CLEC-1 Restrains Acute Inflammatory Response and Recruitment of Neutrophils following Tissue Injury.

The inflammatory response is a key mechanism for the elimination of injurious agents but must be tightly controlled to prevent additional tissue damage and progression to persistent inflammation. C-type lectin receptors expressed mostly by myeloid cells play a crucial role in the regulation of inflammation by recognizing molecular patterns released by injured tissues. We recently showed that the C-type lectin receptor CLEC-1 is able to recognize necrotic cells. However, its role in the acute inflammatory response following tissue damage had not yet been investigated. We show in this study, in a mouse model of liver injury induced by acetaminophen intoxication, that Clec1a deficiency enhances the acute immune response with increased expression of Il1b, Tnfa, and Cxcl2 and higher infiltration of activated neutrophils into the injured organ. Furthermore, we demonstrate that Clec1a deficiency exacerbates tissue damage via CXCL2-dependent neutrophil infiltration. In contrast, we observed that the lack of CLEC-1 limits CCL2 expression and the accumulation, beyond the peak of injury, of monocyte-derived macrophages. Mechanistically, we found that Clec1a-deficient dendritic cells increase the expression of Il1b, Tnfa, and Cxcl2 in response to necrotic cells, but decrease the expression of Ccl2. Interestingly, treatment with an anti-human CLEC-1 antagonist mAb recapitulates the exacerbation of acute immunopathology observed by genetic loss of Clec1a in a preclinical humanized mouse model. To conclude, our results demonstrate that CLEC-1 is a death receptor limiting the acute inflammatory response following injury and represents a therapeutic target to modulate immunity.

TRAIL-Based Therapies Efficacy in Pediatric Bone Tumors Models Is Modulated by TRAIL Non-Apoptotic Pathway Activation via RIPK1 Recruitment.

Despite advances in clinical management, osteosarcoma and Ewing sarcoma, the two most frequent malignant primary bone tumors at pediatric age, still have a poor prognosis for high-risk patients (i.e., relapsed or metastatic disease). Triggering a TRAIL pro-apoptotic pathway represents a promising therapeutic approach, but previous studies have described resistance mechanisms that could explain the declining interest of such an approach in clinical trials. In this study, eight relevant human cell lines were used to represent the heterogeneity of the response to the TRAIL pro-apoptotic effect in pediatric bone tumors and two cell-derived xenograft models were developed, originating from a sensitive and a resistant cell line. The DR5 agonist antibody AMG655 (Conatumumab) was selected as an example of TRAIL-based therapy. In both TRAIL-sensitive and TRAIL-resistant cell lines, two signaling pathways were activated following AMG655 treatment, the canonical extrinsic apoptotic pathway and a non-apoptotic pathway, involving the recruitment of RIPK1 on the DR5 protein complex, activating both pro-survival and pro-proliferative effectors. However, the resulting balance of these two pathways was different, leading to apoptosis only in sensitive cells. In vivo, AMG655 treatment reduced tumor development of the sensitive model but accelerated tumor growth of the resistant one. We proposed two independent strategies to overcome this issue: (1) a proof-of-concept targeting of RIPK1 by shRNA approach and (2) the use of a novel highly-potent TRAIL-receptor agonist; both shifting the balance in favor of apoptosis. These observations are paving the way to resurrect TRAIL-based therapies in pediatric bone tumors to help predict the response to treatment, and propose a relevant adjuvant strategy for future therapeutic development.

SIRPγ-CD47 Interaction Positively Regulates the Activation of Human T Cells in Situation of Chronic Stimulation.

