[Show abstract][Hide abstract] ABSTRACT: Study of myeloid cells endowed with suppressive activity is an active field of research which has particular importance in cancer, in view of the negative regulatory capacity of these cells to the host's immune response. The expansion of these cells, called myeloid-derived suppressor cells (MDSCs), has been documented in many models of tumor-bearing mice and in patients with tumors of various origin, and their presence is associated with disease progression and reduced survival. For this reason, monitoring this type of cell expansion is of clinical importance, and flow cytometry is the technique of choice for their identification. Over the years, it has been demonstrated that MDSCs comprise a group of immature myeloid cells belonging both to monocytic and granulocytic lineages, with several stages of differentiation; their occurrence depends on tumor-derived soluble factors, which guide their expansion and determine their block of differentiation. Because of their heterogeneous composition, accurate phenotyping of these cells requires a multicolor approach, so that the expansion of all MDSC subsets can be appreciated.This review article focuses on identifying MDSCs and discusses problems associated with phenotyping circulating and tumor-associated MDSCs in humans and in mouse models. This article is protected by copyright. All rights reserved.
Cytometry Part B Clinical Cytometry 11/2014; · 2.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We analyzed the interactions between primary cells from pancreatic ductal adenocarcinoma (PDAC) and polymeric scaffolds to develop 3D cancer models useful for mimicking the biology of this tumor. Three scaffold types based on two biocompatible polymeric formulations, such as poly(vinyl alcohol)/gelatin (PVA/G) mixture and poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymer, were obtained via different techniques, namely, emulsion and freeze-drying, compression molding followed by salt leaching, and electrospinning. In this way, primary PDAC cells interfaced with different pore topographies, such as sponge-like pores of different shape and size or nanofiber interspaces. The aim of this study was to investigate the influence played by the scaffold architecture over cancerous cell growth and function. In all scaffolds, primary PDAC cells showed good viability and synthesized tumor-specific metalloproteinases (MMPs) such as MMP-2, and MMP-9. However, only sponge-like pores, obtained via emulsion-based and salt leaching-based techniques allowed for an organized cellular aggregation very similar to the native PDAC morphological structure. Differently, these cell clusters were not observed on PEOT/PBT electrospun scaffolds. MMP-2 and MMP-9, as active enzymes, resulted to be increased in PVA/G and PEOT/PBT sponges, respectively. These findings suggested that spongy scaffolds supported the generation of pancreatic tumor models with enhanced aggressiveness. In conclusion, primary PDAC cells showed diverse behaviors while interacting with different scaffold types that can be potentially exploited to create stage-specific pancreatic cancer models likely to provide new knowledge on the modulation and drug susceptibility of MMPs.
[Show abstract][Hide abstract] ABSTRACT: Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, and its synthetic analog Trehalose-6,6-dibehenate (TDB) bind to the C-type lectin receptors macrophage-inducible C-type lectin (Mincle) and Mcl to activate macrophages. Genetically, the transcriptional response to TDB/TDM has been defined to require FcRγ-Syk-Card9 signaling. However, TDB/TDM-triggered kinase activation has not been studied well, and it is largely unknown which transcriptional regulators bring about inflammatory gene expression. In this article, we report that TDB/TDM caused only weak Syk-phosphorylation in resting macrophages, consistent with low basal Mincle expression. However, LPS-priming caused MYD88-dependent upregulation of Mincle, resulting in enhanced TDB/TDM-induced kinase activation and more rapid inflammatory gene expression. TLR-induced Mincle expression partially circumvented the requirement for Mcl in the response to TDB/TDM. To dissect transcriptional responses to TDB/TDM, we mined microarray data and identified early growth response (Egr) family transcription factors as direct Mincle target genes, whereas upregulation of Cebpb and Hif1a required new protein synthesis. Macrophages and dendritic cells lacking C/EBPβ showed nearly complete abrogation of TDB/TDM responsiveness, but also failed to upregulate Mincle. Retroviral rescue of Mincle expression in Cebpb-deficient cells restored induction of Egr1, but not of G-CSF. This pattern of C/EBPβ dependence was also observed after stimulation with the Dectin-1 ligand Curdlan. Inducible expression of hypoxia-inducible factor 1α (HIF1α) also required C/EBPβ. In turn, HIF1α was not required for Mincle expression, kinase activation, and Egr1 or Csf3 expression, but critically contributed to NO production. Taken together, we identify C/EBPβ as central hub in Mincle expression and inflammatory gene induction, whereas HIF1α controls Nos2 expression. C/EBPβ also connects TLR signals to cord factor responsiveness through MYD88-dependent upregulation of Mincle.
