Phenotypic comparison and the potential antitumor function of immortalized bone marrow-derived macrophages (iBMDMs).

Introduction: Macrophages are an important component of innate immunity and involved in the immune regulation of multiple diseases. The functional diversity and plasticity make macrophages to exhibit different polarization phenotypes after different stimuli. During tumor progression, the M2-like polarized tumor-associated macrophages (TAMs) promote tumor progression by assisting immune escape, facilitating tumor cell metastasis, and switching tumor angiogenesis. Our previous studies demonstrated that functional remodeling of TAMs through engineered-modifying or gene-editing provides the potential immunotherapy for tumor. However, lack of proliferation capacity and maintained immune memory of infused macrophages restricts the application of macrophage-based therapeutic strategies in the repressive tumor immune microenvironment (TIME). Although J2 retrovirus infection enabled immortalization of bone marrow-derived macrophages (iBMDMs) and facilitated the mechanisms exploration and application, little is known about the phenotypic and functional differences among multi kinds of macrophages. Methods: HE staining was used to detect the biosafety of iBMDMs, and real-time quantitative PCR, immunofluorescence staining, and ELISA were used to detect the polarization response and expression of chemokines in iBMDMs. Flow cytometry, scratch assay, real-time quantitative PCR, and crystal violet staining were used to analyze its phagocytic function, as well as its impact on tumor cell migration, proliferation, and apoptosis. Not only that, the inhibitory effect of iBMDMs on tumor growth was detected through subcutaneous tumor loading, while the tumor tissue was paraffin sectioned and flow cytometry was used to detect its impact on the tumor microenvironment. Results: In this study, we demonstrated iBMDMs exhibited the features of rapid proliferation and long-term survival. We also compared iBMDMs with RAW264.7 cell line and mouse primary BMDMs with in vitro and in vivo experiments, indicating that the iBMDMs could undergo the same polarization response as normal macrophages with no obvious cellular morphology changes after polarization. What's more, iBMDMs owned stronger phagocytosis and pro-apoptosis functions on tumor cells. In addition, M1-polarized iBMDMs could maintain the anti-tumor phenotypes and domesticated the recruited macrophages of receptor mice, which further improved the TIME and repressed tumor growth. Discussion: iBMDMs can serve as a good object for the function and mechanism study of macrophages and the optional source of macrophage immunotherapy.

[Clinical Characteristics of Children with Hemophagocytic Syndrome with Different EB Virus DNA Loads].

OBJECTIVE: To analyze the clinical characteristics of hemophagocytic syndrome (HLH) children with different EB virus (EBV) DNA loads, and to explore the relationship between differential indicators and prognosis. METHODS: Clinical data of 73 children with HLH treated in our hospital from January 2015 to April 2022 were collected. According to EBV DNA loads, the children were divided into negative group (≤5×102 copies/ml), low load group (>5×102-<5×105 copies/ml) and high load group (≥5×105copies/ml）. The clinical symptoms and laboratory indexes of the three groups were compared, and the ROC curve was used to determine the best cut-off value of the different indexes. Cox regression model was used to analyze the independent risk factors affecting the prognosis of children, and to analyze the survival of children in each group. RESULTS: The proportion of female children, the swelling rate of liver and spleen lymph nodes and the involvement rate of blood, liver, circulation and central nervous system in the high load group were higher than those in the negative group. The incidence of disseminated intravascular coagulation(DIC) and central nervous system(CNS) involvement in the high load group were higher than those in the low load group. The liver swelling rate and circulatory system involvement rate in the low load group were higher than those in the negative group(P<0.05). PLT counts in the high load group were significantly lower than those in the negative group, and the levels of GGT, TBIL, CK-MB, LDH, TG, SF, and organ involvement were significantly higher than those in the negative group. The levels of CK, LDH, SF and the number of organ involvement in the high load group were significantly higher than those in the low load group. The levels of GGT and TBIL in low load group were significantly higher than those in negative group. In terms of treatment, the proportion of blood purification therapy in the high and low load group was significantly higher than that in the negative group(P<0.01). ROC curve analysis showed that the best cut-off values of PLT, LDH, TG and SF were 49.5, 1139, 3.12 and 1812, respectively. The appellate laboratory indicators were dichotomized according to the cut-off value, and the differential clinical symptoms were included in the Cox regression model. Univariate analysis showed that LDH>1139 U/L, SF>1812 µg/L, dysfunction of central nervous system, number of organ damage, DIC and no blood purification therapy were the risk factors affecting the prognosis of children (P<0.05); Multivariate analysis shows that PLT≤49.5×109/L and dysfunction of central nervous system were risk factors affecting the prognosis of children (P<0.05). Survival analysis showed that there was no significant difference in the survival rate among the three groups. CONCLUSION: The incidence of adverse prognostic factors in children with HLH in the EBV-DNA high load group is higher, and there is no significant difference in the survival rate of the three groups after blood purification therapy. Therefore, early identification and application of blood purification therapy is of great significance for children with HLH in the high load group.

