Myelopoiesis Research Articles

CircFN1 promotes acute myeloid leukemia cell proliferation and invasion but refrains apoptosis via miR-1294/ARHGEF10L axis.

Previous studies have proved circFN1 is highly expressed in acute myeloid leukemia (AML) patients and AML cell lines. This study aims to investigate the impact of circFN1 on AML and its mechanism. Via real-time quantitative PCR to detect circFN1, miR-1294, ARHGEF10L expressions in clinical plasma samples and AML cell lines, AML cells were cultured in vitro and transfected with si-circFN1, pcDNA3.1-circFN1, and si-ARHGEF10L, respectively, or co-transfected pcDNA3.1-circFN1 + miR-1294 mimic and pcDNA3.1-circFN1 + si-ARHGEF10L. Using dual luciferase reporter experiment to detect the relationship between circFN1 and miR-1294, as well as miR-1294 and ARHGEF10L. CCK-8 was used to detect cell proliferation, Transwell to cell invasion, TUNEL staining and flow cytometry to detect cell apoptosis, RT-qPCR to circFN1 RNA, miR-1294, and ARHGEF10L expression levels in HL-60 cells, and western blot to ARHGEF10L protein expression level in HL-60 cells. We found highly expressed circFN1 and ARHGEF10L, as well as low-expressed miR-1294 in AML patients and AML cell lines. In contrast to si-NC group, si-circFN1 group could signally inhibit HL-60 cell proliferation and migration, but promote cell apoptosis; compared with mimic NC group, miR-1294 mimic group could visually inhibit HL-60 cell proliferation and migration, but promote cell apoptosis. miR-1294 was the target of circFN1, and ARHGEF10L was the target of miR-1294. Over-expressing miR-1294 or silencing ARHGEF10L could signally inhibit circFN1 promoting HL-60 cell proliferation and migration and repressing cell apoptosis. circFN1 promotes proliferation and invasion of AML cell and represses cell apoptosis via regulating miR-1294/ARHGEF10L axis, which provides new insight for molecular targeted-treatment for AML.

Distinct microglial transcriptomic signatures within the hippocampus.

Microglia, the resident immune cells of the brain, are crucial in the development of the nervous system. Recent evidence demonstrates that microglia modulate adult hippocampal neurogenesis by inhibiting cell proliferation of neural precursors and survival both in vitro and in vivo, thus maintaining a balance between cell division and cell death in the neural stem cell pool. There are increasing reports suggesting these microglia found in neurogenic niches differ from their counterparts in non-neurogenic areas. Here, we present evidence that hippocampal microglia exhibit transcriptomic heterogeneity, with some cells expressing genes associated with neurogenesis. By comprehensively profiling myeloid lineage cells in the hippocampus using single cell RNA-sequencing, we have uncovered a small, yet distinct population of microglia which exhibit depletion in genes associated with homeostatic microglia and enrichment of genes associated with phagocytosis. Intriguingly, this population also expresses a gene signature with substantial overlap with previously characterized phenotypes, including disease associated microglia (DAM), a particularly unique and compelling microglial state.

The Apoptotic Property of Nymphaea Caerulea Flower Extract on Acute Myeloid Leukaemia Cell Line, THP-1.

Acute Myeloid Leukaemia (AML) is considered to be an extremely heterogeneous malignancy of bone marrow and blood. The first line of therapy for AML is prolonged chemotherapy. Due to the presence of molecular heterogeneity in AML as confirmed by next-generation sequencing, researchers are planning to develop newer strategies of therapy. In the present study we have explored the anti-cancer potentiality of the hydro-ethanolic extract (50% and 70%) of the whole flower of Nymphaea caerulea against the Acute Myeloid Leukaemia cell line, THP-1 with control of normal human kidney epithelial cell line (HEK 293). The present study is a novel contribution to the existing scientific knowledge as at present no study as an anti-leukaemic agent is available on N. caerulea (blue lotus) extract and exploring its action mechanism on in-vitro cell line model. Some targeted cytokine and apoptotic genes genes to deduce the anti-cancer mechanism of action of the crude extract (hydro-ethanolic extract (50% and 70%) of the whole flower) were selected as Interferon (IFN) γ, Interleukins - IL-6, IL-8, IL- 10, IL-1β, Transforming Growth Factor (TGF β1), Tumor Necrosis Factor (TNF α), Caspase 3(CAS 3), Caspase 9 (CAS 9), CD95 (Fas), Tumor Necrosis Factor Receptor 1 (TNFRSF1A) to observe relative fold changes of the expression using Real-Time PCR with housekeeping gene β-actin. Cellular cytopathic effect (CPE), cell viability assay by methylene blue assay, and cell cytotoxicity of the crude extract against the THP-1 cell line were also studied along with it's bio-active compositional analysis of the extract was explored using ultra-performance liquid chromatography followed by mass spectra. The N. caerulea flower extract is capable of inducing apoptosis in AML and it can balance cytokine alterations in such diseases. Nymphaea caerulea flower extract appears to be a good anti-leukemia agent.