Journal of immunology (Baltimore, Md. : 1950). 08/2014;
[Show abstract][Hide abstract] ABSTRACT: By restraining T-cell activation and promoting Treg-cell expansion, myeloid-derived suppressor cells (MDSCs) and tolerogenic dendritic cells (tDCs) can control self-reactive and anti-graft effector T cells in autoimmunity and transplantation. Their therapeutic use and characterization, however, is limited by their scarce availability in the peripheral blood of tumor-free donors. In the present study, we describe and characterize a novel population of human myeloid suppressor cells, named fibrocytic MDSC (f-MDSCs), which are differentiated from umbilical cord blood (UCB) precursors by 4-day culture with FDA-approved cytokines (rh-GM-CSF and rh-G-CSF). This MDSC subset, characterized by the expression of MDSC-, DC-, and fibrocyte-associated markers, promotes Treg-cell expansion and induces normoglycemia in a xenogeneic mouse model of Type 1 diabetes (T1D). In order to exert their pro-tolerogenic function, fibrocytic MDSCs require direct contact with activated T cells, which leads to the production and secretion of IDO. This new myeloid subset may have an important role in the in vitro and in vivo production of Treg cells for the treatment of autoimmune diseases, and in either the prevention or control of allograft rejection.This article is protected by copyright. All rights reserved
European Journal of Immunology 08/2014; · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The dynamic interplay between cancer and host immune system often affects the process of myelopoiesis. As a consequence, tumor-derived factors sustain the accumulation and functional differentiation of myeloid cells, including myeloid-derived suppressor cells (MDSCs), which can interfere with T cell-mediated responses. Since both the phenotype and mechanisms of action of MDSCs appear to be tumor-dependent, it is important not only to determine the presence of all MDSC subsets in each cancer patient, but also which MDSC subsets have clinical relevance in each tumor environment. In this review, we describe the differences between MDSC populations expanded within different tumor contexts and evaluate the prognostic significance of MDSC expansion in peripheral blood and within tumor masses of neoplastic patients.
Annals of the New York Academy of Sciences 06/2014; 1319(1):47-65. · 4.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION: Pancreatic Ductal Adenocarcinoma (PDAC) is a malignant neoplasm of epithelial origin which represents one of the major causes of death in the western world. At present, the Tumor Tissue Microenvironment (TME) is supposed to play a pivotal role on the aggressiveness of pancreatic epithelial tumor cells. Aim of this study was to investigate the interactions between PDAC cells (derived from patients who underwent surgical treatment) and different biocompatible scaffolds to generate new 3D in vitro models for TME studies.
METHODS: To create 3D cellular models, three scaffolds were fabricated with different polymers and techniques: polyvinyl alcohol/gelatin (PVA/G; weight composition ratio of 70/30) obtained via emulsion and freeze-drying; poly(ethylene oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) produced via either compression molding and particle leaching (namely, CM) or electrospinning (namely, ES). Human epithelial primary cultures were derived from PDAC explants. Both tissues and derived cells were genetically characterized for K-ras and TP53. PDAC cells were seeded on the sterile scaffolds at a density of 1×104 cells/mm3 and cultured for 9 days in complete RMPI medium. Sample characterization included: cell viability (using the AlamarBlue assay), cell morphology [via scanning electron microscopy (SEM) and histology with H&E staining), expression of MMP9 and MMP2 proteins via Western Blot (WB) and immunohistochemistry (IHC).
RESULTS: At the endpoint cells were found to be viable in the scaffolds showing AlamarBlue reduction of 47.5% (PVA/G), 40.6% (PEOT/PBT_CM) and 45.1% (PEOT/PBT_ES). SEM analysis detected a good 3D colonization and a very strong cellular adhesion in all three models. The histologic analysis highlighted the formation of PDAC clusters showing a ductal-like morphology only in the PVA/G model. Finally, in the 3D models, a higher expression of MMP9 and MMP2 proteins was observed with respect to 2D cell cultures.
