Tumor microenvironment restricts IL-10 induced multipotent progenitors to myeloid-lymphatic phenotype.

Lymphangiogenesis is induced by local pro-lymphatic growth factors and bone marrow (BM)-derived myeloid-lymphatic endothelial cell progenitors (M-LECP). We previously showed that M-LECP play a significant role in lymphangiogenesis and lymph node metastasis in clinical breast cancer (BC) and experimental BC models. We also showed that differentiation of mouse and human M-LECP can be induced through sequential activation of colony stimulating factor-1 (CSF-1) and Toll-like receptor-4 (TLR4) pathways. This treatment activates the autocrine interleukin-10 (IL-10) pathway that, in turn, induces myeloid immunosuppressive M2 phenotype along with lymphatic-specific proteins. Because IL-10 is implicated in differentiation of numerous lineages, we sought to determine whether this pathway specifically promotes the lymphatic phenotype or multipotent progenitors that can give rise to M-LECP among other lineages. Analyses of BM cells activated either by CSF-1/TLR4 ligands in vitro or orthotopic breast tumors in vivo showed expansion of stem/progenitor population and coincident upregulation of markers for at least four lineages including M2-macrophage, lymphatic endothelial, erythroid, and T-cells. Induction of cell plasticity and multipotency was IL-10 dependent as indicated by significant reduction of stem cell markers and those for multiple lineages in differentiated cells treated with anti-IL-10 receptor (IL-10R) antibody or derived from IL-10R knockout mice. However, multipotent CD11b+/Lyve-1+/Ter-119+/CD3e+ progenitors detected in BM appeared to split into a predominant myeloid-lymphatic fraction and minor subsets expressing erythroid and T-cell markers upon establishing tumor residence. Each sub-population was detected at a distinct intratumoral site. This study provides direct evidence for differences in maturation status between the BM progenitors and those reaching tumor destination. The study results suggest preferential tumor bias towards expansion of myeloid-lymphatic cells while underscoring the role of IL-10 in early BM production of multipotent progenitors that give rise to both hematopoietic and endothelial lineages.

Diet-induced obesity reduces bone marrow T and B cells and promotes tumor progression in a transplantable Vk*MYC model of multiple myeloma.

Obesity is associated with an increased risk of developing multiple myeloma (MM). The molecular mechanisms causing this association is complex and incompletely understood. Whether obesity affects bone marrow immune cell composition in multiple myeloma is not characterized. Here, we examined the effect of diet-induced obesity on bone marrow immune cell composition and tumor growth in a Vk*MYC (Vk12653) transplant model of multiple myeloma. We find that diet-induced obesity promoted tumor growth in the bone marrow and spleen and reduced the relative number of T and B cells in the bone marrow. Our results suggest that obesity may reduce MM immune surveillance and thus may contribute to increased risk of developing MM.

Digital morphology compared to the optical microscope: A validation study on reporting bone marrow aspirates.

INTRODUCTION: This study aims to evaluate the effectiveness and reliability of the utilization for clinical reporting of the evaluation of digital images of bone marrow aspirates by morphologists and their comparability with the classic microscopic morphological evaluation. METHODS: We scanned 180 consecutive bone marrow needle aspirates smears using the "Metafer4 VSlide" whole slide imaging (WSI) digital scanning system. We evaluated the statistical comparability and the risk of bias of the microscopic readings with those performed on the screen on the digitized medullary images. RESULTS: The evaluation of cellularity on the screen was equivalent, with a higher frequency of "normal" than the analysis of digital preparations. The means and medians of the percentage values obtained on the different cell populations with the microscopic and digital reading were comparable as the main categories are concerned, with an average difference equal to 0 for the neutrophilic and eosinophilic granulocytic series, at -0.2% for the total myeloid cells, at 1.2% for the erythroid series, at -0.4% for the lymphocytes and at -0.4% for the blasts. Dysplastic features were consistently identified in 69/71 cell lineages. CONCLUSION: Our study demonstrated that screen evaluation of digitized bone marrow needle aspirates provides quantitative and qualitative results comparable to traditional microscopic analysis of the corresponding slide smears. Digital images offer significant benefits in reducing the workload of experienced operators, reproducibility and sharing of observations, and image preservation. Even in routine diagnostic activities, their use does not alter the quality of the results obtained in evaluating bone marrow needle aspirates.

