Myelopoiesis Research Articles

Design and fabrication of novel microfluidic-based droplets for drug screening on a chronic myeloid leukemia cell line

Background The challenges associated with traditional drug screening, such as high costs and long screening times, have led to an increase in the use of single-cell isolation technologies. Small sample volumes are required for high-throughput, cell-based assays to reduce assay costs and enable rapid sample processing. Using microfluidic chips, single-cell analysis can be conducted more effectively, requiring fewer reagents and maintaining biocompatibility. Due to the chip’s ability to manipulate small volumes of fluid, high-throughput screening assays can be developed that are both miniaturized and automated. In the present study, we employ microfluidic chips for drug screening in chronic myeloid leukemia. This study aimed to establish a robust methodology integrating diverse assays, providing a holistic understanding of drug response. Material and methods Herein, we have used a chronic myeloid leukemia derived cell line (K562) for drug screening with an innovative microfluidic-based drug screening approach to investigate the efficacy of imatinib in K562 cells. Cell viability was assessed using MTT assay. Apoptosis was measured using Annexin/PI staining by flow cytometry. Results Significant increased apoptosis was seen in K562 cells treated with imatinib in the microfluidic device compared to cells treated with imatinib in 24- and 96-well plates. Moreover, in the microfluidic chip, drug screening time was reduced from 48 hours to 24 hours. Conclusion Compared to traditional approaches, microfluidic-based drug screening efficiently evaluates the efficacy of imatinib in K562 cells. This approach is promising for drug discovery and treatment optimization, as it increases sensitivity and streamlines the screening process.

A Humanized Monoclonal Antibody Against CD300A Ameliorates Acute Ischemic Stroke in Humanized Mice.

CD300a and CD300A, among the CD300 immunoglobulin (Ig)-like receptor family members in mice and humans, respectively, are expressed on myeloid cell lineage. The interaction of CD300a and CD300A with their ligands phosphatidylserine and phosphatidylethanolamine, respectively, exposed on the plasma membrane of dead cells mediate an inhibitory signal in myeloid cells. We previously reported that a neutralizing antimouse CD300a monoclonal antibody (mAb) enhanced efferocytosis by macrophages and ameliorated acute ischemic stroke (AIS) in mice. Unlike mouse CD300a, human CD300A has a single nucleotide polymorphism (SNP, rs2272111) encoding a nonsense mutation of glutamine (CD300AQ111) instead of arginine (CD300AR111) at residue 111. In this study, we show that the SNP frequency is 32%-35% for the heterozygous allele and 4%-5% for the homozygous alleles, except Africa. In addition, we developed a humanized antihuman CD300A mAb, named TNAX103, that recognizes both CD300AR111 and CD300AQ111. We show that TNAX103 interfered with the binding of CD300AR111 and CD300AQ111 to dead cells. In addition, the injection of TNAX103 decreased neurological scores and prolonged survival in humanized mice after middle cerebral artery occlusion. These results suggest that TNAX103 may be potentially useful for the treatment of patients expressing either CD300AR111 or CD300AQ111 with AIS.

Comprehensive analysis of DNA methylation and gene expression to identify tumor suppressor genes reactivated by MLN4924 in acute myeloid leukemia.

This study investigated whether the neddylation inhibitor MLN4924 induces aberrant DNA methylation patterns in acute myeloid leukemia and contributes to the reactivation of tumor suppressor genes. DNA methylation profiles of Kasumi-1 and KU812 acute myeloid leukemia cell lines before and after MLN4924 treatment were generated using the 850K Methylation BeadChip. RNA sequencing was used to obtain transcriptomic profiles of Kasumi-1 cells. Target genes were identified through a combined analysis of methylation and transcriptome data. Methylation-specific PCR and quantitative PCR validated the changes in methylation and expression. Prognostic analysis of target genes was performed using databases, and Pearson correlation was used to examine the relationship between methylation and expression levels. In Kasumi-1 and KU812 cells, 301 and 469 differentially methylated sites, respectively, were identified. A total of 4310 differential expression genes were detected in Kasumi-1. Combined analysis revealed that TRIM58 exhibited significant demethylation and upregulation after MLN4924 treatment, as confirmed by quantitative and methylation-specific PCR. Furthermore, database analysis revealed that both down-expression and promoter hypermethylation of TRIM58 were correlated with poor prognosis in acute myeloid leukemia. A negative correlation was observed between TRIM58 methylation and expression levels. This study suggests that MLN4924 alters DNA methylation patterns in acute myeloid leukemia and reactivates TRIM58, a potential tumor suppressor gene, through demethylation.

