Late-rising CD4 T cells resolve mouse cytomegalovirus persistent replication in the salivary gland.

Conventional antiviral memory CD4 T cells typically arise during the first two weeks of acute infection. Unlike most viruses, cytomegalovirus (CMV) exhibits an extended persistent replication phase followed by lifelong latency accompanied with some gene expression. We show that during mouse CMV (MCMV) infection, CD4 T cells recognizing an epitope derived from the viral M09 protein only develop after conventional memory T cells have already peaked and contracted. Ablating these CD4 T cells by mutating the M09 genomic epitope in the MCMV Smith strain, or inducing them by introducing the epitope into the K181 strain, resulted in delayed or enhanced control of viral persistence, respectively. These cells were shown to be unique compared to their conventional memory counterparts; producing higher IFNγ and IL-2 and lower IL-10 levels. RNAseq analyses revealed them to express distinct subsets of effector genes as compared to classical CD4 T cells. Additionally, when M09 cells were induced by epitope vaccination they significantly enhanced protection when compared to conventional CD4 T cells alone. These data show that late-rising CD4 T cells are a unique memory subset with excellent protective capacities that display a development program strongly differing from the majority of memory T cells.

Profiling Human Cytomegalovirus-Specific T Cell Responses Reveals Novel Immunogenic Open Reading Frames.

Despite the prevalence and medical significance of human cytomegalovirus (HCMV) infections, a systematic analysis of the targets of T cell recognition in humans that spans the entire genome and includes recently described potential novel open reading frames (ORFs) is not available. Here, we screened a library of epitopes predicted to bind HLA class II that spans over 350 different HCMV ORFs and includes ∼150 previously described and ∼200 recently described potential novel ORFs by using an ex vivo gamma interferon (IFN-γ) FluoroSpot assay. We identified 235 unique HCMV-specific epitopes derived from 100 ORFs, some previously described as immunodominant and others that were not previously described to be immunogenic. Of those, 41 belong to the set of recently reported novel ORFs, thus providing evidence that at least some of these are actually expressed in vivo in humans. These data reveal that the breadth of the human T cell response to HCMV is much greater than previously thought. The ORFs and epitopes identified will help elucidate how T cell immunity relates to HCMV pathogenesis and instruct ongoing HCMV vaccine research. IMPORTANCE To understand the crucial role of adaptive immunity in controlling cytomegalovirus infection and disease, we systematically analyzed the CMV "ORFeome" to identify new CMV epitopes targeted primarily by CD4 T cells in humans. Our study identified >200 new T cell epitopes derived from both canonical and novel ORFs, highlighting the substantial breadth of the anti-CMV T cell response and providing new targets for vaccine design.

Structure of human cytomegalovirus UL144, an HVEM orthologue, bound to the B and T cell lymphocyte attenuator.

Human cytomegalovirus (HCMV) is a ß-herpesvirus that has co-evolved with the host immune system to establish lifelong persistence. HCMV encodes many immunomodulatory molecules, including the glycoprotein UL144. UL144 is a structural mimic of the tumor necrosis factor receptor superfamily member HVEM (herpesvirus entry mediator), which binds to the various ligands LIGHT, LTα, BTLA, CD160, and gD. However, in contrast to HVEM, UL144 only binds BTLA, inhibiting T-cell activation. Here, we report the crystal structure of the UL144-BTLA complex, revealing that UL144 utilizes residues from its N-terminal cysteine-rich domain 1 (CRD1) to interact uniquely with BTLA. The shorter CRD2 loop of UL144 also alters the relative orientation of BTLA binding with both N-terminal CRDs. By employing structure-guided mutagenesis, we have identified a mutant of BTLA (L123A) that interferes with HVEM binding but preserves UL144 interactions. Furthermore, our results illuminate structural differences between UL144 and HVEM that explain its binding selectivity and highlight it as a suitable scaffold for designing superior, immune inhibitory BTLA agonists.

Cytomegalovirus Evades TRAIL-Mediated Innate Lymphoid Cell 1 Defenses.