Expression, Function, and Significance of NonB Cell-Derived Immunoglobulin in Haematological System.

Acute myeloid leukaemia (AML) is a collection of genetically diverse diseases characterised by abnormal proliferation of immature haematopoietic cells and disruption of normal haematopoiesis. Myeloid cells and lymphocytes originate from different haematopoietic precursors within the bone marrow. It has been traditionally assumed that myeloid cells cannot produce immunoglobulin (Ig), a marker of B cells and plasma cells. However, in recent years, all five Ig classes have been detected in CD34+ haematopoietic stem cells, mature monocytes and neutrophils, differentiated macrophages and tumour-associated macrophages, acute myeloid leukaemia cell lines, as well as myeloblasts of AML. The rearranged V(D)J sequences exhibit unique restricted or biased V gene usage and evidence of somatic mutation. Furthermore, AML-derived Igs could promote cell proliferation, induce apoptosis, and enhance migration. Elevated levels of Ig expression predict inferior clinical outcomes. These findings indicate that AML-derived Ig plays a role in AML pathogenesis and progression, and could serve as a novel biomarker for risk stratification, disease monitoring, and targeted therapy. In this chapter, we provide a comprehensive review of recent literature on the expression, function, and significance of non B cell-derived Ig in the haematological system, with a focus on AML.

Modification of the Antibiotic, Colistin, with Dextrin Causes Enhanced Cytotoxicity and Triggers Apoptosis in Myeloid Leukemia.

Acute myeloid leukemia (AML) remains difficult to treat due to its heterogeneity in molecular landscape, epigenetics and cell signaling alterations. Precision medicine is a major goal in AML therapy towards developing agents that can be used to treat patients with different 'subtypes' in combination with current chemotherapies. We have previously developed dextrin-colistin conjugates to combat the rise in multi-drug resistant bacterial infections and overcome dose-limiting nephrotoxicity. Recent evidence of colistin's anticancer activity, mediated through inhibition of intracellular lysine-specific histone demethylase 1 (LSD1/KDM1A), suggests that dextrin-colistin conjugates could be used to treat cancer cells, including AML. This study aimed to evaluate whether dextrin conjugation (which reduces in vivo toxicity and prolongs plasma half-life) could enhance colistin's cytotoxic effects in myeloid leukemia cell lines and compare the intracellular uptake and localization of the free and conjugated antibiotic. Our results identified a conjugate (containing 8000 g/mol dextrin with 1 mol% succinoylation) that caused significantly increased toxicity in myeloid leukemia cells, compared to free colistin. Dextrin conjugation altered the mechanism of cell death by colistin, from necrosis to caspase 3/7-dependent apoptosis. In contrast, conjugation via a reversible ester linker, instead of an amide, had no effect on the mechanism of the colistin-induced cell death. Live cell confocal microscopy of fluorescently labelled compounds showed both free and dextrin-conjugated colistins were endocytosed and co-localized in lysosomes, and increasing the degree of modification by succinoylation of dextrin significantly reduced colistin internalization. Whilst clinical translation of dextrin-colistin conjugates for the treatment of AML is unlikely due to the potential to promote antimicrobial resistance (AMR) and the relatively high colistin concentrations required for anticancer activity, the ability to potentiate the effectiveness of an anticancer drug by polymer conjugation, while reducing side effects and improving biodistribution of the drug, is very attractive, and this approach warrants further investigation.