Tetrabromobisphenol A, but not bisphenol A, disrupts plasma membrane homeostasis in myeloid cell models - A novel threat from an established persistent organic pollutant.

Bisphenol A (BPA, a plastic polymer component) and tetrabromobisphenol A (TBBPA, a brominated flame retardant) are industrial compounds and representative persistent organic pollutants (POPs) with similar chemical structure. We studied their impact on biological membrane dynamics, which is an emerging and understudied target for environmental contaminants, using a set of state-of-the-art methods. We found that exposure to TBBPA, but not to BPA, leads to disruption of biophysical homeostasis of the plasma membrane in myeloid cell lines HL-60, THP-1 and Mono Mac 6. Applied methods include: pyrene excimer formation, fluorescence anisotropy, solvatochromic shift ratiometry (using di-4-ANEPPDHQ, NR12A and laurdan) and fluorescence recovery after photobleaching. TBBPA increased rotational and lateral mobility and decreased general polarity and lipid order in plasma membranes of myeloid cells, but decreased mobility and increased rigidity in internal membranes of the same cells. Strikingly, BPA had no significant membrane effects in these cells, suggesting a specific molecular interaction mechanism of TBBPA action which may potentially lead to disruption of immune function. Identification of this novel threat from an established pollutant with documented exposure pathways highlights the possibility that immunotoxicity of POPs may contribute to their environmental toxicity burden.

Early differentiation of committed erythroid cells defined by miR-144/451 expression.

Before committing to an erythroid cell lineage, hematopoietic stem cells differentiate along a myeloid cell pathway to generate megakaryocyte-erythroid biopotential progenitor cells in bone marrow. Recent studies suggest that erythroid progenitors (EryPs) could be generated at the level of common myeloid progenitors (CMPs). However, due to a lack of suitable markers, little is known about the early differentiation of these committed EryP cells during CMP development. Herein, using miR-144/451-eGFP knock-in mice, we found that early differentiation of committed erythroid cells could be defined by miR-144/451 expression within CMPs. Single-cell RNA sequencing showed that miR-144/451+ progenitors had obvious differentiation characteristics of erythroid lineage cells and diverged from megakaryocyte and other myeloid cell lineages. These progenitors exclusively gave rise to erythroid cells, both in vitro and in vivo, and the commitment to an erythroid cell lineage was accompanied by loss of CD53 expression. Our findings will facilitate further understanding of the molecular mechanisms governing erythroid development and support the identification of therapeutic targets for diseases related to erythrocyte development.

CTLA4-Ig reduces muscle fiber damage in a model of Duchenne muscular dystrophy by attenuating pro-inflammatory gene expression in myeloid lineage cells.

CTLA4-Ig reduces muscle fiber damage in a model of Duchenne muscular dystrophy by attenuating pro-inflammatory gene expression in myeloid lineage cells.

Mesenchymal Stromal Cell (MSC) Functional Analysis-Macrophage Activation and Polarization Assays.

Stem cell-based therapies have evolved to become a key component of regenerative medicine approaches to human pathologies. Exogenous stem cell transplantation takes advantage of the potential of stem cells to self-renew, differentiate, home to sites of injury, and sufficiently evade the immune system to remain viable for the release of anti-inflammatory cytokines, chemokines, and growth factors. Common to many pathologies is the exacerbation of inflammation at the injury site by proinflammatory macrophages. An increasing body of evidence has demonstrated that mesenchymal stromal cells (MSCs) can influence the immunophenotype and function of myeloid lineage cells to promote therapeutic effects. Understanding the degree to which MSCs can modulate the phenotype of macrophages within an inflammatory environment is of interest when considering strategies for targeted cell therapies. There is a critical need for potency assays to elucidate these intercellular interactions in vitro and provide insight into potential mechanisms of action attributable to the immunomodulatory and polarizing capacities of MSCs, as well as other cells with immunomodulatory potential. However, the complexity of the responses, in terms of cell phenotypes and characteristics, timing of these interactions, and the degree to which cell contact is involved, have made the study of these interactions challenging. To provide a research tool to study the direct interactions between MSCs and macrophages, we developed a potency assay that directly co-cultures MSCs with naïve macrophages under proinflammatory conditions. Using this assay, we demonstrated changes in the macrophage secretome and phenotype, which can be used to evaluate the abilities of the cell samples to influence the cell microenvironment. These results suggest the immunomodulatory effects of MSCs on macrophages while revealing key cytokines and phenotypic changes that may inform their efficacy as potential cellular therapies. Key features • The protocol uses monocytes differentiated into naïve macrophages, which are loosely adherent, have a relatively homogeneous genetic background, and resemble peripheral blood mononuclear cells-derived macrophages. • The protocol requires a plate reader and a flow cytometer with the ability to detect six fluorophores. • The protocol provides a quantitative measurement of co-culture conditions by the addition of a fixed number of freshly thawed or culture-rescued MSCs to macrophages. • This protocol uses assessment of the secretome and cell harvest to independently verify the nature of the interactions between macrophages and MSCs.