Cytomegalovirus (CMV) establishes a lifelong infection facilitated, in part, by circumventing immune defenses mediated by tumor necrosis factor (TNF)-family cytokines. An example of this is the mouse CMV (MCMV) m166 protein, which restricts expression of the TNF-related apoptosis-inducing ligand (TRAIL) death receptors, promoting early-phase replication. We show here that replication of an MCMV mutant lacking m166 is also severely attenuated during viral persistence in the salivary glands (SG). Depleting group I innate lymphoid cells (ILCs) or infecting Trail-/- mice completely restored persistent replication of this mutant. Group I ILCs are comprised of two subsets, conventional natural killer cells (cNK) and tissue-resident cells often referred to as innate lymphoid type I cells (ILC1). Using recently identified phenotypic markers to discriminate between these two cell types, their relative expression of TRAIL and gamma interferon (IFN-γ) was assessed during both early and persistent infection. ILC1 were found to be the major TRAIL expressers during both of these infection phases, with cNK expressing very little, indicating that it is ILC1 that curtail replication via TRAIL in the absence of m166-imposed countermeasures. Notably, despite high TRAIL expression by SG-resident ILC1, IFN-γ production by both ILC1 and cNK was minimal at this site of viral persistence. Together these results highlight TRAIL as a key ILC1-utilized effector molecule that can operate in defense against persistent infection at times when other innate control mechanisms may be muted and highlight the importance for the evolution of virus-employed countermeasures.IMPORTANCE Cytomegalovirus (a betaherpesvirus) is a master at manipulating immune responses to promote its lifelong persistence, a result of millions of years of coevolution with its host. Using a one-of-a-kind MCMV mutant unable to restrict expression of the TNF-related apoptosis-inducing ligand death receptors (TRAIL-DR), we show that TRAIL-DR signaling significantly restricts both early and persistent viral replication. Our results also reveal that these defenses are employed by TRAIL-expressing innate lymphoid type I cells (ILC1) but not conventional NK cells. Overall, our results are significant because they show the key importance of viral counterstrategies specifically neutralizing TRAIL effector functions mediated by a specific, tissue-resident subset of group I ILCs.

Cytomegalovirus: Shape-Shifting the Immune System.

Systems-based based approaches have begun to shed light on extrinsic factors that contribute to immune system variation. Among these, CMV (HHV-5, a ß-herpesvirus) imposes a surprisingly profound impact. Most of the world's population is CMV+, and the virus goes through three distinct infection phases en route to establishing lifelong détente with its host. Immune control of CMV in each phase recruits unique arms of host defense, and in turn the virus employs multiple immune-modulatory strategies that help facilitate the establishment of lifelong persistence. In this review, we explain how CMV shapes immunity and discuss the impact it may have on overall health.

Crystal structure of murine 4-1BB and its interaction with 4-1BBL support a role for galectin-9 in 4-1BB signaling.

4-1BB (CD137) is a TNF receptor superfamily (TNFRSF) member that is thought to undergo receptor trimerization upon binding to its trimeric TNF superfamily ligand (4-1BBL) to stimulate immune responses. 4-1BB also can bind to the tandem repeat-type lectin galectin-9 (Gal-9), and signaling through mouse (m)4-1BB is reduced in galectin-9 (Gal-9)-deficient mice, suggesting a pivotal role of Gal-9 in m4-1BB activation. Here, using sulfur-SAD phasing, we determined the crystal structure of m4-1BB to 2.2-Å resolution. We found that similar to other TNFRSFs, m4-1BB has four cysteine-rich domains (CRDs). However, the organization of CRD1 and the orientation of CRD3 and CRD4 with respect to CRD2 in the m4-1BB structure distinctly differed from those of other TNFRSFs. Moreover, we mapped two Asn residues within CRD4 that are N-linked glycosylated and mediate m4-1BB binding to Gal-9. Kinetics studies of m4-1BB disclosed a very tight nanomolar binding affinity to m4-1BBL with an unexpectedly strong avidity effect. Both N- and C-terminal domains of Gal-9 bound m4-1BB, but with lower affinity compared with m4-1BBL. Although the TNF homology domain (THD) of human (h)4-1BBL forms non-covalent trimers, we found that m4-1BBL formed a covalent dimer via 2 cysteines absent in h4-1BBL. As multimerization and clustering is a prerequisite for TNFR intracellular signaling, and as m4-1BBL can only recruit two m4-1BB monomers, we hypothesize that m4-1BBL and Gal-9 act together to aid aggregation of m4-1BB monomers to efficiently initiate m4-1BB signaling.