Determination of age-dependent bone marrow normocellularity.

OBJECTIVES: Determination of bone marrow cellularity is a key part of bone marrow examination because it provides a small window into a patient's current state of hematopoietic well-being. Traditionally, bone marrow cellularity is estimated semiquantitatively through microscopic examination of core biopsy specimens harvested from the iliac crest of the pelvic bone. Bone marrow cellularity is then designated as hypercellular, normocellular, or hypocellular based on the patient's age. This assessment can have significant clinical impact, but the variation in the age-adjusted normocellularity range is not sufficiently characterized because of a lack of study data, especially in older patients (those older than 70 years of age). This study further established the normal range of bone marrow cellularity, particularly in older adults. METHODS: In this study, 570 benign staging and healthy donor bone marrows from patients 1 year to 93 years of age were analyzed for cellularity. RESULTS: Linear regression modeling demonstrates that cellularity in adults declines approximately 3% per decade, including after the seventh decade of life. The 90% reference interval for normocellularity in United States is 30% to 75% for those aged 18 to 90 years. CONCLUSIONS: The findings revealed a more stable and slower rate of decline in cellularity with age in adults than the widely used linear model of "100% minus the patient age in decades." Normocellularity is better modeled based on age group. In those younger than 20 years of age, normocellularity ranges from 45% to 85% (mean [SD], 65% [20%]), as defined by Friebert et al in 1998. Based on our study finding of a little less than 3% decline per decade of age, the following is our recommendation for normocellularity range: For individuals 20 to 40 years of age, it ranges from 40% to 70% (mean [SD], 55% [15%]); for individuals 40 to 60 years of age, it ranges from 35% to 65% (mean [SD], 50% [15%]); and for individuals older than 60 years of age, it ranges from 30% to 60% (mean [SD], 45% [15%]). Interestingly, those older than 70 years of age do not show a significant decrease from those aged 60 to 69 years.

Stromal bone marrow fibroblasts and mesenchymal stem cells support acute myeloid leukaemia cells and promote therapy resistance.

The bone marrow (BM) is the primary site of adult haematopoiesis, where stromal elements (e.g. fibroblasts and mesenchymal stem cells [MSCs]) work in concert to support blood cell development. However, the establishment of an abnormal clone can lead to a blood malignancy, such as acute myeloid leukaemia (AML). Despite our increased understanding of the pathophysiology of the disease, patient survival remains suboptimal, mainly driven by the development of therapy resistance. In this review, we highlight the importance of bone marrow fibroblasts and MSCs in health and acute myeloid leukaemia and their impact on patient prognosis. We discuss how stromal elements reduce the killing effects of therapies via a combination of contact-dependent (e.g. integrins) and contact-independent (i.e. secreted factors) mechanisms, accompanied by the establishment of an immunosuppressive microenvironment. Importantly, we underline the challenges of therapeutically targeting the bone marrow stroma to improve acute myeloid leukaemia patient outcomes, due to the inherent heterogeneity of stromal cell populations. LINKED ARTICLES: This article is part of a themed issue on Cancer Microenvironment and Pharmacological Interventions. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.2/issuetoc.

Unbiased Single-Cell Sequencing of Hematopoietic and Immune Cells from Aplastic Anemia Reveals the Contributors of Hematopoiesis Failure and Dysfunctional Immune Regulation.

