Analgesic candidate adenosine A 3 receptors are expressed by perineuronal peripheral macrophages in human dorsal root ganglion and spinal cord microglia.

ABSTRACT: Adenosine receptors are a family of purinergic G protein-coupled receptors that are widely distributed in bodily organs and in the peripheral and central nervous systems. Recently, antihyperalgesic actions have been suggested for the adenosine A 3 receptor, and its agonists have been proposed as new neuropathic pain treatments. We hypothesized that these receptors may be expressed in nociceptive primary afferent neurons. However, RNA sequencing across species, eg, rat, mouse, dog, and human, suggests that dorsal root ganglion (DRG) expression of ADORA3 is inconsistent. In rat and mouse, Adora3 shows very weak to no expression in DRG, whereas it is well expressed in human DRG. However, the cell types in human DRG that express ADORA3 have not been delineated. An examination of DRG cell types using in situ hybridization clearly detected ADORA3 transcripts in peripheral macrophages that are in close apposition to the neuronal perikarya but not in peripheral sensory neurons. By contrast, ADORA1 was found primarily in neurons, where it is broadly expressed at low levels. These results suggest that a more complex or indirect mechanism involving modulation of macrophage and/or microglial cells may underlie the potential analgesic action of adenosine A 3 receptor agonism.

Flow cytometric immunophenotypic differentiation patterns of bone marrow eosinophilopoiesis.

BACKGROUND: Flow cytometry has been widely used to study immunophenotypic patterns of maturation of most hematopoietic lineages in normal human bone marrow aspirates, thus allowing identification of changes in patterns in many myeloid malignancies. Eosinophils play an important role in a wide variety of disorders, including some myeloid neoplasms. However, changes in flow cytometric immunophenotypic patterns during normal and abnormal bone marrow eosinophilopoiesis have not been well studied. METHODS: Fresh bone marrow aspirates from 15 healthy donors, 19 patients with hypereosinophilic syndromes (HES), and 11 patients with systemic mastocytosis (SM) were analyzed for candidate markers that included EMR-1, Siglec-8, CCR3, CD9, CD11a, CD11b, CD11c, CD13, CD16, CD29, CD34, CD38, CD45, CD44, CD49d, CD49f, CD54, CD62L, CD69, CD117, CD125 (IL-5Rα), HLA-DR, using 10 parameter flow cytometry. Putative CD34-negative immature and mature normal eosinophil populations were first identified based on changes in expression of the above markers in healthy donors, then confirmed using fluorescence-based cell sorting and morphological evaluation of cytospin preparations. The normal immunophenotypic patterns were then compared to immunophenotypic patterns of eosinophilopoiesis in patients with HES and SM. RESULTS: The eosinophilic lineage was first verified using the human eosinophil-specific antibody EMR-1 in combination with anti-IL-5Rα antibody. Then, a combination of Siglec-8, CD9, CD11b, CCR3, CD49d, and CD49f antibodies was used to delineate normal eosinophilic maturational patterns. Early stages (eosinophilic promyelocytes/myelocytes) were identified as Siglec-8 dim/CD11b dim to moderate/CD9 dim/CCR3 dim/CD49d bright/CD49f dim, intermediate stages (eosinophilic myelocytes/metamyelocytes) as Siglec-8 moderate/CD11b moderate to bright/CD9 moderate/CCR3 moderate/CD49d moderate/CD49f moderate and mature bands/segmented eosinophils as Siglec-8 bright/CD11b bright/CD9 bright/CCR3 bright/CD49d dim/CD49f bright. Overall maturational patterns were also similar in patients with HES and SM; however, the expression levels of several surface markers were altered compared to normal eosinophils. CONCLUSION: A novel flow cytometric antibody panel was devised to detect alterations in immunophenotypic patterns of bone marrow eosinophil maturation and evaluated in normal, HES and SM samples. This approach will allow us to elucidate changes in immunophenotypic patterns of bone marrow eosinophilopoiesis in other hematological diseases.

Venous-plexus-associated lymphoid hubs support meningeal humoral immunity.

