Bone Marrow Extracellular Matrix Research Articles

Newly identified roles for PIEZO1 mechanosensor in controlling normal megakaryocyte development and in primary myelofibrosis.

Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in mouse and human Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Yet, a role for PIEZO1 in megakaryopoiesis and proplatelet formation has never been investigated. Here, we show that activation of PIEZO1 increases the number of immature Mks in mice, while the number of mature Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its activation reduces Mk size, ploidy, maturation, and proplatelet extension. Resulting effects of PIEZO1 activation on Mks resemble the profile in Primary Myelofibrosis (PMF). Intriguingly, Mks derived from Jak2V617F PMF mice show significantly elevated PIEZO1 expression, compared to wild-type controls. Accordingly, Mks isolated from bone marrow aspirates of JAK2V617F PMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2V617F PMF patients are rescued upon PIEZO1 inhibition. Together, our data suggest that PIEZO1 places a brake on Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease.

Newly Identified Roles for PIEZO1 Mechanosensor in Controlling Normal Megakaryocyte Development and in Primary Myelofibrosis

Introduction: Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix (ECM) to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Primary Myelofibrosis (PMF) is a Chromosome Philadelphia-negative Myeloproliferative Neoplasm (MPN), characterized by altered stiffer ECM and megakaryocytosis, often driven by the gain-of-function mutation in JAK2 V617F. Here, we study a role for PIEZO1 in megakaryopoiesis and proplatelet formation under normal physiological conditions and in the context of PMF. Methods: Bone marrow Mks from C57BL/6J control and myelofibrotic mice carrying the human JAK2 V617F+ mutation, or Mks derived from stem cells of patients carrying the same mutation were analyzed at mRNA and protein levels for Piezo1 expression. Effects of Piezo1 activation or inhibition on Mk maturation and ploidy were assessed by flow cytometry, and proplatelet formation by fluorescent microscopy. Human peripheral blood samples from PMF patients, diagnosed according to established criteria, or healthy individuals were used to compare the two categories for Piezo1 relative expression on mRNA and protein levels and platelet biogenesis. Mk-specific double knockout mice of Piezo1/2 were generated to compare platelet counts under normal conditions or bone marrow ablation with 5-FU challenge. Culture of mouse and human Mks in different 3D microenvironments was used to demonstrate in vitro association of Piezo1 expression levels with different ECM stiffness substrates. Results: Mouse Mks express PIEZO1 and negligible levels of PIEZO2. Pharmacological activation of mouse PIEZO1 increases the number of immature CD41 + Mks, while the number of mature CD41 +CD42 + Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its pharmacological activation reduces Mk size, ploidy, maturation, and proplatelet development. Resulting effects of PIEZO1 activation on Mks resemble the profile in PMF. Intriguingly, Mks derived from PMF mice bearing the Jak2 V617F mutation show significantly elevated PIEZO1 expression, compared to wild-type controls. Pharmacological activation of PIEZO1 in these cells significantly augments the number of immature CD41 + Mks, and sharply reduces the number of mature CD41 +CD42 + cells and ploidy level. Accordingly, Mks isolated from bone marrow aspirates of JAK2 V617FPMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Additionally, PIEZO1 was overexpressed in 3D culture of human and mouse Mks in stiffer microenvironments as compared to softer ones and liquid medium. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2 V617FPMF patients are rescued upon PIEZO1 inhibition with GsMTx4. Discussion: Our data show that PIEZO1 might serve as a break of Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease. Our finding that inhibition of PIEZO1 activity partially rescues the aberrant Mk phenotype associated with PMF suggests that GsMTx4 holds potential as a therapeutic strategy to mitigate the pathological effects observed in Mk development in PMF patients.

Immunohistochemistry of bone marrow extracellular matrix in ph-negative myeloproliferative diseases