DISCUSSION: From our findings, the development of 3D cancer tissue-engineered models using biocompatible scaffolds for cell growth seems to be feasible, versatile, low time consuming and representative of PDAC features. This platform could be used as a preliminary attempt before in vivo testing. In the close future, 3D models could be used to screen experimental pharmacological treatments for the modulation of different tumor proteins. Furthermore, in these 3D models an integration among different cellular types involved in PDAC microenvironment could be generated and be more representative of the human TME than the in vitro models used so far.
48th Annual Meeting of the Pancreas Club, Westin Lombard Chicago IL; 05/2014
[Show abstract][Hide abstract] ABSTRACT: Tumor-infiltrating myeloid cells such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) form an important component of the hypoxic tumor microenvironment. Here, we investigated the influence of hypoxia on immune checkpoint receptors (programmed death [PD]-1 and CTLA-4) and their respective ligands (PD-1 ligand 1 [PD-L1], PD-L2, CD80, and CD86) on MDSCs. We demonstrate that MDSCs at the tumor site show a differential expression of PD-L1 as compared with MDSCs from peripheral lymphoid organ (spleen). Hypoxia caused a rapid, dramatic, and selective up-regulation of PD-L1 on splenic MDSCs in tumor-bearing mice. This was not limited to MDSCs, as hypoxia also significantly increased the expression of PD-L1 on macrophages, dendritic cells, and tumor cells. Furthermore, PD-L1 up-regulation under hypoxia was dependent on hypoxia-inducible factor-1α (HIF-1α) but not HIF-2α. Chromatin immunoprecipitation and luciferase reporter assay revealed direct binding of HIF-1α to a transcriptionally active hypoxia-response element (HRE) in the PD-L1 proximal promoter. Blockade of PD-L1 under hypoxia enhanced MDSC-mediated T cell activation and was accompanied by the down-regulation of MDSCs IL-6 and IL-10. Finally, neutralizing antibodies against IL-10 under hypoxia significantly abrogated the suppressive activity of MDSCs. Simultaneous blockade of PD-L1 along with inhibition of HIF-1α may thus represent a novel approach for cancer immunotherapy.
Journal of Experimental Medicine 04/2014; · 13.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tumor microenvironment of solid tumors is characterized by a strikingly high concentration of adenosine and ATP. Physiological significance of this biochemical feature is unknown, but it has been suggested that it may affect infiltrating immune cell responses and tumor progression. There is increasing awareness that many of the effects of extracellular ATP on tumor and inflammatory cells are mediated by the P2X7 receptor (P2X7R). Aim of this study was to investigate whether: (i) extracellular ATP is a component of neuroblastoma (NB) microenvironment, (ii) myeloid-derived suppressor cells (MDSCs) express functional P2X7R and (iii) the ATP/P2X7R axis modulates MDSC functions. Our results show that extracellular ATP was detected in NB microenvironment in amounts that increased in parallel with tumor progression. The percentage of CD11b(+)/Gr-1(+) cells was higher in NB-bearing mice compared with healthy animals. Within the CD11b/Gr-1(+) population, monocytic MDSCs (M-MDSCs) produced higher levels of reactive oxygen species (ROS), arginase-1 (ARG-1), transforming growth factor-β1 (TGF-β1) and stimulated more potently in vivo tumor growth, as compared with granulocytic MDSCs (G-MDSCs). P2X7R of M-MDSCs was localized at the plasma membrane, coupled to increased functionality, upregulation of ARG-1, TGF-β1 and ROS. Quite surprisingly, the P2X7R in primary MDSCs as well as in the MSC-1 and MSC-2 lines was uncoupled from cytotoxicity. This study describes a novel scenario in which MDSC immunosuppressive functions are modulated by the ATP-enriched tumor microenvironment.
Cell Death & Disease 03/2014; 5:e1135. · 5.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study employed mass sequencing of small RNA libraries to identify the repertoire of small noncoding RNAs expressed in normal CD4+ T-cells compared to cells transformed with human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia/lymphoma (ATLL). Results revealed distinct patterns of microRNA expression in HTLV-1-infected CD4+ T-cell lines with respect to their normal counterparts. In addition, a search for viral-encoded microRNAs yielded 2 sequences that originated from the plus strand of the HTLV-1 genome. Several sequences derived from tRNAs were expressed at substantial levels in both uninfected and infected cells. One of the most abundant tRNA fragments (tRF-3019) was derived from the 3' end of tRNA-proline. tRF-3019 exhibited perfect sequence complementarity to the primer binding site of HTLV-1. Results of an in vitro reverse transcriptase assay verified that tRF-3019 was capable of priming HTLV-1 reverse transcriptase. Both tRNA-proline and tRF-3019 were detected in virus particles isolated from HTLV-1-infected cells. These findings suggest that tRF-3019 may play an important role in priming HTLV-1 reverse transcription and could thus represent a novel target to control HTLV-1 infection.