There is increasing interest in how immune cells in the meninges-the membranes that surround the brain and spinal cord-contribute to homeostasis and disease in the central nervous system1,2. The outer layer of the meninges, the dura mater, has recently been described to contain both innate and adaptive immune cells, and functions as a site for B cell development3-6. Here we identify organized lymphoid structures that protect fenestrated vasculature in the dura mater. The most elaborate of these dural-associated lymphoid tissues (DALT) surrounded the rostral-rhinal confluence of the sinuses and included lymphatic vessels. We termed this structure, which interfaces with the skull bone marrow and a comparable venous plexus at the skull base, the rostral-rhinal venolymphatic hub. Immune aggregates were present in DALT during homeostasis and expanded with age or after challenge with systemic or nasal antigens. DALT contain germinal centre B cells and support the generation of somatically mutated, antibody-producing cells in response to a nasal pathogen challenge. Inhibition of lymphocyte entry into the rostral-rhinal hub at the time of nasal viral challenge abrogated the generation of germinal centre B cells and class-switched plasma cells, as did perturbation of B-T cell interactions. These data demonstrate a lymphoid structure around vasculature in the dura mater that can sample antigens and rapidly support humoral immune responses after local pathogen challenge.

Laser Interstitial Thermal Therapy Induces Robust Local Immune Response for Newly Diagnosed Glioblastoma With Long-term Survival and Disease Control.

Laser interstitial thermal therapy (LITT) is a minimally invasive neurosurgical technique used to ablate intra-axial brain tumors. The impact of LITT on the tumor microenvironment is scarcely reported. Nonablative LITT-induced hyperthermia (33-43˚C) increases intra-tumoral mutational burden and neoantigen production, promoting immunogenic cell death. To understand the local immune response post-LITT, we performed longitudinal molecular profiling in a newly diagnosed glioblastoma and conducted a systematic review of anti-tumoral immune responses after LITT. A 51-year-old male presented after a fall with progressive dizziness, ataxia, and worsening headaches with a small, frontal ring-enhancing lesion. After clinical and radiographic progression, the patient underwent stereotactic needle biopsy, confirming an IDH-WT World Health Organization Grade IV Glioblastoma, followed by LITT. The patient was subsequently started on adjuvant temozolomide, and 60 Gy fractionated radiotherapy to the post-LITT tumor volume. After 3 months, surgical debulking was conducted due to perilesional vasogenic edema and cognitive decline, with H&E staining demonstrating perivascular lymphocytic infiltration. Postoperative serial imaging over 3 years showed no evidence of tumor recurrence. The patient is currently alive 9 years after diagnosis. Multiplex immunofluorescence imaging of pre-LITT and post-LITT biopsies showed increased CD8 and activated macrophage infiltration and programmed death ligand 1 expression. This is the first depiction of the in-situ immune response to LITT and the first human clinical presentation of increased CD8 infiltration and programmed death ligand 1 expression in post-LITT tissue. Our findings point to LITT as a treatment approach with the potential for long-term delay of recurrence and improving response to immunotherapy.

Human endogenous retrovirus K contributes to a stem cell niche in glioblastoma.

Human endogenous retroviruses (HERVs) are ancestral viral relics that constitute nearly 8% of the human genome. Although normally silenced, the most recently integrated provirus HERV-K (HML-2) can be reactivated in certain cancers. Here, we report pathological expression of HML-2 in malignant gliomas in both cerebrospinal fluid and tumor tissue that was associated with a cancer stem cell phenotype and poor outcomes. Using single-cell RNA-Seq, we identified glioblastoma cellular populations with elevated HML-2 transcripts in neural progenitor-like cells (NPC-like) that drive cellular plasticity. Using CRISPR interference, we demonstrate that HML-2 critically maintained glioblastoma stemness and tumorigenesis in both glioblastoma neurospheres and intracranial orthotopic murine models. Additionally, we demonstrate that HML-2 critically regulated embryonic stem cell programs in NPC-derived astroglia and altered their 3D cellular morphology by activating the nuclear transcription factor OCT4, which binds to an HML-2-specific long-terminal repeat (LTR5Hs). Moreover, we discovered that some glioblastoma cells formed immature retroviral virions, and inhibiting HML-2 expression with antiretroviral drugs reduced reverse transcriptase activity in the extracellular compartment, tumor viability, and pluripotency. Our results suggest that HML-2 fundamentally contributes to the glioblastoma stem cell niche. Because persistence of glioblastoma stem cells is considered responsible for treatment resistance and recurrence, HML-2 may serve as a unique therapeutic target.

