Bone Marrow-derived Hematopoietic Progenitor Research Articles

Interleukin-17A Directly Signals to Bone Marrow-Derived Hematopoietic Stem and Progenitor Cells to Promote Their Expansion and Differentiation in Vitro

Rationale: IL-17 is a family of six pro-inflammatory cytokines (named from A to F) with distinct patterns of cellular expression. Among these, IL-17A is secreted by lymphoid subsets such as T-helper 17 cells and innate lymphoid cells type 3. Through its action on many tissues, IL-17A is recognized as a key mediator of response to infection and pathogenic states such as cancer and auto-immunity. Until now, the main effect of IL-17A on hematopoiesis has been attributed to an indirect loop through IL-17RA signaling on bone marrow stromal cells, which stimulates secretion of hematopoietic factors such as G-CSF. However, we hypothesized that IL-17A can directly signal to hematopoietic stem and progenitor cells (HSPCs) to determine their self-renewal and differentiation. Methodology: We analyzed the expression of the IL-17 signaling pathway in publicly available human and mouse hematopoietic single-cell RNAseq datasets. Mouse HSPCs were isolated using FACS to perform clonogenic and differentiation assays in Methocult (StemCell Technologies) and StemPro-34 (Gibco). Spectral flow cytometry (SONY ID7000) was used for analysing IL-17RA expression and differentiation of mouse HSPCs. IL-17A levels were measured in blood and bone marrow serum using ELISA (Invitrogen). Results: IL-17RA is expressed at the surface of mouse HSPCs, with greatest levels in common lymphoid progenitors and granulocyte-monocyte progenitors. mRNA expression of IL-17RA signaling pathway is also detectable in human HSPCs, with greatest levels in young individuals. IL-17A supplementation leads to an increase in clonogenic capacity of mouse HSPCs in methylcellulose colony-formation assays. In liquid culture, supplementation with IL-17A leads to a greater expansion of sorted Lin -/s-kit +/Sca-1 + progenitors, accompanied by an increased proportion of myeloid-committed cells.IL-17A is also detected in the bone marrow serum, with possible production from subsets of cells expressing RORγt, a master regulator in development of T helper 17 (Th17) cells. Conclusions: Our work suggests the ability of IL-17A to directly promote clonogenic potential of HSPCs and drive a myeloid-biased lineage commitment in progenitor subsets. Additional studies are required to characterize the response of HSPCs to direct IL-17A signaling in vivo. Considering the important role of IL-17A as a mediator of immune response, and tissue repair, further studies are warranted to elucidate whether modulation of this pathway may be therapeutic in the context of hematopoietic failure or malignancy.

Mechanical loading intensities affect the release of extracellular vesicles from mouse bone marrow-derived hematopoietic progenitor cells and change their osteoclast-modulating effect.

Low-intensity loading maintains or increases bone mass, whereas lack of mechanical loading and high-intensity loading decreases bone mass, possibly via the release of extracellular vesicles by mechanosensitive bone cells. How different loading intensities alter the biological effect of these vesicles is not fully understood. Dynamic fluid shear stress at low intensity (0.7 ± 0.3 Pa, 5 Hz) or high intensity (2.9 ± 0.2 Pa, 1 Hz) was used on mouse hematopoietic progenitor cells for 2 min in the presence or absence of chemical compounds that inhibit release or biogenesis of extracellular vesicles. We used a Receptor activator of nuclear factor kappa-Β ligand-induced osteoclastogenesis assay to evaluate the biological effect of different fractions of extracellular vesicles obtained through centrifugation of medium from hematopoietic stem cells. Osteoclast formation was reduced by microvesicles (10 000× g) obtained after low-intensity loading and induced by exosomes (100 000× g) obtained after high-intensity loading. These osteoclast-modulating effects could be diminished or eliminated by depletion of extracellular vesicles from the conditioned medium, inhibition of general extracellular vesicle release, inhibition of microvesicle biogenesis (low intensity), inhibition of ESCRT-independent exosome biogenesis (high intensity), as well as by inhibition of dynamin-dependent vesicle uptake in osteoclast progenitor cells. Taken together, the intensity of mechanical loading affects the release of extracellular vesicles and change their osteoclast-modulating effect.

