scholarly journals CD47 but Not Calreticulin Is over Expressed in Patients with Myeloproliferative Neoplasms

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5492-5492
Author(s):  
Kristian Boasman ◽  
Matthew J Simmonds ◽  
Ciro R Rinaldi
Keyword(s):  
Cell Surface ◽  
Protein Expression ◽  
Mononuclear Cells ◽  
Immune Escape ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Response To Therapy ◽  
Solid Tumours ◽  
Polycythaemia Vera ◽  
Gene And Protein Expression

Abstract Myeloproliferative neoplasms (MPN) are chronic myeloid cancers characterized by the overproduction of mature blood cells, and the tendency to evolve into acute leukaemia. In solid tumours, calreticulin (CALR) overexpression on the cell surface, produces a pro-phagocytic signal and is counteracted by concomitant expression of anti- phagocytic CD47, reflecting an apoptosis vs survival mechanism. In this study, we investigated the expression of CALR and CD47 in patients with MPN and potential changes induced by treatment. CALR and CD47 gene and protein expression were measured by Real Time PCR and western blotting, in K562 and in mononuclear cells obtained by FICOLL separation, from peripheral blood of 13 MPN patients [4 Polycythaemia Vera, 8 Essential Thrombocythemia, and 1 myelofibrosis; 7 treated (IFN or Hydroxyurea) and 6 untreated] and compared with 4 healthy controls. Cells were also fractionised into 4 compartments: membrane, cytoplasm, cytosol and nucleus and protein fractions analysed. We found a significant increase in CD47 protein expression into the membrane of MPN patients comparing with controls (91.9 % vs 19.7%). Interestingly in treated MPN the CD47 expression increases even further in comparison with untreated (92.6% vs 91.9%). No significant differences were found in total CALR expression comparing with controls (3.23, vs 2.95-fold), however in treated patients we observed a reduction in the expression in the membrane comparing with untreated (26.4 % vs 39.5 %) and an increase into cytoplasm (69.4 % vs 54.6 %). These findings suggest CD47 exposure as a mechanism of defence in MPN cells during treatment. To better understand the potential effects of therapy on CALR and CD47 expression, we incubated K562 with 0.05μM/ml of Ruxolitinib (RUXO), re-dosed at 24 hours and harvested at 48 hours. RUXO induced CALR internalization, reducing its expression into the membrane (RUXO 8.2% vs Untreated: 13.3%), but increasing its expression in cytosol (RUXO 51% vs Untreated 48.4%) and in the cytoplasm (RUXO 13.5% vs Untreated 2.9%). In contrast, CD47 expression remains relatively unmodified, with only slight increases on the membrane (RUXO 43.1% vs Untreated 40.4%) and in the cytoplasm (RUXO 34.5% vs UT 32.5%). In this study we demonstrated that CD47 but not CALR, is overexpressed on the membrane of patients with MPN. This opposes previous studies in solid tumours, which show significant increases of both CALR and CD47. This suggests a role for CD47 as a strong anti-phagocytic signal responsible for immune survival in MPN. We have also shown a potential mechanism adopted by MPN cells in response to therapy, with the internalization of CALR and the enhanced membrane expression of CD47 which remains strongly expressed on cell surface. The addition of Anti-CD47 compounds in combination with conventional therapies might represent a future therapeutically strategy to counteract MPN cells immune-escape mechanism. Disclosures No relevant conflicts of interest to declare.

scholarly journals Expression of CD47 and Calr in Myeloproliferative Neoplasms: Potential New Therapeutical Targets

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4598-4598
Author(s):  
Kristian Boasman ◽  
Matthew J Simmonds ◽  
Ciro R Rinaldi
Keyword(s):  
Cell Membrane ◽  
Protein Expression ◽  
Cell Membranes ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Membrane Expression ◽  
Myeloid Malignancies ◽  
Response To Chemotherapy ◽  
Significant Difference

