Comparison of Three Different NOD/SCID-Related Strains in Preclinical Models of Acute Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2361-2361
Author(s):  
Alice Agliano ◽  
Ines Martin-Padura ◽  
Paola Merighetti ◽  
Patrizia Mancuso ◽  
Cristina Rabascio ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Blood Cells ◽  
Lymphoblastic Leukemia ◽  
Primary Cells ◽  
Preclinical Models ◽  
Immunodeficient Mice ◽  
Nsg Mice ◽  
Different Strains

Abstract Transplantation of human acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) primary cells and cell lines in a variety of different strains of immunodeficient mice has led to preclinical models extensively used to investigate AML and ALL stem cells, biology, and drug sensitivity. We investigated the engraftment kinetics of two AML cell lines (HL-60 and KG-1), two ALL cell lines (MOLT-16 and 697) and AML primary cells from an AML M4 patient (QD1-EIO, described in Fusetti et al, Cancer Res 2000) in 3 different strains of NOD/LtSz-Prkdcscid (NOD/SCID, NS)-related immunodeficient mice. NS, NS/beta2 null (NSB) and NOD/SCID/IL-2Rgamma null (NSG) mice were injected ip with 10x106 AML or ALL cells. Mice were observed daily and sacrificed when leukemia-related symptoms were evident. Overall, leukemia-related symptoms were observed in 71, 84 and 86% of NS, NSB and NSG mice, respectively (n=42, p<0.01), after a median of 53, 49 and 35 days (p<0.001). Leukemia engraftment was investigated in the marrow, the blood and the spleen by means of morphology, flow cytometry and quantitative PCR for human genes. AML HL-60 and KG-1 cells accounted for 10-6, 5-1 and 7-3 % of peripheral blood cells in NS, NSB and NSG mice, respectively. ALL MOLT-16 and 697 cells accounted for 2-12, 22-27 and 1-27 % of blood cells in NS, NSB and NSG mice, respectively. AML primary cells QD1-EIO accounted for <1, <1 and 3% of blood cells in NS, NSB and NSG mice, respectively. Similar engraftment results were observed in the marrow and in the spleen. Leukemia cell-injected NSG mice, compared to NS and NSB, showed a significantly higher increase of circulating endothelial mature cells (CEC, enumerated by flow cytometry as CD45−, CD13+ VEGFR2+ cells) and progenitors (CEP, enumerated by flow cytometry as CD45−, CD13+ VEGFR2+ CD117+ cells, see Shaked et al, Cancer Cell 2005). This CEC and CEP increase paralleled leukemia engraftment. Taken together, our data indicate that the 3 different strains have significantly different leukemia engraftment behavior and kinetics. Engraftment in NSG mice is significantly faster compared to the other two strains, leukemia-related microenvironment is differently modulated, and less leukemic burden might be needed to observe leukemia-related symptoms. These data might have major implications to design future studies on leukemia-initiating stem cells, leukemia biology and preclinical leukemia treatment studies.

Flow Cytometry Detection of Intra-Cellular Tyrosine Kinase Inhibitors (TKI) Showed Variable Uptake in CML CD34+ Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2747-2747
Author(s):  
Céline Bourgne ◽  
Mahchid Bamdad ◽  
Alexandre Janel ◽  
Frédéric Libert ◽  
Agnès Guerci ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Blood Cells ◽  
Second Generation ◽  
Molecular Response ◽  
Primary Cells ◽  
Uv Fluorescence ◽  
Cd34 Cells