Single-cell transcriptomes of dissecting the intra-tumoral heterogeneity of breast cancer microenvironment

ObjectiveTo investigate the mechanism by which heterogeneity in breast cancer developed and acted in single-cell transcriptomes.MethodsThe composition of breast cancer based on the single-cell transcriptomes of 54,055 high-quality cells from clinical specimens of 4 malignant and 4 non-malignant patients were investigated.ResultsWe identified six common expression programs and six subtype-specific expression programs form malignant epithelial cells. The expression program of malignant epithelial cells exhibited activated EMT (Epithelial Mesenchymal Transition) in TME, which might indicate EMT intervention have a general therapeutic effect on various subtypes. Gene set enrichment analysis (GSEA) based on the top 50 highly NMF (non-negative matrix factorization) score genes in each program depicted the distinct function of each program in breast cancer progression. Moreover, we revealed the profound cellular heterogeneity of myeloid cell lineages in breast cancer. In macrophages, two mainly tumor associated macrophages (TAMs), TAM1 and TAM2, were also detected and the highly variable genes in TAM2 were strongly enriched in IFN-α and IFN-γ. The changes of lipid metabolism pathways in macrophages are closely related to the microenvironment of breast cancer.ConclusionWe constructed a comprehensive single-cell transcriptome atlas of 54,055 cells from 4 malignant and 4 nonmalignant patients, providing insights into the mechanisms underlying breast cancer progression and the development of potential therapeutic strategies in breast cancer.

Development of a preclinical model of myeloid tumors with high immune checkpoints expression

Background. Myelodysplastic syndrome is a group of malignant blood diseases with a high risk of transformation into acute myeloid leukemia. One treatment approach is to target immune checkpoints (ICs) that are overexpressed on tumor cells. To develop these drugs, relevant models are needed for highthroughput screening and study of these biologically active substances, since traditionally used models (mouse and patient biomaterials) are difficult to access, financially and laborintensive, and are characterized by poorly reproducible results.Aim. To develop a model based on a human myeloid cell line with increased expression of L1 and TIM3 to study the activity of ICs inhibitors, the presence of which in the tumor microenvironment in patients with myelodysplastic syndrome and acute myeloid leukemia was associated with a high risk and worse prognosis.Materials and methods. Initial testing of the L1 and TIM3 basal expression level was carried out on cell lines: TH1, HL60, OCIAML2, OCIAML5, KG1, MonoMac1. Induction of IC expression was carried out using interferon γ. Analysis of marker expression was carried out 24 hours after induction of ICs expression and addition of MK2206 using flow cytometry.Results. Basal expression of the studied ICs receptors was absent in all of them, except for KG1; TIM3 was present in 88.4 ± 7.1 % of cells, and L1 – in 88 ± 8.5 %. The addition of interferon γ at a concentration of 50 ng/mL to the MonoMac1 culture led to a significant increase in the proportion of TIM3 and L1 expressing cells (53.3 ± 12.2 and 97.3 ± 1.1 % respectively, compared to 0.1 ± 0.1 and 0.1 ± 0.1 % without interferon γ), and for TH1 only L1 expression (87.5 ± 20 %, control 0.1 ± 0.1 %) was observed at the concentration of interferon γ in a medium of 50 ng/mL, while the proportion of cells expressing TIM3 was 6.9 ± 10 % (control 0.1 ± 0.1 %).Conclusion. The KG1 line, which constantly expresses significant levels of target ICs, as well as TH1 and MonoMac1, which are induced by 50 ng/mL interferon γ, were selected as a model with increased L1 and TIM3 expression based on a human myeloid cell line. The model efficiency was confirmed by the rational response to the IC pathway inhibitor.