Abstract Myeloproliferative neoplasms (MPN) are myeloid malignancies characterized by overproduction of mature blood cells, hyperplastic bone marrow and tendency to evolve into acute myeloid leukaemia. In solid tumours, calreticulin (CALR) overexpression produces a pro-phagocytic signal and is counteracted by concomitant expression of anti-phagocytic CD47, reflecting an apoptosis vs survival mechanism. Increases of both CALR and CD47 on the cell membrane have been observed in response to chemotherapy, however their role in myeloid malignancies is poorly understood. Aims: Investigate the expression and cellular localisation of CALR and CD47 in untreated and treated patients with essential thrombocythemia (ET), polycythemia vera (PV) myelofibrosis (MF), in comparison with healthy controls. Methods: Mononuclear cells were collected by Ficoll separation, from peripheral blood of 30 MPN (8 PV, 16 ET, 6 MF); 18 MPN patients received cyto-reductive therapies (Hydroxyurea, Anagrelide or Ruxolitinib); and 4 controls. Cells were fractionised into 4 compartments: membrane, cytoplasm, cytosol and nucleus. Proteins were extracted using TRIzol, with CALR and CD47 protein expression analysed by western blotting. Results: Total CALR and CD47 protein expression increased in MPN samples compared with controls (CALR- 7.9 vs 5.1; CD47- 2.7 vs 2.2 fold, respectively). CD47 showed higher expression of its overall protein on MPN cell membranes when compared with CALR (22% vs 13.9%). We observed a significant reduction of CALR expression in all MPN subtypes when patients were treated with cyto-reductive agents (ET- untreated 43.3% vs treated 2%, PV- 3.6% vs 2.2%, ET- 21% vs 11%). Interestingly we have observed a significant increase in CD47 cell membrane expression after treatment in MF and PV (CD47 in MF- untreated 11.8% vs treated 34.3%, PV-11.4% vs 35.9%), suggesting an anti-phagocytic effect induced by cytotoxic drugs. In ET cell membranes however, CD47 expression is reduced after cyto-reductive treatment (22% vs 16.6%), suggesting instead a prophagocytic effect. Summary/Conclusion: CD47, but not CALR, is overexpressed on the membrane of patients with MPN, suggesting a role for CD47 as a strong antiphagocytic signal responsible for immune survival in MPN. We observed a significant difference in CD47 expression across different MPN subtypes with a significant increase in CD47 expression in PV and MF but not ET when patients were exposed to cytoreduction. The use of anti-CD47 antibodies could represent a new strategy to enhance the treatment response in particular in PV and MF. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Clinical Significance Of Circulating Microparticles In Ph- Myeloproliferative Neoplasms (MPN)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2368-2368 ◽  
Author(s):  
Yue Han ◽  
Shixiang Zhao ◽  
Wenjuan Zhang ◽  
Jiannong Cen ◽  
Wei Zhang ◽  
...  
Keyword(s):  
Plasma Levels ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Jak2v617f Mutation ◽  
Polycythaemia Vera ◽  
Healthy Donors ◽  
Significant Difference ◽  
Thrombotic Complications