Abstract Abstract 2747 Introduction Despite the major benefit of TKI in the treatment of Chronic Myeloid Leukemia (CML), patient response is heterogeneous and it is generally accepted that residual disease and relapse are due to persistent CML cells, considered as leukemic stem cells. Their resistance has been related to lower TKI uptake. The amount of drug penetrating the targeted cells is most likely a major parameter of targeted therapy efficacy since it is essential that the therapeutic molecule be as close as possible to the target molecule. We developed a flow cytometry technique to analyze primary cells. Method To evaluate intracellular imatinib (ICIM) uptake, we developed a patented method based on natural UV fluorescence related to chemical structure. Consequently, since the difference in UV fluorescence units between treated and control cells is proportional to the amount of intra-cellular drugs, we validated this method after incubating K562 and KCL22 cell lines with TKI. The flow cytometry technique was standardized by using Flow-Check Fluorosphere calibrated beads immediately before, and at the end of, each series of analyses with a Coulter Epics Elite™ flow cytometer (Beckman Coulter) equipped with an Innova I90C-4 UV laser (Coherent). Then we analyzed primary blood cells from CML patients in chronic phase before any treatment. After lysis of erythrocytes, nucleated cells were incubated at 1.106 cells/ml with different doses of imatinib (IMA) (n=22), Nilotinib (NIL) (n=20) and Dasatinib (DAS) (n=20) at different times. Whenever possible, CML stem cells were analyzed using CD34-FITC staining. Results In preliminary assays, we checked that there was a significant correlation between additional fluorescence measured by flow cytometry and the amount quantified by physico-chemical analysis after lysing a known number of cells (n=57, r2=0.73, p<0.001), which enabled us to convert UV fluorescence into pg of IMA per cell. Then we confirmed that IMI rapidly penetrated K562 and KCL22 cells (from 5 minutes of incubation) and reached a stable influx in viable cells from 1 hour (T1h). We chose this incubation time for further experiments. Similarly, we choose T2h for second generation TKI. We observed a dose-dependent accumulation in the two cells lines, but with differences at the lowest extra-cellular concentrations (1–5 μM) and not correlated with any membrane pump expression (OCT-1, ABCG2, ABCB1 and ABCC1). ICIM at T1h was correlated with cell sensitivity to IM at T24h expressed by the proportion of dead cells (r2=0.93 and 0.88 for K562 and KCL22 cells, respectively). We then applied our method to primary CML blood cells in comparison with normal blood cells. TKI penetrated all cell subsets, but amounts varied depending on cell sizes (FS/SS characteristics). The first data obtained with IM showed ICIM levels in CML cells that were relatively heterogeneous from one patient to another, ranging from 0.9 to 4 pg/cell for an extracellular concentration of 5 μM, i.e. a higher concentration (x 300) than in culture medium. The ability of the granulocyte cell lineage to store IMA was related to the Sokal prognostic index (p=0.05). We detected variable ICIM levels in CML CD34+ cells from 10/16 patients (0.04–0.7 pg/cell) and no signal for 6/16 patients. Surprisingly, the ability of CD34+ to store second generation TKIs is variable and not necessarily correlated to IMA uptake. Discussion We developed a simple, rapid flow cytometry method directly applicable to primary cells and requiring only few cells which makes it possible to identify target cell subsets, such as CML stem cells. The strong correlation between the ICIM amount and the sensitivity of CML cell lines to TKIs validated the method and suggested that ICIM could be a relevant biomarker for predicting the sensitivity of the CML clone. In our CML series, we observed striking inter-patient variability of the capacity of primary CML cells to store TKI. A correlation with the Sokal score suggests possible predictive value with regard to in vivo CML response to IMA, which could be taken into account when choosing TKI for first-line therapy. Furthermore, we observed marked heterogeneity between CML CD34+ cells for storing TKI that could partially explain the heterogeneity of in vivo response. The relationship between the ability of untreated CML CD34+ cells to store TKI and complete molecular response has to be established. Disclosures: No relevant conflicts of interest to declare.

Evaluation of CD9 as a Candidate Leukemia Stem Cell Marker In Childhood Precursor B Cell Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3241-3241
Author(s):  
Noriko Satake ◽  
Astra Chang ◽  
Bridget McLaughlin ◽  
Sara Bauman ◽  
James Chan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Raman Spectroscopy ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Heterogeneous Group ◽  
Leukemia Cells ◽  
Nsg Mice ◽  
Primary Leukemia

Abstract Abstract 3241 Leukemia cells are believed to arise from leukemia stem cells (LSC). It is also known that LSC are responsible for relapse in certain types of leukemia, such as acute myeloid leukemia (AML). However, the existence and role of LSC in acute lymphoblastic leukemia (ALL) is unclear. CD9 was reported to be a marker for LSC in B-ALL using cell lines (Nishida H. et al., 2009). CD9 is a tetraspanin and is believed to be involved in cell adhesion, motility, and signaling events. It is also involved in metastasis; however, the mechanisms are unknown. Since childhood ALL is a heterogeneous group of diseases and cell lines can be different from primary leukemia cells, we tested the role of CD9 as a candidate LSC marker using primary precursor B (preB) ALL cells from pediatric patients. Two methods, Raman spectroscopy and serial transplantation of sorted leukemia cells in NOD/SCID/IL2R g null (NSG) mice, were used to confirm LSC. Raman spectroscopy is a laser-based technique for the single cell analysis of intrinsic molecular vibrations reflecting cellular biochemical information. It can provide a quantitative assessment of the levels of DNA, RNA, proteins, lipids, and carbohydrates in the cell, as well as molecular-level conformational changes. Previous studies by our group showed that unique Raman fingerprints were identified in normal blood cells, ALL cells, and stem cells, including hematopoietic stem cells and embryonic stem cells. Four preB ALL samples were stained for CD9 and sorted by flow cytometry. ALL samples were obtained from patients at diagnosis or freshly harvested from NSG mice engrafted with primary leukemia samples. All samples showed heterogeneous expression of CD9. CD9 high-positive cells and negative cells were flow sorted. Raman spectra of freshly sorted CD9 high-positive and negative cells were obtained. 10 to 20 cells were analyzed in each sample. CD34 positive cells, which were isolated from normal donors, were also analyzed by Raman spectroscopy as a control. No unique Raman fingerprints were identified to separate CD9 high-positive cells from negative cells using Principal Component Analysis (PCA). Furthermore, CD9 high-positive and negative cells from three preB ALL samples were transplanted into NSG mice via intra-bone marrow injection. Equal cell numbers (5×105 to 1.5×106 cells) were used for positive and negative samples in each injection. The majority of the mice from both groups (transplanted with CD9 high-positive or negative cells) developed leukemia 3 to 4 months after injection. Leukemia phenotype was confirmed to be the same as the original leukemia. In conclusion, although CD9 was shown to be a marker for LSC in B-ALL cell lines, it does not appear to be an LSC marker in primary preB ALL. Since childhood preB ALL is a heterogeneous group of diseases, larger cohorts are necessary to confirm our findings. Raman spectroscopy may be a useful screening tool for analysis of cellular intrinsic markers. Disclosures: No relevant conflicts of interest to declare.