Production, Isolation, and Detection of Poly IC in Extracellular Vesicles from U937 Cells.

Double-stranded RNA is produced by viruses during their replicative cycle. It is a potent immune modulator and indicator of viral infection within the body. Extracellular vesicles (EVs) are lipid-bound particles released from cells homeostatically. Recent studies have shown that a commercially available dsRNA, poly inosinic: poly cytidylic acid (poly IC), can be detected within EVs. This finding opens the door for studying EVs as (1) carriers for dsRNA and (2) indicators of viral infection. To study dsRNA-containing EVs, we must have reliable methods for producing, isolating, and detecting them. This chapter uses U937, a pro-monocytic, human myeloid leukemia cell line, as the EV producer following poly IC treatment, and an immunoblot using an anti-dsRNA antibody (J2) for detection. Two methods for isolating the EVs and two methods for isolating the RNA from these EVs are described. Together, these methods effectively produce, isolate, and detect long dsRNA from EVs.

Inhibitory Effects of the Methanol Extract of the Golden Chanterelle Mushroom, Cantharellus cibarius (Agaricomycetes) in Treating a Human Acute Myeloid Leukemia Cell Line

Currently available treatments for acute myeloid leukemia exhibit side effects that limit their use, with primary and secondary resistance as persistent issues. While edible mushrooms possess nutritional value, they are also an excellent source of bioactive compounds that may have the potential to treat multiple disease states. The aim of the present study was to investigate the <i>in vitro</i> inhibitory effects of chromatographic fractions from the methanol extract of <i>Cantharellus cibarius</i> in a human acute myeloid leukemia (AML) cell line MV4-11. The mushrooms were purchased from a grocery store, the fruiting bodies were triturated and then extracted with 99.9% methanol to generate a crude extract. This crude extract was then redissolved in methanol, filtered over cotton to remove insoluble solids, and then fractionated over Sephadex<sup>®</sup> LH-20. Each fraction was dried and then analyzed by reverse-phase HPLC. A typical UV wavelength was selected for the detection of possible anticancer compounds in <i>C. cibarius </i>based on the major chromophores in main fungal anticancer agents reported. Based on chromatographic profiles, specific fractions that might contain potential anticancer agents were combined, and the inhibitory activity of the combined fractions was assessed against the MV4-11 leukemia cell line. Data confirmed that one combined fraction LH-20F-IV showed the greatest degree of <i>in vitro</i> inhibitory activity against the selected cell line. Fast determination of the potential anticancer compound containing fraction was completed through the application of reported chemoinformatics on HPLC detection of possible chromophores in potential anticancer agents, in combination with cellular bioassays.

CD177 is a novel IgG Fc receptor and CD177 genetic variants affect IgG-mediated function.

CD177 plays an important role in the proliferation and differentiation of myeloid lineage cells including neutrophils, myelocytes, promyelocytes, megakaryocytes, and early erythroblasts in bone marrow. CD177 deficiency is a common phenotype in humans. Our previous studies revealed genetic mechanisms of human CD177 deficiency and expression variations. Up to now, immune functions of CD177 remain undefined. In the current study, we revealed human IgG as a ligand for CD177 by using flow cytometry, bead-rosette formation, and surface plasmon resonance (SPR) assays. In addition, we show that CD177 variants affect the binding capacity of CD177 for human IgG. Furthermore, we show that the CD177 genetic variants significantly affect antibody-dependent cell-mediated cytotoxicity (ADCC) function. The demonstration of CD177 as a functional IgG Fc-receptor may provide new insights into CD177 immune function and genetic mechanism underlying CD177 as biomarkers for human diseases.

Characterization of lysosomal acid lipase in Ly6G+ and CD11c+ myeloid-derived suppressor cells.