[Show abstract][Hide abstract] ABSTRACT: Under many inflammatory contexts, such as tumor progression, systemic and peripheral immune response is tailored by reactive nitrogen species (RNS)-dependent post-translational modifications, suggesting a biological function for these chemical alterations. RNS modify both soluble factors and receptors essential to induce and maintain a tumor-specific immune response, creating a "chemical barrier" that impairs effector T cell infiltration and functionality in tumor microenvironment and supports the escape phase of cancer. RNS generation during tumor growth mainly depends on nitric oxide production by both tumor cells and tumor-infiltrating myeloid cells that constitutively activate essential metabolic pathways of l-arginine catabolism. This review provides an overview of the potential immunological and biological role of RNS-induced modifications and addresses new approaches targeting RNS either in search of novel biomarkers or to improve anti-cancer treatment.
[Show abstract][Hide abstract] ABSTRACT: The spleen is the main filter for blood-borne pathogens and antigens, as well as a key organ for iron metabolism and erythrocyte homeostasis. Also, immune and hematopoietic functions have been recently unveiled for the mouse spleen, suggesting additional roles for this secondary lymphoid organ. Here we discuss the integration of the spleen in the regulation of immune responses locally and in the whole body and present the relevance of findings for our understanding of inflammatory and degenerative diseases and their treatments. We consider whether equivalent activities in humans are known, as well as initial therapeutic attempts to target the spleen for modulating innate and adaptive immunity.
[Show abstract][Hide abstract] ABSTRACT: Tumor progression is accompanied by an altered myelopoiesis causing the accumulation of immunosuppressive cells. Here, we showed that miR-142-3p downregulation promoted macrophage differentiation and determined the acquisition of their immunosuppressive function in tumor. Tumor-released cytokines signaling through gp130, the common subunit of the interleukin-6 cytokine receptor family, induced the LAP(∗) isoform of C/EBPβ transcription factor, promoting macrophage generation. miR-142-3p downregulated gp130 by canonical binding to its messenger RNA (mRNA) 3' UTR and repressed C/EBPβ LAP(∗) by noncanonical binding to its 5' mRNA coding sequence. Enforced miR expression impaired macrophage differentiation both in vitro and in vivo. Mice constitutively expressing miR-142-3p in the bone marrow showed a marked increase in survival following immunotherapy with tumor-specific T lymphocytes. By modulating a specific miR in bone marrow precursors, we thus demonstrated the feasibility of altering tumor-induced macrophage differentiation as a potent tool to improve the efficacy of cancer immunotherapy.
[Show abstract][Hide abstract] ABSTRACT: Despite extensive ex vivo investigation, the spatiotemporal organization of immune cells interacting with virus-infected cells in tissues remains uncertain. To address this, we used intravital multiphoton microscopy to visualize immune cell interactions with virus-infected cells following epicutaneous vaccinia virus (VV) infection of mice. VV infects keratinocytes in epidermal foci and numerous migratory dermal inflammatory monocytes that outlie the foci. We observed Ly6G(+) innate immune cells infiltrating and controlling foci, while CD8(+) T cells remained on the periphery killing infected monocytes. Most antigen-specific CD8(+) T cells in the skin did not interact with virus-infected cells. Blocking the generation of reactive nitrogen species relocated CD8(+) T cells into foci, modestly reducing viral titers. Depletion of Ly6G(+) and CD8(+) cells dramatically increased viral titers, consistent with their synergistic but spatially segregated viral clearance activities. These findings highlight previously unappreciated differences in the anatomic specialization of antiviral immune cell subsets.