Monocyte-derived IL-6 programs microglia to rebuild damaged brain vasculature.

Cerebrovascular injury (CVI) is a common pathology caused by infections, injury, stroke, neurodegeneration and autoimmune disease. Rapid resolution of a CVI requires a coordinated innate immune response. In the present study, we sought mechanistic insights into how central nervous system-infiltrating monocytes program resident microglia to mediate angiogenesis and cerebrovascular repair after an intracerebral hemorrhage. In the penumbrae of human stroke brain lesions, we identified a subpopulation of microglia that express vascular endothelial growth factor A. These cells, termed 'repair-associated microglia' (RAMs), were also observed in a rodent model of CVI and coexpressed interleukin (IL)-6Ra. Cerebrovascular repair did not occur in IL-6 knockouts or in mice lacking microglial IL-6Ra expression and single-cell transcriptomic analyses revealed faulty RAM programming in the absence of IL-6 signaling. Infiltrating CCR2+ monocytes were the primary source of IL-6 after a CVI and were required to endow microglia with proliferative and proangiogenic properties. Faulty RAM programming in the absence of IL-6 or inflammatory monocytes resulted in poor cerebrovascular repair, neuronal destruction and sustained neurological deficits that were all restored via exogenous IL-6 administration. These data provide a molecular and cellular basis for how monocytes instruct microglia to repair damaged brain vasculature and promote functional recovery after injury.

Proteostasis Modulation in Germline Missense von Hippel Lindau Disease.

PURPOSE: Missense mutated von Hippel Lindau (VHL) protein (pVHL) maintains intrinsic function but undergoes proteasomal degradation and tumor initiation and/or progression in VHL disease. Vorinostat can rescue missense mutated pVHL and arrest tumor growth in preclinical models. We asked whether short-term oral vorinostat could rescue pVHL in central nervous system hemangioblastomas in patients with germline missense VHL. PATIENTS AND METHODS: We administered oral vorinostat to 7 subjects (ages 46.0 ± 14.5 years) and then removed symptomatic hemangioblastomas surgically (ClinicalTrials.gov identifier NCT02108002). RESULTS: Vorinostat was tolerated without serious adverse events by all patients. pVHL expression was elevated in neoplastic stromal cells compared with untreated hemangioblastomas from same patients. We found transcriptional suppression of downstream hypoxia-inducible factor (HIF) effectors. Mechanistically, vorinostat prevented Hsp90 recruitment to mutated pVHL in vitro. The effects of vorinostat on the Hsp90-pVHL interaction, pVHL rescue, and transcriptional repression of downstream HIF effectors was independent of the location of the missense mutation on the VHL locus. We confirmed a neoplastic stromal cell-specific effect in suppression of protumorigenic pathways with single-nucleus transcriptomic profiling. CONCLUSIONS: We found that oral vorinostat treatment in patients with germline missense VHL mutations has a potent biologic effect that warrants further clinical study. These results provide biologic evidence to support the use of proteostasis modulation for the treatment of syndromic solid tumors involving protein misfolding. Proteostasis modulation with vorinostat rescues missense mutated VHL protein. Further clinical trials are needed to demonstrate tumor growth arrest.

Cation leak through the ATP1A3 pump causes spasticity and intellectual disability.

ATP1A3 encodes the α3 subunit of the sodium-potassium ATPase, one of two isoforms responsible for powering electrochemical gradients in neurons. Heterozygous pathogenic ATP1A3 variants produce several distinct neurological syndromes, yet the molecular basis for phenotypic variability is unclear. We report a novel recurrent variant, ATP1A3(NM_152296.5):c.2324C>T; p.(Pro775Leu), in nine individuals associated with the primary clinical features of progressive or non-progressive spasticity and developmental delay/intellectual disability. No patients fulfil diagnostic criteria for ATP1A3-associated syndromes, including alternating hemiplegia of childhood, rapid-onset dystonia-parkinsonism or cerebellar ataxia-areflexia-pes cavus-optic atrophy-sensorineural hearing loss (CAPOS), and none were suspected of having an ATP1A3-related disorder. Uniquely among known ATP1A3 variants, P775L causes leakage of sodium ions and protons into the cell, associated with impaired sodium binding/occlusion kinetics favouring states with fewer bound ions. These phenotypic and electrophysiologic studies demonstrate that ATP1A3:c.2324C>T; p.(Pro775Leu) results in mild ATP1A3-related phenotypes resembling complex hereditary spastic paraplegia or idiopathic spastic cerebral palsy. Cation leak provides a molecular explanation for this genotype-phenotype correlation, adding another mechanism to further explain phenotypic variability and highlighting the importance of biophysical properties beyond ion transport rate in ion transport diseases.