Single cell multiomics reveal divergent effects of DNMT3A and TET2 mutant clonal hematopoiesis in inflammatory response.

DNMT3A and TET2 are epigenetic regulator genes commonly mutated in age-related clonal hematopoiesis (CH). Despite having opposed epigenetic functions, these mutations are associated with increased all-cause mortality and a low risk for progression to hematological neoplasms. While individual impacts on the epigenome have been described using different model systems, the phenotypic complexity in humans remains to be elucidated. Here we make use of a natural inflammatory response occurring during coronavirus disease 2019 (COVID-19), to understand the association of these mutations with inflammatory morbidity (acute respiratory distress syndrome-ARDS) and mortality. We demonstrate the age-independent, negative impact of DNMT3A mutant CH on COVID-19-related ARDS and mortality. Using single cell (sc-) proteogenomics we show that DNMT3A mutations involve myeloid and lymphoid lineage cells. Using single cell multiomics sequencing, we identify cell-specific gene expression changes associated with DNMT3A mutations, along with significant epigenomic deregulation affecting enhancer accessibility, resulting in overexpression of IL32, a proinflammatory cytokine that can result in inflammasome activation in monocytes and macrophages. Finally, we show with single cell resolution that the loss of function of DNMT3A is directly associated with increased chromatin accessibility in mutant cells. Hence, we demonstrate the negative prognostic impact of DNMT3Amt CH on COVID-19 related ARDS and mortality. DNMT3Amt CH in the context of COVID-19, was associated with inflammatory transcriptional priming, resulting in overexpression of IL32. This overexpression was secondary to increased chromatic accessibility, specific to DNMT3Amt CH cells. DNMT3Amt CH can thus serve as a potential biomarker for adverse outcomes in COVID-19.

Incorporating immune cell surrogates into a full-thickness tissue equivalent of human skin to characterize dendritic cell activation

In the past decades studies investigating the dendritic cell (DC) activation have been conducted almost exclusively in animal models. However, due to species-specific differences in the DC subsets, there is an urgent need for alternative in vitro models allowing the investigation of Langerhans cell (LC) and dermal dendritic cell (DDC) activation in human tissue. We have engineered a full-thickness (FT) human skin tissue equivalent with incorporated LC surrogates derived from the human myeloid leukemia-derived cell line Mutz-3, and DDC surrogates generated from the human leukemia monocytic cell line THP-1. Topical treatment of the skin models encompassing Mutz-LCs only with nickel sulfate (NiSO4) or 1-chloro-2,4-dinitrobenzene (DNCB) for 24 h resulted in significant higher numbers of CD1a positive cells in the dermal compartment, suggesting a sensitizer-induced migration of LCs. Remarkably, exposure of the skin models encompassing both, LC and DDC surrogates, revealed an early sensitizer-induced response reflected by increased numbers of CD1a positive cells in the epidermis and dermis after 8 h of treatment. Our human skin tissue equivalent encompassing incorporated LC and DDC surrogates allows the investigation of DC activation, subsequent sensitizer identification and drug discovery according to the principles of 3R.

Potential role of bacterial extracellular vesicles in the pathophysiology of systemic lupus erythematosus

Systemic Lupus Erythematosus (SLE) is a rare autoimmune disorder influenced by various factors, including infections. Following bacterial or viral infections, there is an increase in IFN-I production, primarily by plasmacytoid dendritic cells (pDCs). Overproduction of IFN-I has been shown to drive the progression of SLE. Recent findings indicate that the gene expression signature of IFN-I in lupus nephritis patients does not co-occur with pDCs as expected; instead, it is localized in glomerular regions with anastomosing capillaries, suggesting a potential source from the blood. T cells, natural killer cells, and myeloid lineage cells may also secrete IFN in the bloodstream. Bacterial presence in the bloodstream challenges the concept of blood sterility, raising the possibility that cell-free microbial components, such as bacterial extracellular vesicles (BEVs), could trigger excessive IFN production through systemic circulation. While existing studies suggest a role for BEVs in human SLE, experimental evidence has yet to establish a direct association between the entire BEV nanoparticle and the disease. Research has primarily focused on (1) mammalian EVs and (2) purified eDNA and other specific cargoes as contributors to SLE progression. It remains unproven whether these bacterial molecules exert their effects while associated with vesicular membranes, i.e., BEVs. We hypothesize that BEVs, which carry diverse cargoes including nucleic acids, may enter systemic circulation, induce cellular responses leading to IFN overproduction, and exacerbate SLE severity. Clinical studies could investigate the association of BEVs with SLE progression by monitoring their presence and quantity in blood samples and correlating these factors with disease outcomes. Furthermore, understanding the involvement of BEVs may aid in identifying SLE biomarkers, inform infection prevention strategies, and inspire the development of BEV-neutralizing agents.