Abstract Background Microparticles (MPs) are small membrane vesicles that are classified as red blood cell MPs (RMPs), platelet-derived MPs (PMPs), tissue factor MPs (TF+MPs) and endothelial MPs (EMPs) based on their origins. Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-MPN) are disorders characterized by abnormal hematopoiesis, thrombosis, JAK2V617F mutation. Although MPs are considered as biomarkers reflecting procoagulant state in cancer patients, their involvement in the patients with Ph-MPN remains unclear. Our objective in this study was to measure the alterations of the four MPs types in the patients with MPN and to evaluate their correlations with JAK2V617F mutation and some clinical complications, especially for thrombosis and splenomegaly. Methods Sixty-seven patients with MPN were enrolled in this study, including 12 polycythaemia vera (PV), 49 essential thrombocythemia (ET) and 6 primary myelofibrosis (PMF). 30 healthy donors were selected as normal controls. Venous blood was anticoagulated with sodium citrate (1:9). Using flow cytometry, plasma samples were measured for RMPs, PMPs, TF+MPs and EMPs with phycoerythrin (PE)-conjugated monoclonal antibodies CD235a, CD61, CD142, and CD62E, respectively. Forward scatter was set in scale using fluorescent microspheres of 0.8μm and standard fluorescent microbeads (0-0.8μm) in diameter were used to set the microparticle gate. Data were expressed as median (M) and interquartile range (IQR). Meanwhile, genomic DNA was extracted from mononuclear cells and amplified by allele specific polymerase chain reaction (PCR). Results (1) Patients with MPN showed significantly higher plasma levels for all four MPs compared with healthy donors (P<0.05), namely 49.0/μl (15.8-109.5/μl) vs 21.0/μl (13.8-32.6/μl) for RMPs, 181.2/μl(75.8-1111.6/μl) vs 74.9/μl (55.5-115.4/μl) for PMPs, 48.1/μl (13.1-72.4/μl) vs 31.0/μl (14.9-47.6/μl) for TF+MPs and 310.2/μl (128.6-1130.5/μl) vs 155.9/μl (100.3-227.6/μl) for EMPs. (2) Among different subtypes of MPN, PMPs were higher in patients with PMF than patients with PV and ET (P<0.05), but there was no significant difference between PV and ET group. No obvious difference was found in RMPs, TF+MPs and EMPs among different subtypes of MPN patients. (3) MPN patients with JAK2V617F mutation (n=34) were found to have higher plasma levels of TF+MPs and RMPs than those without mutation (P<0.05) and this difference was not found for PMPs and EMPs. (4) MPN patients with various thrombotic complications (n=10) showed higher levels of all four types of MPs than those without thrombotic complications (n=31) (P<0.05). Elevated MP levels were also found in patients with splenomegaly (n=19) compared to those without splenomegaly (n=14) (P<0.05). Conclusion Higher levels of MPs were observed in MPN patients compared with healthy controls, especially in patients complicated with thrombosis and splenomegaly, which reflects a prothrombotic state. Moreover, significantly increased TF+MPs and RMPs were found in MPN patients with JAK2V617F mutatioin. Disclosures: No relevant conflicts of interest to declare.

scholarly journals VEGF Regulation of Angiogenic Factors via Inflammatory Signaling in Myeloproliferative Neoplasms

2021 ◽  
Vol 22 (13) ◽  
pp. 6671
Author(s):  
Tijana Subotički ◽  
Olivera Mitrović Ajtić ◽  
Emilija Živković ◽  
Miloš Diklić ◽  
Dragoslava Đikić ◽  
...  
Keyword(s):  
Protein Expression ◽  
Chronic Inflammation ◽  
Myeloproliferative Neoplasms ◽  
Myeloproliferative Neoplasm ◽  
Angiogenic Factors ◽  
Mtor Signaling ◽  
Angiogenic Factor ◽  
Inflammatory Signaling ◽  
Gene And Protein Expression ◽  
Hel Cells

Background: Chronic inflammation has been recognized in neoplastic disorders, including myeloproliferative neoplasm (MPN), as an important regulator of angiogenesis. Aims: We investigated the influence of vascular endothelial growth factor (VEGF) and pro-inflammatory interleukin-6 (IL-6) on the expression of angiogenic factors, as well as inflammation-related signaling in mononuclear cells (MNC) of patients with MPN and JAK2V617F positive human erythroleukemic (HEL) cells. Results: We found that IL-6 did not change the expression of angiogenic factors in the MNC of patients with MPN and HEL cells. However, IL-6 and the JAK1/2 inhibitor Ruxolitinib significantly increased angiogenic factors—endothelial nitric oxide synthase (eNOS), VEGF, and hypoxia-inducible factor-1 alpha (HIF-1α)—in patients with polycythemia vera (PV). Furthermore, VEGF significantly increased the expression of HIF-1α and eNOS genes, the latter inversely regulated by PI3K and mTOR signaling in the MNC of primary myelofibrosis (PMF). VEGF and inhibitors of inflammatory JAK1/2, PI3K, and mTOR signaling reduced the eNOS protein expression in HEL cells. VEGF also decreased the expression of eNOS and HIF-1α proteins in the MNC of PMF. In contrast, VEGF increased eNOS and HIF-1α protein expression in the MNC of patients with PV, which was mediated by the inflammatory signaling. VEGF increased the level of IL-6 immunopositive MNC of MPN. In summary, VEGF conversely regulated gene and protein expression of angiogenic factors in the MNC of PMF, while VEGF increased angiogenic factor expression in PV mediated by the inflammation-related signaling. Conclusion: The angiogenic VEGF induction of IL-6 supports chronic inflammation that, through positive feedback, further promotes angiogenesis with concomitant JAK1/2 inhibition.

scholarly journals 83 Spatially resolved transcriptomic and proteomic investigation of breast cancer and its immune microenvironment