CXCR7 Is Highly Expressed in Acute Lymphoblastic Leukemia and Potentiates the Chemotactic Response to SDF-1

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1482-1482
Author(s):  
Rita de Cassia Carvalho Melo ◽  
Carolina Bigarella ◽  
Mariana Ozello Baratti ◽  
Fabíola Traina ◽  
Sara T Olalla Saad
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Whitney Test ◽  
Mann Whitney Test ◽  
Cell Chemotaxis

Abstract Abstract 1482 The crosstalk between leukemic cells and their microenvironment provides pivotal signals for the localization and progression of leukemias. The stromal derived factor-1 (SDF-1)/CXCR4 pathway is deregulated in hematology malignances, such as acute lymphoblastic leukemia (ALL). SDF-1 represents the major chemokine for initiating stem cell chemotactic migration. The majority of cytokines that mediate stem cell chemotaxis does so via modulation either of SDF-1 or of its receptor, CXCR4. CXCR4 receptor is expressed in leukemic cells enabling cells to access marrow niches that normally are restricted to quiescent stem cells, thereby ensuring its protection of cell death resulting in a worse prognosis. Recently, CXCR7 was identified as another SDF-1-binding receptor, but its contribution to SDF-1 – mediated effects in hematopoietic cells is still poorly explored, even though the CXCR7 relationship with tumor progression in non-hematopoietic malignancies is well established. Given that SDF-1/CXCR4 signaling is altered in patients with leukemia and that there is little information regarding CXCR7 in leukemia we investigated its expression in patients and leukemia cell lines. In addition, the relationship of CXCR4 and CXCR7 in potentiating the SDF-1 response was also investigated. mRNA expression of CXCR7 was analyzed by Real-time PCR (normalized by GAPDH and HPRT) in bone marrow samples of 29 acute myeloid leukemia (AML), 11 ALL patients and 12 control subjects (healthy donors). CXCR7 protein expression in myeloid cell lines (U937, P39, K562 and KG -1) and lymphoblastic cell lines (Jurkat, Molt-4, Raji e Daudi) was analyzed by western blot. Localization of CXCR4 and CXCR7 proteins was investigated using flow cytometry and confocal microscopic analysis and CXCR7 knockdown cells were obtained by transduction with lentivirus-mediated shRNA. These cells were treated with AMD3100 (antagonist CXCR4) and their chemotactic capacity was analyzed by transwell chemotaxis assay. CXCR7 was significantly higher expressed in ALL compared to AML and control subjects (p=0.0008, Mann-Whitney test). CXCR7 protein (western blot with ABCAM antibody) was also higher expressed in lymphoblastic cell lines (Molt-4 and Jurkat) compared with myeloid cells. The subcellular location of CXCR7 and CXCR4 by confocal microscopy and flow cytometry evidenced CXCR7 in the membrane of Molt-4 cells and in the cytoplasm of Jurkat cells whereas CXCR4 was in the membrane of both cell lines. Interestingly, we also noticed that, after SDF-1 induction, Molt-4 cells have higher chemotactic ability compared with Jurkat (median Molt 4=52.0 ± 5 vs Jurkat=24.1 ± 3, p=0.0079, Mann-Whitney test) which may be related with the membrane availability of CXCR7. In addition, the inhibition of CXCR7 or CXCR4 resulted in significant changes in Molt4 and Jurkat chemotactic response (0.01&gt;p&lt; 0.02, Mann-Whitney test), however, the inhibition of both CXCR7 and CXCR4 resulted in a more significant reduction in cell migration (p=0.0079/Molt-4; p=0.0043/Jurkat, Mann-Whitney test). Increased expression of CXCR7, as here observed in lymphoblastic leukemia cells, is a phenomenon already described in a variety of solid tumor cell lines such as brain, prostate and lung. In solid tumors, CXCR7 mainly increases the proliferation of malignant cells. These results suggest that the biological function of CXCR7 depends on its tissue and organ localization and that, in acute lymphoblastic leukemia may have a role in cell chemotaxis, potentiating CXCR4 response to SDF-1 and thus, could contribute for leukemia initiating cell recruitment to niches once occupied by normal hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals 883 An anti-carcinoma monoclonal antibody (mAb) NEO-201 can also target human acute myeloid leukemia (AML) cell lines in vitro

