Using electron microscopy to localize rare cellular events or structures in complex tissue is challenging. Correlative light and electron microscopy procedures have been developed to link fluorescent ...protein expression with ultrastructural resolution. Here, we present an optimized scanning electron microscopy (SEM) workflow for volumetric array tomography for asymmetric samples and model organisms (
,
,
). We modified a diamond knife to simplify serial section array acquisition with minimal artifacts. After array acquisition, the arrays were transferred to a glass coverslip or silicon wafer support. Using light microscopy, the arrays were screened rapidly for initial recognition of global anatomical features (organs or body traits). Then, using SEM, an in-depth study of the cells and/or organs of interest was performed. Our manual and automatic data acquisition strategies make 3D data acquisition and correlation simpler and more precise than alternative methods. This method can be used to address questions in cell and developmental biology that require the efficient identification of a labeled cell or organelle.
Inhibitor of Growth 3 (ING3) is a candidate tumor suppressor gene whose expression is lost in tumors such as hepatocellular carcinoma, head and neck squamous cell carcinoma and melanoma. In the ...present study, we show that ING3-depleted human cells and yeast cells deleted for its ortholog YNG2 are sensitive to DNA damage suggesting a conserved role in response to such stress. In human cells, ING3 is recruited to DNA double strand breaks and is required for ATM activation. Remarkably, in response to doxorubicin, ATM activation is dependent on ING3 but not on TIP60, whose recruitment to DNA breaks also depends on ING3. These events lead to ATM-mediated phosphorylation of NBS1 and the subsequent recruitment of RNF8, RNF168, 53BP1, and BRCA1, which are major mediators of the DNA damage response. Accordingly, upon genotoxic stress, DNA repair by non-homologous end joining (NHEJ) or homologous recombination (HR) were impaired in absence of ING3. Finally, immunoglobulin class switch recombination (CSR), a physiological mechanism requiring NHEJ repair, was impaired in the absence of ING3. Since deregulation of DNA double strand break repair is associated with genomic instability, we propose a novel function of ING3 as a caretaker tumor suppressor involved in the DNA damage signaling and repair.
BackgroundMembers of the tumor necrosis factor receptor superfamily (TNFRSF) are key co-stimulators of T cells. CD27, a member of the TNFRSF, is expressed only on the surface of lymphocytes, ...including naive and activated CD4+ and CD8+ T cells as well as NK cells. It enhances T cell activation, proliferation, and differentiation of effector and memory T cells after stimulation with its ligand, CD70. The costimulatory signal of CD27 is mediated via the NFkB pathway but also via the phosphatidylinositol 3 kinase and the protein kinase B pathways. CD27 signaling also influences the innate immune response via direct activation of NK cells and subsequent secretion of interferon-gamma (IFNg). Several published preclinical studies demonstrated that anti-CD27 agonistic monoclonal antibodies can promote T-cell activation and antitumor immunity making CD27 an attractive cancer immunotherapy target.MethodsHere we describe the characterization, preclinical development, and selection of our anti-CD27 fully human monoclonal antibody (mAb) lead candidate. We selected this candidate from a library of 147 anti-CD27 mAbs generated after immunization of humanized Trianni® mice with soluble human CD27 extracellular domain (hCD27-ECD).ResultsAnti-CD27 mAbs were tested in an accelerated stability study and showed excellent stability parameters for up to 7 days at 4 and 37°C in commonly used formulation buffers. The selected agonist anti-CD27 mAb demonstrated high affinity binding to both human and cynomolgus monkey CD27 and not to mouse CD27. It also demonstrated high specificity against CD27 with no cross-reactivity detected against other members of the TNFRSF. This lead candidate did not block the binding of CD27 natural ligand, CD70 and induced strong NFkB-mediated CD27 signaling in the absence or presence of cross-linking by Fc gamma receptors or secondary cross-linking antibodies. Moreover, this anti-CD27 mAb mediated NFkB activation is significantly potentiated by the addition of a sub-optimal amount of soluble CD70. The anti-CD27 lead mAb induced T cell proliferation and secretion of pro-inflammatory cytokines only in the presence of sub-optimal TCR stimulation in vitro using primary human T cells. It also activated NK cells demonstrated by CD69 expression induction. The anti-CD27 mAb lead candidate showed extended serum half-life in hCD27-KI mice. It also demonstrated a significant antitumor effect as a single agent in human CD27-Knockin mice (hCD27-KI) subcutaneously implanted with MC38 or in NOD-SCID mice subcutaneously implanted with Raji.ConclusionsThese preclinical results establish that the selected anti-CD27 mAb is a promising drug candidate and we are actively pursuing its development.