Functional Langerinhigh-Expressing Langerhans-like Cells Can Arise from CD14highCD16- Human Blood Monocytes in Serum-Free Condition.

Langerhans cells (LCs) are epithelial APCs that sense danger signals and in turn trigger specific immune responses. In steady-state, they participate in the maintenance of peripheral tolerance to self-antigens whereas under inflammation LCs efficiently trigger immune responses in secondary lymphoid organs. It has been demonstrated in mice that LC-deprived epithelia are rapidly replenished by short half-life langerin-expressing monocyte-derived LCs (MDLCs). These surrogate LCs are thought to be progressively replaced by langerin(high) LCs arising from self-renewing epithelial precursors of hematopoietic origin. How LCs arise from blood monocytes is not fully understood. Hence, we sought to characterize key factors that induce differentiation of langerin(high)-expressing monocyte-derived Langerhans-like cells. We identified GM-CSF and TGF-ß1 as key cytokines to generate langerin(high)-expressing cells but only in serum-free conditions. These cells were shown to express the LC-specific TROP-2 and Axl surface markers and contained Birbeck granules. Surprisingly, E-cadherin was not spontaneously expressed by these cells but required a direct contact with keratinocytes to be stably induced. MDLCs induced stronger allogeneic T cell proliferations but released low amounts of inflammatory cytokines upon TLR stimulation compared with donor-paired monocyte-derived dendritic cells. Immature langerin(high) MDLCs were responsive to MIP-3ß/CCL20 and CTAC/CCL27 chemokine stimulations. Finally, we demonstrated that those cells behaved as bona fide LCs when inserted in a three-dimensional rebuilt epithelium by becoming activated upon TLR or UV light stimulations. Collectively, these results prompt us to propose these langerin(high) MDLCs as a relevant model to address LC biology-related questions.

Loss of connexin43 expression in Ewing's sarcoma cells favors the development of the primary tumor and the associated bone osteolysis.

Ewing's sarcoma (ES) is a primary bone tumor characterized by a chromosomic translocation between the EWS gene and a member of the ETS gene family, mainly FLI1, which leads to an aberrant transcription factor EWS-FLI1 that promotes tumorigenicity. Gap junctions are intercellular channels composed of transmembrane proteins (connexin: Cx), that allow direct intercellular communication between adjacent cells. Numerous studies have shown that tumorigenesis may be associated with a loss of gap junctional intercellular communication (GJIC). Loss of Cx43 expression was observed at the protein and mRNA levels in ES cell lines compared to those measured in human mesenchymal stem cells. A673 ES cells stably transfected with an shRNA targeting EWS-FLI1 showed an increase in Cx43 expression (at the mRNA, protein and transcriptional levels) and GJIC. In an osteolytic murine model of ES, the overexpression of Cx43 in ES cells dramatically reduced tumor growth, leading to a significant increase in animal survival. In vitro assays showed that Cx43 overexpression increases the p27 level with an associated marked decrease of Rb phosphorylation, consistent with the observed blockade of the cell cycle in G0/G1 phase. In addition, the bone microarchitectural parameters, assessed by micro-CT analysis, showed an increased bone volume when Cx43 expression was enhanced. Histological analysis demonstrated that the overexpression of Cx43 in ES tumor cells inhibits osteoclast activity and therefore bone resorption. Our study demonstrated that the loss of Cx43 expression in ES cells plays a crucial role in the development of the primary tumor and the associated bone osteolysis.

Drugs targeting the bone microenvironment: new therapeutic tools in Ewing's sarcoma?