Temozolomide Treatment Induces HMGB1 to Promote the Formation of Glioma Stem Cells via the TLR2/NEAT1/Wnt Pathway in Glioblastoma.

Formation of glioma stem cells (GSCs) is considered as one of the main reasons of temozolomide (TMZ) resistance in glioma patients. Recent studies have shown that tumor microenvironment-derived signals could promote GSCs formation. But the critical molecule and underlying mechanism for GSCs formation after TMZ treatment is not entirely identified. Our study showed that TMZ treatment promoted GSCs formation by glioma cells; TMZ treatment of biopsy-derived glioblastoma multiforme cells upregulated HMGB1; HMGB1 altered gene expression profile of glioma cells with respect to mRNA, lncRNA and miRNA. Furthermore, our results showed that TMZ-induced HMGB1 increased the formation of GSCs and when HMGB1 was downregulated, TMZ-mediated GSCs formation was attenuated. Finally, we showed that the effect of HMGB1 on glioma cells was mediated by TLR2, which activated Wnt/ß-catenin signaling to promote GSCs. Mechanistically, we found that HMGB1 upregulated NEAT1, which was responsible for Wnt/ß-catenin activation. In conclusion, TMZ treatment upregulates HMGB1, which promotes the formation of GSCs via the TLR2/NEAT1/Wnt pathway. Blocking HMGB1-mediated GSCs formation could serve as a potential therapeutic target for preventing TMZ resistance in GBM patients.

Adenovirus infection promotes the formation of glioma stem cells from glioblastoma cells through the TLR9/NEAT1/STAT3 pathway.

BACKGROUND: Glioma stem cells (GSCs) are glioma cells with stemness and are responsible for a variety of malignant behaviors of glioma. Evidence has shown that signals from tumor microenvironment (TME) enhance stemness of glioma cells. However, identification of the signaling molecules and underlying mechanisms has not been completely elucidated. METHODS: Human samples and glioma cell lines were cultured in vitro to determine the effects of adenovirus (ADV) infection by sphere formation, RT-qPCR, western blotting, FACS and immunofluorescence. For in vivo analysis, mouse intracranial tumor model was applied. Bioinformatics analysis, gene knockdown by siRNA, RT-qPCR and western blotting were applied for further mechanistic studies. RESULTS: Infection of patient-derived glioma cells with ADV increases the formation of tumor spheres. ADV infection upregulated stem cell markers and in turn promoted the capacities of self-renewal and multi-lineage differentiation of the infected tumor spheres. These ADV infected tumor spheres had stronger potential to form xenograft tumors in immune-compromised mice. GSCs formation could be promoted by ADV infection via TLR9, because TLR9 was upregulated after ADV infection, and knockdown of TLR9 reduced ADV-induced GSCs. Consistently, MYD88, as well as total STAT3 and phosphorylated (p-)STAT3, were also upregulated in ADV-induced GSCs. Knockdown of MYD88 or pharmaceutical inhibition of STAT3 attenuated stemness of ADV-induced GSCs. Moreover, we found that ADV infection upregulated lncRNA NEAT1. Knockdown of NEAT1 impaired stemness of ADV-induced GSCs. Lastly, HMGB1, a damage associated molecular pattern (DAMP) that triggers TLR signaling, also upregulated stemness markers in glioma cells. CONCLUSION: ADV, which has been developed as vectors for gene therapy and oncolytic virus, promotes the formation of GSCs via TLR9/NEAT1/STAT3 signaling. Video abstract.

A ketogenic diet attenuates proliferation and stemness of glioma stem­like cells by altering metabolism resulting in increased ROS production.