Pituitary adenomas evade apoptosis via noxa deregulation in Cushing's disease.

Sporadic pituitary adenomas occur in over 10% of the population. Hormone-secreting adenomas, including those causing Cushing's disease (CD), cause severe morbidity and early mortality. Mechanistic studies of CD are hindered by a lack of in vitro models and control normal human pituitary glands. Here, we surgically annotate adenomas and adjacent normal glands in 25 of 34 patients. Using single-cell RNA sequencing (RNA-seq) analysis of 27594 cells, we identify CD adenoma transcriptomic signatures compared with adjacent normal cells, with validation by bulk RNA-seq, DNA methylation, qRT-PCR, and immunohistochemistry. CD adenoma cells include a subpopulation of proliferating, terminally differentiated corticotrophs. In CD adenomas, we find recurrent promoter hypomethylation and transcriptional upregulation of PMAIP1 (encoding pro-apoptotic BH3-only bcl-2 protein noxa) but paradoxical noxa downregulation. Using primary CD adenoma cell cultures and a corticotroph-enriched mouse cell line, we find that selective proteasomal inhibition with bortezomib stabilizes noxa and induces apoptosis, indicating its utility as an anti-tumor agent.

Targeting protein arginine methyltransferase 5 sensitizes glioblastoma to trametinib.

Background: The prognosis of glioblastoma (GBM) remains dismal because therapeutic approaches have limited effectiveness. A new targeted treatment using MEK inhibitors, including trametinib, has been proposed to improve GBM therapy. Trametinib had a promising preclinical effect against several cancers, but its adaptive treatment resistance precluded its clinical translation in GBM. Previously, we have demonstrated that protein arginine methyltransferase 5 (PRMT5) is upregulated in GBM and its inhibition promotes apoptosis and senescence in differentiated and stem-like tumor cells, respectively. We tested whether inhibition of PRMT5 can enhance the efficacy of trametinib against GBM. Methods: Patient-derived primary GBM neurospheres (GBMNS) with transient PRMT5 knockdown were treated with trametinib and cell viability, proliferation, cell cycle progression, ELISA, and western blot were analyzed. In vivo, NSG mice were intracranially implanted with PRMT5-intact and -depleted GBMNS, treated with trametinib by daily oral gavage, and observed for tumor progression and mice survival rate. Results: PRMT5 depletion enhanced trametinib-induced cytotoxicity in GBMNS. PRMT5 knockdown significantly decreased trametinib-induced AKT and ERBB3 escape pathways. However, ERBB3 inhibition alone failed to block trametinib-induced AKT activity suggesting that the enhanced antitumor effect imparted by PRMT5 knockdown in trametinib-treated GBMNS resulted from AKT inhibition and not ERBB3 inhibition. In orthotopic murine xenograft models, PRMT5-depletion extended the survival of tumor-bearing mice, and combination with trametinib further increased survival. Conclusion: Combined PRMT5/MEK inhibition synergistically inhibited GBM in animal models and is a promising strategy for GBM therapy.

PET imaging of TSPO expression in immune cells can assess organ-level pathophysiology in high-consequence viral infections.