Lysosomal acid lipase (LAL) is a key enzyme in the metabolic pathway of neutral lipids, whose deficiency (LAL-D) induces the differentiation of myeloid lineage cells into myeloid-derived suppressor cells (MDSCs), which promotes tumor growth and metastasis. This protocol provides detailed procedures for assessment of various LAL biochemical and physiological activities in Ly6G+ and CD11c+ MDSCs, including isolation of Ly6G+ and CD11c+ cells from the bone marrow and blood of mice, assays of LAL-D-induced cellular metabolic and mitochondrial activities, assessment of LAL-D-induced pathogenic immunosuppressive activity and tumor stimulatory activity. Pharmacological inhibition of the LAL activity was also described in both murine myeloid cells and human white blood cells.

PIEZO1 is essential for the survival and proliferation of acute myeloid leukemia cells.

Leukemogenesis is a complex process that interconnects tumoral cells with their microenvironment, but the effect of mechanosensing in acute myeloid leukemia (AML) blasts is poorly known. PIEZO1 perceives and transmits the constraints of the environment to human cells by acting as a non-selective calcium channel, but very little is known about its role in leukemogenesis. For the first time, we show that PIEZO1 is preferentially expressed in healthy hematopoietic stem and progenitor cells in human hematopoiesis, and globally overexpressed in AML cells. In AML subtypes, PIEZO1 expression associates with favorable outcomes as better overall (OS) and disease-free survival (DFS). If PIEZO1 is expressed and functional in THP1 leukemic myeloid cell line, its chemical activation doesn't impact the proliferation, differentiation, nor survival of cells. However, the downregulation of PIEZO1 expression dramatically reduces the proliferation and the survival of THP1 cells. We show that PIEZO1 knock-down blocks the cell cycle in G0/G1 phases of AML cells, impairs the DNA damage response pathways, and critically increases cell death by triggering extrinsic apoptosis pathways. Altogether, our results reveal a new role for PIEZO1 mechanosensing in the survival and proliferation of leukemic blasts, which could pave the way for new therapeutic strategies to target AML cells.

SBDS Gene Mutation Increases ROS Production and Causes DNA Damage as Well as Oxidation of Mitochondrial Membranes in the Murine Myeloid Cell Line 32Dcl3.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutation in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SDS has a variety of clinical features, including exocrine pancreatic insufficiency and hematological dysfunction. Neutropenia is the most common symptom in patients with SDS. SDS is also associated with an elevated risk of developing myelodysplastic syndromes and acute myeloid leukemia. The SBDS protein is involved in ribosome biogenesis, ribosomal RNA metabolism, stabilization of mitotic spindles and cellular stress responses, yet the function of SBDS in detail is still incompletely understood. Considering the diverse function of SBDS, the effect of SBDS seems to be different in different cells and tissues. In this study, we established myeloid cell line 32Dcl3 with a common pathogenic SBDS variant on both alleles in intron 2, 258 + 2T > C, and examined the cellular damage that resulted. We found that the protein synthesis was markedly decreased in the mutant cells. Furthermore, reactive oxygen species (ROS) production was increased, and oxidation of the mitochondrial membrane lipids and DNA damage were induced. These findings provide new insights into the cellular and molecular pathology caused by SBDS deficiency in myeloid cells.

The Chaperone Protein Cct5 is Essential for Hematopoietic Stem Cell Maintenance.

Proper proteostasis is indispensable for the long-term maintenance of hematopoietic stem and progenitor cells (HSPCs). The TRiC/CCT (chaperonin-containing TCP-1) complex, a key constituent of cellular machinery facilitating accurate protein folding, has remained enigmatic in its specific function within HSPCs. Here we show that conditional knockout (KO) of Cct5 significantly impairs the maintenance of murine HSPCs. Primary and secondary transplantation experiments unequivocally demonstrate the incapacity of Cct5 KO HSPCs to reconstitute both myeloid and lymphoid lineage cells in recipient mice, highlighting the pivotal role of the TRiC/CCT complex in governing these cellular lineages. Furthermore, leveraging an integrated approach that merges a Protein-Protein Interaction (PPI) database with RNA sequencing (RNA-seq) data of HSPCs, our analysis reveals intricate interactions between Cct5 and vital transcription factors crucial for HSC homeostasis. Notably, Cct5 engages with MYC, PIAS1, TP53, ESR1, HOXA1, and JUN, intricately regulating the transcriptional landscape governing autophagy, myeloid differentiation, and cytoskeleton organization within HSPCs. Our study unveils the profound significance of TRiC/CCT complex-mediated proteostasis in orchestrating the maintenance and functionality of HSPCs.