Introduction Evaluation of immunohistochemical expression of matrix metalloproteinases 2 and 9 (MMP2, MMP9), their inhibitors (TIMP1, TIMP2), fibroblast growth factor (FGF2), transforming growth factor beta (TGFB1) and collagen type III in the bone marrow of patients with Ph-negative myeloproliferative neoplasms (MPN) is of great importance.The aim of the study was the evaluation of expression of extracellular matrix components (MMP-2, MMP-9, TIMP1, TIMP-2, FGF2, TFGB1, Collagen III) involved in myelofibrosis progression in bone marrow trepan biopsies depending on mutational status of patients with CMPD.Materials and methods We analyzed 108 bone marrow biopsies of patients with MPN, which were divided into 3 groups: JAK2-positive (n=62), CARL-positive (n=25) and triple-negative (n=21). Whole-slide sections were immunostained using antibodies against MMP-2, MMP-9, TIMP-1, TIMP-2, FGF2, TGFB1, collagen type III and scored by ImageJ plugin software. We used Kruskal- Wallis test and Mann-Whitney U-test for comparisons of differences in medians. Spearman’s rank order correlation was calculated. Statistical significance was set at p<0,05.Results and Discussion MMP2 expression was observed in megakaryocytes. MMP9 expression was observed in neutrophils, macrophages and the bone marrow extracellular matrix (EM). TIMP1 expression was observed in the EM. TIMP-2, FGF2, TGFB1 and collagen type III expression was observed in megakaryocytes and the EM. Kruskal-Wallis test determined the differences between all 3 groups (MMP- 2 p< 0,001, MMP-9 p=0,023, TIMP-1 p< 0,001, TIMP-2 p< 0,001, FGF2 p< 0,001, TGFB1 p< 0,001, collagen type III p< 0,001). Mann-Whitney U-test determined the most differences between JAK2- and CALR-groups (MMP-2 p=0,001, MMP-9 p=0,001, TIMP-1 p=0,001, FGF2 p=0,001, TGFB1 p=0,001, collagen type III p=0,001), except TIMP-2. There was the weak and moderate positive correlation between JAK2-mutation and the immunohistochemistry expression of EM components, also the weak negative correlation between CALR-mutation and the immunohistochemistry expression of EM components.Conclusion The bone marrow immunohistochemistry expression of MMP-2, MMP-9, TIMP-1, TIMP-2, FGF2, TGFB1, collagen type III depends on driver mutations. It may be useful for understanding of fibrosis pathogenesis and prognosis estimate of Ph-negative MPN.

Biophysical cues of bone marrow-inspired scaffolds regulate hematopoiesis of hematopoietic stem and progenitor cells

Hematopoietic stem cells (HSCs) are adult multipotential stem cells with the capacity to differentiate into all blood cells and immune cells, which are essential for maintaining hematopoietic homeostasis throughout the lifespan and reconstituting damaged hematopoietic system after myeloablation. However, the clinical application of HSCs is hindered by the imbalance of its self-renewal and differentiation during in vitro culture. Considering the fact that HSC fate is uniquely determined by natural bone marrow microenvironment, various elaborate cues in this hematopoietic micro-niche provide an excellent reference for the regulation of HSCs.Inspired by the bone marrow extracellular matrix (ECM) network, we designed degradable scaffolds by orchestrating the physical parameters to investigate the decoupling effects of Young's modulus and pore size of three-dimensional (3D) matrix materials on the fate of hematopoietic stem and progenitor cells (HSPCs). We ascertained that the scaffold with larger pore size (80 μm) and higher Young's modulus (70 kPa) was more favorable for HSPCs proliferation and the maintenance of stemness related phenotypes. Through in vivo transplantation, we further validated that scaffolds with higher Young's modulus were more propitious in maintaining the hematopoietic function of HSPCs. We systematically screened an optimized scaffold for HSPC culture which could significantly improve the cell function and self-renewal ability compared with traditional two-dimensional (2D) culture. Together, these results indicate the important role of biophysical cues in regulating HSC fate and pave the way for the parameter design of 3D HSC culture system.

Fabrication of a three-dimensional bone marrow niche-like acute myeloid Leukemia disease model by an automated and controlled process using a robotic multicellular bioprinting system

BackgroundAcute myeloid leukemia (AML) is a hematological malignancy that remains a therapeutic challenge due to the high incidence of disease relapse. To better understand resistance mechanisms and identify novel therapies, robust preclinical models mimicking the bone marrow (BM) microenvironment are needed. This study aimed to achieve an automated fabrication process of a three-dimensional (3D) AML disease model that recapitulates the 3D spatial structure of the BM microenvironment and applies to drug screening and investigational studies.MethodsTo build this model, we investigated a unique class of tetramer peptides with an innate ability to self-assemble into stable hydrogel. An automated robotic bioprinting process was established to fabricate a 3D BM (niche-like) multicellular AML disease model comprised of leukemia cells and the BM’s stromal and endothelial cellular fractions. In addition, monoculture and dual-culture models were also fabricated. Leukemia cell compatibility, functionalities (in vitro and in vivo), and drug assessment studies using our model were performed. In addition, RNAseq and gene expression analysis using TaqMan arrays were also performed on 3D cultured stromal cells and primary leukemia cells.ResultsThe selected peptide hydrogel formed a highly porous network of nanofibers with mechanical properties similar to the BM extracellular matrix. The robotic bioprinter and the novel quadruple coaxial nozzle enabled the automated fabrication of a 3D BM niche-like AML disease model with controlled deposition of multiple cell types into the model. This model supported the viability and growth of primary leukemic, endothelial, and stromal cells and recapitulated cell-cell and cell-ECM interactions. In addition, AML cells in our model possessed quiescent characteristics with improved chemoresistance attributes, resembling more the native conditions as indicated by our in vivo results. Moreover, the whole transcriptome data demonstrated the effect of 3D culture on enhancing BM niche cell characteristics. We identified molecular pathways upregulated in AML cells in our 3D model that might contribute to AML drug resistance and disease relapse.ConclusionsOur results demonstrate the importance of developing 3D biomimicry models that closely recapitulate the in vivo conditions to gain deeper insights into drug resistance mechanisms and novel therapy development. These models can also improve personalized medicine by testing patient-specific treatments.Graphical