Liquid biopsies are increasingly used for the diagnosis and follow-up of cancer patients. Urine is a body fluid that can be used to detect cancers and others diseases. It is noninvasive and easy to ...collect. To detect Bladder Cancer (BC), cytology is the first assay used. It is an effective way to detect high grade BC but has a high rate of equivocal results, especially for low grade BC. Furthermore, cystoscopy is used to confirm cytology results and to determine cancer status. Cystoscopy is also effective but highly invasive, and not well accepted by patients, especially for BC follow-up. In this review we survey the numerous assays recently developed in order to diagnose BC at an early stage, and to facilitate the follow-up of patients. We discuss their effectiveness, ease of use, and applications. Finally, we discuss assays that, in the future, could improve the diagnosis and management of BC patients.
ING2 (Inhibitor of Growth 2) is a tumor suppressor gene that has been implicated in critical biological functions (cell-cycle regulation, replicative senescence, DNA repair and DNA replication), most ...of which are recognized hallmarks of tumorigenesis occurring in the cell nucleus. As its close homolog ING1 has been recently observed in the mitochondrial compartment, we hypothesized that ING2 could also translocate into the mitochondria and be involved in new biological functions. In the present study, we demonstrate that ING2 is imported in the inner mitochondrial fraction in a redox-sensitive manner in human cells and that this mechanism is modulated by 14-3-3η protein expression. Remarkably, ING2 is necessary to maintain mitochondrial ultrastructure integrity without interfering with mitochondrial networks or polarization. We observed an interaction between ING2 and mtDNA under basal conditions. This interaction appears to be mediated by TFAM, a critical regulator of mtDNA integrity. The loss of mitochondrial ING2 does not impair mtDNA repair, replication or transcription but leads to a decrease in mitochondrial ROS production, suggesting a detrimental impact on OXPHOS activity. We finally show using multiple models that ING2 is involved in mitochondrial respiration and that its loss confers a protection against mitochondrial respiratory chain inhibition in vitro. Consequently, we propose a new tumor suppressor role for ING2 protein in the mitochondria as a metabolic shift gatekeeper during tumorigenesis.
Lung cancer is one of the most common and deadliest cancers. Preclinical models are essential to study new therapies and combinations taking tumor genetics into account. We have established cell ...lines expressing the luciferase gene from lines with varied genetic backgrounds, commonly encountered in patients with pulmonary adenocarcinoma. We have characterized these lines by testing their response to multiple drugs. Thus, we have developed orthotopic preclinical mouse models of NSCLC with very high engraftment efficiency. These models allow the easy monitoring of tumor growth, particularly in response to treatment, and of tumor cells dissemination in the body. We show that concomitant treatment with osimertinib (3rd generation tyrosine kinase inhibitor targeting mutated EGFR) and bevacizumab (anti-angiogenic targeting VEGF) can have a beneficial therapeutic effect on EGFR-mutated tumors. We also show that the addition of afatinib to osimertinib-treated tumors in escape leads to tumor growth inhibition. No such effect is observed with selumetinib or simvastatin. These preclinical mouse models therefore make it possible to test innovative therapeutic combinations and are also a tool of choice for studying resistance mechanisms.
