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B-lineage acute lymphoblastic leukemia (B-ALL) is one of the most common malignancies in children. Despite advances in treatment, the role of the tumor microenvironment in B-ALL remains poorly understood. Among the key components of the immune microenvironment, macrophages play a critical role in the progression of the disease. However, recent research has suggested that abnormal metabolites may influence the function of macrophages, altering the immune microenvironment and promoting tumor growth. Our previous non-targeted metabolomic detection revealed that the metabolite 1,5-anhydroglucitol (1,5-AG) level in the peripheral blood of children newly diagnosed with B-ALL was significantly elevated. Except for its direct influence on leukemia cells, the effect of 1,5-AG on macrophages is still unclear. Herein, we demonstrated new potential therapeutic targets by focusing on the effect of 1,5-AG on macrophages. We used polarization-induced macrophages to determine how 1,5-AG acted on M1-like polarization and screened out the target gene CXCL14 via transcriptome sequencing. Furthermore, we constructed CXCL14 knocked-down macrophages and a macrophage-leukemia cell coculture model to validate the interaction between macrophages and leukemia cells. We discovered that 1,5-AG upregulated the CXCL14 expression, thereby inhibiting M1-like polarization. CXCL14 knockdown restored the M1-like polarization of macrophages and induced leukemia cells apoptosis in the coculture model. Our findings offer new possibilities for the genetic engineering of human macrophages to rehabilitate their immune activity against B-ALL in cancer immunotherapy., Competing Interests: Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Ovarian granulosa cells (OGCs) play an essential role in the regulation of follicular growth and development. However, previous studies of OGCs have concentrated on traditional 2D cultures. In the present study, we used the hanging drop culture method to culture rat OGCs (rOGCs) and assessed the effects of 3D conditions on their proliferation and gene expression profiles. Compared with those grown in 2D conditions, rOGCs grown in 3D cultures showed a significantly different spatial cell distribution and cell alignment under electron microscopy. In particular, rOGCs in 3D cultures showed abundant rough and microvilli-like structures on their cell surface. Here, we showed that these cells grew slowly following 3D culture; the G0/G1-phase increased and the S- and G2/M-phases decreased. Using whole-transcriptome sequencing analysis, 501 genes were shown to have been significantly upregulated and 502 were shown to have been downregulated. Differentially expressed genes were most enriched in pathways involved in focal adhesion, MAPK, and PI3K/Akt signaling according to Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Western blotting revealed that SPP1 and FGF7 in the PI3K/Akt pathway were significantly upregulated following 3D culture. These findings improve our understanding of OGCs in real 3D environments in vivo and provide possible avenues for future research on OGCs., (© 2023 The Authors. Cell Biology International published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.)

Background: Precursor B-cell acute lymphoblastic leukemia (pre-B ALL) is the most common hematological malignancy in children. Cellular metabolic reorganization is closely related to the progression and treatment of leukemia. We found that the level of 1,5-anhydroglucitol (1,5-AG), which is structurally similar to glucose, was elevated in children with pre-B ALL. However, the effect of 1,5-AG on pre-B ALL was unclear. Here, we aimed to reveal the roles and mechanisms of 1,5-AG in pre-B ALL progression., Methods: The peripheral blood plasma level of children with initial diagnosis of pre-B ALL and that of healthy children was measured using untargeted metabolomic analysis. Cell Counting Kit-8 assay, RNA sequencing, siRNA transfection, real-time quantitative PCR, and western blot were performed using pre-B ALL cell lines Reh and HAL-01. Cell cycle, cell apoptosis, ROS levels, and the positivity rate of CD19 were assessed using flow cytometry. Oxygen consumption rates and extracellular acidification rate were measured using XFe24 Extracellular Flux Analyzer. The lactate and nicotinamide adenine dinucleotide phosphate levels were measured using kits. The effect of 1,5-AG on pre-B ALL progression was verified using the In Vivo Imaging System in a xenotransplantation leukemia model., Results: We confirmed that 1,5-AG promoted the proliferation, viability, and intracellular glycolysis of pre-B ALL cells. Mechanistically, 1,5-AG promotes glycolysis while inhibiting mitochondrial respiration by upregulating pyruvate dehydrogenase kinase 4 (PDK4). Furthermore, high levels of intracellular glycolysis promote pre-B ALL progression by activating the reactive oxygen species (ROS)-dependent mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. Conversely, N-acetylcysteine or vitamin C, an antioxidant, effectively inhibited 1,5-AG-mediated progression of leukemia cells., Conclusions: Our study reveals a previously undiscovered role of 1,5-AG in pre-B ALL, which contributes to an in-depth understanding of anaerobic glycolysis in the progression of pre-B ALL and provides new targets for the clinical treatment of pre-B ALL., (© 2023. The Author(s).)

