As critical regulators of brain homeostasis, microglia are influenced by numerous factors, including sex and genetic mutations. To study the impact of these factors on microglia biology, we employed ...genetically engineered mice that model Neurofibromatosis type 1 (NF1), a disorder characterized by clinically relevant sexually dimorphic differences. While microglia phagocytic activity was reduced in both male and female heterozygous Nf1 mutant (Nf1+/−) mice, purinergic control of phagocytosis was only affected in male Nf1+/− mice. ATP-induced P2Y-mediated membrane currents and P2RY12-dependent laser lesion-induced accumulation of microglial processes were also only impaired in male, but not female Nf1+/−, microglia. These defects resulted from Nf1+/− male-specific defects in cyclic AMP regulation, rather than from changes in purinergic receptor expression. Cyclic AMP elevation by phosphodiesterase blockade restored the male Nf1+/− microglia defects in P2Y-dependent membrane currents and process motility. Taken together, these data establish a sex-by-genotype interaction important to microglia function in the adult mouse brain.
•Neurofibromin 1 knock down (Nf1+/-) impairs microglia phagocytosis•P2RY-mediated membrane currents and lesion-induced process outgrowth are impaired in male but not female Nf1+/- microglia•These defects result from Nf1+/− male-specific defects in cyclic AMP regulation, which are corrected by cyclic AMP elevation•Sex and Nf1 converge to regulate microglia function
Immunotherapy has revolutionized cancer treatment. Despite the recent advances in immunotherapeutic approaches for several tumor entities, limited response has been observed in malignant gliomas, ...including glioblastoma (GBM). Conversely, one of the emerging immunotherapeutic modalities is chimeric antigen receptors (CAR) T cell therapy, which demonstrated promising clinical responses in other solid tumors. Current pre-clinical and interventional clinical studies suggest improved efficacy when CAR-T cells are delivered locoregionally, rather than intravenously. In this review, we summarize possible CAR-T cell administration routes including locoregional therapy, systemic administration with and without focused ultrasound, direct intra-arterial drug delivery and nanoparticle-enhanced delivery in glioma. Moreover, we discuss published as well as ongoing and planned clinical trials involving CAR-T cell therapy in malignant glioma. With increasing neoadjuvant and/or adjuvant combinatorial immunotherapeutic concepts and modalities with specific modes of action for malignant glioma, selection of administration routes becomes increasingly important.
Neurofibromatosis type 1 (NF1) is an autosomal dominant condition caused by germline mutations in the neurofibromin 1 (NF1) gene. Children with NF1 are prone to the development of multiple nervous ...system abnormalities, including autism and brain tumors, which could reflect the effect of NF1 mutation on microglia function. Using heterozygous Nf1-mutant mice, we previously demonstrated that impaired purinergic signaling underlies deficits in microglia process extension and phagocytosis in situ. To determine whether these abnormalities are also observed in human microglia in the setting of NF1, we leveraged an engineered isogenic series of human induced pluripotent stem cells to generate human microglia-like (hiMGL) cells heterozygous for three different NF1 gene mutations found in patients with NF1. Whereas all NF1-mutant and isogenic control hiMGL cells expressed classical microglia markers and exhibited similar transcriptomes and cytokine/chemokine release profiles, only NF1-mutant hiMGL cells had defects in P2X receptor activation, phagocytosis and motility. Taken together, these findings indicate that heterozygous NF1 mutations impair a subset of the functional properties of human microglia, which could contribute to the neurological abnormalities seen in children with NF1.
BackgroundGlioblastoma (GBM) is the most aggressive primary brain tumor with a survival rate of 14–16 months. Several ongoing clinical trials employ vaccine, immune checkpoint inhibitors and/or ...adoptive T cell therapy to treat GBM. However, the impact of irradiation as part of the standard of care on the immune microenvironment and response to immunotherapy is ill-defined.MethodsIn the present study, we employed clinically relevant fractionated irradiation (FIR) at a dose of 4x 2 Gy to investigate the response of experimental orthotopic glioma expressing the tumor-associated antigen glycoprotein (gp)100 in immunocompetent syngeneic mice to a gp100 peptide vaccine as well as to adoptively transferred T cells. We employed single cell RNA sequencing to decipher the post-irradiation changes in the GBM immune microenvironment and elucidate the impact FIR on the efficacy of T cell-mediated immunotherapies.ResultsFIR boosted the response of orthotopically-implanted GL261 tumors to a gp100 vaccine. Furthermore, FIR enhanced the infiltration of GL261-gp100 gliomas by adoptively-transferred gp100 T cell receptor-transgenic T cells, which also displayed a more activated and less exhausted phenotype post-irradiation. Single cell transcriptomic studies of tumor-infiltrating CD45+ leukocytes revealed that FIR promotes the expression of leukocyte-endothelial adhesion molecules in T cells, and transcripts encoding proinflammatory M1-like genes in tumor-associated microglia and macrophages.ConclusionsOur preclinical findings support the use of FIR with to increase response to T cell-based immunotherapies.