Hyaluronic acid–GPRC5C signalling promotes dormancy in haematopoietic stem cells

Bone marrow haematopoietic stem cells (HSCs) are vital for lifelong maintenance of healthy haematopoiesis. In inbred mice housed in gnotobiotic facilities, the top of the haematopoietic hierarchy is occupied by dormant HSCs, which reversibly exit quiescence during stress. Whether HSC dormancy exists in humans remains debatable. Here, using single-cell RNA sequencing, we show a continuous landscape of highly purified human bone marrow HSCs displaying varying degrees of dormancy. We identify the orphan receptor GPRC5C, which enriches for dormant human HSCs. GPRC5C is also essential for HSC function, as demonstrated by genetic loss- and gain-of-function analyses. Through structural modelling and biochemical assays, we show that hyaluronic acid, a bone marrow extracellular matrix component, preserves dormancy through GPRC5C. We identify the hyaluronic acid–GPRC5C signalling axis controlling the state of dormancy in mouse and human HSCs.

Emilin-2 is a component of bone marrow extracellular matrix regulating mesenchymal stem cell differentiation and hematopoietic progenitors

BackgroundDissection of mechanisms involved in the regulation of bone marrow microenvironment through cell–cell and cell–matrix contacts is essential for the detailed understanding of processes underlying bone marrow activities both under physiological conditions and in hematologic malignancies. Here we describe Emilin-2 as an abundant extracellular matrix component of bone marrow stroma.MethodsImmunodetection of Emilin-2 was performed in bone marrow sections of mice from 30 days to 6 months of age. Emilin-2 expression was monitored in vitro in primary and mesenchymal stem cell lines under undifferentiated and adipogenic conditions. Hematopoietic stem cells and progenitors in bone marrow of 3- to 10-month-old wild-type and Emilin-2 null mice were analyzed by flow cytometry.ResultsEmilin-2 is deposited in bone marrow extracellular matrix in an age-dependent manner, forming a meshwork that extends from compact bone boundaries to the central trabecular regions. Emilin-2 is expressed and secreted by both primary and immortalized bone marrow mesenchymal stem cells, exerting an inhibitory action in adipogenic differentiation. In vivo Emilin-2 deficiency impairs the frequency of hematopoietic stem/progenitor cells in bone marrow during aging.ConclusionOur data provide new insights in the contribution of bone marrow extracellular matrix microenvironment in the regulation of stem cell niches and hematopoietic progenitor differentiation.

Biomimetic 3D Environment Based on Microgels as a Model for the Generation of Drug Resistance in Multiple Myeloma.

The development of three-dimensional environments to mimic the in vivo cellular response is a problem in the building of disease models. This study aimed to synthesize and validate three-dimensional support for culturing monoclonal plasma cells (mPCs) as a disease model for multiple myeloma. The three-dimensional environment is a biomimetic microgel formed by alginate microspheres and produced on a microfluidic device whose surface has been functionalized by a layer-by-layer process with components of the bone marrow’s extracellular matrix, which will interact with mPC. As a proof of concept, RPMI 8226 cell line cells were cultured in our 3D culture platform. We proved that hyaluronic acid significantly increased cell proliferation and corroborated its role in inducing resistance to dexamethasone. Despite collagen type I having no effect on proliferation, it generated significant resistance to dexamethasone. Additionally, it was evidenced that both biomolecules were unable to induce resistance to bortezomib. These results validate the functionalized microgels as a 3D culture system that emulates the interaction between tumoral cells and the bone marrow extracellular matrix. This 3D environment could be a valuable culture system to test antitumoral drugs efficiency in multiple myeloma.

Characterizing Naked Nuclei Frequency and Movement in Primary AML Cell Culture Using an ECM-Based Model