The Role of Hematopoietic Stem Cells in Lung Cancer Response to Radiation Therapy

Non-small cell lung carcinoma (NSCLC) comprises approximately 85% of all lung cancer cases in the United States. We demonstrated that after radiation therapy (RT), bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) are recruited to the NSCLC tumor microenvironment (TME). These HSPCs seemed to positively affect tumor regrowth as their numbers directly correlated with regrowth rates. The TME is comprised of tumor tissue, stromal cells, immune cells, bone marrow-derived cells, and extra cellular matrix (ECM) proteins and has emerged as one of the predominant defensive weapons with which cancer can resist various forms of therapeutic intervention. The ability of tumor-associated HSPCs to maintain hematopoietic potency in tumors suggested a prominent role of these cells in tumor physiology, however; what was missing was a mechanistic understanding of: 1) how HSPCs participate in tumor growth post-RT and 2) how HSPCs are maintained within tumors. To answer these questions, we established NSCLC tumors in mice using Lewis Lung Carcinoma (LLC) cells and assessed tumor-associated HSPCs activity and molecular changes in the TME following RT. We observed that RT induces the TME to produce colony stimulating factor (CSF)-1, a factor that promotes the differentiation of HSPCs into tumor supportive M2-macrophages. Adoptive transplantation of dsRed labeled HSPCs into tumor bearing mice followed by RT directly showed the differentiation of HSPCs into M2-macrophages and a concomitant increase in the regrowth of tumors post-RT. Use of a therapeutic inhibitor of the CSF-1 receptor (GW2580) prevented HSPC differentiation in to M2-macrophages and significantly decreasing regrowth rates. We next assessed how tumor-associated HSPCs maintain their hematopoietic potency within the TME. Analysis of tumors demonstrated high expression levels of the laminin 5-1-1 isoform, which is known to bind CD49f (integrin alpha 6), a marker of functional HSPCs. Tumors exposed to RT demonstrated significantly reduced expression of both laminin 5-1-1 and CD49f. When bound, CD49f inhibits phosphorylation of focal adhesion kinase (FAK) resulting in maintenance of pluripotent stem cells. Analysis of tumor-associated HSPCs demonstrated significantly higher levels of phosphorylated FAK following tumor exposure to RT. Since RT induces HSPC differentiation in to M2-macophages, this observation suggests that tumor-associated HSPC functionality is maintained through the interaction between laminin 5-1-1- and CD49f. The identification of single HSPCs that differentiated in to M2-macrophages and which expressed phosphorylated FAK supports this conclusion. Taken together, this data province's evidence of a mechanism wherein RT disrupts laminin 5-1-1/CD49f interactions releasing tumor-associated HSPCs from maintenance signals. Concomitantly, RT induces the production of CSF-1 within the TME, which induces these “released” HSPCs to differentiate into tumor supportive M2-macrophages and promote tumor regrowth post-RT. Overall, these studies define a new understanding of tumor biology in which HSPCs are significant mediators of tumor response to RT. These findings also identify new cellular and molecular pathways to target in the treatment of NSCLC to achieve more optimal clinical outcomes.

The role of the PZP domain of AF10 in acute leukemia driven by AF10 translocations

Chromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10PZP), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10PZP-dependent CALM-AF10-mediated leukemogenesis.

Transforming activities of the NUP98-KMT2A fusion gene associated with myelodysplasia and acute myeloid leukemia.

Inv(11)(p15q23), found in myelodysplastic syndromes and acute myeloid leukemia, leads to expression of a fusion protein consisting of the N-terminal of nucleoporin 98 (NUP98) and the majority of the lysine methyltransferase 2A (KMT2A). To explore the transforming potential of this fusion we established inducible iNUP98-KMT2A transgenic mice. After a median latency of 80 weeks, over 90% of these mice developed signs of disease, with anemia and reduced bone marrow cellularity, increased white blood cell numbers, extramedullary hematopoiesis, and multilineage dysplasia. Additionally, induction of iNUP98-KMT2A led to elevated lineage marker-negative Sca-1+ c-Kit+ cell numbers in the bone marrow, which outcompeted wildtype cells in repopulation assays. Six iNUP98-KMT2A mice developed transplantable acute myeloid leukemia with leukemic blasts infiltrating multiple organs. Notably, as reported for patients, iNUP98-KMT2A leukemic blasts did not express increased levels of the HoxA-B-C gene cluster, and in contrast to KMT2A-AF9 leukemic cells, the cells were resistant to pharmacological targeting of menin and BET family proteins by MI-2-2 or JQ1, respectively. Expression of iNUP98-KMT2A in mouse embryonic fibroblasts led to an accumulation of cells in G1 phase, and abrogated replicative senescence. In bone marrow-derived hematopoietic progenitors, iNUP98-KMT2A expression similarly resulted in increased cell numbers in the G1 phase of the cell cycle, with aberrant gene expression of Sirt1, Tert, Rbl2, Twist1, Vim, and Prkcd, mimicking that seen in mouse embryonic fibroblasts. In summary, we demonstrate that iNUP98-KMT2A has in vivo transforming activity and interferes with cell cycle progression rather than primarily blocking differentiation.