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A925-A925
Author(s):  
Alessandra Romano ◽  
Nunziatina Parrinello ◽  
Sara Marino ◽  
Enrico La Spina ◽  
Massimo Fantini ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Functional Analysis ◽  
Epithelial Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Phase 1 ◽  
Adcc Activity ◽  
Phenotypic Analysis

BackgroundNEO-201 is an IgG1 mAb targeting variants of CEACAM5/6 and has demonstrated tumor sensitivity and specificity in epithelial cells. Functional analysis has revealed that NEO-201 can engage innate immune effector mechanisms including ADCC and CDC to directly kill tumor cells expressing its target. A recent Phase 1 clinical trial at the NCI has determined both safety and recommended Phase 2 dosing. We have also seen the expression of the NEO-201 target on hematologic cells, specifically Tregs and neutrophils. Due to epitope being expressed both on malignant epithelial cells as well as several hematologic cells, we designed this study to explore the reactivity of NEO-201 against hematological neoplastic cells in vitro.MethodsPhenotypic analysis was conducted by flow cytometry. Cell lines used were six AML (HL60, U937, MOLM13, AML2, IMS-M2 and OCL-AML3), two multiple myelomas (MM) (OPM2, MM1.S), two acute lymphoblastic leukemia (ALL) (SUP-B15, RPMI8402) and four mantle cell lymphoma (MCL) (Jeko-1, Z138, JVM2 and JVM13). Markers used for flow cytometry analysis were CD15, CD45, CD38, CD138, CD14, CD19 and NEO-201. Functional analysis was performed by evaluating the ability of NEO-201 to mediate ADCC activity against AML cell lines using human NK cells as effector cells.Results5 of 6 AML cell lines tested bind to NEO-201 and the% of positive cells were 47%, 99.5%,100%,100% and 97.8% for HL60, U937, MOLM13, AML3 and IMS-M2, respectively. The% of positive cells in the two MM cell line were 99% and 18% for OPM2 and MM1.S, respectively. NEO-201 binding was not detected in the two ALL and the four MCL cell lines tested. Functional analysis has demonstrated that NEO-201 can mediate ADCC activity against the AML cell line (HL60) tested.ConclusionsThis study demonstrates that NEO-201 mAb’s target is expressed in most of the AML cell lines tested in vitro. In addition, we have shown it can mediate ADCC activity against HL60 cells (AML). Together, these findings provide a rationale for further investigation of the role of NEO-201 in AML as well as MM, further exploring patient PBMCs and bone marrow samples.

scholarly journals Sustained human hematopoiesis in immunodeficient mice by cotransplantation of marrow stroma expressing human interleukin-3: analysis of gene transduction of long-lived progenitors

Blood ◽  
1994 ◽  
Vol 83 (10) ◽  
pp. 3041-3051 ◽  
Author(s):  
JA Nolta ◽  
MB Hanley ◽  
DB Kohn
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Gene Transduction ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Interleukin 3 ◽  
Human Cd34 ◽  
Time Period

Abstract We have developed a novel cotransplantation system in which gene- transduced human CD34+ progenitor cells are transplanted into immunodeficient (bnx) mice together with primary human bone marrow (BM) stromal cells engineered to produce human interleukin-3 (IL-3). The IL- 3-secreting stroma produced sustained circulating levels of human IL-3 for at least 4 months in the mice. The IL-3-secreting stroma, but not control stroma, supported human hematopoiesis from the cotransplanted human BM CD34+ progenitors for up to 9 months, such that an average of 6% of the hematopoietic cells removed from the mice were of human origin (human CD45+). Human multilineage progenitors were readily detected as colony-forming units from the mouse marrow over this time period. Retroviral-mediated transfer of the neomycin phosphotransferase gene or a human glucocerebrosidase cDNA into the human CD34+ progenitor cells was performed in vitro before cotransplantation. Human multilineage progenitors were recovered from the marrow of the mice 4 to 9 months later and were shown to contain the transduced genes. Mature human blood cells marked by vector DNA circulated in the murine peripheral blood throughout this time period. This xenograft system will be useful in the study of gene transduction of human hematopoietic stem cells, by tracing the development of individually marked BM stem cells into mature blood cells of different lineages.