The catalytically inactive caspase-8-homologous protein, c-FLIP, is a potent antiapoptotic protein highly expressed in various types of cancers. c-FLIP competes with caspase-8 for binding to the ...adaptor protein FADD (Fas-Associated Death Domain) following death receptors' (DRs) activation via the ligands of the TNF-R family. As a consequence, the extrinsic apoptotic signaling pathway involving DRs is inhibited. The inhibition of c-FLIP activity in tumor cells might enhance DR-mediated apoptosis and overcome immune and anticancer drug resistance. Based on an in silico approach, the aim of this work was to identify new small inhibitory molecules able to bind selectively to c-FLIP and block its anti-apoptotic activity. Using a homology 3D model of c-FLIP, an in silico screening of 1880 compounds from the NCI database (National Cancer Institute) was performed. Nine molecules were selected for in vitro assays, based on their binding affinity to c-FLIP and their high selectivity compared to caspase-8. These molecules selectively bind to the Death Effector Domain 2 (DED2) of c-FLIP. We have tested in vitro the inhibitory effect of these nine molecules using the human lung cancer cell line H1703, overexpressing c-FLIP. Our results showed that six of these newly identified compounds efficiently prevent FADD/c-FLIP interactions in a molecular pull-down assay, as well as in a DISC immunoprecipitation assay. The overexpression of c-FLIP in H1703 prevents TRAIL-mediated apoptosis; however, a combination of TRAIL with these selected molecules significantly restored TRAIL-induced cell death by rescuing caspase cleavage and activation. Altogether, our findings indicate that new inhibitory chemical molecules efficiently prevent c-FLIP recruitment into the DISC complex, thus restoring the caspase-8-dependent apoptotic cascade. These results pave the way to design new c-FLIP inhibitory molecules that may serve as anticancer agents in tumors overexpressing c-FLIP.
Immune checkpoint therapies have led to significant breakthroughs in cancer patient treatment in recent years. However, their efficiency is variable, and resistance to immunotherapies is common. ...VISTA is an immune-suppressive checkpoint inhibitor of T cell response belonging to the B7 family and a promising novel therapeutic target. VISTA is expressed in the immuno-suppressive tumor microenvironment, primarily by myeloid lineage cells, and its genetic knockout or antibody blockade restores an efficient antitumor immune response.
Fully human monoclonal antibodies directed against VISTA were produced after immunizing humanized Trianni mice and single B cell sequencing. Anti-VISTA antibodies were evaluated for specificity, cross-reactivity, monocyte and T cell activation, Fc-effector functions, and antitumor efficacy using
and
models to select the KVA12123 antibody lead candidate. The pharmacokinetics and safety profiles of KVA12123 were evaluated in cynomolgus monkeys.
Here, we report the development of a clinical candidate anti-VISTA monoclonal antibody, KVA12123. KVA12123 showed high affinity binding to VISTA through a unique epitope distinct from other clinical-stage anti-VISTA monoclonal antibodies. This clinical candidate demonstrated high specificity against VISTA with no cross-reactivity detected against other members of the B7 family. KVA12123 blocked VISTA binding to its binding partners. KVA12123 induced T cell activation and demonstrated NK-mediated monocyte activation. KVA12123 treatment mediated strong single-agent antitumor activity in several syngeneic tumor models and showed enhanced efficacy in combination with anti-PD-1 treatment. This clinical candidate was engineered to improve its pharmacokinetic characteristics and reduce Fc-effector functions. It was well-tolerated in preclinical toxicology studies in cynomolgus monkeys, where hematology, clinical chemistry evaluations, and clinical observations revealed no indicators of toxicity. No cytokines associated with cytokine release syndrome were elevated.
These results establish that KVA12123 is a promising drug candidate with a distinct but complementary mechanism of action of the first generation of immune checkpoint inhibitors. This antibody is currently evaluated alone and in combination with pembrolizumab in a Phase 1/2 open-label clinical trial in patients with advanced solid tumors.