Cross-talk between T Cells and Hematopoietic Stem Cells during Adoptive Cellular Therapy for Malignant Glioma.

Purpose: Adoptive T-cell immunotherapy (ACT) has emerged as a viable therapeutic for peripheral and central nervous system (CNS) tumors. In peripheral cancers, optimal efficacy of ACT is reliant on dendritic cells (DCs) in the tumor microenvironment. However, the CNS is largely devoid of resident migratory DCs to function as antigen-presenting cells during immunotherapy. Herein, we demonstrate that cellular interactions between adoptively transferred tumor-reactive T cells and bone marrow-derived hematopoietic stem and progenitor cells (HSPCs) lead to the generation of potent intratumoral DCs within the CNS compartment.Experimental Design: We evaluated HSPC differentiation during ACT in vivo in glioma-bearing hosts and HSPC proliferation and differentiation in vitro using a T-cell coculture system. We utilized FACS, ELISAs, and gene expression profiling to study the phenotype and function of HSPC-derived cells ex vivo and in vivo To demonstrate the impact of HSPC differentiation and function on antitumor efficacy, we performed survival experiments.Results: Transfer of HSPCs with concomitant ACT led to the production of activated CD86+CD11c+MHCII+ cells consistent with DC phenotype and function within the brain tumor microenvironment. These intratumoral DCs largely supplanted abundant host myeloid-derived suppressor cells. We determined that during ACT, HSPC-derived cells in gliomas rely on T-cell-released IFNγ to differentiate into DCs, activate T cells, and reject intracranial tumors.Conclusions: Our data support the use of HSPCs as a novel cellular therapy. Although DC vaccines induce robust immune responses in the periphery, our data demonstrate that HSPC transfer uniquely generates intratumoral DCs that potentiate T-cell responses and promote glioma rejection in situClin Cancer Res; 24(16); 3955-66. ©2018 AACR.

Stromal Cell-Derived Factor-1α Promotes Endothelial Colony-Forming Cell Migration Through the Ca2+-Dependent Activation of the Extracellular Signal-Regulated Kinase 1/2 and Phosphoinositide 3-Kinase/AKT Pathways.

Stromal cell-derived factor-1α (SDF-1α) drives endothelial colony-forming cell (ECFC) homing and incorporation within neovessels, thereby restoring tissue perfusion in ischemic tissues and favoring tumor vascularization and metastasis. SDF-1α stimulates ECFC migration by activating the Gi-protein-coupled receptor, CXCR4, and then engaging the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. Sporadic evidence showed that SDF-1α may also act through an increase in intracellular Ca2+ concentration ([Ca2+]i) in bone marrow-derived hematopoietic progenitor cells and fully differentiated endothelial cells. Of note, recent evidence demonstrated that intracellular Ca2+ signals play a key role in controlling the proangiogenic activity of ECFCs. The present investigation was, therefore, undertaken to assess whether and how SDF-1α induces ECFC motility by triggering intracellular Ca2+ signals. We found that SDF-1α caused a dose-dependent increase in [Ca2+]i that was inhibited by ADM3100, a selective CXCR4 antagonist. Pharmacological manipulation revealed that the Ca2+ response to [Ca2+]i was shaped by an initial intracellular Ca2+ release through inositol-1,4,5-trisphosphate receptors (InsP3Rs), followed by a sustained phase of extracellular Ca2+ entry through store-operated Ca2+ channels. InsP3-dependent Ca2+ release and store-operated Ca2+ entry (SOCE) were both necessary for SDF-1α-induced extracellular signal-regulated kinases 1/2 (ERK 1/2) and AKT phosphorylation. Finally, SDF-1α employed intracellular Ca2+ signals, ERK 1/2, and PI3K/AKT to promote ECFC migration in vitro and neovessel formation in vivo. These data, therefore, provide the first evidence that SDF-1α induces ECFC migration through the Ca2+-dependent activation of the ERK 1/2 and PI3K/AKT pathways.