Ebola virus (EBOV) disease is characterized by lymphopenia, breach in vascular integrity, cytokine storm, and multiorgan failure. The pathophysiology of organ involvement, however, is incompletely understood. Using [18F]-DPA-714 positron emission tomography (PET) imaging targeting the translocator protein (TSPO), an immune cell marker, we sought to characterize the progression of EBOV-associated organ-level pathophysiology in the EBOV Rhesus macaque model. Dynamic [18F]-DPA-714 PET/computed tomography imaging was performed longitudinally at baseline and at multiple time points after EBOV inoculation, and distribution volumes (Vt) were calculated as a measure of peripheral TSPO binding. Using a mixed-effect linear regression model, spleen and lung Vt decreased, while the bone marrow Vt increased over time after infection. No clear trend was found for liver Vt. Multiple plasma cytokines correlated negatively with lung/spleen Vt and positively with bone marrow Vt. Multiplex immunofluorescence staining in spleen and lung sections confirmed organ-level lymphoid and monocytic loss/apoptosis, thus validating the imaging results. Our findings are consistent with EBOV-induced progressive monocytic and lymphocytic depletion in the spleen, rather than immune activation, as well as depletion of alveolar macrophages in the lungs, with inefficient reactive neutrophilic activation. Increased bone marrow Vt, on the other hand, suggests hematopoietic activation in response to systemic immune cell depletion and leukocytosis and could have prognostic relevance. In vivo PET imaging provided better understanding of organ-level pathophysiology during EBOV infection. A similar approach can be used to delineate the pathophysiology of other systemic infections and to evaluate the effectiveness of newly developed treatment and vaccine strategies.

IDH-mutated gliomas promote epileptogenesis through d-2-hydroxyglutarate-dependent mTOR hyperactivation.

BACKGROUND: Uncontrolled seizures in patients with gliomas have a significant impact on quality of life and morbidity, yet the mechanisms through which these tumors cause seizures remain unknown. Here, we hypothesize that the active metabolite d-2-hydroxyglutarate (d-2-HG) produced by the IDH-mutant enzyme leads to metabolic disruptions in surrounding cortical neurons that consequently promote seizures. METHODS: We use a complementary study of in vitro neuron-glial cultures and electrographically sorted human cortical tissue from patients with IDH-mutant gliomas to test this hypothesis. We utilize micro-electrode arrays for in vitro electrophysiological studies in combination with pharmacological manipulations and biochemical studies to better elucidate the impact of d-2-HG on cortical metabolism and neuronal spiking activity. RESULTS: We demonstrate that d-2-HG leads to increased neuronal spiking activity and promotes a distinct metabolic profile in surrounding neurons, evidenced by distinct metabolomic shifts and increased LDHA expression, as well as upregulation of mTOR signaling. The increases in neuronal activity are induced by mTOR activation and reversed with mTOR inhibition. CONCLUSION: Together, our data suggest that metabolic disruptions in the surrounding cortex due to d-2-HG may be a driving event for epileptogenesis in patients with IDH-mutant gliomas.

Identification of circulating CD31+CD45+ cell populations with the potential to differentiate into erythroid cells.

Erythro-myeloid progenitors (EMP) are found in a population of cells expressing CD31 and CD45 markers (CD31+CD45+). A recent study indicated that EMPs persist until adulthood and can be a source of endothelial cells. We identified two sub-populations of EMP cells, CD31lowCD45low and CD31highCD45+, from peripheral blood that can differentiate into cells of erythroid lineage. Our novel findings add to the current knowledge of hematopoietic lineage commitment, and our sequential, dual-step, in vitro culture model provides a platform for the study of the molecular and cellular mechanisms underlying human hematopoiesis and erythroid differentiation.

The Persistent Pain Transcriptome: Identification of Cells and Molecules Activated by Hyperalgesia.

During persistent pain, the dorsal spinal cord responds to painful inputs from the site of injury, but the molecular modulatory processes have not been comprehensively examined. Using transcriptomics and multiplex in situ hybridization, we identified the most highly regulated receptors and signaling molecules in rat dorsal spinal cord in peripheral inflammatory and post-surgical incisional pain models. We examined a time course of the response including acute (2 hours) and longer term (2 day) time points after peripheral injury representing the early onset and instantiation of hyperalgesic processes. From this analysis, we identify a key population of superficial dorsal spinal cord neurons marked by somatotopic upregulation of the opioid neuropeptide precursor prodynorphin, and 2 receptors: the neurokinin 1 receptor, and anaplastic lymphoma kinase. These alterations occur specifically in the glutamatergic subpopulation of superficial dynorphinergic neurons. In addition to specific neuronal gene regulation, both models showed induction of broad transcriptional signatures for tissue remodeling, synaptic rearrangement, and immune signaling defined by complement and interferon induction. These signatures were predominantly induced ipsilateral to tissue injury, implying linkage to primary afferent drive. We present a comprehensive set of gene regulatory events across 2 models that can be targeted for the development of non-opioid analgesics. PERSPECTIVE: The deadly impact of the opioid crisis and the need to replace morphine and other opioids in clinical practice is well recognized. Embedded within this research is an overarching goal of obtaining foundational knowledge from transcriptomics to search for non-opioid analgesic targets. Developing such analgesics would address unmet clinical needs.

