Extracellular adenosine is a potent immunosuppressor that accumulates during tumor growth. We performed proof-of-concept studies investigating the therapeutic potential and mechanism of action of monoclonal antibody (mAb)-based therapy against CD73, an ecto-enzyme overexpressed on breast-cancer cells that catalyzes the dephosphorylation of adenosine monophosphates into adenosine. We showed that anti-CD73 mAb therapy significantly delayed primary 4T1.2 and E0771 tumor growth in immune-competent mice and significantly inhibited the development of spontaneous 4T1.2 lung metastases. Notably, anti-CD73 mAb therapy was essentially dependent on the induction of adaptive anti-tumor immune responses. Knockdown of CD73 in 4T1.2 tumor cells confirmed the tumor-promoting effects of CD73. In addition to its immunosuppressive effect, CD73 enhanced tumor-cell chemotaxis, suggesting a role for CD73-derived adenosine in tumor metastasis. Accordingly, administration of adenosine-5′-N-ethylcarboxamide to tumorbearing mice significantly enhanced spontaneous 4T1.2 lung metastasis. Using selective adenosine-receptor antagonists, we showed that activation of A2B adenosine receptors promoted 4T1.2 tumorcell chemotaxis in vitro and metastasis in vivo. In conclusion, our study identified tumor-derived CD73 as a mechanism of tumor immune escape and tumor metastasis, and it also established the proof of concept that targeted therapy against CD73 can trigger adaptive anti-tumor immunity and inhibit metastasis of breast cancer.adenosine | cancer | chemotaxis | immunosuppression | regulatory

It has been suggested that marrow stromal cells (MSCs) may be immunoprivileged and can engraft in allogeneic recipients with intact immune systems. We determined if the implantation of murine MSCs engineered to release erythropoietin (Epo) would be feasible in major histocompatibility complex (MHC)-mismatched allogeneic mice without immunosuppression, and we monitored hematocrit (Hct) as a reporter of MSC graft survival. MSCs from C57Bl/6 mice were engineered to release murine Epo (Epo ؉ MSCs) and implanted subcutaneously in either syngeneic C57Bl/6 mice or MHC-mismatched Balb/c mice. In syngeneic recipients, the Hct rapidly rose from baseline level and remained higher than .88 (88%) for more than 200 days. However, in MHCmismatched recipient Balb/c mice, the Hct rose transiently and rapidly declined to baseline values. Repeat implantations in these same mice were associated with an acquired refractoriness in the Hct response consistent with alloimmunization to donor Epo ؉ MSCs. Allogeneic MSC implants had an increased proportion of host-derived lymphoid CD8 ؉ , natural killer T (NKT), and NK infiltrating cells compared with syngeneic controls, and splenocytes isolated from Balb/c mice that had received implants also displayed a significant interferon-gamma (IFN␥) response to C57Bl/6 MSCs in vitro. These IntroductionBone marrow stromal cells (MSCs), 1 also sometimes referred to as mesenchymal stem cells, are promising for regenerative medicine due to their innate ability to differentiate into various cell types. [2][3][4][5] Thus, autologous MSCs and their genetically engineered progeny have potential use in several preclinical regenerative medicine scenarios, such as cardiovascular regeneration, 6-14 brain and spinal cord regeneration, 15,16 as well as bone and cartilage repair. [17][18][19][20][21][22][23] Furthermore, genetically engineered MSCs may operate as cellular vehicles for the delivery of therapeutic proteins in hereditary and acquired metabolic, endocrine, and malignant diseases. 4,[24][25][26][27][28][29][30] In vitro studies on human, baboon, and murine MSCs have revealed that MSCs are immunosuppressive. [31][32][33][34] Depending upon the experimental circumstances, suppression of mixed lymphocyte reaction (MLR) in vitro between major histocompatibility complex (MHC)-mismatched stimulator and responder cells by MSCs appears to arise from both contact-dependent 35 and soluble factors including, but not limited to, [36][37][38] hepatocyte growth factor (HGF) and transforming growth factor ␤1 (TGF-␤1). 32 However, alternative experimental settings suggest that MSCs may also behave as nonprofessional antigen-presenting cells (APCs). 39,40 In support of their in vivo immunosuppressive features are the observations that allogeneic MSCs may prolong skin allograft survival in immunocompetent baboons, 31 prevent the rejection of allogeneic B16 mouse melanoma cells in immunocompetent C3H mice, 34 and attenuate graft-versus-host disease in mice and humans. 38,41 The sum of these observations supports the...