Heterodimerization of AML1/ETO with CBF\u03b2 is required for leukemogenesis but not for myeloproliferation

The AML1/Runx1 transcription factor and its heterodimerization partner CBFβ are essential regulators of myeloid differentiation. The chromosomal translocation t(8;21), fusing the DNA binding domain of AML1 to the corepressor eight-twenty-one (ETO), is frequently associated with acute myeloid leukemia and generates the AML1/ETO (AE) fusion protein. AE represses target genes usually activated by AML1 and also affects the endogenous repressive function of ETO at Notch target genes. In order to analyze the contribution of CBFβ in AE-mediated leukemogenesis and deregulation of Notch target genes, we introduced two point mutations in a leukemia-initiating version of AE in mice, called AE9a, that disrupt the AML1/CBFβ interaction (AE9aNT). We report that the AE9a/CBFβ interaction is not required for the AE9a-mediated aberrant expression of AML1 target genes, while upregulation/derepression of Notch target genes does require the interaction with CBFβ. Using retroviral transduction to express AE9a in murine adult bone marrow-derived hematopoietic progenitors, we observed that both AE9a and AE9aNT lead to increased myeloproliferation in vivo. However, both development of leukemia and long-term replating capacity are only observed with AE9a but not with AE9aNT. Thus, deregulation of both AML1 and Notch target genes is required for the development of AE9a-driven leukemia.

Pathways of Retinoid Synthesis in Mouse Bone Marrow-Derived Macrophages and Hematopoietic Progenitors

Introduction:Retinoid receptors are nuclear hormone receptors which are dynamically regulated during terminal myeloid maturation. Retinoic acid receptor α (RARA) is the target of at least ten fusion proteins that lead to acute promyelocytic leukemia (APL). All trans-retinoic acid (ATRA) has been thought to be the principle natural ligand for RARs and it has been used for the treatment of patients with APL. However, the enzymatic pathways that regulate natural retinoids metabolism in hematopoietic cells have not been well defined.ATRA is synthesized from vitamin A through two sequential steps. Vitamin A is oxidized by an alcohol dehydrogenase to yield retinal, which is then irreversibly oxidized by an aldehyde dehydrogenase (ALDH) to generate retinoic acid (RA). At least 19 different human ALDHs have been identified. Among them, ALDH1A1, ALDH1A2 and ALDH1A3 have been shown to oxidize all trans-retinal to ATRA with high affinity, which can be inhibited by diethylaminobenzaldehyde (DEAB). Whether other ALDHs participate in RA metabolism is unknown. Our study identified two distinct retinoid metabolism pathways that are active in bone marrow (BM) progenitors and in macrophages (Mφ).Methods:Gal4-UAS reporter system was used to detect natural RARA ligands in mouse primary hematopoietic cells. We transduced UAS-GFP mouse BM cells with retrovirus that expresses a fusion protein containing the DNA binding domain of Gal4 (which recognizes the UAS promoter) and the ligand binding domain of RARA. An IRES mCherry cassette was included for normalization purposes. Transduced cells were cultured in vitro, or transplanted into lethally irradiated recipient mice. Cells with intracellular RARA ligands activate GFP expression. GFP and mCherry expression were evaluated by flow cytometry. Real-time PCR and Affymetrix array were used to quantify ALDH expression. We identified RARA ligands by mass spectrometry (MS).Results:In vitro, we found that both mouse BM Kit+ cells (Kit+ progenitors) and BM-derived macrophages (BMMφ) could synthesize active RARA ligands via different pathways, but only when the cell culture media was supplemented with vitamin A. Kit+ progenitors utilize DEAB-sensitive ALDH pathways, whereas BMMφ use DEAB-insensitive pathways. By real-time PCR we found Kit+ progenitors have high expression of Aldh1a1, Aldh1a2 and Aldh1a3, whereas BMMφ have no detectable expression of these enzymes. We compared gene expression in Kit+ progenitors and BMMφ by Affymetrix profiling and found that Aldh3b1 was overexpressed in BMMφ. Ectopic expression of Aldh3b1 in 293T cells resulted activation of the same GFP reporter, which could be abrogated by two different antagonist, Ro41-5253 and BMS493, suggesting that Aldh3b1 generated an RARA specific ligand, which we subsequently identified as ATRA via MS. Reciprocally, we found that siRNA knock down of Aldh3b1 in BMMφ reduced the transactivation of the RARA-dependent GFP reporter.The X-RARA fusions have been proposed to act via dominant-negative mechanisms, decreasing retinoid-dependent transcription and myeloid maturation. Surprisingly, in vivo, only rare GFPdim cells were observed in BM cells and no GFP positive cells in peritoneal Mφ of UAS-GFP/Gal4-RARA transplant mice. As positive control, we treated mice with ATRA and observed a dose-dependent GFP increase in both cell types, suggesting that the in vivo reporter can respond to ATRA, but ATRA is not synthesized during adult hematopoiesis, and that dominant-negative inhibition of ATRA-dependent transcription may not be the predominant pathogenic effect of the X-RARA fusion oncoproteins.Conclusion:We have found that at least two distinct enzymatic pathways may be utilized in primary hematopoietic cells to synthesize active RARA ligands from vitamin A. Mouse BM Kit+ progenitors predominantly employ a set of DEAB-sensitive enzymes (Aldh1a1, Aldh1a2 and Aldh1a3), whereas Mφ utilize DEAB-insensitive pathways. We identified Aldh3b1 as a likely candidate and shown that it is capable of ATRA synthesis in vitro. Although these enzymes are expressed in primary BM cells, we found that this does not result in active intracellular RARA ligands in monocytes or Mφ in vivo, suggesting that the rate-limiting step in retinoid synthesis in vivo is likely to involve additional enzymes required for intracellular transport of protein-bound, serum-available vitamin A. DisclosuresNo relevant conflicts of interest to declare.