A numerous number of positive and negative signals via various molecules modulate T-cell activation. Within the various transmembrane proteins, SIRPγ is of interest since it is not expressed in rodents. SIRPγ interaction with CD47 is reevaluated in this study. Indeed, we show that the anti-SIRPγ mAb clone LSB2.20 previously used by others has not been appropriately characterized. We reveal that the anti-SIRPα clone KWAR23 is a Pan anti-SIRP mAb which efficiently blocks SIRPα and SIRPγ interactions with CD47. We show that SIRPγ expression on T cells varies with their differentiation and while being expressed on Tregs, is not implicated in their suppressive functions. SIRPγ spatial reorganization at the immune synapse is independent of its interaction with CD47. In vitro SIRPα-γ/CD47 blockade with KWAR23 impairs IFN-γ secretion by chronically activated T cells. In vivo in a xeno-GvHD model in NSG mice, the SIRPγ/CD47 blockade with the KWAR23 significantly delays the onset of the xeno-GvHD and deeply impairs human chimerism. In conclusion, we have shown that T-cell interaction with CD47 is of importance notably in chronic stimulation.

ICG-001, an Inhibitor of the ß-Catenin and cAMP Response Element-Binding Protein Dependent Gene Transcription, Decreases Proliferation but Enhances Migration of Osteosarcoma Cells.

High-grade osteosarcomas are the most frequent malignant bone tumors in the pediatric population, with 150 patients diagnosed every year in France. Osteosarcomas are associated with low survival rates for high risk patients (metastatic and relapsed diseases). Knowing that the canonical Wnt signaling pathway (Wnt/ß-catenin) plays a complex but a key role in primary and metastatic development of osteosarcoma, the aim of this work was to analyze the effects of ICG-001, a CBP/ß-catenin inhibitor blocking the ß-catenin dependent gene transcription, in three human osteosarcoma cell lines (KHOS, MG63 and 143B). The cell proliferation and migration were first evaluated in vitro after ICG-001 treatment. Secondly, a mouse model of osteosarcoma was used to establish the in vivo biological effect of ICG-001 on osteosarcoma growth and metastatic dissemination. In vitro, ICG-001 treatment strongly inhibits osteosarcoma cell proliferation through a cell cycle blockade in the G0/G1 phase, but surprisingly, increases cell migration of the three cell lines. Moreover, ICG-001 does not modulate tumor growth in the osteosarcoma mouse model but, rather significantly increases the metastatic dissemination to lungs. Taken together, these results highlight, despite an anti-proliferative effect, a deleterious pro-migratory role of ICG-001 in osteosarcoma.

Selective SIRPα blockade reverses tumor T cell exclusion and overcomes cancer immunotherapy resistance.

T cell exclusion causes resistance to cancer immunotherapies via immune checkpoint blockade (ICB). Myeloid cells contribute to resistance by expressing signal regulatory protein-α (SIRPα), an inhibitory membrane receptor that interacts with ubiquitous receptor CD47 to control macrophage phagocytosis in the tumor microenvironment. Although CD47/SIRPα-targeting drugs have been assessed in preclinical models, the therapeutic benefit of selectively blocking SIRPα, and not SIRPγ/CD47, in humans remains unknown. We report a potent synergy between selective SIRPα blockade and ICB in increasing memory T cell responses and reverting exclusion in syngeneic and orthotopic tumor models. Selective SIRPα blockade stimulated tumor nest T cell recruitment by restoring murine and human macrophage chemokine secretion and increased anti-tumor T cell responses by promoting tumor-antigen crosspresentation by dendritic cells. However, nonselective SIRPα/SIRPγ blockade targeting CD47 impaired human T cell activation, proliferation, and endothelial transmigration. Selective SIRPα inhibition opens an attractive avenue to overcoming ICB resistance in patients with elevated myeloid cell infiltration in solid tumors.

In vitro and in vivo discrepancy in inducing apoptosis by mesenchymal stromal cells delivering membrane-bound tumor necrosis factor-related apoptosis inducing ligand in osteosarcoma pre-clinical models.