Synergistic Effects of Inecalcitol With Imatinib and Dasatinib on Chronic Myeloid Leukemia Cell Lines.

According to American Cancer Society's recent estimates, about 5,330 men and 3,950 women are diagnosed each year with chronic myeloid leukemia (CML). Roughly, 750 men and 530 women are predicted to die as a result of CML, establishing a dire need for advancements in CML treatment. Tyrosine kinase inhibitors (TKIs) imatinib and dasatinib have profound effects for prognosis and delaying survival from CML. The role of inecalcitol in the treatment and prevention of various cancers including leukemia has been established. The aim of this study was to analyze the putative synergistic treatment effects of inecalcitol in combination with imatinib or dasatinib on various cell lines including AR-230, LAMA-84-s, KCL-22, and U-937. Cells grown in plates were treated alone or with varying combinations of inecalcitol, imatinib, and dasatinib and incubated for 48 h at 37°C. Cell death was determined using MTT assay. KCL-22 and U-937 were resistant to both combination treatments, whereas AR-230 exhibited a maximal antiproliferative effect (24%) with the combined treatment of imatinib (0.325 μM) and inecalcitol (15.8 μM) (p<0.001). With dasatinib (0.456 nM) and inecalcitol (15.8 μM), AR-230 exhibited a 34% antiproliferative effect (p<0.001). In drastic contrast, LAMA84-s exhibited a 45% antiproliferative effect (with the combined treatment of imatinib (0.325 μM) and inecalcitol (15.8 μM) (p<0.006). Notably, with dasatinib (0.456 nM) and inecalcitol (15.8 μM), LAMA84-s exhibited approximately 78% cell killing (p<0.007). The study suggests that the synergistic effects of inecalcitol with imatinib on cell killing are more drastic in some cells than others. These varying effects may be due to differences in cell metabolism.

Effects of ERK1/2 Inhibitors on the Growth of Acute Leukemia Cells.

Extracellular signal-regulated kinases (ERK)1/2 are important regulatory proteins that control cell proliferation and survival, playing a significant role in cancer progression, metastasis, and chemoresistance. This study investigated the effects of ERK1/2 inhibitors on the in vitro growth of acute leukemia cell lines. Three ERK1/2 inhibitors were used: SCH772984, temuterkib (LY3214996), and ulixertinib (BVD-523). Four acute myeloid leukemia cell lines (OCI/AML3, HL-60, THP-1, and U-937) and two T-lymphoblastic leukemia cell lines (Jurakt and KOPT-K1) were treated with these inhibitors. Cell growth was assessed using a colorimetric assay, and cell-cycle progression and apoptosis were analyzed using flow cytometry. The expression of intracellular signaling proteins was evaluated via immunoblotting. The effects of small interfering RNA (siRNA)-mediated ERK1/2 knockdown were also evaluated. The inhibitors suppressed the growth of three leukemia cell lines (OCI/AML3, HL-60, and THP-1) harboring neuroblastoma rat sarcoma virus (NRAS) mutations. Growth suppression occurred through G0/G1 arrest in all three cell lines and through apoptosis in OCI/AML3 cells. Immunoblotting demonstrated that these inhibitors suppressed the expression of MYC proto-oncogene, bHLH transcription factor (MYC), in the three cell lines. The additional molecular mechanisms of growth suppression varied depending on the specific inhibitor and cell line. The inhibitors had milder suppressive effects on normal lymphocytes compared to the leukemia cell lines. ERK1/2 inhibitors may serve as novel molecular-targeted drugs for treating leukemia with NRAS mutations.