T15: Circulating tumor cells and bone marrow progenitor cells in the blood of breast cancer patients in the dynamics of neoadjuvant chemotherapy

Background Despite the abundance of theoretical evidences displaying the considerable role of the “soil” in metastasis progression, the majority of cancer research and cancer therapy pathogenetic methods focused mainly on the tumor cells. There is still no clear clinical data showing the role of “soil” in the metastasis. Identification of factors and mechanisms driving the conditions promoting tumor dissemination may lead to therapeutic strategy to detect and prevent metastases at the earliest step. It has been established that bone marrow-derived hematopoietic progenitor cells home to pre-metastatic sites before tumor cells infiltrate in these sites. These hematopoietic progenitor cells form regulatory cell cluster of stromal microenvironment (premetastatic niche) to promote secondary tumor growth. In this context, the aim of our study was to evaluate the level of different pools of circulating tumor cells and bone marrow progenitor cells in the blood of patients with breast cancer in the dynamics of neoadjuvant chemotherapy. Materials and methods Patients (prospective study) with newly diagnosed invasive breast cancer in the age range 18–50 years and tumor volume of 2.0(> or =) cm, referred at the Tomsk Cancer Research Institute for treatment were included into the study. Eligibility criteria were as follows: the patient’s informed consent to participate in research; morphologically verified diagnosis of invasive carcinoma of non-specific type; luminal B-1, -2, triple negative (basal-like subtype included), HER2 positive breast cancer; T2-4N0-3M0; preserved menstrual function; satisfactory performance status (on a scale ( Results The study showed that each succeeding course of NACHT increased blood levels of circulating tumor cells, and bone marrow progenitor cells with a phenotype characteristic of EPC (endothelial progenitor cells) (CD34 + CD45–VEGFR + CD133 + CD202 +) and MSC (mesenchymal progenitor stem cells fibroblasts) (CD34–CD90 + VEGFR1–CD45–CD202–). Influence of the NACHT on hematopoietic stem cells HSC (VEGFR1 + CD34 + CD45lowSD202–) and progenitor cells HPC – CD34 + CD45 + CD90–VEGFR1 + CD133 – was ambiguous. Conclusion Neoadjuvant chemotherapy increases blood levels of circulating tumor cells, endothelial progenitor cells and mesenchymal stem cells progenitor fibroblasts, thus promoting the formation of premetastatic niche. The study was conducted with financial support from the Council for Grants of the President of the Russian Federation (Russia) for the state support of young philosophy doctors, agreements of SC 114.120.14.168-MD Russian Scientific Foundation (Russia), Grant No. 14-15-00318 (sample collection) and Russian Foundation for Basic Research (Russia), Grant No. 15-34-20864 . We acknowledge support of this work by the Tomsk State University Competitiveness Improvement Program (Russia).

Lineage-related and particle size-dependent cytotoxicity of chitosan nanoparticles on mouse bone marrow-derived hematopoietic stem and progenitor cells

Chitosan nanoparticles (CSNPs) have potential applications in stem cell research. In this study, ex vivo cytotoxicity of CSNPs on mouse bone marrow-derived (MBMCs) hematopoietic stem and progenitor cells (HSPCs) was determined. MBMCs were exposed to CSNPs of different particle sizes at various concentrations for up to 72 h. Cytotoxicity effect of CSNPs on MBMCs was determined using MTT, Live/Dead Viability/Cytotoxicity assays and flow cytometry analysis of surface antigens on HSCs (Sca-1+), myeloid-committed progenitors (CD11b+, Gr-1+), and lymphoid-committed progenitors (CD45+, CD3e+). At 24 h incubation, MBMCs' viability was not affected by CSNPs. At 48 and 72 h, significant reduction was detected at higher CSNPs concentrations. Small CSNPs (200 nm) significantly reduced MBMCs' viability while medium-sized particle (∼400 nm) selectively promoted MBMCs growth. Surface antigen assessment demonstrated lineage-dependent effect. Significant decrease in Sca-1+ cells percentage was observed for medium-sized particle at the lowest CSNPs concentration. Meanwhile, reduction of CD11b+ and Gr-1+ cells percentage was detected at high and intermediate concentrations of medium-sized and large CSNPs. Percentage of CD45+ and CD3e+ cells along with ROS levels were not significantly affected by CSNPs. In conclusion, medium-sized and large CSNPs were relatively non-toxic at lower concentrations. However, further investigations are necessary for therapeutic applications.