The mosquito effect: regulatory and effector T cells acquire cytoplasmic material from tumor cells through intercellular transfer.

The phenomenon of intercellular transfer of cellular material, including membranes, cytoplasm, and even organelles, has been observed for decades. The functional impact and molecular mechanisms of such transfer in the immune system remain largely elusive due to the absence of a robust in vivo model. Here, we introduce a new tumor mouse model, where tumor cells express the soluble ultra-bright fluorescent protein ZsGreen, which allows detection and measurement of intercellular transfer of cytoplasm from tumor cells to infiltrating immune cells. We found that in addition to various types of myeloid lineage cells, a large fraction of T regulatory cells and effector CD8 T cells acquire tumor material. Based on the distribution of tumor-derived ZsGreen, the majority of T cells integrate captured cytoplasm into their own, while most myeloid cells store tumor material in granules. Furthermore, scRNA-seq analysis revealed significant alterations in transcriptomes of T cells that acquired tumor cell cytoplasm, suggesting potential impact on T cell function. We identified that the participation of T cells in intercellular transfer requires cell-cell contact and is strictly dependent on the activation status of T lymphocytes. Finally, we propose to name the described phenomenon of intercellular transfer for tumor infiltrating T cells the "mosquito effect".

The NR4A Orphan Receptor Modulator C-DIM12 Selectively Alters Inflammatory Mediators in Myeloid Cells

Orphan nuclear receptor subfamily 4A (NR4A) are key regulators of inflammatory responses, largely by their interactions with NF-κB. Over the last decade, several NR4A modulators have been developed, and they are showing potential as therapeutics, although their widespread use in laboratory settings is limited. Here, we have examined, using myeloid cell line THP-1, whether the NR4A modulator 3-[(4-Chlorophenyl)-(1H-indol-3-yl)methyl]-1H-indole (C-DIM12) can alter the inflammatory outcome of six inflammatory ligands: lipopolysaccharide (LPS), tumour necrosis factor alpha (TNFα), interleukin-1 beta (IL-1β), flagellin (FL), lipoteichoic acid (LTA), and zymosan (ZY). We demonstrate that C-DIM12 (10 µM) selectively alters the secretion of inflammatory chemokine MCP-1 following exposure to distinct inflammatory ligands in a concentration-dependent manner. Furthermore, data obtained from THP-1 Lucia cell experiments show that 10 µM C-DIM12, and not 1 µM C-DIM12, can significantly attenuate the increased NF-κB transcriptional activity observed following the exposure to several inflammatory ligands (LPS, FL, TNFα, LTA, and ZY). Lastly, experimental analysis confirms that the cellular action(s) of C-DIM12 is independent of changes in metabolic parameters. Thus, these data contribute to the understanding of how the NR4A modulator C-DIM12 alters inflammatory responses in a myeloid cell following exposure to multiple ligands.

Asperuloside regulates the proliferation, apoptosis, and differentiation of chronic myeloid leukemia cell line K562 through the RAS/MEK/ERK pathway

ContextChronic myeloid leukemia (CML) is a malignant hematopoietic stem cell disease caused by excessive proliferation and abnormal differentiation of hematopoietic stem cells. Asperuloside (ASP) is considered to have good biological activity and may be a good anti-CML drug. ObjectiveThis study aimed to explore the effects and possible mechanisms of ASP on the biological behavior of K562 cells based on RNA-seq. Materials and methodsThe IC50 of ASP in K562 cells was calculated by the concentration-effect curve. Cell viability, apoptosis, and differentiation were detected by CCK8, flow cytometry, benzidine staining, and WB analysis, respectively. Further, RNA-seq was used to analyze the possible mechanism of ASP regulating K562 cells. ResultsASP significantly inhibited the proliferation, and promoted apoptosis and differentiation of K562 cells. A total of 117 differentially expressed genes were screened by RNA-seq, mainly involved in the RAS/MEK/ERK pathway. PD98059 was used to inhibit the RAS/MEK/ERK pathway in K562 cells, and results confirmed that PD98059 could not only inhibit the RAS/MEK/ERK pathway, but also inhibit the regulation of ASP on the proliferation and differentiation of K562 cells. ConclusionASP inhibited the proliferation, promoted apoptosis and differentiation of K562 cells by regulating the RAS/MEK/ERK pathway, and played a good anti-CML role.

