NY-ESO-1 is a "cancer-testis" antigen frequently expressed in epithelial ovarian cancer (EOC) and is among the most immunogenic tumor antigens defined to date. In an effort to understand in vivo tolerance mechanisms, we assessed the phenotype and function of NY-ESO-1-specific CD8 + T cells derived from peripheral blood lymphocytes (PBLs), tumor-infiltrating lymphocytes (TILs), and tumorassociated lymphocytes (TALs) of EOC patients with NY-ESO-1-expressing tumors, with or without humoral immunity to NY-ESO-1. Whereas NY-ESO-1-specific CD8 + T cells were readily detectable ex vivo with tetramers in TILs and TALs of seropositive patients, they were only detectable in PBLs following in vitro stimulation. Compared with PBLs, tumor-derived NY-ESO-1-specific CD8 + T cells demonstrated impaired effector function, preferential usage of dominant T-cell receptor, and enriched coexpression of inhibitory molecules LAG-3 and PD-1. Expression of LAG-3 and PD-1 on CD8 + T cells was up-regulated by IL-10, IL-6 (cytokines found in tumor ascites), and tumor-derived antigen-presenting cells. Functionally, CD8 + LAG-3 + PD-1 + T cells were more impaired in IFN-γ/TNF-α production compared with LAG-3 + PD-1 − or LAG-3 − PD-1 − subsets. Dual blockade of LAG-3 and PD-1 during T-cell priming efficiently augmented proliferation and cytokine production by NY-ESO-1-specific CD8 + T cells, indicating that antitumor function of NY-ESO-1-specific CD8 + T cells could potentially be improved by therapeutic targeting of these inhibitory receptors.tumor-infiltrating lymphocytes | IL-6 | IL-10 | T cell receptor T he presence of tumor-infiltrating lymphocytes within the tumor microenvironment is considered to be an indication of the host immune response to tumor antigens and is thought to reflect the dynamic process of "cancer immunoediting" (1). In epithelial ovarian cancer (EOC), support for the role of immune surveillance of tumors comes from observations indicating that the presence of intraepithelial CD8 + -infiltrating T lymphocytes in tumors is associated with improved survival of patients with the disease (2). Although several lines of evidence have shown that spontaneous or vaccine-induced tumor-antigen-specific CD8 + T cells can recognize EOC targets (3), prolongation of survival in patients treated with immunization has only rarely been observed. This is probably a reflection of several in vivo immunosuppressive mechanisms in EOC-bearing hosts (4). Therefore, understanding factors that regulate the function(s) of tumor-antigen-specific CD8 + T cells is critical for effective control of tumor recurrence.The NY-ESO-1 tumor antigen is a major target of CD8 + T cell recognition in EOC, eliciting both cellular and humoral immune responses in a proportion of patients with advanced NY-ESO-1-expressing tumors (5). However, similar to infectious disease models, chronic antigenic stimulation may result in exhaustion of antigen-specific CD8 + T cells (6) and loss of ability to produce key cytokines that are critical for the maintenance of CD8 + T ce...

Cells with sphere forming capacity, spheroid cells, are present in the malignant ascites of patients with epithelial ovarian cancer (EOC) and represent a significant impediment to efficacious treatment due to their putative role in progression, metastasis and chemotherapy resistance. The exact mechanisms that underlie EOC metastasis and drug resistance are not clear. Understanding the biology of sphere forming cells may contribute to the identification of novel therapeutic opportunities for metastatic EOC. Here we generated spheroid cells from human ovarian cancer cell lines and primary ovarian cancer. Xenoengraftment of as few as 2000 dissociated spheroid cells into immune-deficient mice allowed full recapitulation of the original tumor, whereas >105 parent tumor cells remained non-tumorigenic. The spheroid cells were found to be enriched for cells with cancer stem cell-like characteristics such as upregulation of stem cell genes, self-renewal, high proliferative and differentiation potential, and high aldehyde dehydrogenase (ALDH) activity. Furthermore, spheroid cells were more aggressive in growth, migration, invasion, scratch recovery, clonogenic survival, anchorage-independent growth, and more resistant to chemotherapy in vitro. 13C-glucose metabolic studies revealed that spheroid cells route glucose predominantly to anaerobic glycolysis and pentose cycle to the detriment of re-routing glucose for anabolic purposes. These metabolic properties of sphere forming cells appear to confer increased resistance to apoptosis and contribute to more aggressive tumor growth. Collectively, we demonstrated that spheroid cells with cancer stem cell-like characteristics contributed to tumor generation, progression and chemotherapy resistance. This study provides insight into the relationship between tumor dissemination and metabolic attributes of human cancer stem cells and has clinical implications for cancer therapy.