BackgroundThe metabolism of tryptophan to kynurenines (KYN) by indoleamine-2,3-dioxygenase or tryptophan-2,3-dioxygenase is a key pathway of constitutive and adaptive tumor immune resistance. The ...immunosuppressive effects of KYN in the tumor microenvironment are predominantly mediated by the aryl hydrocarbon receptor (AhR), a cytosolic transcription factor that broadly suppresses immune cell function. Inhibition of AhR thus offers an antitumor therapy opportunity via restoration of immune system functions.MethodsThe expression of AhR was evaluated in tissue microarrays of head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). A structure class of inhibitors that block AhR activation by exogenous and endogenous ligands was identified, and further optimized, using a cellular screening cascade. The antagonistic properties of the selected AhR inhibitor candidate BAY 2416964 were determined using transactivation assays. Nuclear translocation, target engagement and the effect of BAY 2416964 on agonist-induced AhR activation were assessed in human and mouse cancer cells. The immunostimulatory properties on gene and cytokine expression were examined in human immune cell subsets. The in vivo efficacy of BAY 2416964 was tested in the syngeneic ovalbumin-expressing B16F10 melanoma model in mice. Coculture of human H1299 NSCLC cells, primary peripheral blood mononuclear cells and fibroblasts mimicking the human stromal-tumor microenvironment was used to assess the effects of AhR inhibition on human immune cells. Furthermore, tumor spheroids cocultured with tumor antigen-specific MART-1 T cells were used to study the antigen-specific cytotoxic T cell responses. The data were analyzed statistically using linear models.ResultsAhR expression was observed in tumor cells and tumor-infiltrating immune cells in HNSCC, NSCLC and CRC. BAY 2416964 potently and selectively inhibited AhR activation induced by either exogenous or endogenous AhR ligands. In vitro, BAY 2416964 restored immune cell function in human and mouse cells, and furthermore enhanced antigen-specific cytotoxic T cell responses and killing of tumor spheroids. In vivo, oral application with BAY 2416964 was well tolerated, induced a proinflammatory tumor microenvironment, and demonstrated antitumor efficacy in a syngeneic cancer model in mice.ConclusionsThese findings identify AhR inhibition as a novel therapeutic approach to overcome immune resistance in various types of cancers.
•Tnc controls microglial phagocytosis at postnatal age partially via TLR4.•Tnc-KO microglia exhibit reduced HDAC1 protein levels at postnatal age.•Tnc induces HDAC1 expression in cultured microglia ...in part via TLR4.•Tnc regulates microglial cytokines, chemotaxis, phagocytosis via TLR4 in vitro.
Tenascin C (Tnc) is an extracellular matrix glycoprotein, expressed in the CNS during development, as well as in the setting of inflammation, fibrosis and cancer, which operates as an activator of Toll-like receptor 4 (TLR4). Although TLR4 is highly expressed in microglia, the effect of Tnc on microglia has not been elucidated to date. Herein, we demonstrate that Tnc regulates microglial phagocytic activity at an early postnatal age (P4), and that this process is partially dependent on microglial TLR4 expression. We further show that Tnc regulates proinflammatory cytokine/chemokine production, chemotaxis and phagocytosis in primary microglia in a TLR4-dependent fashion. Moreover, Tnc induces histone-deacetylase 1 (HDAC1) expression in microglia, such that HDAC1 inhibition by MS-275 decreases Tnc-induced microglial IL-6 and TNF-α production. Finally, Tnc−/− cortical microglia have reduced HDAC1 expression levels at P4. Taken together, these findings establish Tnc as a regulator of microglia function during early postnatal development.