INTRODUCTION: Ewing's sarcoma (ES) is the second most frequent malignant primary bone tumour in children, adolescents and young adults. The overall survival is 60 - 70% at 5 years but still very poor for patients with metastases, disease relapse or for those not responding to chemotherapy. For these high risk patients, new therapeutic approaches are needed beyond conventional therapies (chemotherapy, surgery and radiation) such as targeted therapies. AREAS COVERED: Transcriptomic and genomic analyses in ES have revealed alterations in genes that control signalling pathways involved in many other cancer types. To set up more specific approaches, it is reasonable to think that the particular microenvironment of these bone tumours is essential for their initiation and progression, including in ES. To support this hypothesis, preclinical studies using drugs targeting bone cells (bisphosphonate zoledronate, anti-receptor activator of NF-κB ligand strategies) showed promising results in animal models. This review will discuss the new targeted therapeutic options in ES, focusing more particularly on the ones modulating the bone microenvironment. EXPERT OPINION: Targeting the microenvironment represents a new option for patients with ES. The proof-of-concept has been demonstrated in preclinical studies using relevant animal models, especially for zoledronate, which induced a strong inhibition of tumour progression in an orthotopic bone model.

Therapeutic efficacy of soluble receptor activator of nuclear factor-kappa B-Fc delivered by nonviral gene transfer in a mouse model of osteolytic osteosarcoma.

Osteosarcoma is the most frequent primary bone tumor that develops mainly during youth, the median age of diagnosis being 18 years. Despite improvement in osteosarcoma treatment, survival rate is only 30% after 5 years for patients with pulmonary metastases at diagnosis. This warrants exploration of new therapeutic options. The anti-bone resorption molecule receptor activator of NF-kappaB (RANK) is very promising, as it may block the vicious cycle between bone resorption and tumor proliferation that takes place during tumor development in bone site. The cDNA encoding murine RANK-Fc (mRANK-Fc) was administered by gene transfer using an amphiphilic polymer in a mouse model of osteolytic osteosarcoma. Clinical and bone microarchitecture variables were assessed by radiography and micro-CT analyses. In vitro experiments were designed to determine the mechanism of action of RANK-Fc on tumor cell proliferation (XTT assays), apoptosis (caspase activation), cell cycle distribution (fluorescence-activated cell sorting analysis), or gene expression (reverse transcription-PCR). RANK-Fc was effective in preventing the formation of osteolytic lesions associated with osteosarcoma development and in reducing the tumor incidence, the local tumor growth, and the lung metastases dissemination leading to a 3.9-fold augmentation of mice survival 28 days after implantation. On the contrary, mRANK-Fc did not prevent the development of nonosseous tumor nodules, suggesting that bone environment is necessary for mRANK-Fc therapeutic efficacy. Furthermore, mRANK-Fc has no direct activity on osteosarcoma cells in vitro. mRANK-Fc exerts an indirect inhibitory effect on osteosarcoma progression through inhibition of bone resorption.

Interleukin-17D and Nrf2 mediate initial innate immune cell recruitment and restrict MCMV infection.

Innate immune cells quickly infiltrate the site of pathogen entry and not only stave off infection but also initiate antigen presentation and promote adaptive immunity. The recruitment of innate leukocytes has been well studied in the context of extracellular bacterial and fungal infection but less during viral infections. We have recently shown that the understudied cytokine Interleukin (IL)-17D can mediate neutrophil, natural killer (NK) cell and monocyte infiltration in sterile inflammation and cancer. Herein, we show that early immune cell accumulation at the peritoneal site of infection by mouse cytomegalovirus (MCMV) is mediated by IL-17D. Mice deficient in IL-17D or the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an inducer of IL-17D, featured an early decreased number of innate immune cells at the point of viral entry and were more susceptible to MCMV infection. Interestingly, we were able to artificially induce innate leukocyte infiltration by applying the Nrf2 activator tert-butylhydroquinone (tBHQ), which rendered mice less susceptible to MCMV infection. Our results implicate the Nrf2/IL-17D axis as a sensor of viral infection and suggest therapeutic benefit in boosting this pathway to promote innate antiviral responses.

N-glycosylation of mouse TRAIL-R and human TRAIL-R1 enhances TRAIL-induced death.