Inhibiting protein phosphatase 2A increases the antitumor effect of protein arginine methyltransferase 5 inhibition in models of glioblastoma.

BACKGROUND: Despite multi-model therapy of maximal surgical resection, radiation, chemotherapy, and tumor-treating fields, the median survival of glioblastoma (GBM) patients is less than 15 months. Protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetric dimethylation of arginine residues and is overexpressed in GBM. Inhibition of PRMT5 causes senescence in stem-like GBM tumor cells. LB100, a first-in-class small molecular inhibitor of protein phosphatase 2A (PP2A), can sensitize therapy-resistant tumor cells. Here, we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. METHODS: Patient-derived primary GBM neurospheres (GBMNS), transfected with PRMT5 target-specific siRNA, were treated with LB100 and subjected to in vitro assays including PP2A activity and western blot. The intracranial mouse xenograft model was used to test the in vivo antitumor efficacy of combination treatment. RESULTS: We found that PRMT5 depletion increased PP2A activity in GBMNS. LB100 treatment significantly reduced the viability of PRMT5-depleted GBMNS compared to PRMT5-intact GBMNS. LB100 enhanced G1 cell cycle arrest induced by PRMT5 depletion. Combination therapy also increased the expression of phospho-MLKL. Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100, indicating that necroptosis caused the enhanced cytotoxicity of combination therapy. In the in vivo mouse tumor xenograft model, LB100 treatment combined with transient depletion of PRMT5 significantly decreased tumor size and prolonged survival, while LB100 treatment alone had no survival benefit. CONCLUSION: Overall, combined PRMT5 and PP2A inhibition had significantly greater antitumor effects than PRMT5 inhibition alone.

Regulation of stem cell function and neuronal differentiation by HERV-K via mTOR pathway.

Stem cells are capable of unlimited proliferation but can be induced to form brain cells. Factors that specifically regulate human development are poorly understood. We found that human stem cells expressed high levels of the envelope protein of an endogenized human-specific retrovirus (HERV-K, HML-2) from loci in chromosomes 12 and 19. The envelope protein was expressed on the cell membrane of the stem cells and was critical in maintaining the stemness via interactions with CD98HC, leading to triggering of human-specific signaling pathways involving mammalian target of rapamycin (mTOR) and lysophosphatidylcholine acyltransferase (LPCAT1)-mediated epigenetic changes. Down-regulation or epigenetic silencing of HML-2 env resulted in dissociation of the stem cell colonies and enhanced differentiation along neuronal pathways. Thus HML-2 regulation is critical for human embryonic and neurodevelopment, while it's dysregulation may play a role in tumorigenesis and neurodegeneration.

MerTK inhibition decreases immune suppressive glioblastoma-associated macrophages and neoangiogenesis in glioblastoma microenvironment.