miRNA Profiling of Pediatric ALL and Non-Hodgkin Lymphomas.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2719-2719
Author(s):  
Pablo Landgraf ◽  
Amanda Rice ◽  
Nicola Iovino ◽  
Valerio Fulci ◽  
Robert Sheridan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Burkitt Lymphoma ◽  
Lymphoblastic Leukemia ◽  
Expression Patterns ◽  
Critical Role ◽  
Mrna Translation ◽  
Cell Types ◽  
Two Samples ◽  
Hematopoetic Stem Cells

Abstract MicroRNAs (miRNAs) are conserved 21−23 nt non-coding RNA molecules that regulate gene expression either by mRNA cleavage or by repression of mRNA translation. miRNAs regulate many different processes, including apoptosis and cell proliferation and may therefore also play a critical role in oncogenic transformation. To date, most miRNAs have been discovered by cDNA cloning and sequencing, though other profiling methods, such as miRNA micro-arrays, have recently been applied. Profiling of miRNA expression by cloning has the advantage of identifying new miRNA genes, and if a large number of clones are sequenced, to also be quantitative. In addition the exact sequence is determined and polymorphisms and mutations in any miRNAs can readily be detected. To get an insight in the role of miRNAs in the differentiation and maturation of hematopoetic cells as well as their contribution to oncogenesis in ALL and lymphomas, we cloned and sequenced various cell lines and patient samples:five cell lines (B-ALL, AML, Burkitt Lymphoma); samples from sorted blood cells covering pluripotent stem cells, B-, T-, NK- cells, monocytes and granulocytes; eight patient samples with ALL (2 pro-B-ALL, 2 pre-B-ALL, 2 cALL, 2 T-ALL) at the time point of diagnosis; four additional samples of these patients with B-ALL and two samples of T-ALL patients each after 36 days of treatment according to the protocol of the German Cooperative Acute Lymphoblastic Leukemia study group (COALL-07-03). We also recorded the small RNA profiles of three patients with various forms of AML at diagnosis and after the first induction according to the protocol of the AML-BFM 2004 study; two Burkitt lymphoma samples and one B-Non Hodgkin Lymphoma (B-NHL) sample. We report here the identification of over 20 novel human miRNAs in these samples. To determine specific expression patterns, the miRNA profiles were compared to a reference set of 22 different human tissue types. Some miRNAs are expressed in a cell or tissue specific manner, others have a more general expression pattern between different cell types and tissues. For example human miRNA miR-142 is ubiquitously expressed in cells of the hematopoetic lineage, whereas human miR-150 is only expressed in differentiated hematopoetic cells, but not in hematopoetic stem cells. In hematopoetic stem cells human miR-126 is 3 to more than 10 times higher expressed than in differentiated hematopoetic cells. The existence of the latter two in humans are first described in this study. miR-16 on the other hand is expressed in all cell types examined including non-hematopoetic. Furthermore, miRNAs are up/down-regulated in ALL and NHL patient samples. In conclusion, this study identifies miRNAs that might be involved in hematopoetic cell differentiation and maturation and is important to identify miRNAs that might contribute to oncogenesis in leukemia and lymphomas.

Stimulation of TNF-α Production from Monocytes by Anti-Moesin Antibodies in the Serum of Patients with Aplastic Anemia: A Novel Mechanism of Myelosuppression by Autoantibodies.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 976-976
Author(s):  
Hiroyuki Takamatsu ◽  
Xingmin Feng ◽  
Xuzhang Lu ◽  
Tatsuya Chuhjo ◽  
Katsuya Okawa ◽  
...  
Keyword(s):  
Cell Lines ◽  
Western Blotting ◽  
Blood Cells ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Surface Proteins ◽  
Healthy Individuals ◽  
Monocytic Leukemia ◽  
Leukemia Cell Lines ◽  
Tnf Α