APO2L/TRAIL (TNF-related apoptosis-inducing ligand) induces death of tumor cells through two agonist receptors, TRAIL-R1 and TRAIL-R2. We demonstrate here that N-linked glycosylation (N-glyc) plays also an important regulatory role for TRAIL-R1-mediated and mouse TRAIL receptor (mTRAIL-R)-mediated apoptosis, but not for TRAIL-R2, which is devoid of N-glycans. Cells expressing N-glyc-defective mutants of TRAIL-R1 and mouse TRAIL-R were less sensitive to TRAIL than their wild-type counterparts. Defective apoptotic signaling by N-glyc-deficient TRAIL receptors was associated with lower TRAIL receptor aggregation and reduced DISC formation, but not with reduced TRAIL-binding affinity. Our results also indicate that TRAIL receptor N-glyc impacts immune evasion strategies. The cytomegalovirus (CMV) UL141 protein, which restricts cell-surface expression of human TRAIL death receptors, binds with significant higher affinity TRAIL-R1 lacking N-glyc, suggesting that this sugar modification may have evolved as a counterstrategy to prevent receptor inhibition by UL141. Altogether our findings demonstrate that N-glyc of TRAIL-R1 promotes TRAIL signaling and restricts virus-mediated inhibition.

Unique phenotypes and clonal expansions of human CD4 effector memory T cells re-expressing CD45RA.

The expression of CD45RA is generally associated with naive T cells. However, a subset of effector memory T cells re-expresses CD45RA (termed TEMRA) after antigenic stimulation with unknown molecular characteristics and functions. CD4 TEMRA cells have been implicated in protective immunity against pathogens such as dengue virus (DENV). Here we show that not only the frequency but also the phenotype of CD4 TEMRA cells are heterogeneous between individuals. These cells can be subdivided into two major subsets based on the expression of the adhesion G protein-coupled receptor GPR56, and GPR56+ TEMRA cells display a transcriptional and proteomic program with cytotoxic features that is distinct from effector memory T cells. Moreover, GPR56+ TEMRA cells have higher levels of clonal expansion and contain the majority of virus-specific TEMRA cells. Overall, this study reveals the heterogeneity of CD4 TEMRA cells and provides insights into T-cell responses against DENV and other viral pathogens.

Osteoprotegerin inhibits bone resorption and prevents tumor development in a xenogenic model of Ewing's sarcoma by inhibiting RANKL.

Ewing's sarcoma (ES) associated with high osyeolytic lesions typically arises in the bones of children and adolescents. The development of multi-disciplinary therapy has increased current long-term survival rates to greater than 50% but only 20% for high risk group patients (relapse, metastases, etc.). Among new therapeutic approaches, osteoprotegerin (OPG), an anti-bone resorption molecule may represent a promising candidate to inhibit RANKL-mediated osteolytic component of ES and consequently to limit the tumor development. Xenogenic orthotopic models of Ewing's sarcoma were induced by intra-osseous injection of human TC-71 ES cells. OPG was administered in vivo by non-viral gene transfer using an amphiphilic non ionic block copolymer. ES bearing mice were assigned to controls (no treatment, synthetic vector alone or F68/empty pcDNA3.1 plasmid) and hOPG treated groups. A substantial but not significant inhibition of tumor development was observed in the hOPG group as compared to control groups. Marked bone lesions were revealed by micro-computed tomography analyses in control groups whereas a normal bone micro-architecture was preserved in the hOPG treated group. RANKL over-expressed in ES animal model was expressed by tumor cells rather than by host cells. However, TRAIL present in the tumor microenvironment may interfere with OPG effect on tumor development and bone remodeling via RANKL inhibition. In conclusion, the use of a xenogenic model of Ewing's sarcoma allowed discriminating between the tumor and host cells responsible for the elevation of RANKL production observed in this tumor and demonstrated the relevance of blocking RANKL by OPG as a promising therapy in ES.

TRAIL receptor signaling and therapeutic option in bone tumors: the trap of the bone microenvironment.

Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL/TNFSF10) has been reported to specifically induce malignant cell death being relatively nontoxic to normal cells. Since its identification 15 years ago, the antitumor activity and therapeutic value of TRAIL have been extensively studied. Five receptors quickly emerged, two of them being able to induce programmed cell death in tumor cells. This review takes a comprehensive look at this ligand and its receptors, and its potential role in primary bone tumors (osteosarcoma and Ewing's sarcoma) therapy. The main limit of clinical use of TRAIL being the innate or acquired resistance mechanisms, different possibilities to sensitize resistant cells are discussed in this review, together with the impact of bone microenvironment in the regulation of TRAIL activity.

