integral to interrogate endogenous antitumor responses and to advance downstream therapeutic applications. Multiple biomarkers are reported to identify endogenous tumor-specific tumor-infiltrating lymphocytes (TILs), namely CD137, PD-1, CD103, and CD39; however, a direct comparison of these molecules has yet to be performed. We evaluated these biomarkers in primary human ovarian tumor samples using single-cell mass cytometry to compare their relative phenotypic profiles, and examined their response to autologous tumor cells ex vivo. PD-1

Topics: Antigens, CD; Apyrase; Biomarkers, Tumor; Female; Humans; Integrin alpha Chains; Interferon-gamma; Lymphocytes, Tumor-Infiltrating; Ovarian Neoplasms; Programmed Cell Death 1 Receptor; Tumor Necrosis Factor Receptor Superfamily, Member 9

Phenotypic characterization of γδ T cells in the MALs (malignant ascites lymphocytes), TILs (tumor infiltrating lymphocytes), and PBLs (peripheral blood lymphocytes) of ovarian cancer (OvCA) patients is lacking. Therefore, we quantified γδ T cell prevalence in MAL, TIL, and PBL specimens from

Topics: Apyrase; Carcinoma, Ovarian Epithelial; Female; Hepatitis A Virus Cellular Receptor 2; Humans; Ovarian Neoplasms; Programmed Cell Death 1 Receptor; Receptors, Immunologic

Tumor antigen-specific CD4 T cells accumulate at tumor sites, evoking their involvement in antitumor effector functions in situ. Contrary to CD8 cytotoxic T lymphocyte exhaustion, that of CD4 T cells remains poorly appreciated. Here, using phenotypic, transcriptomic, and functional approaches, we characterized CD4 T cell exhaustion in patients with head and neck, cervical, and ovarian cancer. We identified a CD4 tumor-infiltrating lymphocyte (TIL) population, defined by high PD-1 and CD39 expression, which contained high proportions of cytokine-producing cells, although the quantity of cytokines produced by these cells was low, evoking an exhausted state. Terminal exhaustion of CD4 TILs was instated regardless of TIM-3 expression, suggesting divergence with CD8 T cell exhaustion. scRNA-Seq and further phenotypic analyses uncovered similarities with the CD8 T cell exhaustion program. In particular, PD-1hiCD39+ CD4 TILs expressed the exhaustion transcription factor TOX and the chemokine CXCL13 and were tumor antigen specific. In vitro, PD-1 blockade enhanced CD4 TIL activation, as evidenced by increased CD154 expression and cytokine secretion, leading to improved dendritic cell maturation and consequently higher tumor-specific CD8 T cell proliferation. Our data identify exhausted CD4 TILs as players in responsiveness to immune checkpoint blockade.

Topics: Antigens, Neoplasm; Apyrase; CD8-Positive T-Lymphocytes; Female; Gene Expression; Head and Neck Neoplasms; Humans; Immune Tolerance; Immunity, Cellular; In Vitro Techniques; Lymphocyte Activation; Lymphocyte Cooperation; Lymphocytes, Tumor-Infiltrating; Male; Ovarian Neoplasms; Programmed Cell Death 1 Receptor; RNA, Messenger; T-Lymphocytes, Helper-Inducer; Tumor Escape; Uterine Cervical Neoplasms

Ovarian cancer is the most lethal gynecological malignancy, with serous histotype as the most prevalent epithelial ovarian cancer (EOC). Peritoneal ascites is a frequent comorbidity in advanced EOC. EOC-associated ascites provide a reliable sampling source for studying lymphocytes directly from tumor environment. Herein, we carried out flow cytometry-based analysis to readdress issues on NK and T lymphocyte subsets in women with advanced EOC, additionally evaluating phenotypic modulation of their intracellular pathways involved in interleukin (IL)-2 and IL-15 signaling. Results depicted ascites as an inflammatory and immunosuppressive environment, presenting significantly (

Topics: Adult; Aged; Aged, 80 and over; Apyrase; Ascites; CD56 Antigen; Cystadenocarcinoma, Serous; Female; Gene Expression Regulation, Neoplastic; Humans; Immunophenotyping; Interleukin-10; Interleukin-15; Interleukin-2; Interleukin-6; K562 Cells; Killer Cells, Natural; Middle Aged; Neoplasm Grading; Neoplasm Staging; Ovarian Neoplasms; Programmed Cell Death 1 Receptor; Signal Transduction; STAT5 Transcription Factor; T-Lymphocytes; Tumor Microenvironment