Abnormal metabolism serves a critical role in the development and progression of different types of malignancies including glioblastoma (GBM), and may therefore serve as a promising target for treatment of cancer. Preclinical studies have indicated that a ketogenic diet (KD) may exhibit beneficial effects in patients with GBM; however, the underlying mechanisms remain incompletely understood. The aim of the present study was to evaluate the effects of a KD on glioma stem­like cells (GSCs), by culturing patient­derived primary GSCs as well as a GSC cell line in glucose­restricted, ß­hydroxybutyrate­containing medium (BHB­Glow) which was used to mimic clinical KD treatment. GSCs cultured in BHB­Glow medium exhibited reduced proliferation and increased apoptosis compared with cells grown in the control medium. Furthermore, decreased expression of stem cell markers, diminished self­renewal in vitro, and reduced tumorigenic capacity in vivo, providing evidence that the stemness of GSCs was compromised. Mechanistically, culturing in BHB­Glow medium reduced glucose uptake and inhibited glycolysis in GSCs. Furthermore, culturing in the BHB­Glow medium resulted in morphological and functional disturbances to the mitochondria of GSCs. These metabolic changes may have reduced ATP production, promoted lactic acid accumulation, and thus, increased the production of reactive oxygen species (ROS) in GSCs. The expression levels and activation of mammalian target of rapamycin, hypoxia­inducible factor 1 and B­cell lymphoma 2 were decreased, consistent with the reduced proliferation of GSCs in BHB­Glow medium. ROS scavenging reversed the inhibitory effects of a KD on GSCs. Taken together, the results demonstrate that treatment with KD inhibited proliferation of GSCs, increased apoptosis and attenuated the stemness in GSCs by increasing ROS production.

Stem cells: a promising candidate to treat neurological disorders.

Neurologic impairments are usually irreversible as a result of limited regeneration in the central nervous system. Therefore, based on the regenerative capacity of stem cells, transplantation therapies of various stem cells have been tested in basic research and preclinical trials, and some have shown great prospects. This manuscript overviews the cellular and molecular characteristics of embryonic stem cells, induced pluripotent stem cells, neural stem cells, retinal stem/progenitor cells, mesenchymal stem/stromal cells, and their derivatives in vivo and in vitro as sources for regenerative therapy. These cells have all been considered as candidates to treat several major neurological disorders and diseases, owing to their self-renewal capacity, multi-directional differentiation, neurotrophic properties, and immune modulation effects. We also review representative basic research and recent clinical trials using stem cells for neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and age-related macular degeneration, as well as traumatic brain injury and glioblastoma. In spite of a few unsuccessful cases, risks of tumorigenicity, and ethical concerns, most results of animal experiments and clinical trials demonstrate efficacious therapeutic effects of stem cells in the treatment of nervous system disease. In summary, these emerging findings in regenerative medicine are likely to contribute to breakthroughs in the treatment of neurological disorders. Thus, stem cells are a promising candidate for the treatment of nervous system diseases.

Expression and purification of mouse Ttyh1 fragments as antigens to generate Ttyh1-specific monoclonal antibodies.

Ttyh1 is a murine homolog of the Drosophila Tweety and is predicted as a five-pass transmembrane protein. The Ttyh1 mRNA is expressed in mouse brain tissues with a restricted pattern and in human glioma cells. Ttyh1 protein may function as a large-conductance chloride channel, however, the role of Ttyh1 in normal neural development and tumorigenesis has been largely unknown, at least partially due to the lack of effective antibodies. Here we report the expression in E. coli and purification of two recombinant Ttyh1 protein fragments corresponding to one of the predicted extracellular domains and the carboxyl terminus of the mouse Ttyh1. With these Ttyh1 protein products, a set of monoclonal antibodies (mAbs) against the mouse Ttyh1 protein was established by using conventional hybridoma techniques. The specificity of the anti-Ttyh1 mAbs was determined based on their activities in Western blotting and immunofluorescent analysis using embryonic brain tissues and cultured mouse neural stem cells (NSCs). We also show that the mouse Ttyh1 protein was expressed in cultured NSCs, most likely in membrane and cytoplasm. In mouse embryonic brains, it appeared that the Ttyh1 protein was specifically expressed in the apical edge of the ventricular zone as puncta-like structures, as determined by using immunofluorescence. Taken together, our study provided a useful tool for further exploration of the biological functions and pathological significance of Ttyh1 in mice.

Inhibition of tumor angiogenesis and tumor growth by the DSL domain of human Delta-like 1 targeted to vascular endothelial cells.