INTRODUCTIONNaked nuclei (NN) are observed upon examining bone marrow aspirate slides from healthy individuals and from those with hematologic malignancies, but are not well understood. Without characteristic findings like cytoplasm or plasma membrane, NN are considered remnants of slide preparation or are discounted as cells of undetermined significance. NN have been associated with poor prognosis in several solid cancers. Understanding the significance of NN in hematologic malignancies such as acute myelogenous leukemia (AML) may elucidate valuable diagnostic and prognostic knowledge. Decellularized Wharton's jelly matrix (DWJM) is an extra cellular matrix (ECM)-based in vitro model that shares similar elements with the bone marrow ECM and can be used as scaffolding to culture leukemia cells. We hypothesize that NN exist in AML and interact with other cells, suggesting potential biological relevance within the bone marrow microenvironment.METHODSPrimary AML samples obtained by leukapheresis were cultured in suspension with growth media or in the presence of DWJM submerged in growth media. In samples grown with DWJM, cells that were non-adherent to the matrix were collected first and then adherent cells were isolated by treating DWJM with collagenase. Live cells were stained with CellVue Maroon (CVM) for membrane, CellTracker Green (CTG) for cytoplasm, and Hoechst 33342 for nucleus, followed by analysis with Amnis/Luminex ImageStream-X imaging flow cytometer (Figure 1A). NN were defined as events positive for nuclear stain and negative for cytoplasmic and membrane stains (Figure 1B). 3-D movement of adherent AML cells in DWJM was captured in real time using confocal microscopy. Fixed cells from leukemia cell line K562 served as a control for movement. NN (Hoechst positive only), non-nucleated (Hoechst negative/CTG positive), and nucleated cells (Hoechst and CTG positive) were identified by fluorescent labeling. NN were also observed after isolation by cell sorting. Cell speed, cell displacement from origin, and change in distance to closest neighboring cell over time were measured. Additionally, flow sorted NN were examined by immunohistochemistry (IHC).RESULTSAdherent populations contained significantly more NN than non-adherent and suspension populations. The frequency of NN in matrix adherent cells ranged from 0.4-2.4% at day 3 and 0.5-5.4% at day 7 of culture (Figure 1C). Through confocal microscopic analysis, we observed NN, nucleated, and non-nucleated cells moving at speeds ranging from 0.002-0.08 µm/sec. Fixed cells showed no discernible movement in DWJM. The average speed of NN [0.019 mm/s, SD 0.011] significantly differed from the average speed of nucleated and non-nucleated cells [0.027 mm/s, SD 0.016] (p=0.004) (Figure 1E). To demonstrate directional movement, we measured change in distance between NN and closest neighboring nucleated or non-nucleated cells over time. Cells (nucleated and non-nucleated) and NN moved closer to each other over time suggesting directional movement (p=0.001) (Figure 1D). NN also showed movement in DWJM after isolation by cell sorting. IHC analysis showed sorted NN stained positive for nuclear lamin A/C, which are considered markers of nuclear membrane (Figure 1F).CONCLUSIONSOur findings confirm that NN are present in primary AML cells cultured in vitro using ourECM-based model and that they can be isolated using flow cytometry. Additionally, NN display directional movement in DWJM suggesting that they interact with other cells and may be biologically relevant structures in the bone marrow microenvironment. [Display omitted] DisclosuresBaran: AstraZeneca/Acerta: Research Funding. Chu: Pfizer: Current equity holder in publicly-traded company; Acerta/AstraZeneca: Research Funding; TG Therapeutics: Research Funding.

Tonsil-derived mesenchymal stem cells enhance allogeneic bone marrow engraftment via collagen IV degradation

BackgroundCo-transplantation of bone marrow cells (BMCs) and mesenchymal stem cells (MSCs) is used as a strategy to improve the outcomes of bone marrow transplantation. Tonsil-derived MSCs (TMSCs) are a promising source of MSCs for co-transplantation. Previous studies have shown that TMSCs or conditioned media from TMSCs (TMSC-CM) enhance BMC engraftment. However, the factors in TMSCs that promote better engraftment have not yet been identified.MethodsMice were subjected to a myeloablative regimen of busulfan and cyclophosphamide, and the mRNA expression in the bone marrow was analyzed using an extracellular matrix (ECM) and adhesion molecule-targeted polymerase chain reaction (PCR) array. Nano-liquid chromatography with tandem mass spectrometry, real-time quantitative PCR, western blots, and enzyme-linked immunosorbent assays were used to compare the expression levels of metalloproteinase 3 (MMP3) in MSCs derived from various tissues, including the tonsils, bone marrow, adipose tissue, and umbilical cord. Recipient mice were conditioned with busulfan and cyclophosphamide, and BMCs, either as a sole population or with control or MMP3-knockdown TMSCs, were co-transplanted into these mice. The effects of TMSC-expressed MMP3 were investigated. Additionally, Enzchek collagenase and Transwell migration assays were used to confirm that the collagenase activity of TMSC-expressed MMP3 enhanced BMC migration.ResultsMice subjected to the myeloablative regimen exhibited increased mRNA expression of collagen type IV alpha 1/2 (Col4a1 and Col4a2). Among the various extracellular matrix-modulating proteins secreted by TMSCs, MMP3 was expressed at higher levels in TMSCs than in other MSCs. Mice co-transplanted with BMCs and control TMSCs exhibited a higher survival rate, weight recovery, and bone marrow cellularity compared with mice co-transplanted with BMCs and MMP3-knockdown TMSCs. Control TMSC-CM possessed higher collagenase activity against collagen IV than MMP3-knockdown TMSC-CM. TMSC-CM also accelerated BMC migration by degrading collagen IV in vitro.ConclusionsCollectively, these results indicate that TMSCs enhance BMC engraftment by the secretion of MMP3 for the modulation of the bone marrow extracellular matrix.