BACKGROUND: Glioblastoma-associated macrophages and microglia (GAMs) are the predominant immune cells in the tumor microenvironment. Activation of MerTK, a receptor tyrosine kinase, polarizes GAMs to an immunosuppressive phenotype, promoting tumor growth. Here, the role of MerTK inhibition in the glioblastoma microenvironment is investigated in vitro and in vivo. METHODS: Effects of MRX-2843 in glioblastoma microenvironment regulation were determined in vitro by cell viability, cytokine array, in vitro tube formation, Western blotting, and wound healing assays. A syngeneic GL261 orthotopic glioblastoma mouse model was used to evaluate the survival benefit of MRX-2843 treatment. Multiplex fluorescent immunohistochemistry was used to evaluate the expression of CD206, an anti-inflammatory marker on GAMs, and angiogenesis in murine brain tumor tissues. RESULTS: MRX-2843 inhibited cell growth and induced apoptosis in human glioblastoma cells and decreased protein expression of phosphorylated MerTK, AKT, and ERK, which are essential for cell survival signaling. Interleukin-8 and C-C motif chemokine ligand 2, the pro-glioma and pro-angiogenic cytokines, were decreased by MRX-2843. Decreased vascular formation and numbers of immunosuppressive (CD206+) GAMs were observed following MRX-2843 treatment in vivo, suggesting that in addition to alleviating immunosuppression, MRX-2843 also inhibits neoangiogenesis in the glioma microenvironment. These results were supported by a prolonged survival in the syngeneic mouse orthotopic GL261 glioblastoma model following MRX-2843 treatment. CONCLUSION: Our findings suggest that MRX-2843 has a therapeutic benefit via promoting GAM polarization away from immunosuppressive condition, inhibiting neoangiogenesis in the glioblastoma microenvironment and inducing tumor cell death.

GL261 luciferase-expressing cells elicit an anti-tumor immune response: an evaluation of murine glioma models.

Preclinical models that reliably recapitulate the immunosuppressive properties of human gliomas are essential to assess immune-based therapies. GL261 murine glioma cells are widely used as a syngeneic animal model of glioma, however, it has become common practice to transfect these cells with luciferase for fluorescent tumor tracking. The aim of this study was to compare the survival of mice injected with fluorescent or non-fluorescent GL261 cells and characterize the differences in their tumor microenvironment. Mice were intracranially implanted with GL261, GL261 Red-FLuc or GL261-Luc2 cells at varying doses. Cytokine profiles were evaluated by proteome microarray and Kaplan-Meier survival analysis was used to determine survival differences. Median survival for mice implanted with 5 × 104 GL261 cells was 18 to 21 days. The GL261 Red-FLuc implanted mice cells did not reach median survival at any tumor dose. Mice injected with 3 × 105 GL261-Luc2 cells reached median survival at 23 days. However, median survival was significantly prolonged to 37 days in mice implanted with 5 × 104 GL261-Luc2 cells. Additionally, proteomic analyses revealed significantly elevated inflammatory cytokines in the supernatants of the GL261 Red-FLuc cells and GL261-Luc2 cells. Our data suggest that GL261 Red-FLuc and GL261-Luc2 murine models elicit an anti-tumor immune response by increasing pro-inflammatory modulators.

Overexpression of fibroblast growth factor-21 (FGF-21) protects mesenchymal stem cells against caspase-dependent apoptosis induced by oxidative stress and inflammation.

The clinical application of stem cells offers great promise as a potential avenue for therapeutic use in neurodegenerative diseases. However, cell loss after transplantation remains a major challenge, which currently plagues the field. On the basis of our previous findings that fibroblast growth factor 21 (FGF-21) protected neurons from glutamate excitotoxicity and that upregulation of FGF-21 in a rat model of ischemic stroke was associated with neuroprotection, we proposed that overexpression of FGF-21 protects bone marrow-derived mesenchymal stem cells (MSCs) from apoptosis. To test this hypothesis, we examined whether the detrimental effects of apoptosis can be mitigated by the transgenic overexpression of FGF-21 in MSCs. FGF-21 was transduced into MSCs by lentivirus and its overexpression was confirmed by quantitative polymerase chain reaction. Moreover, FGF-21 overexpression did not stimulate the expression of other FGF family members, suggesting it does not activate a positive feedback system. The effects of hydrogen peroxide (H2 O2 ), tumor necrosis factor-α (TNF-α), and staurosporine, known inducers of apoptosis, were evaluated in FGF-21 overexpressing MSCs and mCherry control MSCs. Caspases 3 and 7 activity was markedly and dose-dependently increased by all three stimuli in mCherry MSCs. FGF-21 overexpression robustly suppressed caspase activation induced by H2 O2 and TNF-α, but not staurosporine. Moreover, the assessment of apoptotic morphological changes confirmed the protective effects of FGF-21 overexpression. Taken together, these results provide compelling evidence that FGF-21 plays a crucial role in protecting MSCs from apoptosis induced by oxidative stress and inflammation and merits further investigation as a strategy for enhancing the therapeutic efficacy of stem cell-based therapies.