Microglial cells are considered as sensors of brain pathology by detecting any sign of brain lesions, infections, or dysfunction and can influence the onset and progression of neurological diseases. ...They are capable of sensing their neuronal environment via many different signaling molecules, such as neurotransmitters, neurohormones and neuropeptides. The neuropeptide VGF has been associated with many metabolic and neurological disorders. TLQP21 is a VGF-derived peptide and has been shown to signal via C3aR1 and C1qBP receptors. The effect of TLQP21 on microglial functions in health or disease is not known. Studying microglial cells in acute brain slices, we found that TLQP21 impaired metabotropic purinergic signaling. Specifically, it attenuated the ATP-induced activation of a K
conductance, the UDP-stimulated phagocytic activity, and the ATP-dependent laser lesion-induced process outgrowth. These impairments were reversed by blocking C1qBP, but not C3aR1 receptors. While microglia in brain slices from male mice lack C3aR1 receptors, both receptors are expressed in primary cultured microglia. In addition to the negative impact on purinergic signaling, we found stimulating effects of TLQP21 in cultured microglia, which were mediated by C3aR1 receptors: it directly evoked membrane currents, stimulated basal phagocytic activity, evoked intracellular Ca
transient elevations, and served as a chemotactic signal. We conclude that TLQP21 has differential effects on microglia depending on C3aR1 activation or C1qBP-dependent attenuation of purinergic signaling. Thus, TLQP21 can modulate the functional phenotype of microglia, which may have an impact on their function in health and disease.
The neuropeptide VGF and its peptides have been associated with many metabolic and neurological disorders. TLQP21 is a VGF-derived peptide that activates C1qBP receptors, which are expressed by microglia. We show here, for the first time, that TLQP21 impairs P2Y-mediated purinergic signaling and related functions. These include modulation of phagocytic activity and responses to injury. As purinergic signaling is central for microglial actions in the brain, this TLQP21-mediated mechanism might regulate microglial activity in health and disease. We furthermore show that, in addition to C1qBP, functional C3aR1 responses contribute to TLQP21 action on microglia. However, C3aR1 responses were only present in primary cultures but not
, suggesting that the expression of these receptors might vary between different microglial activation states.
•Quercetin exerts antiproliferative activity on breast cancer cells.•Quercetin enhances the anticancer effect of Doxorubicin in vitro as proved by in vitro assays and FTIR microspectroscopy.•The ...effect was closely studied using MCF7 cell line and both in vitro tests and FTIR microspectroscopy were carried out.
Breast cancer is the most common type of cancer among females worldwide. Doxorubicin (Dox) is one of the main chemotherapy drugs used in neoadjuvant and adjuvant breast cancer therapy. Dox-induced cardiotoxicity limits its use to a definite period and a definite cumulative dose. On the other hand, quercetin (Quer), a natural antioxidant, has been successfully reported to protect cardiomyocytes from Dox toxicity. Our aim is to optimize Dox regimen by assessing Quer effect on Dox cytotoxicity in a breast cancer cell model, MCF7 using a classic in vitro technique as well as Fourier Transform Infrared (FTIR) microspectroscopy. The cancer cells were exposed to Dox, Quer, or the combination of both agents for 72 incubation hours. Cell proliferation was assessed by using the classical colorimetric Sulforhodamine B (SRB) assay and JC1 dye. MCF7cells were further investigated by FTIR microspectroscopy to correlate SRB results with the changes in the distinctive IR spectra of the cells. Our results show that Quer exerts an antiproliferative effect and enhances the cytotoxicity of Dox in MCF7 cells. Results obtained from both the in vitro assays and FTIR microspectroscopy showed that Quer potentiates the effect of Dox in breast cancer cells.
Breast cancer is the leading cause of cancer death among women and the second in humans worldwide. Many published studies have suggested an association between MDR1 polymorphisms and breast cancer ...risk. Our aim was to study the association between genetic polymorphism of MDR1 at three sites (C3435T, G2677A/T, and C1236T) and their haplotype and the risk of breast cancer in Jordanian females.
A case-control study involving 150 breast cancer cases and 150 controls was conducted. Controls were age-matched to cases. The polymerase chain reaction/restriction fragment length polymorphism (PCR- RFLP) technique and sequencing were performed to analyse genotypes.
The distribution of MDR1 C3435T genotypes differed between cases and controls cases, CC 45.3%, CT 41.3%, and TT 13.3%; controls, CC 13.4%, CT 43.3%, and TT 30.2%, p < 0.001. Similarly, the distribution of G2677A/T significantly differed cases, GG 43.1 %, GT+GA 50.9% and AA+TT 6%; controls, GG 29.6 %, GT+GA 50.9%, and AA+TT 19.4%, p = 0.004. On the other hand, genotype and allelotype distribution of C1236T was not statistically different between cases and controls (p=0.56 and 0.26, respectively). The CGC haplotype increased the risk to breast cancer by 2.5-fold compared to others, while TGC and TTC haplotypes carried 2.5- and 5-fold lower risk of breast cancer, respectively.
Genetic polymorphisms of MDR1 C3435T and G2677A/T, but not C1236T, are associated with increased risk of breast cancer. In addition, CGC, TGC and TTC haplotypes have different impacts on the risk of breast cancer. Future, larger studies are needed to validate these findings.
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