Abstract 6204: Prostate cancer exosomes alter extracellular matrix composition in bone marrow and enhance metastasis

Abstract Introduction: Exosomes produced by neoplastic cells can promote the development of a pro-tumorigenic microenvironment via intercellular communication, and in some cases promote metastasis. The role of exosomes in the spread of prostate cancer, however, remains poorly understood. Here, we show that exosomes derived from enzalutamide resistant prostate cancer cells (EnzR exos) mediate intercellular communication with bone marrow-resident cells, alter the expression of extracellular matrix (ECM) proteins, and enhance metastasis in a clinically relevant mouse model of metastatic prostate cancer. Methods: Exosomes were isolated via ultracentrifugation of conditioned media from enzalutamide resistant CWR-R1 cells. Mice were primed with exosomes via three systemic injections of 10 ug exosomal protein. Exosome-mediated changes in bone marrow ECM composition were assessed via RNA sequencing of CD11b+ bone marrow cells and immunohistochemistry of formalin-fixed, decalcified, paraffin-embedded mouse femurs and tibias. For metastasis studies, intracardiac injection of luciferase-expressing enzalutamide resistant CWR-R1 cells into C.B.-17 SCID mice was used to establish metastatic tumor burden, and animals were monitored via bioluminescence imaging. Exosomal RNA sequencing was performed on EnzR exos and clinical specimens, with exosomes from normal prostate epithelial cells (PNT2) used as a control. Results: Mice primed with EnzR exos developed significantly enhanced metastatic tumor burden compared to unprimed controls. EnzR exo priming also led to decreased expression of thrombospondin-1 in the bone marrow compartment, and increased expression of versican, as determined by RNA sequencing and immunohistochemistry. Exosomal RNA sequencing revealed that five miRNAs were significantly enriched in EnzR exos compared to PNT2 exosome controls. One of these, miR-4443, was found to target thrombospondin-1. Prostate cancer patient serum samples were also enriched in miR-4443 over healthy controls, and exosomal miR-4443 correlated with disease progression. Conclusions: These data indicate that exosomes derived from enzalutamide resistant prostate cancer cells can alter ECM composition in the bone marrow compartment in a pro-tumorigenic manner. Evidence for the clinical relevance of these data was observed in both serum samples and bone biopsies from prostate cancer patients, demonstrating abundant serum exosomal miR-4443 and similarly altered bone marrow ECM composition in advanced prostate cancer patients. Exosomal miR-4443 may be a promising diagnostic and prognostic marker, as well as a potential therapeutic target. Citation Format: Stephen E. Henrich, Kaylin McMahon, Michael Plebanek, Fabio Tavora, Andre De Souza, Anthony Mega, Benedito Carneiro, C. Shad Thaxton. Prostate cancer exosomes alter extracellular matrix composition in bone marrow and enhance metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6204.

Integrin expression and adhesivity to fibronectin in primary acute myeloid leukemia cells: Impact of NPM1 and FLT3 mutations.

Interaction of leukemia cells with the bone marrow extracellular matrix promotes cell survival and resistance to chemotherapy. In this work, we analyzed integrin expression and adhesivity to fibronectin in primary cells from patients with acute myeloid leukemia. Surface expression of integrins β1 and αVβ3 on primary leukemia cells (N=46) was correlated with the stem cell marker CD34, as well as with cell adhesivity to fibronectin. The results were analyzed with regard to the mutational status of NPM1 and FLT3 genes. The integrin β1 was omnipresent, whereas αVβ3 was often more expressed on CD34-positive cells. In particular, higher αVβ3 expression on CD34+ cells was associated with NPM1 mutation (P=.0018). Monocytic leukemias had significantly higher αVβ3 expression compared to less maturated cases (P=.0008). Cells from patients with internal tandem duplications in FLT3 (FLT3-ITD) had lower adhesivity to fibronectin compared to cells with wild-type FLT3 (P=.031), specifically in less differentiated myeloblasts. Inhibition of a putative FLT3-ITD target, EZH2, increased cell adhesivity in MV4-11 cell line (P=.024). The integrin αVβ3 is expressed in particular on CD34+ cells with NPM1 mutation and might have a prognostic value in patients with mutated NPM1. FLT3-ITD is associated with lower cell adhesivity, especially in patients with less differentiated leukemias.

Remodelling of the bone marrow microenvironment by stromal hyaluronan modulates the malignancy of breast cancer cells

BackgroundHyaluronan (HA) is an abundant component of the bone marrow (BM) extracellular matrix. Here, we investigated the abnormal deposition of HA in the BM microenvironment and its remodelling in mediating the malignancy of breast cancer cells (BCCs).MethodsBCCs were transplanted into nude mice by intracardiac injection. The BCCs were cocultured with BM-derived stromal HS5 cells. Then, the abnormal metabolism of HA and its correlation with the malignant growth and the intracellular signalling pathways of the BCCs were investigated. After knockdown/out of the HA receptor CD44 in cancer cells by shRNA and CRISPR/Cas9, the mechanism was investigated in vivo through intratibial inoculation and in vitro by coculture with HS5 cells.ResultsThe malignancy of cancer cells was highly related to the degree of accumulation of HA in the BM. Further, stromal cell-derived HA, especially the mixed complex, significantly promoted the growth of BCCs and osteolysis by binding to the CD44 receptor. Additionally, the investigation of the underlying mechanism revealed that the PI3K, Cyclin D1, and CDK4 pathways were involved in the effect of bone stromal cell-derived HA on the BCC activities.ConclusionThese data suggested that HA in abnormal BM stroma might be a therapeutic candidate for bone metastasis of breast cancer.19kbyyRgNofaNy4kV-u_efVideo Graphical abstract