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A91-A91
Author(s):  
Jennifer Chew ◽  
Cedric Uytingco ◽  
Rapolas Spalinskas ◽  
Yifeng Yin ◽  
Joe Shuga ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Tumor Microenvironment ◽  
Protein Expression ◽  
Protein Detection ◽  
Response To Therapy ◽  
Pathological Examination ◽  
Multi Drug Resistance ◽  
Spatially Resolved ◽  
Gene And Protein Expression

BackgroundThe tumor microenvironment (TME) is composed of highly heterogeneous extracellular structures and cell types such as endothelial cells, immune cells, and fibroblasts that dynamically influence and communicate with each other. The constant interaction between a tumor and its microenvironment plays a critical role in cancer development and progression and can significantly affect a tumor’s response to therapy and capacity for multi-drug resistance. High resolution analyses of gene and protein expression with spatial context can provide deeper insights into the interactions between tumor cells and surrounding cells within the TME, where a better understanding of the underlying biology can improve treatment efficacy and patient outcomes. Here, we demonstrated the ability to perform streamlined multi-omic tumor analyses by utilizing the 10X Genomics Visium Spatial Gene Expression Solution for FFPE with multiplex protein enablement. This technique simultaneously assesses gene and protein expression to elucidate the immunological profile and microenvironment of different breast cancer samples in conjunction with standard pathological methods.MethodsSerial (5 µm) sections of FFPE human breast cancer samples were placed on Visium Gene Expression (GEX) slides. The Visium GEX slides incorporate ~5,000 molecularly barcoded, spatially encoded capture spots onto which tissue sections are placed, stained, and imaged. Following incubation with a human whole transcriptome, probe-based RNA panel and an immuno-oncology oligo-tagged antibody panel, developed with Abcam conjugated antibodies, the tissues are permeabilized and the representative probes are captured. Paired GEX and protein libraries are generated for each section and then sequenced on an Illumina NovaSeq at a depth of ~50,000 reads per spot. Resulting reads from both libraries are aligned and overlaid with H&E-stained tissue images, enabling analysis of both mRNA and protein expression. Additional analyses and data visualizations were performed on the Loupe Browser v4.1 desktop software.ConclusionsSpatial transcriptomics technology complements pathological examination by combining histological assessment with the throughput and deep biological insight of highly-multiplexed protein detection and RNA-seq. Taken together, our work demonstrated that Visium Spatial technology provides a spatially-resolved, multi-analyte view of the tumor microenvironment, where a greater understanding of cellular behavior in and around tumors can help drive discovery of new biomarkers and therapeutic targets.

Mechanisms of Co-Operation of DNMT3A Mutations with JAK2 V617F Through Histone H4 Arginine 3 Provides New Insights in MPN Disease Pathogenesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2823-2823
Author(s):  
Nisha Rao ◽  
Carolyn M Butcher ◽  
Petra J Neufing ◽  
Sarah Bray ◽  
Chris N Hahn ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Somatic Mutations ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Chronic Phase ◽  
Site Directed Mutagenesis ◽  
Hek293 Cells ◽  
Histone H4 ◽  
Critical Residue