A functional, new short isoform of death receptor 4 in Ewing's sarcoma cell lines may be involved in TRAIL sensitivity/resistance mechanisms.

Ewing's sarcoma (ES) is a high-grade neoplasm arising in bones of children and adolescents. Survival rate decreases from greater than 50% to only 20% after 5 years for patients not responding to treatment or presenting metastases at diagnosis. TRAIL, which has strong antitumoral activity, is a promising therapeutic candidate. To address TRAIL sensitivity, 7 human ES cell lines were used. Cell viability experiments [3'[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro-)benzene sulfonic acid hydrate (XTT) assay] showed that 4 of the 7 ES cell lines were resistant to TRAIL. Western blotting and flow cytometry analyses revealed that DR5 was uniformly expressed by all ES cell lines, whereas DR4 levels were higher in sensitive cell lines. In TRAIL-sensitive TC-71 cells, knockdown of TNFRSF10A/DR4 by short hairpin RNA (shRNA) was associated with a loss of sensitivity to TRAIL, in spite of DR5 presence. Interestingly, we identified a new transcript variant that results from an alternative splicing and encodes a 310-amino acid protein which corresponds to the 468 aa of DR4 original isoform but truncated of aa 11 to 168 within the extracellular TRAIL-binding domain. According to modeling studies, the contact of this new DR4 isoform (bDR4) with TRAIL seemed largely preserved. The overexpression of bDR4 in a TRAIL-resistant cell line restored TRAIL sensitivity. TRAIL resensitization was also observed after c-FLIP knockdown by shRNA in two TRAIL-resistant cell lines, as shown by XTT assay and caspase-3 assay. The results presented in this study showed that DR4, both as the complete form or as its new short isoform, is involved in TRAIL sensitivity in ES.

Impact of oncopediatric dosing regimen of zoledronic acid on bone growth: preclinical studies and case report of an osteosarcoma pediatric patient.

Osteosarcoma and Ewing sarcoma represent the two most frequent primary bone tumors that arise in the pediatric population. Despite recent improvement in their therapeutic management, no improvement in survival rate has been achieved since early 1980 s. Among new therapeutic approaches, bisphosphonates are promising candidates as potent inhibitors of bone resorption. However, their effects on bone growth must be studied at dosing regimen corresponding to pediatric protocols. To this aim, several protocols using zoledronic acid (ZOL) were developed in growing mice (50 µg/kg every 2 days × 10). Parameters of bone remodeling and bone growth were investigated by radiography, micro-computed tomography, histology, and biologic analyses. Extramedullar hematopoiesis was searched for in spleen tissue. A transient inhibitory effect of ZOL was observed on bone length, with a bone-growth arrest during treatment owing to an impressive increase in bone formation at the growth plate level (8- to 10-fold increase in BV/TV). This sclerotic band then shifted into the diaphysis as soon as endochondral bone formation started again after the end of ZOL treatment, revealing that osteoclasts and osteoblasts are still active at the growth plate. In conclusion, endochondral bone growth is transiently disturbed by high doses of ZOL corresponding to the pediatric treatment of primary bone tumors. These preclinical observations were confirmed by a case report in a pediatric patient treated in the French OS2006 protocol over 10 months who showed a growth arrest during the ZOL treatment period with normal gain in size after the end of treatment.

Preclinical evidence that use of TRAIL in Ewing's sarcoma and osteosarcoma therapy inhibits tumor growth, prevents osteolysis, and increases animal survival.