[Show abstract][Hide abstract] ABSTRACT: Tumors frequently induce immature myeloid cells (iMC), which suppress specific and unrelated cytotoxic T lymphocyte (CTL) responses and are termed myeloid derived suppressor cells (MDSC). Mainly analyzed by in vitro assays in tumor transplantation models, little is known about their function in autochthonous tumor models in vivo. We analyzed iMC in three SV40 large T (Tag)-driven conditional autochthonous cancer models with drastically different immune status: 1. Early Tag-specific CTL competence and rare stochastic Tag activation leading to sporadic cancer, which induces an aberrant immune response and CTL tolerance. 2. Cre/LoxP recombinase-mediated hepatocellular carcinoma (HCC) development in neonatal Tag-tolerant mice. 3. Tag-activation through Cre recombinase-encoding viruses in the liver and HCC development with systemic anti-Tag CTL immunity. In the first but not the two latter models, tumors induced CTL hypo-responsiveness to tumor unrelated antigens. Regardless of the model, tumors produced IL-6 and VEGF but not GM-CSF and induced iMC (CD11b(+)Gr-1(int)) that suppressed CTL responses in vitro. None of the iMC from the different tumor models suppressed CTL responses in adoptive cell transfer experiments, unless GM-CSF was provided in vivo. Together, iMC expand independent of the type of anti-tumor response and are not immune-suppressive in a cell-autonomous fashion.
[Show abstract][Hide abstract] ABSTRACT: Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by two molecularly distinct self-renewing leukemic stem cell (LSC) populations most closely related to normal progenitors and organized as a hierarchy. A requirement for WNT/β-catenin signaling in the pathogenesis of AML has recently been suggested by a mouse model. However, its relationship to a specific molecular function promoting retention of self-renewing leukemia-initiating cells (LICs) in human remains elusive. To identify transcriptional programs involved in the maintenance of a self-renewing state in LICs, we performed the expression profiling in normal (n = 10) and leukemic (n = 33) human long-term reconstituting AC133(+) cells, which represent an expanded cell population in most AML patients. This study reveals the ligand-dependent WNT pathway activation in AC133(bright) AML cells and shows a diffuse expression and release of WNT10B, a hematopoietic stem cell regenerative-associated molecule. The establishment of a primary AC133(+) AML cell culture (A46) demonstrated that leukemia cells synthesize and secrete WNT ligands, increasing the levels of dephosphorylated β-catenin in vivo. We tested the LSC functional activity in AC133(+) cells and found significant levels of engraftment upon transplantation of A46 cells into irradiated Rag2(-/-)γc(-/-) mice. Owing to the link between hematopoietic regeneration and developmental signaling, we transplanted A46 cells into developing zebrafish. This system revealed the formation of ectopic structures by activating dorsal organizer markers that act downstream of the WNT pathway. In conclusion, our findings suggest that AC133(bright) LSCs are promoted by misappropriating homeostatic WNT programs that control hematopoietic regeneration.
Neoplasia (New York, N.Y.) 12/2012; 14(12):1236-2124. · 5.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: One obstacle in eliciting potent anti-tumor immune responses is the induction of tolerance to tumor antigens. TCRlo mice bearing a TCR transgene specific for the melanoma antigen Tyrosinase-related protein-2 (TRP-2, Dct) harbor T cells that maintain tumor antigen responsiveness but lack the ability to control melanoma outgrowth. We used this model to determine whether higher avidity T cells could control tumor growth without becoming tolerized. As part of the current study, we developed a second TRP-2-specific TCR transgenic mouse line (TCRhi) that bears higher avidity T cells and spontaneously developed autoimmune depigmentation. In contrast to TCRlo T cells, which were ignorant of tumor-derived antigen, TCRhi T cells initially delayed subcutaneous B16 melanoma tumor growth. However, persistence in the tumor microenvironment resulted in reduced IFN-γ production and CD107a (Lamp1) mobilization, hallmarks of T cell tolerization. IFN-γ expression by TCRhi T cells was critical for up-regulation of MHC-I on tumor cells and control of tumor growth. Blockade of PD-1 signals prevented T cell tolerization and restored tumor immunity. Depletion of tumor-associated dendritic cells (TADCs) reduced tolerization of TCRhi T cells and enhanced their antitumor activity. In addition, TADCs tolerized TCRhi T cells but not TCRlo T cells in vitro. Our findings demonstrate that T cell avidity is a critical determinant of not only tumor control but also susceptibility to tolerization in the tumor microenvironment. For this reason, care should be exercised when considering T cell avidity in designing cancer immunotherapeutics.