Despite its expression by subsets of various types of malignancies, PSMA is still considered to be fairly sensitive and highly specific for PaC.

NY-ESO-1 is a ''cancer-testis'' antigen expressed in epithelial ovarianHLA-DP4 ͉ peptide epitope ͉ tumor recognition ͉ vaccine T here is increasing evidence that the immune system has the ability to recognize tumor-associated antigens expressed in human malignancies and to induce antigen-specific humoral and cellular immune responses to these targets. In epithelial ovarian cancer (EOC), support for the role of immune surveillance of tumors comes from our recent observation indicating that the presence of intraepithelial CD8 ϩ -infiltrating T lymphocytes in tumors is associated with improved survival of patients with the disease (1). Although the majority of women with advanced-stage ovarian cancer respond to first-line chemotherapy, most of these responses are not durable, and Ͼ70% of patients die of recurrent disease within 5 years of diagnosis. Therefore, the development of strategies to enhance the potential of tumor antigen-specific CD8 ϩ T and CD4 ϩ T cells is urgently needed for extending remission rates in this disease. In this regard, cancer-testis antigens, a unique class of antigens that demonstrate high levels of expression in adult male germ cells but generally not in other normal adult tissues and aberrant expression in a variable proportion of a wide range of different cancer types, are promising candidates for immunotherapy. Among cancer-testis antigens, NY-ESO-1 (2) is one of the most spontaneously immunogenic tumor antigens described so far. Previously, we reported that NY-ESO-1 is a promising target for specific immunotherapy of EOC (3).Although the majority of cancer vaccine trials have focused on eliciting antigen-specific CD8 ϩ T cells, a growing body of evidence indicates that CD4 ϩ T cells play a pivotal role in orchestrating these responses. The multiple roles of antigen-specific CD4 ϩ T cells include the provision of help to CD8 ϩ T cells during the primary and secondary immune responses, direct cytolysis, and activation of B cells for production of tumor antigen-specific Abs. Therefore, we have focused on the NY-ESO-1 epitope, ESO 157-170 , a naturally processed helper epitope that is recognized by CD4 ϩ T cells in the context of HLA-DPB1*0401 and *0402 (4), prevalent MHC class II alleles present in Ϸ43-70% of Caucasians. Moreover, the NY-ESO-1 HLA-DP4 epitope has HLA-A2 (ESO 157-165 ) (5) and HLA-A24 (ESO 158-166 ) (6) motifs embedded in its natural sequence. In this study, we evaluated whether active immunization with ESO 157-170 would elicit NY-ESO-1-specific CD4 ϩ and CD8 ϩ T cell responses in ovarian cancer patients with minimal disease burden. In addition, we characterized NY-ESO-1-specific CD8 ϩ and CD4 ϩ T cell receptor (TCR) repertoires in conjunction with functional analysis of vaccine-elicited T cell clones.

The cancer-testis/cancer-germline antigen NY-ESO-1 is a vaccine target in epithelial ovarian cancer (EOC), but its limited expression is a barrier to vaccine efficacy. As NY-ESO-1 is regulated by DNA methylation, we hypothesized that DNA methyltransferase (DNMT) inhibitors may augment NY-ESO-1 vaccine therapy. In agreement, global DNA hypomethylation in EOC was associated with the presence of circulating antibodies to NY-ESO-1. Pre-clinical studies using EOC cell lines showed that decitabine treatment enhanced both NY-ESO-1 expression and NY-ESO-1-specific CTL-mediated responses. Based on these observations, we performed a phase I dose-escalation trial of decitabine, as an addition to NY-ESO-1 vaccine and doxorubicin liposome (doxorubicin) chemotherapy, in 12 patients with relapsed EOC. The regimen was safe, with limited and clinically manageable toxicities. Both global and promoter-specific DNA hypomethylation occurred in blood and circulating DNAs, the latter of which may reflect tumor cell responses. Increased NY-ESO-1 serum antibodies and T cell responses were observed in the majority of patients, and antibody spreading to additional tumor antigens was also observed. Finally, disease stabilization or partial clinical response occurred in 6/10 evaluable patients. Based on these encouraging results, evaluation of similar combinatorial chemo-immunotherapy regimens in EOC and other tumor types is warranted.