Starting with mouse splenocytes, the purities of the start and final isolated fractions in the above example are 20.5% and 91.6%, respectively, using a PEconjugated anti-mouse CD4 antibody and EasySep™ Mouse PE Positive Selection Kit II.
Enzymatic Preparation of 2'-5',3'-5'-Cyclic Dinucleotides, Their Binding Properties to Stimulator of Interferon Genes Adaptor Protein, and Structure/Activity Correlations. B. Novotn\'a et al.
Journal of medicinal chemistry 2019 dec
Abstract
Cyclic dinucleotides are second messengers in the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway,which plays an important role in recognizing tumor cells and viral or bacterial infections. They bind to the STING adaptor protein and trigger expression of cytokines via TANK binding kinase 1 (TBK1)/interferon regulatory factor 3 (IRF3) and inhibitor of nuclear factor-$\kappa$B (I$\kappa$B) kinase (IKK)/nuclear factor-$\kappa$B (NF$\kappa$B) signaling cascades. In this work,we describe an enzymatic preparation of 2'-5',3'-5'-cyclic dinucleotides (2'3'CDNs) with use of cyclic GMP-AMP synthases (cGAS) from human,mouse,and chicken. We profile substrate specificity of these enzymes by employing a small library of nucleotide-5'-triphosphate (NTP) analogues and use them to prepare 33 2'3'CDNs. We also determine affinity of these CDNs to five different STING haplotypes in cell-based and biochemical assays and describe properties needed for their optimal activity toward all STING haplotypes. Next,we study their effect on cytokine and chemokine induction by human peripheral blood mononuclear cells (PBMCs) and evaluate their cytotoxic effect on monocytes. Additionally,we report X-ray crystal structures of two new CDNs bound to STING protein and discuss structure-activity relationship by using quantum and molecular mechanical (QM/MM) computational modeling.
B7-H3 Suppresses Antitumor Immunity via the CCL2-CCR2-M2 Macrophage Axis and Contributes to Ovarian Cancer Progression. T. Miyamoto et al.
Cancer immunology research 2022 jan
Abstract
New approaches beyond PD-1/PD-L1 inhibition are required to target the immunologically diverse tumor microenvironment (TME) in high-grade serous ovarian cancer (HGSOC). In this study,we explored the immunosuppressive effect of B7-H3 (CD276) via the CCL2-CCR2-M2 macrophage axis and its potential as a therapeutic target. Transcriptome analysis revealed that B7-H3 is highly expressed in PD-L1-low,nonimmunoreactive HGSOC tumors,and its expression negatively correlated with an IFN$\gamma$ signature,which reflects the tumor immune reactivity. In syngeneic mouse models,B7-H3 (Cd276) knockout (KO) in tumor cells,but not in stromal cells,suppressed tumor progression,with a reduced number of M2 macrophages and an increased number of IFN$\gamma$+CD8+ T cells. CCL2 expression was downregulated in the B7-H3 KO tumor cell lines. Inhibition of the CCL2-CCR2 axis partly negated the effects of B7-H3 suppression on M2 macrophage migration and differentiation,and tumor progression. In patients with HGSOC,B7-H3 expression positively correlated with CCL2 expression and M2 macrophage abundance,and patients with B7-H3-high tumors had fewer tumoral IFN$\gamma$+CD8+ T cells and poorer prognosis than patients with B7-H3-low tumors. Thus,B7-H3 expression in tumor cells contributes to CCL2-CCR2-M2 macrophage axis-mediated immunosuppression and tumor progression. These findings provide new insights into the immunologic TME and could aid the development of new therapeutic approaches against the unfavorable HGSOC phenotype.
Multiple metabolic pathways fuel the truncated tricarboxylic acid cycle of the prostate to sustain constant citrate production and secretion. L. Fr\'egeau-Proulx et al.
Molecular metabolism 2022 aug
Abstract
OBJECTIVE The prostate is metabolically unique: it produces high levels of citrate for secretion via a truncated tricarboxylic acid (TCA) cycle to maintain male fertility. In prostate cancer (PCa),this phenotype is reprogrammed,making it an interesting therapeutic target. However,how the truncated prostate TCA cycle works is still not completely understood. METHODS We optimized targeted metabolomics in mouse and human organoid models in ex vivo primary culture. We then used stable isotope tracer analyses to identify the pathways that fuel citrate synthesis. RESULTS First,mouse and human organoids were shown to recapitulate the unique citrate-secretory program of the prostate,thus representing a novel model that reproduces this unusual metabolic profile. Using stable isotope tracer analysis,several key nutrients were shown to allow the completion of the prostate TCA cycle,revealing a much more complex metabolic profile than originally anticipated. Indeed,along with the known pathway of aspartate replenishing oxaloacetate,glutamine was shown to fuel citrate synthesis through both glutaminolysis and reductive carboxylation in a GLS1-dependent manner. In human organoids,aspartate entered the TCA cycle at the malate entry point,upstream of oxaloacetate. Our results demonstrate that the citrate-secretory phenotype of prostate organoids is supported by the known aspartate-oxaloacetate-citrate pathway,but also by at least three additional pathways: glutaminolysis,reductive carboxylation,and aspartate-malate conversion. CONCLUSIONS Our results add a significant new dimension to the prostate citrate-secretory phenotype,with at least four distinct pathways being involved in citrate synthesis. Better understanding this distinctive citrate metabolic program will have applications in both male fertility as well as in the development of novel targeted anti-metabolic therapies for PCa.
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