Integrin-Mediated Adhesion to Extracellular Matrix Protein Fibronectin Drives Megakaryocytosis in JAK2V617F+ Primary Myelofibrosis

Excessive accumulation of extracellular matrix (ECM) is a hallmark of bone marrow (BM) milieu in Primary Myelofibrosis (PMF). Myelofibrosis was long regarded as a bystander of disease in PMF. However, as cells have the ability to sense the surrounding ECM through integrin receptors, we examined the hypothesis that abnormal ECM in myelofibrosis, mediated by integrin activation, contributes to BM megakaryocytosis in JAK2V617F+ PMF. Due to its complex genetic landscape, modeling PMF in animals is challenging. While there is no ideal model to replicate the human disease, Vav1-hJAK2V617F transgenic mice (JAK2V617F+) (Xing S et al. Blood. 2008; 111:5109-5117) are most appropriate for our studies because they express the most frequent mutation in PMF and show megakaryocytosis in BM and a predictable development of myelofibrosis throughout the animal's life. Fibronectin (FN) is a dimeric ECM glycoprotein produced by a variety of cells in BM. FN secretion by BM stromal cells from PMF patients correlates with fibrosis and disease severity. Analysis of myelofibrotic BM from JAK2V617F+ mice revealed elevated levels of FN in BM ECM. Integrins are heterodimeric cell adhesion receptors of α and β subunits that connect the cell cytoskeleton to ECM. Integrin engagement and subsequent signaling affect cell differentiation, proliferation, migration and survival. Integrins α5β1, α4β1, αIIbβ3, and αυβ3 are FN-binding integrins known to be expressed in megakaryocytes. Expression analysis detected significantly elevated expression of α5, αIIb, αυ and β3 subunits in CD41+ megakaryocytes fromJAK2V617F+ mice in vitro and in vivo. Expression of β1 integrin was unchanged in megakaryocytes fromJAK2V617F+ mice, but expression of 9EG7, an antibody clone that detects the high-affinity conformation of β1 integrin, was elevated in JAK2V617F+ CD41+ megakaryocytes. Levels of the high-affinity form of β1 integrin decrease in response to inhibition of α5 integrin function (using Hmα5-1 antibody), indicating a high degree of correlation between affinity to FN and β1 activation in megakaryocytes fromJAK2V617F+ mice. As suggested by expression studies, in-vitro differentiated JAK2V617F+ megakaryocytes showed increased affinity to FN in adhesion assays. To ascertain the contribution of adhesion to FN in development of megakaryocytosis, megakaryocytes were differentiated in vitro on FN, with and without the presence of α5 integrin inhibitory antibody Hmα5-1. Culture on FN had a positive effect on the number of CD41+ megakaryocytes after four days of culture, especially in JAK2V617F+ BM. Importantly, inhibition of α5 integrin decreased the number of CD41+ megakaryocytes in JAK2V617F+ BM to nearly wild-type levels. To determine the effect of in vivo inhibition of α5 integrin on megakaryocyte numbers, the antibody clone 5H10-27, or MFR-5, previously reported to be effective in vivo, was used. Effective targeting in vivo of BM megakaryocytes by the 5H10-27 antibody was confirmed using a fluorochrome-labelled secondary antibody specific to the isotype of the injected clone (Rat IgG2a, κ). The same method was used to determine minimum required dose and interval of administration. JAK2V617F+ mice were treated with three doses of 5H10-27 or isotype control antibody (1mg/Kg IV every 48 hours). Analysis of treated animals on day 5 detected a decrease in megakaryocyte α5 integrin expression in animals treated with 5H10-27 antibody relative to control isotype antibody-treated animals. Platelet counts in peripheral blood and percentage of BM CD41+ megakaryocytes were consistently, albeit non-significantly, lower in 5H10-27 antibody-treated than in isotype antibody-treated animals. Corroborating our findings in mice, analysis of megakaryocytes from patients carrying the JAK2V617F mutation revealed elevated cell surface expression of α5 integrin subunit and increased adhesion to FN, which was dampened by an anti-α5 integrin antibody (Sam-1). Our results uncovered a global de-regulation of FN-binding integrin expression in megakaryocytes carrying the JAK2V617F mutation and the central role of FN-α5 integrin in development of megakaryocytosis in PMF. These results challenge the current paradigm by bringing ECM and myelofibrosis from a bystander position to center stage in the pathology of PMF. Disclosures No relevant conflicts of interest to declare.