Mastocytosis in the skin in children and adults

Background Mastocytosis is a rare disease of bone marrow-derived hematopoietic progenitor cells, which is characterized by abnormal growth and excessive accumulation of mast cells in various tissues. Cutaneous Mastocytosis is a skin limited disease, whereas Systemic Mastocytosis usually involves bone marrow, spleen, liver, lymph nodes and gastrointestinal tract and may present with or without skin involvement. The skin is one of the most commonly affected organ both in children and adults. Mastocytosis in the skin (MIS) is defined by a typical exanthema and monomorphic mast cell infiltrate. MIS is an initial diagnosis which requires further diagnostic steps leading to the final diagnosis of Cutaneous Mastocytosis or Systemic Mastocytosis.

Human adipose-derived stem cells ameliorate cigarette smoke-induced murine myelosuppression via secretion of TSG-6.

Bone marrow-derived hematopoietic stem and progenitor cells (HSC/HPC) are critical to homeostasis and tissue repair. The aims of this study were to delineate the myelotoxicity of cigarette smoking (CS) in a murine model, to explore human adipose-derived stem cells (hASC) as a novel approach to mitigate this toxicity, and to identify key mediating factors for ASC activities. C57BL/6 mice were exposed to CS with or without i.v. injection of regular or siRNA-transfected hASC. For in vitro experiments, cigarette smoke extract was used to mimic the toxicity of CS exposure. Analysis of bone marrow HPC was performed both by flow cytometry and colony-forming unit assays. In this study, we demonstrate that as few as 3 days of CS exposure results in marked cycling arrest and diminished clonogenic capacity of HPC, followed by depletion of phenotypically defined HSC/HPC. Intravenous injection of hASC substantially ameliorated both acute and chronic CS-induced myelosuppression. This effect was specifically dependent on the anti-inflammatory factor TSG-6, which is induced from xenografted hASC, primarily located in the lung and capable of responding to host inflammatory signals. Gene expression analysis within bone marrow HSC/HPC revealed several specific signaling molecules altered by CS and normalized by hASC. Our results suggest that systemic administration of hASC or TSG-6 may be novel approaches to reverse CS-induced myelosuppression.

Cell-Cell Communication Networks Propose a Modulation of the Hematopoietic Stem Cell Niche by Invading Breast Carcinoma Cells

Background: The human bone marrow can become a target of disseminated tumor cells in a relevant proportion of breast cancer patients. However, the underlying pathophysiology is incompletely understood. This study aims to identify and characterize potential mechanisms modulating the bone marrow hematopoietic microenvironment by invading breast cancer cells (BCC) as a basis for experimental evaluation. Methods: Static cell-cell communication networks, representing the integrated signaling among breast carcinoma cell lines (MCF-7 or MDA-MB-231), bone marrow-derived mesenchymal stromal cells (MSC) and hematopoietic stem and progenitor cells (HSPC), were constructed in-silico by combining differentially overexpressed genes of the involved cell populations with known ligand-receptor interactions. Using the networks as guidance, pathophysiological relevance of the analyzed populations to breast cancer-initiated hematologic abnormalities was appraised by systematic literature mining. In-vitro co-culture modeling was performed to evaluate the paracrine effects of BCC on MSC-HSPC signaling and to validate main implications of the exposed signaling network. Results: Breast cancer cells exhibited intensive bidirectional intercellular signaling with MSC and to a lesser extent with HSPC. BCC-derived signals were reported to recruit MSC to sites of breast cancer, activate tumor associated fibroblasts (TAF) and modify MSC differentiation. Hematopoietic microenvironment-derived signals were predominantly associated with BCC attraction and metastatic progression. Potential ligands that protect from metastases were exclusively HSPCderived. In-vitro co-culture modeling revealed that BCC mediated loss of the niche-derived hematopoiesis-supporting factor SDF-1 and the emergence of FGF-2 in the MSC-HSPC interaction. Conclusion: We propose a modulation of MSC by BCC, inter alia via the FGF-2/FGFR1 pathway, resulting in activation of TAF, generation of a vascularized tumor stroma, breast cancer progression and consequential impairment of hematopoiesis by reduction of SDF-1 levels. Those indirect changes in the HSC niche upon BCC invasion might increase the vulnerability for bone metastasis in breast cancer patients.