PURPOSE: Osteosarcoma and Ewing's sarcoma are high-grade neoplasms typically arising in the bones of children and adolescents. Despite improvement in therapy, the five-year survival rate is only 20% for patients not responding to treatment or presenting with metastases. Among new therapeutic strategies, the efficacy of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily with strong antitumoral activity and minimal toxicity to most normal cells and tissues, was investigated by complementary approaches both in vitro and in preclinical models. EXPERIMENTAL DESIGN: The sensitivity of osteosarcoma and Ewing's sarcoma cell lines to TRAIL was investigated in vitro by determining TRAIL receptor expression together with TRAIL effects on cell viability and apoptosis. Complementary preclinical studies were carried out in respective tumor models by inoculation of osteosarcoma or Ewing's sarcoma tumor cells in paraosseous location. In addition, a model of lung nodule dissemination was developed by i.v. injection of osteosarcoma cells. RESULTS: In vitro, both osteosarcoma and Ewing's sarcoma cells that express the TRAIL death receptors were highly sensitive to TRAIL-induced caspase-8-mediated apoptosis. TRAIL administered in vivo by nonviral gene therapy inhibited primary bone tumor incidence and growth by 87% and prevented tumor-induced osteolysis, leading to a significant 2-fold increase in animal survival 40 days after tumor induction. Furthermore, TRAIL inhibited tumor nodule dissemination in lungs and increased survival in an osteosarcoma model. CONCLUSION: These findings suggest that TRAIL is a promising candidate for the development of new therapeutic strategies in the most frequent malignant primary bone tumors.

Zoledronic acid as a new adjuvant therapeutic strategy for Ewing's sarcoma patients.

Ewing's sarcoma (ES) is the second most frequent pediatric bone tumor also arising in soft tissues (15% of cases). The prognosis of patients with clinically detectable metastases at diagnosis, not responding to therapy or with disease relapse, is still very poor. Among new therapeutic approaches, bisphosphonates represent promising adjuvant molecules to chemotherapy to limit the osteolytic component of bone tumors and to protect from bone metastases. The combined effects of zoledronic acid and mafosfamide were investigated on cell proliferation, viability, apoptosis, and cell cycle distribution of human ES cell lines differing in their p53 and p16/ink4 status. ES models were developed to reproduce both soft tissue and intraosseous tumor development. Mice were treated with 100 µg/kg zoledronic acid (two or four times per week) and/or ifosfamide (30 mg/kg, one to three cycles of three injections). ES cell lines showed different sensitivities to zoledronic acid and mafosfamide at the cell proliferation level, with no correlation with their molecular status. Both drugs induced cell cycle arrest, but in the S or G(2)M phase, respectively. In vivo, zoledronic acid had no effect on soft tissue tumor progression, although it dramatically inhibited ES development in bone. When combined with ifosfamide, zoledronic acid exerted synergistic effects in the soft tissue model: Its combination with one cycle of ifosfamide resulted in an inhibitory effect similar to three cycles of ifosfamide alone. This very promising result could allow clinicians to diminish the doses of chemotherapy.

Glycosaminoglycans as potential regulators of osteoprotegerin therapeutic activity in osteosarcoma.

Osteosarcoma is the most frequent primary bone malignant tumor that develops mainly in children and adolescents. Despite recent improvements in chemotherapy and surgery, survival rate is approximately 50% after 5 years. Osteoprotegerin (OPG) is a potent inhibitor of osteoclast differentiation and activation, but its use as therapeutic agent in cancer-associated osteolysis remains controversial due to its ability to bind and inhibit the apoptotic effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on tumor cells. The therapeutic effects of full-length OPG (1-401) and OPG 1-194 lacking its heparin-binding domain delivered by nonviral gene therapy were compared in a murine model of osteolytic osteosarcoma. Tumor incidence, progression, and associated bone lesions were significantly diminished in the OPG 1-194 group, but not in the OPG 1-401 group, compared with controls. As receptor activator of nuclear factor-kappaB ligand (RANKL), TRAIL, and glycosaminoglycans (GAG) were shown to be overexpressed in osteosarcoma environment compared with control tissue, OPG 1-401 bioactivity may be modulated by one of these protagonists. Surface plasmon resonance analyses performed with OPG, TRAIL, and GAGs revealed that TRAIL binds both forms of OPG with the same affinity. In addition, as OPG 1-194 and OPG 1-401 similarly inhibit TRAIL-induced apoptosis, it suggests that TRAIL is not involved in the modulation of OPG bioactivity. However, as GAGs inhibit OPG 1-401 but not OPG 1-194 binding to TRAIL or to RANKL, they may represent potent regulators of OPG availability and antitumor activity in bone tumor microenvironment.