Recent advances in mechanisms of megakaryopoiesis and platelet formation

Platelets are specialized cells, which are functioned to prevent bleeding and minimize blood vessel injury, and are also the predominating factor in the processes of hemostasis and thrombosis, while implicate in other processes including inflammation, innate immunity, angiogenesis and tumor metastasis. Among current mechanisms of platelet formation, platelets are produced by megakaryocytes, which are themselves generated by a process of controlled differentiation and maturation of bone marrow hematopoietic stem cells (HSC) and progenitor cells. Megakaryocytes release platelets by extending long, branching proplatelets, into sinusoidal blood vessels. Bone marrow cavity and extracellular matrix composition together with cytokines as thrombopoietin (TPO) are key regulators of megakaryopoiesis by supporting cell differentiation and platelet formation. Understanding the mechanisms of megakaryocyte differentiation and platelet formation can provide a solid theoretical basis for the treatment of platelet-related diseases. This review summarizes the current scientific research progress in the mechanisms of megakaryopoiesis and thrombopoiesis. Key words: Megakaryocytes; Cell differentiation; Thrombopoiesis; Transcription factors; Apoptosis; Megakaryopoiesis

Hyaluronan arrests human breast cancer cell growth by prolonging the G0/G1 phase of the cell cycle.

In clinical breast cancer patients, quiescent disseminated tumor cells (DTCs) can persist for a long time in the bone marrow (BM) under the influence of microenvironmental cues. As a high molecular weight polysaccharide, hyaluronan (HA) not only has been shown to regulate cancer processes including cell invasion, metastasis, migration, and proliferation, but also is a major component of the BM extracellular matrix. Here, we tested whether HA promotes breast cancer cell quiescence through detecting cell proliferation, cell cycle phase distribution, and the expression of cell cycle-related regulator proteins. In our results, HA slowed the growth and prolonged the G0/G1 phase of the highly metastatic, bone-seeking human breast cancer MDA-MB-231BO cell line, which is consistent with results that HA activated p38α/β, inhibited phospho-ERK1/2 levels and reduced the ERK/p38 signaling ratio. Additionally, we examined the crucial cell cycle factors p21cip1 and Cyclin D1, both of which influence the transition from G1 to S phase. The results revealed that p21cip1 expression was up-regulated by HA, which was consequently accompanied by a decrease in Cyclin D1 level. Further research with a 3D culture model indicated that HA maintained low Ki-67 and high p21cip1 expression levels in MDA-MB-231BO cells. In summary, our work revealed that HA might contribute to DTC quiescence.

A novel extracellular matrix-based leukemia model supports leukemia cells with stem cell-like characteristics

Acute myeloid leukemia (AML) relapse results from the survival of chemotherapy-resistant and quiescent leukemia stem cells (LSC). These LSCs reside in the bone marrow microenvironment, comprised of other cells and extracellular matrix (ECM), which facilitates LSC quiescence through expression of cell adhesion molecules. We used decellularized Wharton’s jelly matrix (DWJM), the gelatinous material in the umbilical cord, as a scaffolding material to culture leukemia cells, because it contains many components of the bone marrow extracellular matrix, including collagen, fibronectin, lumican, and hyaluronic acid (HA). Leukemia cells cultured in DWJM demonstrated decreased proliferation without undergoing significant differentiation. After culture in DWJM, these cells also exhibited changes in morphology, acquiring a spindle-shaped appearance, and an increase in the ALDH+ cell population. When treated with a high-dose of doxorubicin, leukemia cells in DWJM demonstrated less apoptosis compared with cells in suspension. Serial colony forming unit (CFU) assays indicated that leukemia cells cultured in DWJM showed increased colony-forming ability after both primary and secondary plating. Leukemia cell culture in DWJM was associated with increased N-cadherin expression by flow cytometry. Our data suggest that DWJM could serve as an ECM-based model to study AML stem cell-like cell behavior and chemotherapy sensitivity.

Murine hematopoietic stem cell reconstitution potential is maintained by osteopontin during aging

In adult mammals, hematopoietic stem cells (HSCs) reside in the bone marrow and are in part regulated by the bone marrow microenvironment, called the stem cell niche. We have previously identified the bone marrow morphogen osteopontin (OPN), which is abundantly present in the bone marrow extracellular matrix, as a negative regulator of the size of the HSC pool under physiological conditions. Here, we study the impact of OPN on HSC function during aging using an OPN-knockout mouse model. We show that during aging OPN deficiency is associated with an increase in lymphocytes and a decline in erythrocytes in peripheral blood. In a bone marrow transplantation setting, aged OPN-deficient stem cells show reduced reconstitution ability likely due to insufficient differentiation of HSCs into more mature cells. In serial bone marrow transplantation, aged OPN−/− bone marrow cells fail to adequately reconstitute red blood cells and platelets, resulting in severe anemia and thrombocytopenia as well as premature deaths of recipient mice. Thus, OPN has different effects on HSCs in aged and young animals and is particularly important to maintain stem cell function in aging mice.