Abstract Although aplastic anemia (AA) is a T-cell mediated disease, recent studies have revealed the presence of antibodies (Abs) specific to proteins derived from hematopoietic progenitor cells in the serum of AA patients. It is as yet unclear whether these auto-Abs play some roles in the pathophysiology of AA. We previously demonstrated that Abs specific to moesin, a membrane-cytoskeleton linker protein in the cytoplasm, were detectable in approximately 37% of AA patients. Some reports identified moesin-like molecules on the surface of blood cells such as T cells and macrophages. It is therefore conceivable that anti-moesin Ab in AA patients may bind these immune cells and modulate hematopoietic function of AA patients. To test these hypotheses, we first studied the expression of moesin on various types of blood cells using monoclonal Ab specific to moesin (clone 38/87). Flow cytometry detected the expression of the protein recognized by anti-moesin Ab on T cells and monocytes from healthy individuals, acute monocytic leukemia cells lines including U937 and THP-1, and an acute T-lymphoblastic leukemia cell line, Molt-4, but failed to detect the molecule on CD34+ cells from healthy individuals and myeloid leukemia cell lines as well as B-lymphoblastic leukemia cell lines. Treatment of THP-1 cells with phorbol 12-myristate 13-acetate (PMA)/lipopolysaccharide (LPS) augmented the expression level of moesin. To confirm the expression of the moesin-like protein on the surface of monocytic leukemia cell lines, Molt-4 and THP-1 were treated with sulfo-NHS-SS-biotin, and the cell surface proteins were isolated with avidin-fixed column, and were subjected to Western blotting and peptide mass fingerprinting. Western blotting with anti-moesin monoclonal Abs showed two clear bands of proteins (75 kD and 80 kD); an amino acid sequence compatible with moesin was confirmed in the protein eluted from the 80 kD band. Next, we purified anti-moesin Abs from AA patients’ sera using affinity chromatography with recombinant moesin protein. Western blotting showed binding of the serum-derived Abs to a fraction of surface proteins of Molt-4, U937 and THP-1. When THP-1 cells were incubated in the presence of PMA and LPS with 5 αg/ml of control IgG or anti-moesin Abs derived from an AA patient’s serum, TNF-α production from THP-1 cells stimulated by anti-moesin Abs was 1.9–2.3 times as much as that from the control culture depending on the concentration of LPS. Incubation of THP-1 cells in the presence of monoclonal anti-moesin Abs showed the similar augmentation of TNF-α production. These results indicate that anti-moesin Abs may be involved in the suppression of hematopoiesis of AA patients by stimulating TNF-α production from monocytes.

Expression and Function of the CXCR7 Chemokine Receptor in Leukemias

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2423-2423
Author(s):  
Sergej Konoplev ◽  
Hongbo Lu ◽  
Michael A Fiegl ◽  
Zhihong Zeng ◽  
Wenjing Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Human Leukemia ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines ◽  
And Function

Abstract Background: Bone marrow produced stromal-derived factor-1a (SDF-1a) is a key chemokine involved in chemotaxis, homing, mobilization, and expansion of hematopoietic stem and progenitor cells. While the majority of well-defined functions of SDF-1a are mediated via its receptor CXCR4, recent studies have characterized CXCR7 as an alternative receptor capable of binding SDF-1a. Although the functions of CXCR7 are still incompletely understood, the receptor was reported to promote migration and adhesion in certain cell types and function as a pro-survival factor in breast cancer cells. CXCR7 expression and function in human leukemia cells has not been characterized. In this study, we examined CXCR7 expression in leukemia cell lines and primary samples from patients with acute lymphoblastic leukemia (ALL) and utilized a small molecule inhibitor of CXCR7 to probe CXCR7’s function. Materials and methods: CXCR4 and CXCR7 expression was determined by flow cytometry, real-time PCR (RT-PCR) and immunocytochemistry (ICC) in leukemic cell lines including AML (OCI-AML2, OCI-AML3, HL60, U937 NB4, Molm13), ALL (REH, Raji, RS4; 11, Nalm6, Molt4) and CML (KBM5, K562) cells. In primary ALL patient samples, CD34+CD19+ gating was applied to detect CXCR7 expression on pre-B leukemic cells by flow cytometry. The migration of leukemic cells towards SDF-1a was studied using a transwell system. CXCR4 inhibitor AMD3100 was purchased from Sigma, and CXCR7 inhibitor CCX-733 was provided by ChemoCentryx Inc., Mountain View, CA. Results: CXCR4 was found to be ubiquitously expressed on the cell surface of all leukemic cell lines tested. CXCR7 mRNA and protein expression was detectable only in Burkitt lymphoma Raji cells, as analyzed by flow cytometry (clone 11G8, R&D systems), RT-PCR and ICC. Curiously, CXCR7 expression was significantly induced in MOLM13 cells under hypoxic (6% O2) conditions (p=0.01). Low levels of surface CXCR7 were found in 8 of the 9 primary ALL samples by flow cytometry. To determine the respective roles of CXCR4 and CXCR7 in migration of leukemic cells, we utilized CXCR4 inhibitor AMD3100 and CXCR7 inhibitor CCR733 in Raji (CXCR7 positive) and RS4;11 (CXCR7 negative) cells. AMD3100 at 25μM significantly inhibited SDF-1a induced migration (from 38.5% to 12%); CCR733 at 10μM also inhibited SDF-1a induced migration (from 38.5% to 24%) and the combination of AMD3100 and CCR733 resulted in 81% inhibition of migration (from 38.5% to 7.2%). AMD3100 blocked SDF-1a induced migration of CXCR4+CXCR7− RS4;11 cells (from 36.5% to 15.8%), while CCR733 had no effect (36.5% and 39.2%). In conclusion, these studies demonstrate functional expression of the SDF-1 receptor CXCR-7 on Raji and primary ALL cells and suggest that CXCR7 plays an active role in the migration of leukemic cells. CXCR-7 may serve as an alternative receptor to CXCR4. Studies addressing the role of CXCR7 in adhesion, SDF-1a-mediated signaling and survival of leukemic cells are in progress.

