CD73 expression and clinical significance in human metastatic melanoma.

CD73 is an ectoenzyme involved in the production of adenosine. It exerts immunosuppressive and protumoral roles and has emerged as a potential immuno-oncology target. CD73 expression was detected in TC in 54% of melanoma metastases, involving < 50% TC in the majority of the cases, with variable intensity. CD73 expression was significantly associated with a lower Breslow's depth of the primary lesion and was more frequent in patients having received prior non-surgical therapies. In an adjusted analysis, CD73 expression in TC (H-score > 37.5 or intensity > 1) significantly correlated to decreased overall survival (OS) from biopsy. Of the samples containing TIMC, 35% presented CD73+ TIMC. Highly infiltrated tumors were more likely to contain CD73+ TIMC. CD73 expression in TIMC (percentage ≥1%) significantly correlated with improved OS from biopsy. Immunohistochemistry detected CD73 expression in more than half of metastatic melanomas. While CD73 expression in TC significantly correlated with decreased OS, CD73 expression in TIMC significantly associated with improved OS. These results encourage the study of anti-CD73 therapies for metastatic melanoma patients. CD73 expression was assessed by immunohistochemistry in metastatic melanomas from 114 patients. Immunostainings were evaluated in tumor cells (TC) (percentage, intensity (1-3) and H-score) and in tumor-infiltrating mononuclear cells (TIMC) (percentage)

CD73 expression in normal and pathological human hepatobiliopancreatic tissues.

The tumor-expressed CD73 ectonucleotidase generates immune tolerance and promotes invasiveness via adenosine production from degradation of AMP. While anti-CD73 blockade treatment is a promising tool in cancer immunotherapy, a characterization of CD73 expression in human hepatobiliopancreatic system is lacking. CD73 expression was investigated by immunohistochemistry in a variety of non-neoplastic and neoplastic conditions of the liver, pancreas, and biliary tract. CD73 was expressed in normal hepatobiliopancreatic tissues with subcellular-specific patterns of staining: canalicular in hepatocytes, and apical in cholangiocytes and pancreatic ducts. CD73 was present in all hepatocellular carcinoma (HCC), in all pancreatic ductal adenocarcinoma (PDAC), and in the majority of intra and extrahepatic cholangiocellular carcinomas, whereas it was detected only in a subset of pancreatic neuroendocrine neoplasms and almost absent in acinar cell carcinoma. In addition to the canonical pattern of staining, an aberrant membranous and/or cytoplasmic expression was observed in invasive lesions, especially in HCC and PDAC. These two entities were also characterized by a higher extent and intensity of staining as compared to other hepatobiliopancreatic neoplasms. In PDAC, aberrant CD73 expression was inversely correlated with differentiation (p < 0.01) and was helpful to identify isolated discohesive tumor cells. In addition, increased CD73 expression was associated with reduced overall survival (HR 1.013) and loss of E-Cadherin. Consistent CD73 expression supports the rationale for testing anti-CD73 therapies in patients with hepatobiliopancreatic malignancies. Specific patterns of expression could also be of help in the routine diagnostic workup

Autocrine Adenosine Regulates Tumor Polyfunctional CD73⁺CD4⁺ Effector T Cells Devoid of Immune Checkpoints.

The production of CD73-derived adenosine (Ado) by Tregs has been proposed as a resistance mechanism to anti-PD-1 therapy in murine tumor models. We reported that human Tregs express the ectonucleotidase CD39, which generates AMP from ATP, but do not express the AMPase CD73. In contrast, CD73 defined a subset of effector CD4 <sup>+</sup> T cells (Teffs) enriched in polyfunctional Th1.17 cells characterized by expression of CXCR3, CCR6, and MDR1, and production of IL17A/IFNγ/IL22/GM-CSF. CD39 <sup>+</sup> Tregs selectively targeted CD73 <sup>+</sup> Teffs through cooperative degradation of ATP into Ado inhibiting and restricting the ability of CD73 <sup>+</sup> Teffs to secrete IL17A. CD73 <sup>+</sup> Teffs infiltrating breast and ovarian tumors were functionally blunted by Tregs expressing upregulated levels of CD39 and ATPase activity. Moreover, tumor-infiltrating CD73 <sup>+</sup> Teffs failed to express inhibitory immune checkpoints, suggesting that CD73 might be selected under pressure from immune checkpoint blockade therapy and thus may represent a nonredundant target for restoring antitumor immunity.Significance: Polyfunctional CD73 <sup>+</sup> T-cell effectors lacking other immune checkpoints are selectively targeted by CD39 overexpressing Tregs that dominate the breast tumor environment. Cancer Res; 78(13); 3604-18. ©2018 AACR

Adenosine mediates functional and metabolic suppression of peripheral and tumor-infiltrating CD8⁺ T cells.