A highly potent anti-VISTA antibody KVA12123 -a new immune checkpoint inhibitor and a promising therapy against poorly immunogenic tumors.

Immune checkpoint therapies have led to significant breakthroughs in cancer patient treatment in recent years. However, their efficiency is variable, and resistance to immunotherapies is common. VISTA is an immune-suppressive checkpoint inhibitor of T cell response belonging to the B7 family and a promising novel therapeutic target. VISTA is expressed in the immuno-suppressive tumor microenvironment, primarily by myeloid lineage cells, and its genetic knockout or antibody blockade restores an efficient antitumor immune response. Fully human monoclonal antibodies directed against VISTA were produced after immunizing humanized Trianni mice and single B cell sequencing. Anti-VISTA antibodies were evaluated for specificity, cross-reactivity, monocyte and T cell activation, Fc-effector functions, and antitumor efficacy using in vitro and in vivo models to select the KVA12123 antibody lead candidate. The pharmacokinetics and safety profiles of KVA12123 were evaluated in cynomolgus monkeys. Here, we report the development of a clinical candidate anti-VISTA monoclonal antibody, KVA12123. KVA12123 showed high affinity binding to VISTA through a unique epitope distinct from other clinical-stage anti-VISTA monoclonal antibodies. This clinical candidate demonstrated high specificity against VISTA with no cross-reactivity detected against other members of the B7 family. KVA12123 blocked VISTA binding to its binding partners. KVA12123 induced T cell activation and demonstrated NK-mediated monocyte activation. KVA12123 treatment mediated strong single-agent antitumor activity in several syngeneic tumor models and showed enhanced efficacy in combination with anti-PD-1 treatment. This clinical candidate was engineered to improve its pharmacokinetic characteristics and reduce Fc-effector functions. It was well-tolerated in preclinical toxicology studies in cynomolgus monkeys, where hematology, clinical chemistry evaluations, and clinical observations revealed no indicators of toxicity. No cytokines associated with cytokine release syndrome were elevated. These results establish that KVA12123 is a promising drug candidate with a distinct but complementary mechanism of action of the first generation of immune checkpoint inhibitors. This antibody is currently evaluated alone and in combination with pembrolizumab in a Phase 1/2 open-label clinical trial in patients with advanced solid tumors.

Single-cell RNA-seq analyses inform necroptosis-associated myeloid lineages influence the immune landscape of pancreas cancer.

Tumor-infiltrating myeloid cells (TIMs) are key regulators in tumor progression, but the similarity and distinction of their fundamental properties in pancreatic ductal adenocarcinoma (PDAC) remain elusive. In this study, we conducted scRNA-seq data analysis of cells from 12 primary tumor (PT) tissues, 4 metastatic (Met) tumor tissues, 3 adjacent normal pancreas tissues (Para), and PBMC samples across 16 PDAC patients, and revealed a heterogeneous TIMs environment in PDAC. Systematic comparisons between tumor and non-tumor samples of myeloid lineages identified 10 necroptosis-associated genes upregulated in PDAC tumors compared to 5 upregulated in paratumor or healthy peripheral blood. A novel RTM (resident tissue macrophages), GLUL-SQSTM1- RTM, was found to act as a positive regulator of immunity. Additionally, HSP90AA1+HSP90AB1+ mast cells exhibited pro-immune characteristics, and JAK3+TLR4+ CD16 monocytes were found to be anti-immune. The findings were validated through clinical outcomes and cytokines analyses. Lastly, intercellular network reconstruction supported the associations between the identified novel clusters, cancer cells, and immune cell populations. Our analysis comprehensively characterized major myeloid cell lineages and identified three subsets of myeloid-derived cells associated with necroptosis. These findings not only provide a valuable resource for understanding the multi-dimensional characterization of the tumor microenvironment in PDAC but also offer valuable mechanistic insights that can guide the design of effective immuno-oncology treatment strategies.