Disease Propagating Blasts in Standard and High Risk Acute Lymphoblastic Leukemia Are Frequent and of Diverse Immunophenotype.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1421-1421 ◽  
Author(s):  
Klaus Rehe ◽  
Kerrie Wilson ◽  
Hesta McNeill ◽  
Martin Schrappe ◽  
Julie Irving ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
High Risk ◽  
Cancer Stem Cells ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Color Flow ◽  
Nsg Mice ◽  
Limiting Dilution

Abstract Abstract 1421 Poster Board I-444 Conflicting results in the field of cancer stem cells have reignited debate regarding the frequency and identity of cells with the ability to self renew and to propagate the complete phenotype of the malignancy. Initially it was suggested by different studies that cancer stem cells represent only a small minority of the malignant population and that the immunophenotypes of these cells resemble a rather immature type in the cell hierarchy. More recent data from our own and other groups have challenged these findings by demonstrating that cells at different maturity levels within the leukemic hierarchy have cancer stem cell abilities and that the frequency of the leukemia maintaining cell is higher than previously thought (Cancer Cell 2008, 14(1), p47-58). We use an in vivo NOD/scid IL2Rγnull (NSG) mouse intra-femoral transplant model to determine the clonogenicity of sorted candidate leukemic stem cell populations, characterized by specific immunophenotypes. We selected the surface markers CD10 and CD20, in order to differentiate between rather immature and more mature cells. Furthermore we carried out limiting dilution experiments on sorted (CD20) and unsorted leukemic blasts to investigate the frequency of the proposed leukemic stem cells. Flow sorted ALL blasts of CD19+CD20low and CD19+CD20high as well as of CD19+CD10low and CD19+CD10high immunophenotype were transplanted into NSG mice. Sorts were performed on primary patient material and on leukemic blasts that had been harvested following prior passage in mice. Different subtypes of ALL were included (high risk: BCR/ABL (t9;22) positive (patients L4967, L4951, L49101, L8849, L2510), high hyperdiploid/MRD positive high risk (L754, L835), intermediate risk: high WBC/MRD negative (L736, L784), age >10 years (L803)). CD20 sorts were performed on primary patient material (L4951, L49101, L754, L835 and L776), on secondary samples harvested from engrafted primary mice (L4967, L4951, L2510, L736 and L754) and on tertiary samples harvested from engrafted secondary mice (L4967 and L736). In total 151 mice were transplanted, with 122 showing engraftment in consecutive bone marrow punctures or in bone marrow harvests. CD10 sorts were performed on primary patient material (L784 and L49101) and on secondary samples harvested from engrafted primary mice (L4951, L8849, L2510 and L803) with 31 out of 52 mice transplanted with sorted material showing engraftment as seen with CD20 sorted cells. Blasts of all selected immunophenotypes were able to engraft the leukemia in unconditioned NSG mice as determined by 5 color flow cytometry. In particular, sorted cells of both fractions were able to reconstitute the complete phenotype of the leukemia. Harvested cells from engrafted mice could then be re-sorted into high and low antigen expressing fractions and successfully re-engrafted on secondary and tertiary mice. Cell purities of transplanted cells were usually higher than 90% (range 67-100%). The ability of all populations to serially engraft mice demonstrates long-term self-renewal capacity. Two additional patients were used in the limiting dilution assays (high WBC/t(4;11) high risk (L826); low WBC/MRD negative low risk (L792)) and experiments were performed on primary unsorted and secondary sorted material. Cell numbers necessary for ALL engraftment differed between individual leukemias but as little as 100 cells proved to be sufficient in one unsorted and in both the CD19+CD20low and CD19+CD20high fractions (Table 1). Mice transplanted with 10 cells only are still under observation. Table 1 Patient Transplant Population Cell dose Mice engrafted/transplanted L4951 Secondary CD20 high 500 3/3 CD20 low 3/3 CD20 high 100 3/3 CD20 low 3/3 L2510 Secondary CD20 high 3,000 2/4 CD20 low 4/4 CD20 high 300 0/4 CD20 low 1/4 L49101 Primary Unsorted 500 3/4 100 0/4 L792 Primary Unsorted 1,000 5/5 100 1/5 L826 Primary Unsorted 1,000 3/4 100 0/4 In conclusion we present strong evidence that leukemia-propagating cells are much more prevalent than previously thought and that blasts of diverse immunophenotype are able to serially reconstitute the complete leukemia in immune-deficient mice. Disclosures: No relevant conflicts of interest to declare.