Aplastic anemia (AA) is a bone marrow (BM) failure syndrome mediated by hyperactivated T-cells with heterogeneous pathogenic factors. The onset of BM failure cannot be accurately determined in humans; therefore, exact pathogenesis remains unclear. In this study, a cellular atlas and microenvironment interactions is established using unbiased single-cell RNA-seq, along with multi-omics analyses (mass cytometry, cytokine profiling, and oxidized fatty acid metabolomics). A new KIR+ CD8+ regulatory T cells (Treg) subset is identified in patients with AA that engages in immune homeostasis. Conventional CD4+ T-cells differentiate into highly differentiated T helper cells with type 2 cytokines (IL-4, IL-6, and IL-13), GM-SCF, and IL-1ß. Immunosuppressive homeostasis is impaired by enhanced apoptosis of activated Treg cells. Pathological Vδ1 cells dominated the main fraction of γδ T-cells. The B/plasma, erythroid, and myeloid lineages also exhibit substantial pathological features. Interactions between TNFSF12-TNFRSF12A, TNF-TNFRSF1A, and granzyme-gasdermin are associated with the cell death of hematopoietic stem/progenitor (HSPCs), Treg, and early erythroid cells. Ferroptosis, a major driver of HSPCs destruction, is identified in patients with AA. Furthermore, a case of twins with AA is reported to enhance the persuasiveness of the analysis. These results collectively constitute the cellular atlas and microenvironment interactions in patients with AA and provide novel insights into the development of new therapeutic opportunities.

Engagement of Mesenchymal Stromal Cells in the Remodeling of the Bone Marrow Microenvironment in Hematological Cancers.

Mesenchymal stromal cells (MSCs) are a subset of heterogeneous, non-hematopoietic fibroblast-like cells which play important roles in tissue repair, inflammation, and immune modulation. MSCs residing in the bone marrow microenvironment (BMME) functionally interact with hematopoietic stem progenitor cells regulating hematopoiesis. However, MSCs have also emerged in recent years as key regulators of the tumor microenvironment. Indeed, they are now considered active players in the pathophysiology of hematologic malignancies rather than passive bystanders in the hematopoietic microenvironment. Once a malignant event occurs, the BMME acquires cellular, molecular, and epigenetic abnormalities affecting tumor growth and progression. In this context, MSC behavior is affected by signals coming from cancer cells. Furthermore, it has been shown that stromal cells themselves play a major role in several hematological malignancies' pathogenesis. This bidirectional crosstalk creates a functional tumor niche unit wherein tumor cells acquire a selective advantage over their normal counterparts and are protected from drug treatment. It is therefore of critical importance to unveil the underlying mechanisms which activate a protumor phenotype of MSCs for defining the unmasked vulnerabilities of hematological cancer cells which could be pharmacologically exploited to disrupt tumor/MSC coupling. The present review focuses on the current knowledge about MSC dysfunction mechanisms in the BMME of hematological cancers, sustaining tumor growth, immune escape, and cancer progression.

The impact of erythroblast enucleation efficiency on the severity of anemia in patients with myelodysplastic syndrome.

Anemia is the most common manifestation in myelodysplastic syndrome (MDS) patients, but the cause of ineffective hematopoiesis is not fully understood. Enucleation is an important event in the maturation process of erythroblasts. According to a series of morphological phenotypes of the pathological development of MDS erythroblasts, we speculate that there may be enucleation disorders. To verify this hypothesis, we cultured MDS bone marrow CD34+ cells in vitro and induced erythroblast development. The results showed that erythroblast enucleation in MDS was significantly lower than that in the normal group, and the rate of enucleation was positively correlated with hemoglobin concentration. Risk stratification of MDS was performed to further analyze the differences in enucleation among the normal group, low-middle risk group and high-risk group. The results showed that the enucleation rate of the high risk group was higher than that of the low-middle risk group but still lower than that of the normal group. Moreover, the expression of pERK and pAKT in MDS erythroblasts in the high risk group was higher than that in the normal group, while the expression of pERK and pAKT in the low-middle risk group was lower than that in the normal group. Furthermore, the enucleation of MDS was positively correlated with the phosphorylation degree of ERK and AKT. In conclusion, this study reveals that the enucleation of erythroblasts is one of the possible causes of anemia in MDS. Video Abstract.

Abnormal adipogenic signaling in the bone marrow mesenchymal stem cells contributes to supportive microenvironment for leukemia development.