The growth of solid tumors depends on neovascularization. Several therapies targeting tumor angiogenesis have been developed. However, poor response in some tumors and emerging resistance necessitate further investigations of new drug targets. Notch signal pathway plays a pivotal role in vascular development and tumor angiogenesis. Either blockade or forced activation of this pathway can inhibit angiogenesis. As blocking Notch pathway results in the formation of vascular neoplasm, activation of Notch pathway to prevent tumor angiogenesis might be an alternative choice. However, an in vivo deliverable reagent with highly efficient Notch-activating capacity has not been developed. Here, we generated a polypeptide, hD1R, which consists of the Delta-Serrate-Lag-2 fragment of the human Notch ligand Delta-like 1 and an arginine-glycine-aspartate (RGD) motif targeting endothelial cells (ECs). We showed that hD1R could bind to ECs specifically through its RGD motif and effectively triggered Notch signaling in ECs. We demonstrated both in vitro and in vivo that hD1R inhibited angiogenic sprouting and EC proliferation. In tumor-bearing mice, the injection of hD1R effectively repressed tumor growth, most likely through increasing tumor hypoxia and tissue necrosis. The amount and width of vessels reduced remarkably in tumors of mice treated with hD1R. Moreover, vessels in tumors of mice treated with hD1R recruited more NG2(+) perivascular cells and were better perfused. Combined application of hD1R and chemotherapy with cisplatin and teniposide revealed that these two treatments had additive antitumor effects. Our study provided a new strategy for antiangiogenic tumor therapy.

Endothelium-targeted Delta-like 1 promotes hematopoietic stem cell expansion ex vivo and engraftment in hematopoietic tissues in vivo.

BACKGROUND: Notch ligands enhance ex vivo expansion of hematopoietic stem cells (HSCs). But to use Notch ligands in HSC therapies of human diseases, efforts are required to improve ex vivo expansion efficiency and in vivo transplant engraftment. DESIGN AND METHODS: We designed and produced an endothelium-targeted soluble Notch ligand, the DSL domain of Delta-like 1 fused with a RGD motif (D1R), and examined the effects of this protein on HSCs ex vivo and in vivo. RESULTS: D1R efficiently promoted ex vivo expansion of both mouse bone marrow (BM) and human umbilical cord blood HSCs. HSCs expanded with D1R up-regulated many of the stemness-related genes, and showed high BM engraftment efficacy with long-term repopulation capacity after transplantation. Moreover, in vivo administration of D1R increased the number of BM HSCs in mice, and facilitated BM recovery of mice after irradiation. Injection of D1R significantly improved HSC engraftment and myeloid recovery after BM transplantation in irradiated mice. D1R enhanced HSC engraftment not only in BM, but also in the liver and spleen after BM transplantation in mice. D1R induced the formation of compact cell clusters containing the transplanted HSCs in close contact with endothelial cells, reminiscent of HSC niches, in the liver and spleen. CONCLUSIONS: D1R might be applied in improving both HSC expansion ex vivo and HSC engraftment in vivo in transplantation.

Differential regulation of bone marrow-derived endothelial progenitor cells and endothelial outgrowth cells by the Notch signaling pathway.

Endothelial progenitor cells (EPCs) are heterogeneous populations of cells that participate in vasculogenesis and promote tissue regeneration. However the different roles of EPC populations in vasculogenesis and tissue regeneration, as well as their regulation and mechanisms remain elusive. In the present study, we cultured bone marrow (BM)-derived early EPCs (EEPCs) and endothelial outgrowth cells (EOCs), and investigated their roles in liver regeneration and their regulation by the Notch signaling pathway. We found that Notch signaling exhibited different effects on the proliferation and migration of EEPCs and EOCs. Our results also showed that while EEPCs failed to form vessel-like structures in a three dimensional sprouting model in vitro, EOCs could sprout and form endothelial cords, and this was regulated by the Notch signaling. We further showed that, by using a conditional knockout model of RBP-J (the critical transcription factor mediating Notch signaling), Notch signaling differentially regulates EEPCs and EOCs. In a partial hepatectomy (PHx) model, EEPCs Notch-dependently benefitted liver regeneration with respect to liver function and hepatocyte proliferation and apoptosis. In contrast, EOCs appeared not directly involved in the recovery of liver function and the increase of hepatocytes. These data suggested that the RBP-J-mediated Notch signaling differentially regulated the two types of EPCs, which showed different roles in liver regeneration.