To increase the potential and effectiveness of three-dimensional (3D) mesenchymal stem cells (MSCs) for clinical applications, this study explored the effects of short cryo-temperature pretreatment on MSC function. Adipose-derived MSCs (A-MSCs) were cultured via the ordinary monolayer method and 3D hanging drop spheroid method. When the cells adhered to the wall or formed a spheroid, they were subjected to hypothermic stress at 4°C for 1 h and then divided into three recovery periods at 37°C, specifically 0, 12, and 24 h. The control group was not subjected to any treatment throughout the study. Monolayer and 3D spheroid A-MSCs were analyzed via RNA sequencing after hypothermic stress at 4°C for 1 h. Subsequently, each group of cells was collected and subjected to phenotype identification via flow cytometry, and mRNA expression was detected via reverse transcription-quantitative polymerase chain reaction analysis. Western blot analysis was performed to analyze the PI3K-AKT signaling pathway in A-MSCs. The effects of A-MSCs on angiogenesis in vivo were examined using a chick chorioallantoic membrane assay. Transwell assays were performed to determine whether the culture supernatant from each group could induce the chemotaxis of human umbilical vein endothelial cells (HUVECs). Three-dimensional spheroid culture did not change the phenotype of A-MSCs. The expression of fibroblast growth factors, hepatocyte growth factors, and other angiogenesis-related factors in A-MSCs was upregulated. A-MSCs subjected to hypothermic stress promoted angiogenesis under both monolayer and 3D spheroid cultures. Moreover, the chemotaxis of HUVECs to the 3D spheroid culture supernatant increased substantially. Short cryo-temperature pretreatment could stimulate 3D spheroid A-MSCs and activate the PI3K-AKT pathway. This approach has the advantages of promoting angiogenesis and maintaining cell viability.

Adipose-derived mesenchymal stem cells (A-MSCs) are promising cellular therapies for the treatment of immune-mediated diseases. Non-gene editing technologies can improve the immune regulatory function of A-MSCs. Our preliminary experiments revealed that an active form of vitamin B6-pyridoxal-5'-phosphate (PLP)-plays an important role in regulating gene expression and cytokine secretion in A-MSCs in vivo . To further clarify the effect of PLP on receptors and cytokines related to the immune regulatory function of A-MSCs, a series of experiments were designed to verify the relationships between PLP and A-MSCs in vitro . Initially, A-MSCs were obtained, and cytokine secretion and the expression of IDO1, NF- κ B, and Toll-like receptors in PLP-stimulated A-MSCs were evaluated. In addition, coculture was used to detect A-MSCs-mediated apoptosis of CD3 + CD8 + T lymphocytes. These results showed that A-MSCs stimulated with PLP were highly proliferative, consistent with their pluripotent capacity. Further, the surface receptors TLR3, TLR4, IDO1, and NF- κ B were upregulated, while TLR6 was downregulated. Concurrently, A-MSCs preconditioned with PLP had the greatest inhibitory effect on CD3 + CD8 + T lymphocyte proliferation, indicating that PLP altered the immune regulatory function of A-MSCs through the regulation of TLRs and IDO1 expression., Competing Interests: The authors report no conflict of interest., (Copyright © 2019 Cong Li et al.)