Abstract Abstract 2823 Myeloproliferative neoplasms (MPN), including Polycythemia vera (PV), Essential Thrombocythemia (ET) and Primary myelofibrosis (PMF), are clonal stem cell disorders characterised by an excess of mature cells of one or more myeloid lineages in the chronic phase, and an associated risk of progression to myelofibrosis and leukemic transformation. Highly specific somatic mutations of JAK2 and MPL are found in the majority of MPN patients, while numerous other somatic mutations have now been identified to be common between MPN and de novo acute myeloid leukemia (AML), including somatic mutations of TET2, ASXL1, CBL, IKZF1, EZH2, IDH1, IDH2 (reviewed in Tefferi, 2010 (Tefferi)), and most recently the DNA methyltransferase, DNMT3A (Abdel-Wahab, et al, Ley, et al, Stegelmann, et al, Walter, et al, Yan et al). We identified somatic heterozygous mutations in DNMT3A affecting amino acids R882 (R882C) and M880 (M880V), detected in peripheral blood mononuclear cells from two JAKV617F-positive PV patients (Rao et al, 2011, accepted for publication). At the level of chromatin and gene regulation, cooperation of DNMT3A mutations with JAK2V617F can occur through direct interaction of DNMT3A with the chromatin modifying protein PRMT5 which is a direct phosphorylation target of JAK2V617F (Liu F et al, 2011). PRMT5 is an arginine methyltransferase which associates with chromatin and methylates the key arginine (R) residue at the N-terminus of histone H4 (R3), which in turn recruits a DNMT3A complex resulting in methylation of adjacent CpG residues and gene-silencing (Zhao Q et al., 2009). To test the functional affects of DNMT3A mutations M880V and R882C, we have performed site- directed mutagenesis to generate GFP-tagged DNMT3A wild-type and mutant retroviral constructs. These DNMT3A clones will be transduced into cord blood CD34+ve cells and GFP-positive cells selected with flow cytometry. For functional analysis, we will examine DNA methyltransferase activity, changes in histone H4 modifications and CpG methylation. In a complementary analysis we have identified a change in a JAK2V617F+ve PV patient in the highly conserved HIST1H4C gene resulting in a substitution of Arg3 for Cys in Histone H4. Although there are 15 histone H4 genes encoding identical proteins, gene expression varies significantly in tissues and in cancer cells (W.F Holmes et. al, J.Biol Chem, 2005). We have used Q-PCR, using long primers to differentiate H4 transcripts and measured relative expression levels. We have confirmed that HIST1H4C is the major contributor of the H4 pool making ≥50% of the total H4 RNA in normal CD34+ve cells, Monocytes, lymphocytes and a variety of haemopoietic cell lines, indicating that the change to this critical residue in this gene may be functionally important. An analysis of a matched buccal sample showed that the HIST1H4C R3C variant in the PV patient was germline. Subsequent sequencing of the entire open reading frame of HIST1H4C in 232 normal individuals did not identify any other amino acid substitutions affecting this gene. Given the interaction of this residue with DNMT3A, we are currently investigating functional effects associated with this histone H4 variant. We have generated HEK293 cells with 4-Hydroxy tamoxifen (4-HT) inducible expression of the H4 R3C variant, and following the addition of 4-HT, we are initially assessing changes in histone H4 post-translational modifications, and markers of DNA damage such as gamma H2AX serine 139 phosphorylation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression levels of MCP-1, HGF, and IGF-1 in endometriotic patients compared with non-endometriotic controls

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sahel Heidari ◽  
Roya Kolahdouz-Mohammadi ◽  
Sepideh Khodaverdi ◽  
Nader Tajik ◽  
Ali-Akbar Delbandi
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Protein Expression ◽  
Mononuclear Cells ◽  
Endometrial Stromal Cells ◽  
Peripheral Blood Mononuclear ◽  
Monocyte Chemoattractant Protein 1 ◽  
Gene And Protein Expression ◽  
Its Gene ◽  
Hepatocyte Growth

Abstract Background To study the concentrations of monocyte chemoattractant protein-1 (MCP-1), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1) in peritoneal fluid (PF) and serum, and to evaluate their expressions by PF and peripheral blood mononuclear cells (PFMCs and PBMCs, respectively), and ectopic and eutopic endometrial stromal cells of patients with endometriosis (EESCs and EuESCs, respectively) compared with controls. Methods The concentrations of mentioned cytokines in serum and PF, as well as their expression in PBMCs, PFMCs, EuESCs and EESCs from endometriosis patients and controls were assessed. Results The levels of MCP-1, HGF, and IGF-1 in serum and PF in women with endometriosis were significantly higher than the controls (P < 0.05–P < 0.001). Gene expression of MCP-1 and IGF-1 in the PFMCs, PBMCs and EESCs also showed an increased level compared to controls (P < 0.05–P < 0.01). The protein expression of MCP-1 and IGF-1 by PFMCs was statistically higher in endometriotic women (P < 0.05 and P < 0.01, respectively). The gene and protein expression of HGF in PFMCs and its gene expression by EESCs were significantly higher in endometriotic women compared to controls (P < 0.05–P < 0.01). Conclusions The higher concentrations of mentioned cytokines in serum and PF and their higher expression by PFMCs and EESCs in endometriosis patients may contribute to the development of endometriosis.