Several mechanisms are present in the tumor microenvironment (TME) to impair cytotoxic T cell responses potentially able to control tumor growth. Among these, the accumulation of adenosine (Ado) contributes to tumor progression and represents a promising immunotherapeutic target. Ado has been shown to impair T cell effector function, but the role and mechanisms employed by Ado/Ado receptors (AdoRs) in modulating human peripheral and tumor-infiltrating lymphocyte (TIL) function are still puzzling. CD8 <sup>+</sup> T cell cytokine production following stimulation was quantified by intracellular staining and flow cytometry. The cytotoxic capacity of tumor infiltrating lymphocytes (TILs) was quantified by the chromium release assay following co-culture with autologous or anti-CD3-loaded tumor cell lines. The CD8 <sup>+</sup> T cell metabolic fitness was evaluated by the seahorse assay and by the quantification of 2-NBDG uptake and CD71/CD98 upregulation upon stimulation. The expression of AdoRs was assessed by RNA flow cytometry, a recently developed technology that we validated by semiquantitative RT-PCR (qRT-PCR), while the impact on T cell function was evaluated by the use of selective antagonists and agonists. The influence of Ado/AdoR on the PKA and mTOR pathways was evaluated by phosphoflow staining of p-CREB and p-S6, respectively, and validated by western blot. Here, we demonstrate that Ado signaling through the A2A receptor (A2AR) in human peripheral CD8 <sup>+</sup> T cells and TILs is responsible for the higher sensitivity to Ado-mediated suppression of T central memory cells. We confirmed that Ado is able to impair peripheral and tumor-expanded T cell effector functions, and we show for the first time its impact on metabolic fitness. The Ado-mediated immunosuppressive effects are mediated by increased PKA activation that results in impairment of the mTORC1 pathway. Our findings unveil A2AR/PKA/mTORC1 as the main Ado signaling pathway impairing the immune competence of peripheral T cells and TILs. Thus, p-CREB and p-S6 may represent useful pharmacodynamic and efficacy biomarkers of immunotherapies targeting Ado. The effect of Ado on T cell metabolic fitness reinforces the importance of the adenosinergic pathway as a target for next-generation immunotherapy

Renal cell carcinoma induces interleukin 10 and prostaglandin E2 production by monocytes

Immunotherapy with interleukin 2 (IL-2) is not an effective anti-cancer treatment in the majority of patients with renal cell carcinoma (RCC), suggesting that the activation of cytotoxic T cells or NK cells may be impaired in vivo in these patients. The production of immunosuppressive factors by RCC was investigated. Using immunohistochemistry, IL-10 was detectable in 10 of 21 tumour samples tested. IL-10 was undetectable in the supernatant of cell lines derived from these RCCs. However, these cell lines or their conditioned medium (RCC CM), but not normal renal epithelial cells adjacent to the RCC or breastcarcinoma cell lines, were found to induce IL-10, as well as prostaglandin E2 (PGE2) and tumour necrosis factor (TNF)α production by autologous or allogeneic peripheral blood mononuclear cells (PBMCs) and monocytes. IL-10 production induced by RCC CM was found to be dependent on TNF-α and PGE2 since an anti-TNF-α antibody (Ab) inhibited 40–70% of IL-10 production by monocytes, and the combination of anti-TNF-α Ab and indomethacin, an inhibitor of PGE2 production, inhibited 80–94% of RCC CM-induced IL-10 production by monocytes. The RCC CM of the five cell lines tested were found to induce a down-regulation of the expression of HLA-DR and CD86, as well as a strong inhibition of mannose receptor-dependent endocytosis by monocytes. The blockade of HLA-DR and CD86 expression was partially abrogated by indomethacin and anti-IL-10 Ab respectively, and completely abrogated by an anti-TNF-α Ab. The inhibition of mannose receptor-dependent endocytosis was partially abrogated by an anti-IL-10 Ab and completely abrogated by an anti-TNF-α Ab. These esults indicate that RCCs induce IL-10, PGE2 and TNF-α production by monocytes, which down-regulate the expression of cell-surface molecules involved in antigen presentation as well as their endocytic capacity. © 1999 Cancer Research Campaig

Targeting Adenosine in Cancer Immunotherapy to Enhance T-Cell Function.