Osimertinib is a third generation tyrosine kinase inhibitor (TKI) that targets the epidermal growth factor receptor (EGFR) in lung cancer. However, although this molecule is not subject to some of ...the resistance mechanisms observed in response to first generation TKIs, ultimately, patients relapse because of unknown resistance mechanisms. New relevant non-small cell lung cancer (NSCLC) mice models are therefore required to allow the analysis of these resistance mechanisms and to evaluate the efficacy of new therapeutic strategies.
Briefly, PC-9 cells, previously modified for luciferase expression, were injected into the tail vein of mice. Tumor implantation and longitudinal growth, almost exclusively localized in the lung, were evaluated by bioluminescence. Once established, the tumor was treated with osimertinib until tumor escape and development of bone metastases.
Micro-metastases were detected by bioluminescence and collected for further analysis.
We describe an orthotopic model of NSCLC protocol that led to lung primary tumor nesting and, after osimertinib treatment, by metastases dissemination, and that allow the isolation of these small osimertinib-resistant micro-metastases. This model provides new biological tools to study tumor progression from the establishment of a lung tumor to the generation of drug-resistant micro-metastases, mimicking the natural course of the disease in human NSCLC patients.
BackgroundMultiple cancer immunotherapies of T-cell checkpoint inhibitors have been approved worldwide. However, most patients with advanced solid tumors do not benefit, or relapse after T-cell ...checkpoint blockade. Myeloid checkpoint inhibition is a new approach to cancer immunotherapy. V-domain Ig Suppressor of T-cell Activation (VISTA), a promising therapeutic target, is an immune checkpoint primarily expressed by myeloid cells. Its expression in tumors is associated with high myeloid infiltration. Its presence is clearly associated with an immunosuppressive tumor microenvironment, and it may contribute to resistance to anti-CTLA-4 and anti-PD-(L)1 therapies.KVA12123, an engineered IgG1 fully human monoclonal antibody, specifically binds to VISTA with high affinity and blocks VISTA interactions with its ligands. In vitro, KVA12123 increases pro-inflammatory responses and enhances antigen presenting cell phenotypes. In vivo, it exhibits strong single agent anti-tumor activity amplified in combination with anti-PD1 in multiple tumor models. Toxicology studies performed in cynomolgus monkeys at doses up to 100 mg/kg administered intravenously once weekly for a total of 4 doses showed that KVA12123 was well-tolerated at all dose levels.MethodsThe no-observed-adverse-effect-level (NOAEL) of 100 mg/kg in NHP provides a safety margin for KVA12123 administration to humans at a starting dose of 3 mg, which was derived using receptor occupancy and a MABEL (minimum anticipated biological effect level) approach. This trial is a first-in-human, Phase 1/2, multicenter, open-label, dose-escalation, safety, pharmacokinetic (PK), and pharmacodynamic evaluation of intravenously administered KVA12123, both as monotherapy and in combination with pembrolizumab, in adult patients with solid tumors that have failed standard of care therapies (NCT05708950). Up to 60 patients will be enrolled in the Phase 1 of the study. KVA12123 is administered every 2 weeks at escalating doses over a 6-week cycle and pembrolizumab every 6 weeks at a 400mg fixed dose in the combination arm. The dose-limiting toxicity (DLT) evaluation period is 21 days.ResultsSafety, PK, clinical activity using iRECIST and exploratory biomarkers are evaluated in this trial. Preliminary results will be presented. The primary objective of the study is to assess safety and tolerability at increasing dose levels of KVA12123 in successive cohorts of adult patients (alone and in combination with pembrolizumab) with advanced relapsed or refractory solid tumors, to estimate the maximal tolerated dose (MTD) or select the recommended phase 2 doses (RP2Ds). Secondary objectives include characterizing PK, immunogenicity, and preliminary anti-tumor activity. Biomarker evaluation includes receptor occupancy, changes in immune cell markers and VISTA expression on collected biopsies.Trial RegistrationNCT05708950Ethics ApprovalInstitutional review boards’ approval is requested and obtained from each of the 10 clinical sites engaged in this Phase 1/2 clinical trial. Informed consents are collected for every patient enrolled in the trial
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