The metastasis-promoting roles of tumor-associated immune cells

Tumor metastasis is driven not only by the accumulation of intrinsic alterations in malignant cells, but also by the interactions of cancer cells with various stromal cell components of the tumor microenvironment. In particular, inflammation and infiltration of the tumor tissue by host immune cells, such as tumor-associated macrophages, myeloid-derived suppressor cells, and regulatory T cells, have been shown to support tumor growth in addition to invasion and metastasis. Each step of tumor development, from initiation through metastatic spread, is promoted by communication between tumor and immune cells via the secretion of cytokines, growth factors, and proteases that remodel the tumor microenvironment. Invasion and metastasis require neovascularization, breakdown of the basement membrane, and remodeling of the extracellular matrix for tumor cell invasion and extravasation into the blood and lymphatic vessels. The subsequent dissemination of tumor cells to distant organ sites necessitates a treacherous journey through the vasculature, which is fostered by close association with platelets and macrophages. Additionally, the establishment of the pre-metastatic niche and specific metastasis organ tropism is fostered by neutrophils and bone marrow-derived hematopoietic immune progenitor cells and other inflammatory cytokines derived from tumor and immune cells, which alter the local environment of the tissue to promote adhesion of circulating tumor cells. This review focuses on the interactions between tumor cells and immune cells recruited to the tumor microenvironment and examines the factors allowing these cells to promote each stage of metastasis.

Monitoring the glioma tropism of bone marrow-derived progenitor cells by 2-photon laser scanning microscopy and positron emission tomography

Intracerebral experimental gliomas attract intravenously injected murine or human bone marrow-derived hematopoietic progenitor and stem cells (HPC) in vitro, ex vivo, and in vivo, indicating that these progenitor cells might be suitable vehicles for a cell-based delivery of therapeutic molecules to malignant gliomas. With regard to therapeutic application, it is important to investigate cell fates in vivo (i.e., the time-dependent intratumoral and systemic distribution after intravenously injection). Conventional histological analysis has limitations in this regard because longitudinal monitoring is precluded. Here, we used 2-photon laser scanning microscopy (2PLSM), positron emission tomography (PET), and MRI to study the fate of intravenously injected HPC carrying fluorescence, bioluminescence, and PET reporter genes in glioma-bearing mice. Our 2PLSM-based monitoring studies revealed that HPC homing to intracerebral experimental gliomas occurred already within the first 6 h and was most efficient within the first 24 h after intravenous injection. The highest PET signals were detected in intracerebral gliomas, whereas the tracer uptake in other organs, notably spleen, lung, liver, and muscle, remained at background levels. The results have important implications for designing schedules for therapeutic cell-based anti-glioma approaches. Moreover, the PET reporter-based imaging technique will allow noninvasive monitoring of cell fate in future cell-based therapeutic antiglioma approaches.

Hematopoietic progenitor cells (HPCs) in node-negative invasive breast carcinomas: Immunohistochemical analysis and clinico-pathological correlations

Using immunohistochemistry, we investigated 603 negative lymph nodes from 51 patients affected by invasive breast cancer (BC) to recognize bone marrow-derived hematopoietic progenitor cells (HPCs). HPC aggregates, revealed by CD34, CD133, VEGFR1, and CD117 antisera, were determined by an intensity-distribution score (ID). Cases with an ID-score >3 at least for one marker were considered to strongly express HPCs. Twenty-five of 51 (49%) high expressor patients were identified by CD34 antiserum, while 24/51 (47.1%), 17/51 (33.3%), and 15/51 (29.4%) were identified by CD117, CD133, and VEGFR1, respectively. No significant relationships were found between HPCs status and histotype, tumor grade, stage, and hormone receptors, as determined at the moment of the first diagnosis. A significant correlation was recorded for Ki-67 values, as well as for death from invasive BC. No statistical significance was achieved regarding HER2 status, although a tendency toward a statistically significant P value was obtained. A significant relationship ( P < 0.001) was found between high expressors of HPC and progression of disease, documented by the development of distant metastases. An equivalent P value was ascertained for osseous localizations, with a lesser value in other metastatic sites. Regarding the appearance of distant metastases, the greatest efficiency value was obtained by CD133 (85.7%). Overall survival (OS) and distant metastases-free survival (DMFS) revealed a high statistical significance for HPC expression, Ki-67 values, and HER2 status. By multivariate analysis, HPC expression and Ki-67 values emerged as the higher independent prognostic variables in the analysis of DMFS and OS, respectively.