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematological malignancy, associated with unfavorable patient outcome, primarily due to disease relapse. Mesenchymal stem cells (MSCs) residing in the bone marrow (BM) niche are the source of mesenchyma-derived subpopulations, including adipocytes, and osteocytes, that are critical for normal hematopoiesis. This study aimed to characterize BM-derived adipocyte/osteocyte fractions and their crosstalk with AML cells as a potential mechanism underlying leukemogenesis. METHODS: BM cell subpopulations derived from primary AML patients were evaluated using humanized ex-vivo and in-vivo models, established for this study. The models comprised AML blasts, normal hematopoietic stem and progenitor cells and mesenchymal stromal subpopulations. ELISA, FACS analysis, colony forming unit assay, whole exome sequencing and real-time qPCR were employed to assess the differentiation capacity, genetic status, gene expression and function of these cell fractions. To explore communication pathways between AML cells and BM subpopulations, levels of signaling mediators, including cytokines and chemokines, were measured using the ProcartaPlex multiplex immunoassay. RESULTS: The study revealed deficiencies in adipogenic/osteogenic differentiation of BM-MSCs derived from AML patients, with adipocytes directly promoting survival and clonogenicity of AML cells in-vitro. In whole exome sequencing of BM-MSC/stromal cells, the AHNAK2 gene, associated with the stimulation of adipocyte differentiation, was found to be mutated and significantly under-expressed, implying its abnormal function in AML. The evaluation of communication pathways between AML cells and BM subpopulations demonstrated pronounced alterations in the crosstalk between these cell fractions. This was reflected by significantly elevated levels of signaling mediators cytokines/chemokines, in AML-induced adipocytes/osteocytes compared to non-induced MSCs, indicating abnormal hematopoiesis. Furthermore, in-vivo experiments using a fully humanized 3D scaffold model, showed that AML-induced adipocytes were the dominant component of the tumor microenvironment, providing preferential support to leukemia cell survival and proliferation. CONCLUSIONS: This study has disclosed direct contribution of impaired functional, genetic and molecular properties of AML patient-derived adipocytes to effective protection of AML blasts from apoptosis and to stimulation of their growth in vitro and in vivo, which overall leads to disease propagation and relapse. The detected AHNAK2 gene mutations in AML-MSCs point to their involvement in the mechanism underlying abnormal adipogenesis. Video Abstract.

Therapeutic Effect of Human Bone Marrow- and Adipose-Derived Stem Cells on Intervertebral Disc Degeneration: A Comparative Study on Rats.

BACKGROUND: Intervertebral disc degeneration (IVD) is a pain-inflicting disorder, posing a serious threat to the elderly, and new therapies are urgently needed. In this study, we examined the potential therapeutic effect of mesenchymal stem cells (MSCs) transplantation on IVD. METHODS: Both human adipose-derived stem cells (hADSCs) and human bone marrow mesenchymal stem cells (hBMSCs) provided by a volunteer were non-contact co-cultured with the human nucleus pulposus cells (hNPCs) to determine the efficacy of hNPCs-oriented differentiation. Flow cytometry was used to characterize the purity of hADSCs/hBMSCs. We determined the expression of surface antigen molecules, such as CD73, CD105, CD90, CD31, HLA-DR, CD34 and CD45, using flow cytometry. Osteogenic and lipogenic differentiations demonstrated by the cells were identified with Alizarin red and Oil red O staining, respectively, and changes in type II collagen and proteoglycan levels were detected by immunofluorescence. Myeloid cell-related mRNA and protein expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. The therapeutic effect of hADSCs and hBMSCs on IVD was evaluated in experimental rats, in which degeneration was induced by needling the annulus fibrosus of the caudal intervertebral disc. RESULTS: As evidenced by the presence of hNPCs-like morphology, both hBMSCs and hADSC could effectively differentiate into hNPCs. Using flow cytometry assays, we found high expression of type II collagen (COL2) and aggrecan (ACAN) protein in the hNPCs-like tissue. Treatment with hADSCs and hBMSCs attenuated IVD progression in the rats, and most importantly, there was no significant difference between the therapeutic effects of both types of cells on IVD, on the basis of the COL2 and SRY-Box Transcription Factor 9 (SOX9) protein expression and the histological results. Findings from the animal studies also suggested that both hADSCs and hBMSCs transplantation could be applied in IVD treatment. CONCLUSIONS: In summary, both hADSCs and hBMSCs can attenuate the progression of IVD by delaying, rather than completely reversing the deterioration of disc degeneration, and there is no significant difference between hADSCs and hBMSCs on the therapeutic effects.