T cells play a critical role in cancer control, but a range of potent immunosuppressive mechanisms can be upregulated in the tumor microenvironment (TME) to abrogate their activity. While various immunotherapies (IMTs) aiming at re-invigorating the T-cell-mediated anti-tumor response, such as immune checkpoint blockade (ICB), and the adoptive cell transfer (ACT) of natural or gene-engineered ex vivo expanded tumor-specific T cells, have led to unprecedented clinical responses, only a small proportion of cancer patients benefit from these treatments. Important research efforts are thus underway to identify biomarkers of response, as well as to develop personalized combinatorial approaches that can target other inhibitory mechanisms at play in the TME. In recent years, adenosinergic signaling has emerged as a powerful immuno-metabolic checkpoint in tumors. Like several other barriers in the TME, such as the PD-1/PDL-1 axis, CTLA-4, and indoleamine 2,3-dioxygenase (IDO-1), adenosine plays important physiologic roles, but has been co-opted by tumors to promote their growth and impair immunity. Several agents counteracting the adenosine axis have been developed, and pre-clinical studies have demonstrated important anti-tumor activity, alone and in combination with other IMTs including ICB and ACT. Here we review the regulation of adenosine levels and mechanisms by which it promotes tumor growth and broadly suppresses protective immunity, with extra focus on the attenuation of T cell function. Finally, we present an overview of promising pre-clinical and clinical approaches being explored for blocking the adenosine axis for enhanced control of solid tumors

Immune cell dysfunctions in breast cancer patients detected through whole blood multi-parametric flow cytometry assay

International audienceMonitoring functional competence of immune cell populations in clinical routine represents a major challenge. We developed a whole-blood assay to monitor functional competence of peripheral innate immune cells including NK cells, dendritic and monocyte cell subsets through their ability to produce specific cytokines after short-term stimulation, detected through intra-cytoplasmic staining and multi-parametric flow-cytometry. A PMA/ionomycin T cell activation assay complemented this analysis. Comparing cohorts of healthy women and breast cancer (BC) patients at different stages, we identified significant functional alteration of circulating immune cells during BC progression prior to initiation of treatment. Of upmost importance, as early as the localized primary tumor (PT) stage, we observed functional alterations in several innate immune populations and T cells i.e. (i) reduced TNFalpha production by BDCA-1+ DC and non-classical monocytes in response to Type-I IFN, (ii) a strong drop in IFNgamma production by NK cells in response to either Type-I IFN or TLR7/8 ligand, and (iii) a coordinated impairment of cytokine (IL-2, IFNgamma, IL-21) production by T cell subpopulations. Overall, these alterations are further accentuated according to the stage of the disease in first-line metastatic patients. Finally, whereas we did not detect functional modification of DC subsets in response to TLR7/8 ligand, we highlighted increased IL-12p40 production by monocytes specifically at first relapse (FR). Our results reinforce the importance of monitoring both innate and adaptive immunity to better evaluate dysfunctions in cancer patients and suggest that our whole-blood assay will be useful to monitor response to treatment, particularly for immunotherapeutic strategie

Disequilibrium of BMP2 levels in the breast stem cell niche launches epithelial transformation by overamplifying BMPR1B cell response

Understanding the mechanisms of cancer initiation will help to prevent and manage the disease. At present, the role of the breast microenvironment in transformation remains unknown. As BMP2 and BMP4 are important regulators of stem cells and their niches in many tissues, we investigated their function in early phases of breast cancer. BMP2 production by tumor microenvironment appeared to be specifically upregulated in luminal tumors. Chronic exposure of immature human mammary epithelial cells to high BMP2 levels initiated transformation toward a luminal tumor-like phenotype, mediated by the receptor BMPR1B. Under physiological conditions, BMP2 controlled the maintenance and differentiation of early luminal progenitors, while BMP4 acted on stem cells/myoepithelial progenitors. Our data also suggest that microenvironment-induced overexpression of BMP2 may result from carcinogenic exposure. We reveal a role for BMP2 and the breast microenvironment in the initiation of stem cell transformation, thus providing insight into the etiology of luminal breast cancer.Marion Chapellier, Elodie Bachelard-Cascales, Xenia Schmidt, Flora Clement ... Jean-Yves Blay, Richard Iggo ... et al