Transferrin Receptor 2 Protein Is Expressed in Mitochondrial-Associated Lysosomes During Human Erythropoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 628-628
Author(s):  
Seung-Jae Noh ◽  
Yun-Ping Wu ◽  
Colleen Byrnes ◽  
Y. Terry Lee ◽  
Jeffery L. Miller
Keyword(s):  
Protein Expression ◽  
Transferrin Receptor ◽  
Cellular Localization ◽  
Conflicts Of Interest ◽  
Correlation Coefficients ◽  
Jurkat Cells ◽  
Divalent Metal Transporter ◽  
Golgi Stack ◽  
Gene And Protein Expression ◽  
Transferrin Receptor 2

Abstract Abstract 628 Regulated expression of transferrin receptor 1 (TFR1) provides for the major mechanism of iron binding and absorption by endocytosis in erythroblasts. In contrast, the expression of transferrin receptor 2 (TFR2) in erythroid cells is not well understood. Fourteen-day cultures of human CD34+ cells were utilized to explore TFR2 gene and protein expression during erythropoiesis. Transcriptome analyses revealed an expression of the TFR2 gene on culture days 7 and 14. Protein analyses were performed with a TFR2 antibody that demonstrated a doublet band at 105 Kd in K562 and HepG2 cells, but no band in Jurkat cells. Western analyses of the primary erythroblasts demonstrated maximum TFR2 levels on culture days 6-8 coinciding with the onset of high-level glycophorin A expression and just prior to hemoglobin accumulation in the cells. Cellular localization of TFR2 was assessed by dual-staining confocal microscopy. More than 15 cells were analyzed for the calculation of correlation coefficients from 4-6 separate pictures per each sample. Unlike TFR1, expression of TFR2 on the plasma membrane was low or absent at all stages of erythroblast maturation. Expression of both proteins was detected in the endosomes throughout the cytoplasm on culture days 2-6. After culture day 6, TFR1 and TFR2 localization became divergent, with TFR2 localized almost exclusively to a single perinuclear compartment near the Golgi stack in the region of the centrosome. Co-localization experiments indicated that TFR2 was most highly co-localized with LAMP1, a lysosome marker. DMT1, the divalent metal transporter was also colocalized with the TFR2-lysosomes. The TFR2-lysosomes were additionally surrounded by and closely associated with mitochondria. Live cell imaging revealed dynamic interactions between the lysosomes and surrounding mitochondria. Pulse-chase experiments using Alexafluor 594-labeled transferrin (Tf) showed that endocytosed Tf was initially co-localized with TFR1. However, Tf was transported to the TFR2 localized region after a chase period of 10 minutes. In summary, TFR2 protein expression is highly regulated during erythropoiesis and localized to centrosomal lysosomes. These studies further suggest that iron regulation in erythroblasts includes trafficking to TFR2 and DMT1 containing lysosomes, which are dynamically associated with mitochondria. Disclosures: No relevant conflicts of interest to declare.

Circulating Mesenchymal Stromal Cells As a New Prospective Cancer Marker,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3404-3404
Author(s):  
Mikhail Kolonin ◽  
Yan Zhang ◽  
Paul J. Simmons ◽  
Charles Bellows
Keyword(s):  
Endothelial Cells ◽  
Progenitor Cells ◽  
Cancer Patients ◽  
Cancer Progression ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Surrogate Marker ◽  
Response To Therapy ◽  
Peripheral Blood Mononuclear