Identifying tumor antigen-specific T cells from cancer patients has important implications for immunotherapy diagnostics and therapeutics. Here, we show that CD103

Topics: Adenocarcinoma of Lung; Antigens, CD; Apyrase; Carcinoma, Squamous Cell; CD8 Antigens; CD8-Positive T-Lymphocytes; Female; Humans; Immunophenotyping; Integrin alpha Chains; Lymphocytes, Tumor-Infiltrating; Male; Melanoma; Ovarian Neoplasms; Receptors, Antigen, T-Cell, alpha-beta; Squamous Cell Carcinoma of Head and Neck; Survival Analysis; Transcriptome

Extracellular nucleotides and nucleosides have emerged as important elements regulating tissue homeostasis. Acting through specific receptors, have the ability to control gene expression patterns to direct cellular fate. We observed that SKOV-3 cells express the ectonucleotidases: ectonucleotide pyrophosphatase 1 (ENPP1), ecto-5'-nucleotidase (NT5E), and liver alkaline phosphatase (ALPL). Strikingly, in pulse and chase experiments supplemented with ATP, SKOV-3 cells exhibited low catabolic efficiency in the conversion of ADP into AMP, but they were efficient in converting AMP into adenosine. Since these cells release ATP, we proposed that the conversion of ADP into AMP is a regulatory node associated with the migratory ability and the mesenchymal characteristics shown by SKOV-3 cells under basal conditions. The landscape of gene expression profiles of SKOV-3 cell cultures treated with apyrase or adenosine demonstrated similarities (e.g., decrease FGF16 transcript) and differences (e.g., the negative regulation of Wnt 2, and 10B by adenosine). Thus, in SKOV-3 we analyzed the migratory ability and the expression of epithelium to mesenchymal transition (EMT) markers in response to apyrase. Apyrase-treatment favored the epithelial-like phenotype, as revealed by the re-location of E-cadherin to the cell to cell junctions. Pharmacological approaches strongly suggested that the effect of Apyrase involved the accumulation of extracellular adenosine; this notion was strengthened when the incubation of the SKOV-3 cell with α,β-methylene ADP (CD73 inhibitor) or adenosine deaminase was sufficient to abolish the effect of apyrase on cell migration. Overall, adenosine signaling is a fine tune mechanism in the control of cell phenotype in cancer. J. Cell. Biochem. 118: 4468-4478, 2017. © 2017 Wiley Periodicals, Inc.

Topics: Apyrase; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Female; Humans; Neoplasm Proteins; Ovarian Neoplasms; Purines

We investigated the effect of platelets on ovarian cancer and the role of adenosine diphosphate (ADP) receptors (P2Y12 and P2Y1) on platelets in the growth of primary ovarian cancer tumors. We showed that in murine models of ovarian cancer, a P2Y12 inhibitor (ticagrelor) reduced tumor growth by 60% compared with aspirin and by 75% compared with placebo. In P2Y12

Topics: Adenosine; Adoptive Transfer; Animals; Antigens, CD; Apoptosis; Apyrase; Blood Platelets; Cell Proliferation; Female; Gene Knockdown Techniques; Hematopoiesis; Humans; Mice, Inbred C57BL; Ovarian Neoplasms; Receptors, Purinergic P2Y1; Receptors, Purinergic P2Y12; Ticagrelor

Live regulatory T cells (T

Topics: 5'-Nucleotidase; Adenosine; Animals; Antigens, CD; Apoptosis; Apyrase; B7-H1 Antigen; CTLA-4 Antigen; Female; GPI-Linked Proteins; Humans; Immune Tolerance; Interleukin-10; Interleukins; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-E2-Related Factor 2; Ovarian Neoplasms; Oxidative Stress; Oxygen; Receptor, Adenosine A2A; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Microenvironment

The cell surface nucleotidase CD73 is an immunosuppressive enzyme involved in tumor progression and metastasis. Although preclinical studies suggest that CD73 can be targeted for cancer treatment, the clinical impact of CD73 in ovarian cancer remains unclear. In this study, we investigated the prognostic value of CD73 in high-grade serous (HGS) ovarian cancer using gene and protein expression analyses. Our results demonstrate that high levels of CD73 are significantly associated with shorter disease-free survival and overall survival in patients with HGS ovarian cancer. Furthermore, high levels of CD73 expression in ovarian tumor cells abolished the good prognosis associated with intraepithelial CD8(+) cells. Notably, CD73 gene expression was highest in the C1/stromal molecular subtype of HGS ovarian cancer and positively correlated with an epithelial-to-mesenchymal transition gene signature. Moreover, in vitro studies revealed that CD73 and extracellular adenosine enhance ovarian tumor cell growth as well as expression of antiapoptotic BCL-2 family members. Finally, in vivo coinjection of ID8 mouse ovarian tumor cells with mouse embryonic fibroblasts showed that CD73 expression in fibroblasts promotes tumor immune escape and thereby tumor growth. In conclusion, our study highlights a role for CD73 as a prognostic marker of patient survival and also as a candidate therapeutic target in HGS ovarian cancers.