Lateralized response of skull bone marrow via osteopontin signaling in mice after ischemia reperfusion

Skull bone marrow is thought to be an immune tissue closely associated with the central nervous system (CNS). Recent studies have focused on the role of skull bone marrow in central nervous system disorders. In this study, we performed single-cell RNA sequencing on ipsilateral and contralateral skull bone marrow cells after experimental stroke and then performed flow cytometry and analysis of cytokine expression. Skull marrow showed lateralization in response to stroke. Lateralization is demonstrated primarily by the proliferation and differentiation of myeloid and lymphoid lineage cells in the skull bone marrow adjacent to the ischemic region, with an increased proportion of neutrophils compared to monocytes. Analysis of chemokines in the skull revealed marked differences in chemotactic signals between the ipsilateral and contralateral skull, whereas sympathetic signals innervating the skull did not affect cranial bone marrow lateralization. Osteopontin (OPN) is involved in region-specific activation of the skull marrow that promotes inflammation in the meninges, and inhibition of OPN expression improves neurological function.Graphical

Methods to Investigate Thrombin Cleavage of Osteopontin (OPN).

Osteopontin (OPN) is a matricellular protein containing binding sites for a variety of ligands including an RGD sequence for binding to αvβ3 integrins. OPN is a conserved substrate for thrombin, the effector protease of the coagulation cascade. Thrombin cleaves OPN at a single site revealing new functionalities such as a previously cryptic α4β1 and α9β1 integrin-binding site. That integrin-binding site is abolished upon treatment with a basic carboxypeptidase. The thrombin cleavage of OPN has been demonstrated to play a role in regulating tumor growth.This report describes methods for production of full-length OPN as well as the enzymatically cleaved OPN fragments resulting from thrombin and carboxypeptidase treatments. Quantification procedures for the various OPN proteins are described as well as functional assays on mouse melanoma and myeloid cell lines.

Alterations in the mammary gland and tumor microenvironment of formerly obese mice

BackgroundObesity is a risk factor for breast cancer, and women with obesity that develop breast cancer have a worsened prognosis. Within the mammary gland, obesity causes chronic, macrophage-driven inflammation and adipose tissue fibrosis. Weight loss is a recommended intervention to resolve obesity, but the impact of weight loss on the mammary gland microenvironment and in tumors has not been well identified.MethodsTo examine the effects of weight loss following obesity, mice were fed a high-fat diet for 16 weeks to induce obesity, then switched to a low-fat diet for 6 weeks. We examined changes in immune cells, including fibrocytes, which are myeloid lineage cells that have attributes of both macrophages and myofibroblasts, and collagen deposition within the mammary glands of non-tumor-bearing mice and within the tumors of mice that were transplanted with estrogen receptor alpha positive TC2 tumor cells. ResultsIn formerly obese mice, we observed reduced numbers of crown-like structures and fibrocytes in mammary glands, while collagen deposition was not resolved with weight loss. Following transplant of TC2 tumor cells into the mammary glands of lean, obese, and formerly obese mice, diminished collagen deposition and cancer-associated fibroblasts were observed in tumors from formerly obese mice compared to obese mice. Within tumors of obese mice, increased myeloid-derived suppressor cells and diminished CD8+ T cells were identified, while the microenvironment of tumors of formerly obese mice were more similar to tumors from lean mice. When TC2 tumor cells were mixed with CD11b+CD34+ myeloid progenitor cells, which are the cells of origin for fibrocytes, and transplanted into mammary glands of lean and obese mice, collagen deposition within the tumors of both lean and obese was significantly greater than when tumor cells were mixed with CD11b+CD34− monocytes or total CD45+ immune cells. ConclusionsOverall, these studies demonstrate that weight loss resolved some of the microenvironmental conditions within the mammary gland that may contribute to tumor progression. Additionally, fibrocytes may contribute to early collagen deposition in mammary tumors of obese mice leading to the growth of desmoplastic tumors.