Abstract Abstract 3404 Mobilization of progenitor cells is implicated in pathology and can be indicative of disease progression. Recently, we reported the influence of body mass index (BMI) on level of circulating progenitor cells (Bellows et al., Obesity 2011). Comparative analysis of peripheral blood mononuclear cells (PBMC) from 12 non-obese (BMI < 30) and 14 obese (BMI > 30) disease-free donors by flow cytometry revealed that obesity is associated with a 10-fold increased frequency of circulating mesenchymal stromal progenitor cells (MSC), which circulate at a very low level in healthy lean individuals. We showed that obesity is also associated with a 5-fold increased frequency of circulating progenitor cells (CPC), a population consisting of hematopoietic and endothelial precursors, while the frequencies of mature endothelial cells (EC) and CD34-bright leukocytes (CD34b LC) are unaffected by BMI. Here, we followed up on the assessment of circulating MSC as a potential surrogate pathology marker by analyzing the frequency of circulating CD34-positive progenitor and endothelial cells in a cohort of colorectal cancer patients. PBMC were collected from 45 obese and lean cancer patients and compared to control cancer-free donors. Flow cytometric enumeration of cells was performed based on established immunophenotypes: CD34brightCD31dimCD45dim (CPC), CD34dimCD31brightCD45- (EC), CD34brightCD31-CD45- (MSC) and CD34brightCD45bright CD34b (LC). Groups were compared using multivariate regression analysis. After adjusting for co-founders such as age and BMI, the mean frequencies of MSC and CD34bLC, but not of CPC and EC, were found to be significantly higher in the circulation of CRC patients compared to cancer-free donors. Interestingly, the frequency of circulating MSC, but not of the other cell populations, was also found to be significantly higher in the circulation of obese CRC patients compared to lean CRC patients and obese cancer-free controls. We conclude that markedly increased frequency of MSC in the peripheral blood may represent a new diagnostic CRC marker. BMI-dependent changes in circulating MSC, potentially mobilized from adipose tissue may reveal their trafficking to tumors, which could be one of the mechanistic links between obesity and cancer progression. Validation of MSC as a new surrogate marker of cancer could provide a tool for determining prognosis, predicting response to therapy, and detecting relapse following treatment. Disclosures: No relevant conflicts of interest to declare.

Erythropoietin Treatment Enhanced Cell Surface CXCR4 Expression On Neonatal CD34-Positive Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4724-4724
Author(s):  
Norioki Ohno ◽  
Seiichi Hayakawa ◽  
Shuhei Karakawa ◽  
Masao Kobayashi
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Cell Surface ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Cxcr4 Expression ◽  
Stem Cell Marker ◽  
Cell Marker ◽  
Low Oxygen ◽  
Hematopoietic Stem

Abstract Abstract 4724 Erythropoietin (EPO), which is used to treat anemic immature neonates, is thought to be effective for treating neonatal hypoxic encephalopathy by stimulating nervous system cells or vascular stem cells. On stem cells, CXCR4 is an important chemokine receptor for cell migration and proliferation. Previously, we reported that increased expression of CXCR4 on cord blood (CB)-derived CD34-positive cells, caused by low oxygen tension, enhanced cell homing activity and engraftment (Ohno et al., ASH. 2010). In human endothelial stem cells, CXCR4 expression is controlled by hypoxia-inducible factor-1α (HIF-1α), and EPO expression is enhanced by hypoxia via HIF-1α. In this study, we examined the effect of EPO treatment on CXCR4 expression on premature neonatal endothelial stem cells and cultured CB-derived CD34-positive cells. First, we examined the cell surface markers CD34, CD133, and CXCR4 in preterm infants diagnosed with anemia and treated with EPO. The 10 enrolled neonates were first administered EPO when they were over 32 weeks old and were not given oxygen therapy. Blood samples were collected twice before and once 24 h after EPO administration. Mononuclear cells were isolated from peripheral blood and stained for the hematopoietic stem cell marker CD34, vascular endothelial progenitor cell marker CD133, and surface CXCR4. The cells were analyzed by flow cytometry. No change in the proportions of CD34- and CD133-positive cells were found after the treatment (CD34 positive: 0.59%→0.57%; CD133 positive: 0.52%→0.49%). The proportion of cell surface CXCR4 on the CD133-positive cells did not change after EPO administration, whereas the proportion of cell surface CXCR4 on the CD34-positive cells increased significantly after EPO was administered (average: 7.6%→11.0%, p < 0.01). In particular, slightly CD34-positive cells showed enhanced cell surface CXCR4. Next, we examined CXCR4 enhancement in EPO-treated CB-derived CD34-positive enriched samples. Samples were divided into three aliquots and cultured. The first aliquot was incubated for 24 h in RPMI-1640 medium alone as a control; the second, for 24 h in RPMI-1640 medium with EPO (10 U/ml); and the third, for 24 h in RPMI-1640 medium with EPO (10 U/ml) containing the specific HIF-1 antagonist rapamycin. Flow cytometry revealed significantly increased surface CXCR4 expression on CD34-positive cells after incubation with EPO compared with the control expression (average: 51.7% vs. 30.3%, p< 0.05). This enhancement was inhibited completely by the addition of rapamycin. Intracellular HIF-1 was enhanced significantly in the EPO-treated cells compared with the control expression on flow cytometry. The enhanced CXCR4 expression on CD34-positive cells reflects an amplification of the chemotactic and homing abilities of stem cells. The CXCR4 enhancement caused by EPO may increase tissue migration of CD34-positive cells, although this is probably a result of an active HIF-1 pathway. Disclosures: No relevant conflicts of interest to declare.