Bone marrow laminins influence hematopoietic stem and progenitor cell cycling and homing to the bone marrow

Hematopoietic stem and progenitor cell (HSPC) functions are regulated by a specialized microenvironment in the bone marrow - the hematopoietic stem cell niche - of which the extracellular matrix (ECM) is an integral component. We describe here the localization of ECM molecules, in particular the laminin α4, α3 and α5 containing isoforms in the bone marrow. Laminin 421 (composed of laminin α4, β2, γ1 chains) is identified as a major component of the bone marrow ECM, occurring abundantly surrounding venous sinuses and in a specialized reticular fiber network of the intersinusoidal spaces of murine bone marrow (BM) in close association with HSPC. Bone marrow from Lama4−/− mice is significantly less efficient in reconstituting the hematopoietic system of irradiated wildtype (WT) recipients in competitive bone marrow transplantation assays and shows reduced colony formation in vitro. This is partially due to retention of Lin−c-kit+Sca-1+CD48− long-term and short-term hematopoietic stem cells (LT-HSC/ST-HSC) in the G0 phase of the cell cycle in Lama4−/− bone marrow and hence a more quiescent phenotype. In addition, the extravasation of WT BM cells into Lama4−/− bone marrow is impaired, influencing the recirculation of HSPC. Our data suggest that these effects are mediated by a compensatory expression of laminin α5 containing isoforms (laminin 521/522) in Lama4−/− bone marrow. Collectively, these intrinsic and extrinsic effects lead to reduced HSPC numbers in Lama4−/− bone marrow and reduced hematopoietic potential.

Effects of bone marrow stromal cells on the chemotherapeutic sensitivity of acute lymphoblastic leukemia cells

Objective: To investigate the influences of bone marrow stromal cells, components of extracellular matrix and cytokine secreted by stromal cells on the chemotherapeutic sensitivity of acute lymphoblastic leukemia cells to cytosine arabinoside (Ara-C). Methods: The co-culture model of acute lymphoblastic leukemia cell Sup-B15 and bone marrow stromal cell OP9 was constructed. Sup-B15 cells were cultured alone or co-cultured with OP9 cells, inactivated OP9 cells, the conditional medium (CM) of co-cultured OP9 cells and Sup-B15 cells, the CM of OP9 cells alone or Sup-B15 cells alone, respectively. The effects of different concentrations of Ara-C on the proliferation of each Sup-B1 cell group mentioned above were detected by cell counting kit-8 (CCK-8) method. The effects of different concentrations of Ara-C on the apoptosis of each group were detected by flow cytometry (FCM). The expressions of Bcl-2 protein in each group were detected by western blot. Results: The results of CCK-8 test showed that the inhibitory efficiency of Ara-C was in a dose-dependent manner. With different concentrations of Ara-C treatment for 48 hours, the half maximal inhibitory concentrations (IC(50)) of Sup-B15 and OP9 co-cultured group, Sup-B15 and inactivated OP9 co-cultured group were 0.510 and 0.339 μg/ml, respectively, significantly higher than 0.091 μg/ml of Sup-B15 cultured alone group (P<0.05). The IC(50) of CM of Sup-B15 and OP9 co-cultured group was 0.204 μg/ml, significantly higher than 0.087 μg/ml of the CM of OP9 cultured alone group (P<0.05) and 0.097 μg/ml of the CM of Sup-B15 cultured alone group (P<0.05). The results of flow cytometry showed that with 0.10 μg/ml Ara-C treatment for 24 hours, the early apoptotic cell percentages of Sup-B15 and OP9 co-cultured group, Sup-B15 and inactivated OP9 co-cultured group and Sup-B15 cultured alone group were (6.67±2.19) %, (8.95±3.04) % and (20.46±2.63) %, respectively. The early apoptotic cell percentages of Sup-B15 and OP9 co-cultured group, Sup-B15 and inactivated OP9 co-cultured group were significantly lower than that of Sup-B15 cultured alone group (P<0.05). The early apoptotic cell percentages of the CM of Sup-B15 and OP9 co-cultured group, the CM of OP9 cultured alone group and the CM of Sup-B15 cultured alone group were (11.16±2.97)%, (22.08±2.71)% and (19.25±1.57)%, respectively, the former two of which were significantly lower than the last one (P<0.05). The results of western blot showed that the relative expression levels of Bcl-2 protein of Sup-B15 cultured alone group, Sup-B15 and OP9 co-cultured group, Sup-B15 and inactivated OP9 co-cultured group, the CM of Sup-B15 and OP9 co-cultured group, the CM of OP9 cultured alone group and the CM of Sup-B15 cultured alone group were 1.00±0.00, 1.53±0.03, 1.38±0.01, 1.26±0.05, 1.03±0.01 and 0.98±0.02, respectively. The expression levels of bcl-2 protein of three combined groups were significantly higher than that of Sup-B15 cultured alone group (P<0.05). while no statistically significant difference was observed between the CM of OP9 cultured alone group and the CM of Sup-B15 cultured alone group (P>0.05). Conclusion: Bone marrow stromal cell OP9, the components of bone marrow extracellular matrix and cytokine secreted by stromal cells are involved in the induction of the chemotherapeutic resistance of Sup-B15 cells to Ara-C.