BACKGROUND: Osteosarcoma (OS) is the most frequent pediatric malignant bone tumor. OS patients have not seen any major therapeutic progress in the last 30 years, in particular in the case of metastatic disease, which requires new therapeutic strategies. The pro-apoptotic cytokine Tumor necrosis factor (TNF)-Related Apoptosis Inducing Ligand (TRAIL) can selectively kill tumor cells while sparing normal cells, making it a promising therapeutic tool in several types of cancer. However, many OS cell lines appear resistant to recombinant human (rh)TRAIL-induced apoptosis. We, therefore, hypothesized that TRAIL presentation at the membrane level of carrier cells might overcome this resistance and trigger apoptosis. METHODS: To address this, human adipose mesenchymal stromal cells (MSCs) transfected in a stable manner to express membrane-bound full-length human TRAIL (mbTRAIL) were co-cultured with several human OS cell lines. RESULTS: This induced apoptosis by cell-to-cell contact even in cell lines initially resistant to rhTRAIL. In contrast, mbTRAIL delivered by MSCs was not able to counteract tumor progression in this OS orthotopic model. DISCUSSION: This was partly due to the fact that MSCs showed a potential to support tumor development. Moreover, the expression of mbTRAIL did not show caspase activation in adjacent tumor cells.

Bone microenvironment has an influence on the histological response of osteosarcoma to chemotherapy: retrospective analysis and preclinical modeling.

Osteosarcoma, the most common malignant primary bone tumor, is currently treated with chemotherapy and surgery. The effectiveness of chemotherapy is evaluated by means of histological analysis of tumor necrosis, known as "the Huvos score". However, 25% of the patients initially considered good responders will relapse. In our practice, strong tissue heterogeneity around the residual viable cells of the osteosarcoma is observed, but this is not taken into account by the Huvos score, as it is only an average. The objective is to determine whether heterogeneity in the osteosarcoma's microenvironment can play a role in the histological response to chemotherapy. Two complementary approaches have been developed: (i) the therapeutic response to several monotherapies (ifosfamide, cisplatin, doxorubicin) has been compared to tumor growth and the necrosis levels in different preclinical syngeneic osteosarcoma models, mimicking various microenvironments by injecting the tumor cells into subcutaneous, intra-muscular paratibial, or intra-osseous sites; (ii) a retrospective analysis was performed on patients' osteoblastic osteosarcoma biopsies. Tissue localization mapping of residual live tumor cell colonies was evaluated for potential correlation with overall survival. The results of the preclinical studies showed a difference in tumor growth depending on the osteosarcoma model, with a higher rate in bone sites compared to subcutaneous tumors. For the therapeutic response, a higher response to doxorubicin was observed in the intra-osseous model compared to the intra-muscular model for tumor growth (P = 0.013) and necrosis (P = 0.007). These data strongly suggest that the microenvironment plays a role in how osteosarcoma responds to chemotherapy. The retrospective analysis showed no significant survival difference between residual cell sites, although the soft tissues may be seen as a potential negative factor.

TRAIL delivered by mesenchymal stromal/stem cells counteracts tumor development in orthotopic Ewing sarcoma models.

Ewing sarcoma (EWS) is the second most frequent pediatric malignant bone tumor. EWS patients have not seen any major therapeutic progress in the last 30 years, in particular in the case of metastatic disease, which requires new therapeutic strategies. The pro-apoptotic cytokine TNF-Related Apoptosis Inducing Ligand (TRAIL) can selectively kill tumor cells while sparing normal cells, making it a promising therapeutic tool in several types of cancer. However, certain EWS cell lines appear resistant to recombinant human (rh) TRAIL-induced apoptosis. We therefore hypothesized that a TRAIL presentation at the surface of the carrier cells might overcome this resistance and trigger apoptosis. For this purpose, human adipose mesenchymal stromal/stem cells (MSC) transfected in a stable manner to express full-length human TRAIL were co-cultured with several human EWS cell lines, inducing apoptosis by cell-to-cell contact even in cell lines initially resistant to rhTRAIL or AMG655, an antibody agonist to the death receptor, DR5. In vivo, TRAIL delivered by MSCs was able to counteract tumor progression in two orthotopic models of Ewing sarcoma, associated with caspase activation, indicating that a cell-based delivery of a potent apoptosis-inducing factor could be relevant in EWS.