Automated bone marrow cell classification through dual attention gates dense neural networks.

PURPOSE: The morphology of bone marrow cells is essential in identifying malignant hematological disorders. The automatic classification model of bone marrow cell morphology based on convolutional neural networks shows considerable promise in terms of diagnostic efficiency and accuracy. However, due to the lack of acceptable accuracy in bone marrow cell classification algorithms, automatic classification of bone marrow cells is now infrequently used in clinical facilities. To address the issue of precision, in this paper, we propose a Dual Attention Gates DenseNet (DAGDNet) to construct a novel efficient, and high-precision bone marrow cell classification model for enhancing the classification model's performance even further. METHODS: DAGDNet is constructed by embedding a novel dual attention gates (DAGs) mechanism in the architecture of DenseNet. DAGs are used to filter and highlight the position-related features in DenseNet to improve the precision and recall of neural network-based cell classifiers. We have constructed a dataset of bone marrow cell morphology from the First Affiliated Hospital of Chongqing Medical University, which mainly consists of leukemia samples, to train and test our proposed DAGDNet together with the bone marrow cell classification dataset. RESULTS: When evaluated on a multi-center dataset, experimental results show that our proposed DAGDNet outperforms image classification models such as DenseNet and ResNeXt in bone marrow cell classification performance. The mean precision of DAGDNet on the Munich Leukemia Laboratory dataset is 88.1%, achieving state-of-the-art performance while still maintaining high efficiency. CONCLUSION: Our data demonstrate that the DAGDNet can improve the efficacy of automatic bone marrow cell classification and can be exploited as an assisting diagnosis tool in clinical applications. Moreover, the DAGDNet is also an efficient model that can swiftly inspect a large number of bone marrow cells and offers the benefit of reducing the probability of an incorrect diagnosis.

The inhibitor of MyoD Family A (I-MFA) regulates megakaryocyte lineage commitment and terminal differentiation.

Hematopoiesis and lineage commitment are regulated by several conserved cell-intrinsic signaling pathways, including MAPKs and ß-catenin/TCF/LEF. The Inhibitor of MyoD Family A (I-MFA), a transcriptional repressor and tumor suppressor gene, interacts with these pathways and is dysregulated in chronic and acute myeloid leukemias, suggesting it may play a role in development and differentiation during hematopoiesis. To study this, immune cell populations in the bone marrow (BM) and periphery were analyzed in mice lacking Mdfi, encoding I-MFA (I-MFA-/-), and wild type (WT) controls. I-MFA-/- mice had reduced spleen and BM cellularity, with significant hyposplenism, compared to WT mice. In blood, total red blood cells and platelet counts were significantly reduced in I-MFA-/- mice, accompanied by a reduction in megakaryocyte (MK)/erythrocyte progenitor cells and an increase in myeloid progenitors in BM compared to WT mice. The K562 cell line exhibits PMA-induced MK differentiation, and shRNA knockdown of I-MFA resulted in reduced differentiation compared to control, with an increase and prolongation in phospho-JNK and phospho-ERK signaling. Overexpression of I-MFA promoted MK differentiation. These results suggest I-MFA plays a cell-intrinsic role in the response to differentiation signals, an effect that can be explored in the context of hematological cancers or other blood proliferative disorders.

Ultraviolet radiation shapes dendritic cell leukaemia transformation in the skin.