Downregulation of the CXC chemokine receptor 4/stromal cell–derived factor 1 pathway enhances myocardial neovascularization, cardiomyocyte survival, and functional recovery after myocardial infarction

Although adequate numbers of hematopoietic progenitor cells reside in the human bone marrow, the extent of endogenous neovascularization after myocardial infarction remains insufficient. The aim of this study was to identify the role of the CXC chemokine receptor 4/stromal cell-derived factor 1 axis in the mobilization and homing of hematopoietic progenitor cells in the ischemic heart. Human bone marrow-derived hematopoietic progenitor cells or saline were injected systemically into athymic nude rats 48 hours after myocardial infarction. Myocardial and bone marrow expression of stromal cell-derived factor 1 and chemotaxis of hematopoietic progenitor cells were measured in vitro in the presence or absence of stromal cell-derived factor 1. The role of the CXC chemokine receptor 4/stromal cell-derived factor 1 axis was investigated by means of antibody blockade or systemic administration of granulocyte colony-stimulating factor. Morphologic analysis included measurement of the infarct area, capillary density, and apoptosis, whereas left ventricular function was measured by means of echocardiographic analysis. Expression of postinfarct stromal cell-derived factor 1 was increased by 67% in the bone marrow and decreased by 43% in myocardium. Disruption of bone marrow stromal cell-derived factor 1/CXC chemokine receptor 4 interactions by antibody blockade resulted in a redirection of human hematopoietic progenitor cells from the bone marrow to the ischemic heart and augmented neovascularization and cardiomyocyte survival. Similarly, systemic administration of granulocyte colony-stimulating factor to block CXC chemokine receptor 4/stromal cell-derived factor 1 interaction resulted in increased mobilization and homing of hematopoietic progenitor cells to the ischemic heart, which translated to augmented myocardial neovascularization, prevention of apoptosis, and improved cardiac function. Bone marrow stromal cell-derived factor 1 upregulation after myocardial ischemia prevents mobilization of endogenous hematopoietic progenitor cells. We provide evidence that disruption of stromal cell-derived factor 1/CXC chemokine receptor 4 interactions allows redirection of hematopoietic progenitor cells to ischemic myocardium and enhances recovery of left ventricular function.

Expression and Function of P-selectin and its ligand PSGL1 on hematopoietic progenitors (36.4)

Abstract T cells develop in the thymus from bone marrow-derived hematopoietic progenitors that settle the thymus via the blood. The molecular basis for progenitor entry into the thymus is poorly understood, but p-selectin and P-selectin glycoprotein ligand 1 (PSGL1) are suggested to be involved. We investigated the expression of functional PSGL1 able to bind p-selectin. Our preliminary results suggest that functional PSGL1 is expressed at lower levels on primitive hematopoietic stem cells and downstream multipotential progenitors, but at higher levels on downstream early lymphoid progenitors in the bone marrow. These data indicate that P selectin binding is developmentally regulated. Using competitive thymus reconstitution assays we confirmed that PSGL1 is a functionally important component of the thymic homing process. Our current experiments are focused on understanding the mechanism by which PSGL1 is selectively upregulated on thymus homing progenitors.

Vascular endothelial growth factor receptor‐1 in human cancer

The human vascular endothelial growth factor receptor-1 (VEGFR-1, or Flt-1) is widely expressed in normal and pathologic tissue and contributes to the pathogenesis of both neoplastic and inflammatory diseases. In human cancer, VEGFR-1 mediated signaling is responsible for both direct tumor activation and angiogenesis. VEGFR-1 mediated activation of nonmalignant supporting cells, particularly stromal, dendritic, hematopoietic cells, and macrophages, is also likely important for cancer pathogenesis. VEGFR-1 is also hypothesized to enable the development of cancer metastases by means of activation and premetastatic localization in distant organs of bone marrow-derived hematopoietic progenitor cells, which express VEGFR-1. IMC-18F1 is a fully human IgG(1) antibody that binds to VEGFR-1 and has been associated with the inhibition of cancer growth in multiple in vitro and human tumor xenograft models. The preliminary results of phase 1 investigations have also indicated a favorable safety profile for IMC-18F1 at doses that confer antibody concentrations that are associated with relevant antitumor activity in preclinical models.