Derangements of Toll-like Receptors, Inflammatory Cytokines, and Reactive Oxygen Species in Philadelphia Chromosome-Negative Myeloproliferative Neoplasm: Implicate Roles of Inflammation in the Pathogenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1945-1945 ◽  
Author(s):  
Guanfang Shi ◽  
Hui Chen ◽  
Cherif Abdelmalek ◽  
Andrei Bandarchuk ◽  
Abdullah Khawer Mahmood ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dendritic Cells ◽  
Inflammatory Cytokines ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Philadelphia Chromosome ◽  
Toll Like Receptors ◽  
Clonal Proliferation ◽  
Inflammatory Processes

Abstract Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) including essential thrombocythemia(ET), polycythemia vera(PV), and myelofibrosis (MF) have been regarded as a stem cell diseases with malignant clonal proliferation; but recently, inflammatory processes have been proposed as playing found a major role in the pathogenesis of MPN. Toll-like receptors (TLRs) are a family of pattern-recognition receptors that function as key initiators of innate immunity signaling, then induce inflammatorycytokines, and ROS formation. Therefore we measured TLRs, inflammatory cytokines, and ROS in patients with MPN to assess the role of inflammation in MPN. Methods: TLR assay.TLR-2, 4, 7, 9 quantification was performed by immuno-staining of 1×106 mononuclear cells which were incubated with fluorescence-conjugated anti-TLR-2, 4, 7, 9 antibodies and assayed by flow cytometry. Monocytes culture and dendritic cells differentiation.Human monocytes were isolated from human peripheral blood mononuclear cells (PBMNC) using Pan Monocyte Isolation Kit. Isolated monocytes were incubated with IL-4 and GM-CSF in cultures to differentiate to dendric cells . Immature dendritic cells were either left untreated or stimulated with Pam3CSK4. The maturation of dendritic cells was determined by flow cytometry using CD80, CD83, CD11c, and HLA-DR. Multiplex ELISA. Human plasma and cell culture supernatants were analyzed in duplicates by Meso Scale Discovery Multi-Spot Assay system. In total, ten cytokines were assayed: IFN-g, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-a. Cell ROS measurement. Cellular ROS was determined by a dichlorofluorescein (DCF) assay. PBMNCs were incubated with 10 µM 2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) (Life Technology) for 30 min at 37°C. The oxidation of H2DCFDA was measured and analyzed by flowcytometery. Results. 1)TLR2 was the only TLR found to be elevated in PV or ET patients (Fig 1). 2) PlasmaIL-1b levels were elevated in TLR2 high patients than TLR-2 low patients. 3) No difference between TLR-2 high and low patients in the maturation of monocytes to dendritic cells. 4) Monocyte derived dendritic cells with high TLR-2 patientsreleased more IL-8 and TNF-a after Pam3CSK4 stimulation (Fig 2). 5) ROS were more elevated in MF patients than PV, ET patients, and controls (Fig 3). Conclusion. 1) TLR 2 is significantly elevated in PV and some ET patients and TLR-2 high patients were found to have elevated plasma IL-1b,and IL-8, and TNF-α in monocytes-derived dendric cell cultures afterPam3CSK4 stimulation than TLR-2 low patients. This confirms that TLR-2 is deranged in PV and ET. 2) ROS is elevated in MF patients compared to ET and PV patients, or controls. Thus, this study suggests that inflammatory processes likely play a role in the pathogenesis of Ph- MPN through first TLR-2 derangement in PV and ET , then through years of chronic inflammatory process , with the accumulation of more ROS seen in MF, which caused more damage to the DNA resulting more malignant clonal proliferation. A similar phenomenon was observed that in JAK2V617F mutation , allele-burden was observed gradually increased from ET , PV then to MF . Disclosures No relevant conflicts of interest to declare.

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