Using gene-expression data from over 6,000 breast cancer patients, we report herein that high CD73 expression is associated with a poor prognosis in triple-negative breast cancers (TNBC). Because anthracycline-based chemotherapy regimens are standard treatment for TNBC, we investigated the relationship between CD73 and anthracycline efficacy. In TNBC patients treated with anthracycline-only preoperative chemotherapy, high CD73 gene expression was significantly associated with a lower rate of pathological complete response or the disappearance of invasive tumor at surgery. Using mouse models of breast cancer, we demonstrated that CD73 overexpression in tumor cells conferred chemoresistance to doxorubicin, a commonly used anthracycline, by suppressing adaptive antitumor immune responses via activation of A2A adenosine receptors. Targeted blockade of CD73 enhanced doxorubicin-mediated antitumor immune responses and significantly prolonged the survival of mice with established metastatic breast cancer. Taken together, our data suggest that CD73 constitutes a therapeutic target in TNBC.ectonucleotidase | immunogenic cell death | immunotherapy T riple-negative breast cancer (TNBC), as defined by the absence of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2) expression, accounts for 15-20% of all breast cancers (1). Compared with other breast cancer subtypes, TNBC is characterized by a worse prognosis and increased risk of metastasis to vital organs (1). There are currently no known molecular targets for this subgroup of breast cancer patients and there seem to be few therapeutic options on the horizon, especially given that the development of poly (ADP ribose) polymerase and EGFR inhibitors have been thus far disappointing (2). Hence, the treatment of TNBC is a significant challenge in today's clinical practice and the identification of therapeutic targets an area of urgent clinical need (3).Cytotoxic chemotherapy, particularly anthracycline-based regimens, therefore remains the mainstay of treatment for TNBC today. Although TNBC is associated with a poor prognosis, some patients seem to respond well to anthracycline-based chemotherapy, reflecting a significant degree of molecular heterogeneity within this subgroup (3-5). Unfortunately, the molecular mechanisms underlying this heterogeneity and its relationship to treatment response are still poorly understood. Recently, new light has been shed on the mechanism-of-action of anthracyclines, which may help elucidate new mechanisms of chemoresistance. Accumulating data suggest that anthracyclines mediate their anticancer activity not only by direct cytotoxic effects but also through activation of adaptive antitumor immune responses (6) by inducing a type of tumor cell death that is "immunogenic" (7-10). In mice, chemotherapy with anthracyclines requires IFN-γ-producing CD8 + T cells for optimal activity. This finding is further supported by correlative clinical studies that report that high intratumoral levels of I...

Purpose: Monoclonal antibodies (mAb) that block programmed death (PD)-1 or cytotoxic T lymphocyte antigen (CTLA-4) receptors have been associated with durable clinical responses against a variety of cancer types and hold great potential as novel cancer therapeutics. Recent evidence suggest that targeted blockade of multiple immunosuppressive pathways can induce synergistic antitumor responses.Experimental Design: In this study, we investigated whether targeted blockade of CD73, an ectonucleotidase that catabolizes the hydrolysis of extracellular adenosine monophosphate (AMP) to adenosine, can enhance the antitumor activity of anti-CTLA-4 and anti-PD-1 mAbs against transplanted and chemically induced mouse tumors.Results: Anti-CD73 mAb significantly enhanced the activity of both anti-CTLA-4 and anti-PD-1 mAbs against MC38-OVA (colon) and RM-1 (prostate) subcutaneous tumors, and established metastatic 4T1.2 breast cancer. Anti-CD73 mAb also significantly enhanced the activity of anti-PD-1 mAb against 3-methylcholanthrene (MCA)-induced fibrosarcomas. Gene-targeted mice revealed that single-agent therapies and combinatorial treatments were dependent on host IFN-g and CD8 þ T cells, but independent of perforin. Interestingly, anti-CD73 mAb preferentially synergized with anti-PD-1 mAb. We investigated the effect of extracellular adenosine on tumor-infiltrating T cells and showed that activation of A2A adenosine receptor enhances PD-1 expression, but not CTLA-4 expression, on tumor-specific CD8þ T cells and CD4þ Foxp3þ T regulatory cells. Conclusions: Taken together, our study revealed that targeted blockade of CD73 can enhance the therapeutic activity of anti-PD-1 and anti-CTLA-4 mAbs and may thus potentiate therapeutic strategies targeting immune checkpoint inhibitors in general.