CD52 Expression In Leukemic Stem/Progenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2743-2743 ◽  
Author(s):  
Vivian G. Oehler ◽  
Roland B. Walter ◽  
Carrie Cummings ◽  
Olga Sala-Torra ◽  
Derek L. Stirewalt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Cell Populations ◽  
Cell Surface Glycoprotein ◽  
Hematopoietic Stem

Abstract Abstract 2743 CD52 is a cell surface glycoprotein of unknown function that is expressed in B and T lymphocytes, macrophages, and monocytes, but is not expressed in normal hematopoietic stem/progenitor cells. CD52 is also expressed in chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia (ALL), and some cases of T-ALL. Alemtuzumab, a recombinant humanized monoclonal antibody, targets CD52 and is used to treat CLL. In contrast to normal hematopoietic stem/progenitor cells, CD52 expression has been described in acute myeloid leukemia (AML) and in blast crisis (BC) chronic myeloid leukemia (CML). Based on these observations we were curious whether CD52 expression distinguished normal from malignant or more mature from immature stem/progenitors cells, and whether these cells were sensitive to alemtuzumab. CD52 expression was examined in three blast cell populations (CD34+/CD38-, CD34+/CD38+, and CD34-) in patients with myeloid (44) and lymphoid (18) neoplasms, and normal patients (6). In normal hematopoietic cells, stems cells are enriched in the first population; more mature cells are characterized by increasing CD38 expression and loss of CD34 expression. In AML and CML leukemia stem cells may arise within either CD34+ population and possibly in the CD34- population. Relative to normal lymphocytes average CD52 expression could be characterized as low to moderate. Using an expression cutoff of > 20%, in contrast to normal patients, CD52 was detected in at least one of three blast populations in almost all patients. Using a more stringent cutoff of > 50%, CD52 was expressed in CD34+/CD38- cells in 7/11 B-ALL and 6/7 T-ALL cases and was concordantly expressed in the other two populations. Using the same criteria in myeloid malignancies (Table 1), expression occurred more frequently in AML, AML arising from myelodysplastic syndrome (MDS), and BC CML. In AML and AML arising from MDS, CD52 was expressed in the 34+/38- population in 7/15 cases (47%) and 4/7 cases (57%), respectively; it was expressed in both BC CML patients. In AML and BC CML patients, CD52 was expressed at similar levels in the CD34+/CD38+ fraction. No clear association between CD52 expression and cytogenetic abnormalities was found. We then examined whether CD52 expression differentiated normal from malignant blasts (CD34+/CD38- and CD34+/CD38+) in two CML myeloid BC patients. FISH and quantitative PCR demonstrated that BCR-ABL was expressed in all 4 populations, which were also morphologically distinct. Colony forming unit (CFU) assays demonstrated a significantly decreased ability to form CFU (on average 5–20 fold decrease) in CD52+/CD34+/CD38- CML cells suggesting CD52 cells may be more mature. Lastly and not previously described, we found that several BC CML cell lines express CD52, and complement-mediated cell cytotoxicity was similar in the highest expressing cell lines to that seen in EHEB (B-CLL) cells known to be targeted by alemtuzumab. Thus, alemtuzumab may have clinical efficacy in BC CML. In conclusion, CD52 is expressed on blast populations enriched for leukemic stem cells. Whether the absence or presence of CD52 more precisely segregates a leukemia stem cell containing population currently remains unknown and requires functional testing in a murine model. Our preliminary experiments in CML suggest CD52 may not differentiate between normal and malignant stem/progenitor cells. However, CD52 expression may distinguish normal and malignant stem cell populations in cases where CD52 and CD38 are more highly expressed. The observation that CD52 expression is increased in acute vs. chronic leukemias raises the intriguing possibility that CD52, if not directly involved, may be a marker for genes or pathways contributing to the block in differentiation seen with progression to acute leukemia. Furthermore, given that CD52 expression is heterogeneous in chronic disorders, it is possible that CD52 expression within these populations may correlate with poor prognosis or impending leukemic conversion. Table 1. The proportion of patients (44) expressing CD52 at levels > 50% in 3 blast populations. Three populations were present in most, but not all patients. Gray shading indicates chronic myeloid diseases. MPN is myeloproliferative neoplasm; NOS is not otherwise specified; ET is essential thrombocythemia; CMML is chronic myelomonocytic leukemia; and an arrow represents progressed to. Disclosure: Oehler: Pfizer: Research Funding. Radich:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

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