L-MTP-PE and zoledronic acid combination in osteosarcoma: preclinical evidence of positive therapeutic combination for clinical transfer.

Osteosarcoma, the most frequent malignant primary bone tumor in pediatric patients is characterized by osteolysis promoting tumor growth. Lung metastasis is the major bad prognosis factor of this disease. Zoledronic Acid (ZA), a potent inhibitor of bone resorption is currently evaluated in phase III randomized studies in Europe for the treatment of osteosarcoma and Ewing sarcoma. The beneficial effect of the liposomal form of Muramyl-TriPeptide-Phosphatidyl Ethanolamine (L-mifamurtide, MEPACT®), an activator of macrophage populations has been demonstrated to eradicate lung metastatic foci in osteosarcoma. The objective of this study was to evaluate the potential therapeutic benefit and the safety of the ZA and L-mifamurtide combination in preclinical models of osteosarcoma, as a prerequisite before translation to patients. The effects of ZA (100 µg/kg) and L-mifamurtide (1 mg/kg) were investigated in vivo in xenogeneic and syngeneic mice models of osteosarcoma, at clinical (tumor proliferation, spontaneous lung metastases development), radiological (bone microarchitecture by microCT analysis), biological and histological levels. No interference between the two drugs could be observed on ZA-induced bone protection and on L-mifamurtide-induced inhibition of lung metastasis development. Unexpectedly, ZA and L-mifamurtide association induced an additional and in some cases synergistic inhibition of primary tumor progression. L-mifamurtide has no effect on tumor proliferation in vitro or in vivo, and macrophage population was not affected at the tumor site whatever the treatment. This study evidenced for the first time a significant inhibition of primary osteosarcoma progression when both drugs are combined. This result constitutes a first proof-of-principle for clinical application in osteosarcoma patients.

TRAIL-based therapy in pediatric bone tumors: how to overcome resistance.

Osteosarcoma and Ewing's sarcoma, the two most frequent malignant primary tumors preferentially arise in children and young adults, and have a poor prognosis. TRAIL represents a promising therapeutic approach for most cancers but in the case of primary bone tumors, osteosarcoma cell lines are highly resistant to this pro-apoptotic cytokine. In addition, another signaling pathway mediating cell proliferation and migration may be even activated in this subset of resistant cells leading to protumoral effect. Therapeutic perspectives are linked to possibility to overcome TRAIL resistance by combining other drugs with TRAIL or death receptors agonistic antibodies. We hypothesized that the bone microenvironment may provide a favorable niche for TRAIL resistance that might be targeted by new resensitizing agents.

Intramyocardial delivery of mesenchymal stem cell-seeded hydrogel preserves cardiac function and attenuates ventricular remodeling after myocardial infarction.

BACKGROUND: To improve the efficacy of bone marrow-derived mesenchymal stem cell (MSC) therapy targeted to infarcted myocardium, we investigated whether a self-setting silanized hydroxypropyl methylcellulose (Si-HPMC) hydrogel seeded with MSC (MSC+hydrogel) could preserve cardiac function and attenuate left ventricular (LV) remodeling during an 8-week follow-up study in a rat model of myocardial infarction (MI). METHODOLOGY/PRINCIPAL FINDING: Si-HPMC hydrogel alone, MSC alone or MSC+hydrogel were injected into the myocardium immediately after coronary artery ligation in female Lewis rats. Animals in the MSC+hydrogel group showed an increase in cardiac function up to 28 days after MI and a mid-term prevention of cardiac function alteration at day 56. Histological analyses indicated that the injection of MSC+hydrogel induced a decrease in MI size and an increase in scar thickness and ultimately limited the transmural extent of MI. These findings show that intramyocardial injection of MSC+hydrogel induced short-term recovery of ventricular function and mid-term attenuation of remodeling after MI. CONCLUSION/SIGNIFICANCE: These beneficial effects may be related to the specific scaffolding properties of the Si-HPMC hydrogel that may provide the ability to support MSC injection and engraftment within myocardium.