Tumours most often arise from progression of precursor clones within a single anatomical niche. In the bone marrow, clonal progenitors can undergo malignant transformation to acute leukaemia, or differentiate into immune cells that contribute to disease pathology in peripheral tissues1-4. Outside the marrow, these clones are potentially exposed to a variety of tissue-specific mutational processes, although the consequences of this are unclear. Here we investigate the development of blastic plasmacytoid dendritic cell neoplasm (BPDCN)-an unusual form of acute leukaemia that often presents with malignant cells isolated to the skin5. Using tumour phylogenomics and single-cell transcriptomics with genotyping, we find that BPDCN arises from clonal (premalignant) haematopoietic precursors in the bone marrow. We observe that BPDCN skin tumours first develop at sun-exposed anatomical sites and are distinguished by clonally expanded mutations induced by ultraviolet (UV) radiation. A reconstruction of tumour phylogenies reveals that UV damage can precede the acquisition of alterations associated with malignant transformation, implicating sun exposure of plasmacytoid dendritic cells or committed precursors during BPDCN pathogenesis. Functionally, we find that loss-of-function mutations in Tet2, the most common premalignant alteration in BPDCN, confer resistance to UV-induced cell death in plasmacytoid, but not conventional, dendritic cells, suggesting a context-dependent tumour-suppressive role for TET2. These findings demonstrate how tissue-specific environmental exposures at distant anatomical sites can shape the evolution of premalignant clones to disseminated cancer.

Myeloid-like tumor hybrid cells in bone marrow promote progression of prostate cancer bone metastasis.

BACKGROUND: Bone metastasis is the leading cause of death in patients with prostate cancer (PCa) and currently has no effective treatment. Disseminated tumor cells in bone marrow often obtain new characteristics to cause therapy resistance and tumor recurrence. Thus, understanding the status of disseminated prostate cancer cells in bone marrow is crucial for developing a new treatment. METHODS: We analyzed the transcriptome of disseminated tumor cells from a single cell RNA-sequencing data of PCa bone metastases. We built a bone metastasis model through caudal artery injection of tumor cells, and sorted the tumor hybrid cells by flow cytometry. We performed multi-omics analysis, including transcriptomic, proteomic and phosphoproteomic analysis, to compare the difference between the tumor hybrid cells and parental cells. In vivo experiments were performed to analyze the tumor growth rate, metastatic and tumorigenic potential, drug and radiation sensitivity in hybrid cells. Single cell RNA-sequencing and CyTOF were performed to analyze the impact of hybrid cells on tumor microenvironment. RESULTS: Here, we identified a unique cluster of cancer cells in PCa bone metastases, which expressed myeloid cell markers and showed a significant change in pathways related to immune regulation and tumor progression. We found that cell fusion between disseminated tumor cells and bone marrow cells can be source of these myeloid-like tumor cells. Multi-omics showed the pathways related to cell adhesion and proliferation, such as focal adhesion, tight junction, DNA replication, and cell cycle, were most significantly changed in these hybrid cells. In vivo experiment showed hybrid cells had a significantly increased proliferative rate, and metastatic potential. Single cell RNA-sequencing and CyTOF showed tumor-associated neutrophils/monocytes/macrophages were highly enriched in hybrid cells-induced tumor microenvironment with a higher immunosuppressive capacity. Otherwise, the hybrid cells showed an enhanced EMT phenotype with higher tumorigenicity, and were resistant to docetaxel and ferroptosis, but sensitive to radiotherapy. CONCLUSION: Taken together, our data demonstrate that spontaneous cell fusion in bone marrow can generate myeloid-like tumor hybrid cells that promote the progression of bone metastasis, and these unique population of disseminated tumor cells can provide a potential therapeutic target for PCa bone metastasis.

[Latest Findings on the Mechanism of the Interaction Between Multiple Myeloma Cells and Bone Marrow Microenvironment].