Several studies have demonstrated that marrow stromal cells (MSCs) can suppress allogeneic T-cell responses. However, the effect of MSCs on syngeneic immune responses has been largely overlooked. We describe here that primary MSCs derived from C57BL/6 mice behave as conditional antigenpresenting cells (APCs) and can induce antigen-specific protective immunity. Interferon gamma (IFN␥)-treated C57BL/6 MSCs, but not unstimulated MSCs, cocultured with ovalbumin-specific major histocompatibility (MHC) class II-restricted hybridomas in the presence of soluble ovalbumin-induced significant production of interleukin-2 (IL-2) in an antigen dose-dependent manner (P < .005). IFN␥-treated MSCs could further activate in vitro ovalbumin-specific primary transgenic CD4 ؉ T cells. C57BL/6 MSCs, however, were unable to induce antigen cross-presentation via the MHC class I pathway. When syngeneic mice were immunized intraperitoneally with ovalbumin-pulsed IFN␥-treated MSCs, they developed antigenspecific cytotoxic CD8 ؉ T cells and became fully protected (10 of 10 mice) against ovalbumin-expressing E.G7 tumors. Human MSCs were also studied for antigen-presenting functions. IFN␥-treated DR1-positive human MSCs, but not unstimulated human MSCs, induced significant production of IL-2 when cocultured with DR1-restricted influenzaspecific humanized T-cell hybridomas in the presence of purified influenza matrix protein 1. Taken together, our data strongly suggest that MSCs behave as conditional APCs in syngeneic immune responses.

+ T cells distinguishes between Treg and pathogenic cellular populations that secrete proinflammatory cytokines such as IFNc and IL-17. These latter cell populations are increased, with a concomitant decrease in the CD4 + CD25 + CD39 + Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell-populations to allow tracking of these in health and disease, as in renal allograft rejection.

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 diseasefree 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 þ 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.Cancer Res; 75(21); 4494-503. Ó2015 AACR.

Purpose: CD73 is an adenosine-generating ecto-enzyme that suppresses antitumor immunity in mouse models of cancer, including prostate cancer. Although high levels of CD73 are associated with poor prognosis in various types of cancer, the clinical impact of CD73 in prostate cancer remains unclear.Experimental Design: We evaluated the prognostic value of CD73 protein expression and CD8 þ cell density in 285 cases of prostate cancer on tissue microarray (TMA). Normal adjacent and tumor tissues were evaluated in duplicates.Results: Univariate and multivariate analyses revealed that high levels of CD73 in normal adjacent prostate epithelium were significantly associated with shorter biochemical recurrence (BCR)-free survival. Notably, CD73 expression in normal epithelium conferred a negative prognostic value to prostate-infiltrating CD8 þ cells. Surprisingly, high levels of CD73 in the tumor stroma were associated with longer BCR-free survival in univariate analysis. In vitro studies revealed that adenosine signaling inhibited NF-kB activity in human prostate cancer cells via A2B adenosine receptors. Consistent with these results, CD73 expression in the prostate tumor stroma negatively correlated with p65 expression in the nuclei of prostate tumor cells. Conclusions: Our study revealed that CD73 is an independent prognostic factor in prostate cancer. Our data support a model in which CD73 expression in the prostate epithelium suppresses immunosurveillance by CD8 þ T cells, whereas CD73 expression in the tumor stroma reduces NF-kB signaling in tumor cells via A2B adenosine receptor signaling. CD73 expression, including in normal adjacent prostate epithelium, can thus effectively discriminate between aggressive and indolent forms of prostate cancer.