Novel Pyrrolo-Pyrimidine Derivatives Bearing Amide Functionality as Potential Anticancer Agents: Synthesis and Molecular Docking Studies

Current investigation presents the synthesis, molecular docking and anticancer evaluation of novel pyrrolo-pyrimidine derivatives designed to target Janus kinase 1 (JAK1), Janus kinase 2 (JAK2) and cyclin-dependent kinase 4 (CDK4). The synthesis route commenced with 2-amino-4,6-dichloropyrimidine-5-carbaldehyde, proceeding through sequential steps involving intermediate formations and coupling reactions to yield a diverse array of pyrrolo-pyrimidine derivatives. Characterization using spectroscopic techniques (1H NMR, 13C NMR and HRMS) confirmed the chemical structures of the synthesized compounds. Molecular docking studies against JAK1, JAK2 and CDK4 demonstrated substantial binding affinities, remarkably compounds 7k, 7l and 7d, which displayed promising interactions within the active sites of the proteins. Anticancer evaluation against breast cancer (MCF-7), chronic myeloid leukemia (SET-2), colorectal carcinoma (HCT-116) and HEK-293 cell lines revealed diverse cytotoxic profiles. Specifically, compounds 7k and 7f exhibited potent activity against breast cancer (MCF-7), 7k and 7f showed efficacy against chronic myeloid leukemia (SET-2) and compounds 7k and 7l demonstrated effectiveness against colorectal carcinoma (HCT-116). These findings underscore the potential of these pyrrolo-pyrimidine derivatives as selective and effective anticancer agents, prompting further exploration for therapeutic development.

Emerging functions of FMNL1 in myeloid neoplasms: insights from bioinformatics to biological and pharmacological landscapes.

Myeloid neoplasms encompass disorders characterized by abnormal myeloid cell proliferation and differentiation, including myelodysplastic syndromes (MDS), myeloproliferative neoplasms, acute myeloid leukemia (AML), and chronic myeloid leukemia (CML). Formin-like protein 1 (FMNL1) is involved in the regulation of the actin cytoskeleton and is predominantly expressed in hematopoietic cells. Given its role in leukemia cell proliferation, survival, migration, and invasion, this study investigates FMNL1 expression in normal hematopoiesis and myeloid neoplasms and explores associations with clinical-laboratory characteristics, mutational status, and survival outcomes in AML. Transcript levels of FMNL1 from several blood-forming cell populations and myeloid neoplasms were extracted from publicly available databases. Myeloid neoplasm cell lines were used for gene/protein expression and cell differentiation studies. Functional genomics analysis was performed using RNA-seq data from The Cancer Genome Atlas (TCGA) AML study, and drug sensitivity predictions were investigated using Beat AML and Genomics of Drug Sensitivity in Cancer (GDSC) datasets. Statistical analyses assessed the impact of FMNL1 expression on clinical outcomes. FMNL1 was highly expressed in metamyelocytes, neutrophils, and monocytes compared to hematopoietic stem cells, and its expression increased with granulocytic differentiation. FMNL1 expression was elevated in AML and CML patients compared to healthy donors. FMNL1 expression was not significantly associated with clinical-laboratory characteristics or survival outcomes but showed a higher frequency of WT1 transcription factor (WT1) mutations with low FMNL1 expression in AML patients. High FMNL1 expression in AML correlated with immune response and inflammatory activity pathways. FMNL1 mRNA levels influenced drug sensitivity in AML models, with correlations observed for specific antineoplastic agents. FMNL1 plays a potential role in granulocyte differentiation and function, and its differential expression is linked to critical signaling pathways in leukemogenesis and inflammation. These findings highlight FMNL1's potential therapeutic implications in myeloid neoplasia, warranting further investigation.

Direct Interaction Between CD34+ Hematopoietic Stem Cells and Mesenchymal Stem Cells Reciprocally Preserves Stemness.

A specialized microenvironment in the bone marrow, composed of stromal cells including mesenchymal stem cells (MSCs), supports hematopoietic stem cell (HSC) self-renewal, and differentiation bands play an important role in leukemia development and progression. The reciprocal direct interaction between MSCs and CD34+ HSCs under physiological and pathological conditions is yet to be fully characterized. Here, we established a direct co-culture model between MSCs and CD34+ HSCs or MSCs and acute myeloid leukemia cells (THP-1, Molm-13, and primary cells from patients) to study heterocellular communication. Following MSCs-CD34+ HSCs co-culture, the expression of adhesion markers N-Cadherin and connexin 43 increased in both cell types, forming gap junction channels. Moreover, the clonogenic potential of CD34+ HSCs was increased. However, direct contact of acute myeloid leukemia cells with MSCs reduced the expression levels of connexin 43 and N-Cadherin in MSCs. The impairment in gap junction formation may potentially be due to a defect in the acute myeloid leukemia-derived MSCs. Interestingly, CD34+ HSCs and acute myeloid leukemia cell lines attenuated MSC osteoblastic differentiation upon prolonged direct cell-cell contact. In conclusion, under physiological conditions, connexin 43 and N-Cadherin interaction preserves stemness of both CD34+ HSCs and MSCs, a process that is compromised in acute myeloid leukemia, pointing to the possible role of gap junctions in modulating stemness.