Multiple myeloma (MM) is a hematologic malignancy of terminally differentiated plasma cells. The mechanisms of the pathogenesis and progression of MM include genetic abnormalities of the MM cells and the interaction between MM cells and bone marrow microenvironment (BMME). MM cells start malignant proliferation in BMME and contribute to the pathogenesis and progression of MM through direct or indirect interactions between cells and the extracellular matrix. Exploring the mechanism of interaction between MM cells and the microenvironment is crucial to improving our understanding of the pathogenesis and progression of MM and early diagnosis and treatment. In addition, the metabolic reprogramming of tumors is one of the key issues of oncology research. Herein, we summarized published findings on the the altered metabolic reprogramming of MM and the characteristics of MM metabolic-microbial interactions in order to gain an in-depth understanding of MM pathogenesis and progression and drug resistance mechanisms, and ultimately to explore for new strategies for MM treatment.

Discrepancy in transcriptomic profiling between CD34 + stem cells and primary bone marrow cells in myelodysplastic neoplasm.

Differentially expressed genes (DEGs) biomarkers can be used to help diagnose and monitor the disease, as well as to determine which treatments are most effective. So, given the complexity of Myelodysplastic neoplasm (MDS), it is difficult to determine the impact and disparities of DEGs between CD34+ HSC (hematopoietic stem cells) or primary bone marrow cells (PBMC) in MDS pathogenesis, and therefore it remains largely unknown. Here, we performed an in-silico transcriptome analysis on CD34+ HSC and PBMC from 1092 MDS patients analyzing the divergences between differential gene expression patterns in these two cell types as potential pathogenic biomarkers for MDS. Initially, we observed a difference of 7117 expressed transcripts between PBMC (n = 40,165) and CD34 +HSC (n = 33,048). Also, we identified that CD34+ HSC and PBMC samples showed 240 and 2948 DEGs, respectively. In summary, we identified DEGs disparities in CD34+ HSC and PBMC cell types. However, there was a certain similarity of the activated pathways in both cellular samples based on Gene Ontology and KEGG pathways enrichment analyses. Our results provide novel insights into novel DEGs biomarkers to MDS pathogenesis with clinical significance. AVAILABILITY OF DATA AND MATERIALS: All microarray databases were obtained from Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/). To evaluate the biological function of differentially expressed genes, the DAVID (Database for Annotation, Visualization and Integrated Discovery tool was used) (https://david.ncifcrf.gov/).

Selective homing of CAR-CIK cells to the bone marrow niche enhances control of the acute myeloid leukemia burden.

Acute myeloid leukemia (AML) is a hematological malignancy derived from neoplastic myeloid progenitor cells characterized by abnormal clonal proliferation and differentiation. Although novel therapeutic strategies have recently been introduced, the prognosis of AML is still unsatisfactory. So far, the efficacy of chimeric antigen receptor (CAR)-T-cell therapy in AML has been hampered by several factors, including the poor accumulation of the blood-injected cells in the leukemia bone marrow (BM) niche in which chemotherapy-resistant leukemic stem cells reside. Thus, we hypothesized that overexpression of CXCR4, whose ligand CXCL12 is highly expressed by BM stromal cells within this niche, could improve T-cell homing to the BM and consequently enhance their intimate contact with BM-resident AML cells, facilitating disease eradication. Specifically, we engineered conventional CD33.CAR-cytokine-induced killer cells (CIKs) with the wild-type (wt) CXCR4 and the variant CXCR4R334X, responsible for leukocyte sequestration in the BM of patients with warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis syndrome. Overexpression of both CXCR4wt and CXCR4mut in CD33.CAR-CIKs resulted in significant improvement of chemotaxis toward recombinant CXCL12 or BM stromal cell-conditioned medium, with no observed impairment of cytotoxic potential in vitro. Moreover, CXCR4-overexpressing CD33.CAR-CIKs showed enhanced in vivo BM homing, associated with a prolonged retention for the CXCR4R334X variant. However, only CD33.CAR-CIKs coexpressing CXCR4wt but not CXCR4mut exerted a more sustained in vivo antileukemic activity and extended animal survival, suggesting a noncanonical role for CXCR4 in modulating CAR-CIK functions independent of BM homing. Taken together, these data suggest that arming CAR-CIKs with CXCR4 may represent a promising strategy for increasing their therapeutic potential for AML.