Topics: 5'-Nucleotidase; Adult; Aged; Aged, 80 and over; Animals; Antigens, CD; Apyrase; Biomarkers, Tumor; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Proliferation; Disease-Free Survival; Epithelial-Mesenchymal Transition; Female; Fibroblasts; Gene Expression Regulation, Neoplastic; GPI-Linked Proteins; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Small Interfering; Tumor Escape

P2X7 is a purinergic receptor-channel; its activation by ATP elicits a broad set of cellular actions, from apoptosis to signals for survival. Here, P2X7 expression and function was studied in human ovarian carcinoma (OCA) cells, and biopsies from non-cancerous and cancer patients were analyzed by immunohistochemistry. Ovarian surface epithelium in healthy tissue expressed P2X7 at a high level that was maintained throughout the cancer. The cell lines SKOV-3 and CAOV-3 were used to investigate P2X7 functions in OCA. In SKOV-3 cells, selective stimulation of P2X7 by 2'(3')-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate (BzATP) induced a dose-dependent increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) but not cell death. Instead, BzATP increased the levels of phosphorylated ERK and AKT (pERK and pAKT), with an EC(50) of 44 ± 2 and 1.27 ± 0.5 μM, respectively; 10 μM BzATP evoked a maximum effect within 15 min that lasted for 120 min. Interestingly, basal levels of pERK and pAKT were decreased in the presence of apyrase in the medium, strongly suggesting an endogenous, ATP-mediated phenomenon. Accordingly: (i) mechanically stimulated cells generated a [Ca(2+)](i) increase that was abolished by apyrase; (ii) apyrase induced a decrease in culture viability, as measured by the MTS assay for mitochondrial activity; and (iii) incubation with 10 μM AZ10606120, a specific P2X7 antagonist and transfection with the dominant negative P2X7 mutant E496A, both reduced cell viability to 70.1 ± 8.9% and to 76.5 ± 5%, respectively, of control cultures. These observations suggested that P2X7 activity was auto-induced through ATP efflux; this increased pERK and pAKT levels that generated a positive feedback on cell viability.

Topics: Adenosine Triphosphate; Adult; Aged; Apyrase; Calcium; Cell Line, Tumor; Cell Proliferation; Female; Humans; MAP Kinase Signaling System; Middle Aged; Ovarian Neoplasms; Paracrine Communication; Receptors, Purinergic P2X7

Ovarian cancer is a prevalent gynecological malignancy with potent immune-suppression capabilities; regulatory T cells (Tregs) are significant contributors to this immune-suppression. As ovarian cancer patients present with high levels of TNF and Tregs expressing TNFR2 are associated with maximal suppressive capacity, we investigated TNFR2+ Tregs within these patients. Indeed, TNFR2+ Tregs from tumor-associated ascites were the most potent suppressor T cell fraction. They were abundantly present within the ascites and more suppressive than peripheral blood TNFR2+ Tregs in patients. The increased suppressive capacity can be explained by a distinct cell surface expression profile, which includes high levels of CD39, CD73, TGF-β and GARP. Additionally, CD73 expression level on TNFR2+ Tregs was inversely correlated with IFN-γ production by effector T cells. This Treg fraction can be selectively recruited into the ascites from the peripheral blood of patients. Targeting TNFR2+ Tregs may offer new approaches to enhance the poor survival rates of ovarian cancer.