Cell-cell interactions mediating primary and metastatic breast cancer dormancy.

Breast cancer remains one of the leading causes of death in women around the world. A majority of deaths from breast cancer occur due to cancer cells colonizing distant organ sites. When colonizing these distant organ sites, breast cancer cells have been known to enter into a state of dormancy for extended periods of time. However, the mechanisms that promote dormancy as well as dormant-to-proliferative switch are not fully understood. The tumor microenvironment plays a key role in mediating cancer cell phenotype including regulation of the dormant state. In this review, we highlight cell-cell interactions in the tumor microenvironment mediating breast cancer dormancy at the primary and metastatic sites. Specifically, we describe how immune cells from the lymphoid lineage, tumor-associated myeloid lineage cells, and stromal cells of non-hematopoietic origin as well as tissue resident stromal cells impact dormancy vs. proliferation in breast cancer cells as well as the associated mechanisms. In addition, we highlight the importance of developing model systems and the associated considerations that will be critical in unraveling the mechanisms that promote primary and metastatic breast cancer dormancy mediated via cell-cell interactions.

Cellular signatures in human blood track bone mineral density in postmenopausal women.

Osteoclasts are the sole bone-resorbing cells and are formed by the fusion of osteoclast precursor cells (OCPs) derived from myeloid lineage cells. Animal studies reveal that circulating OCPs (cOCPs) in blood travel to bone and fuse with bone-resident osteoclasts. However, the characteristics of human cOCPs and their association with bone diseases remain elusive. We have identified and characterized human cOCPs and found a positive association between cOCPs and osteoclast activity. Sorted cOCPs have a higher osteoclastogenic potential than other myeloid cells and effectively differentiate into osteoclasts. cOCPs exhibit distinct morphology and transcriptomic signatures. The frequency of cOCPs in the blood varies among treatment-naive postmenopausal women and has an inverse correlation with lumbar spine bone density and a positive correlation with serum CTX, a bone resorption marker. The increased cOCPs in treatment-naive patients with osteoporosis were significantly diminished by denosumab, a widely used antiresorptive therapy. Our study reveals the distinctive identity of human cOCPs and the potential link between the dynamic regulation of cOCPs and osteoporosis and its treatment. Taken together, our study enhances our understanding of human cOCPs and highlights a potential opportunity to measure cOCPs through a simple blood test, which could potentially identify high-risk individuals.

Modulatory Effect of Cucurbitacin D from Elaeocarpus hainanensis on ZNF217 Oncogene Expression in NPM-Mutated Acute Myeloid Leukemia.

Background/Objectives: The expression of oncogene zinc-finger protein 217 (ZNF217) has been reported to play a central role in cancer development, resistance, and recurrence. Therefore, targeting ZNF217 has been proposed as a possible strategy to fight cancer, and there has been much research on compounds that can target ZNF217. The present work investigates the chemo-preventive properties of cucurbitacin D, a compound with a broad range of anticancer effects, in hematological cancer cells, specifically with regard to its ability to modulate ZNF217 expression. Methods: Different cucurbitacins were isolated from the Vietnamese plant Elaeocarpus hainanensis. The purified compounds were tested on nucleophosmin-mutated acute myeloid leukemia and other hematological cancer cell lines to assess their effects on the cell cycle, cell viability and apoptosis, and the expression of ZNF217. Results: Cucurbitacin D resulted in a reduction in the number of acute myeloid leukemia cells by inducing an increase in apoptosis and blocking cell cycle progression. It also led to a significant decrease in ZNF217 expression in the nucleophosmin-mutated acute myeloid leukemia cell line but not in the other hematologic cancer cell lines. The reduction in ZNF217 expression contributed significantly to the blocking of cell cycle progression but did not affect apoptosis. Conclusions: The obtained results suggest that cucurbitacin D is a promising molecule for targeting mutated nucleophosmin or its pathway in acute myeloid leukemia cells, although further studies are needed for in-depth investigations into its specific mechanisms.