Topics: 5'-Nucleotidase; Aged; Antigens, CD; Apyrase; Ascites; CTLA-4 Antigen; Cytokines; Female; Forkhead Transcription Factors; GPI-Linked Proteins; Humans; Leukocytes, Mononuclear; Membrane Proteins; Middle Aged; Ovarian Neoplasms; Receptors, Tumor Necrosis Factor, Type II; T-Lymphocytes, Regulatory; Tumor Microenvironment

Tumour cells activate and aggregate platelets [tumour cell-induced platelet aggregation (TCIPA)] and this process plays an important role in the successful metastasis of cancer cells. To date, most studies on TCIPA have been conducted under no-flow conditions. In this study, we have investigated TCIPA in real time under flow conditions, using an ultrasound standing wave trap that allows formation and levitation of cancer cell clusters in suspension, thus mimicking the conditions generated by flowing blood.. Using 59M adenocarcinoma and HT1080 fibrosarcoma cells and human platelets, cancer cell cluster-platelet aggregates were imaged in real time using epi-fluorescence microscopy (F-actin) and investigated in detail using confocal microscopy (matrix metalloproteinase-2-GPIIb/IIIa co-localization) and scanning electron and helium-ion microscopy (<1 nm resolution). The release of gelatinases from aggregates was studied using zymography.. We found that platelet activation and aggregation takes place on the surface of cancer cells (TCIPA), leading to time-dependent disruption of cancer cell clusters. Pharmacological modulation of TCIPA revealed that EDTA, prostacyclin, o-phenanthroline and apyrase significantly down-regulated TCIPA and, in turn, delayed cell cluster disruption, However, EGTA and aspirin were ineffective. Pharmacological inhibition of TCIPA correlated with the down-regulation of platelet activation as shown by flow-cytometry assay of platelet P-selectin.. Our results show for the first time, that during TCIPA, platelet activation disrupts cancer cell clusters and this can contribute to metastasis. Thus, selective targeting of platelet aggregate-cancer cell clusters may be an important strategy to control metastasis.

Topics: Actins; Adenocarcinoma; Apyrase; Blood Platelets; Cell Communication; Cell Line, Tumor; Down-Regulation; Edetic Acid; Epoprostenol; Female; Fibrosarcoma; Flow Cytometry; Humans; Matrix Metalloproteinase 2; Microscopy, Confocal; Microscopy, Fluorescence; Ovarian Neoplasms; P-Selectin; Phenanthrolines; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Tumor Cells, Cultured; Ultrasonics; Ultrasonography

The ectonucleotidases CD39 and CD73 degrade immune stimulatory ATP to adenosine that inhibits T and NK cell responses via the A(2A) adenosine receptor (ADORA2A). This mechanism is used by regulatory T cells (T(reg)) that are associated with increased mortality in OvCA. Immunohistochemical staining of human OvCA tissue specimens revealed further aberrant expression of CD39 in 29/36 OvCA samples, whereas only 1/9 benign ovaries showed weak stromal CD39 expression. CD73 could be detected on 31/34 OvCA samples. While 8/9 benign ovaries also showed CD73 immunoreactivity, expression levels were lower than in tumour specimens. Infiltration by CD4(+) and CD8(+) T cells was enhanced in tumour specimens and significantly correlated with CD39 and CD73 levels on stromal, but not on tumour cells. In vitro, human OvCA cell lines SK-OV-3 and OaW42 as well as 11/15 ascites-derived primary OvCA cell cultures expressed both functional CD39 and CD73 leading to more efficient depletion of extracellular ATP and enhanced generation of adenosine as compared to activated T(reg). Functional assays using siRNAs against CD39 and CD73 or pharmacological inhibitors of CD39, CD73 and ADORA2A revealed that tumour-derived adenosine inhibits the proliferation of allogeneic human CD4(+) T cells in co-culture with OvCA cells as well as cytotoxic T cell priming and NK cell cytotoxicity against SK-OV3 or OAW42 cells. Thus, both the ectonucleotidases CD39 and CD73 and ADORA2A appear as possible targets for novel treatments in OvCA, which may not only affect the function of T(reg) but also relieve intrinsic immunosuppressive properties of tumour and stromal cells.

Topics: 5'-Nucleotidase; Adenosine; Antigens, CD; Apyrase; Cell Line, Tumor; Cell Separation; Cytotoxicity, Immunologic; Female; Flow Cytometry; GPI-Linked Proteins; Humans; Immunohistochemistry; Killer Cells, Natural; Ovarian Neoplasms; Receptor, Adenosine A2A; RNA Interference; T-Lymphocytes

Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Apyrase; Arachidonic Acid; Cell Degranulation; Cell Survival; Drug Synergism; Epithelial-Mesenchymal Transition; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Neovascularization, Pathologic; Oligonucleotide Array Sequence Analysis; Ovarian Neoplasms; Peptide Fragments; Platelet Adhesiveness; Receptors, Purinergic P2Y1; Receptors, Purinergic P2Y12; Receptors, Thrombin; Reproducibility of Results; Signal Transduction