Technetium-99m and ICG-labeled HPG (hyperbranched polyglycerol) as a SPECT/FL dual imaging nanoprobe for imaging blood cells: in vitro investigation using myelogenous leukemia cells

Author(s):  
Sibel Ay ◽  
Volkan Yasakçı ◽  
Ömer Aras ◽  
Perihan Ünak
2001 ◽  
Vol 44 (S1) ◽  
pp. S639-S641 ◽  
Author(s):  
S. R. F. Moreno ◽  
G. D. Feliciano ◽  
R. S. Freitas ◽  
D. M. Mattos ◽  
M. L. Gomes ◽  
...  

2019 ◽  
Vol 45 (4) ◽  
pp. 683-688 ◽  
Author(s):  
Sonia Mendoza-Cardozo ◽  
Luis D. Pedro-Hernández ◽  
Ulises Organista-Mateos ◽  
Luis Isaac Allende-Alarcón ◽  
Elena Martínez-Klimova ◽  
...  

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1788-1788
Author(s):  
Yiwen Li ◽  
Hongli Li ◽  
Mei-Nai Wang ◽  
Rajiv Bassi ◽  
Dale Ludwig ◽  
...  

Abstract The receptor tyrosine kinase FLT3 is overexpressed in blasts of ~90% of acute myelogenous leukemia (AML) and the majority of B-lymphoid leukemia patients. Internal tandem duplications (ITDs) in the juxtamembrane region and point mutations in the kinase domain of FLT3 are found in ~37% of AML patients and are associated with a poor prognosis. We have recently developed a fully human monoclonal antibody (IMC-EB10) which binds with high affinity to FLT3 receptor on human leukemia cells. In the present study, a novel auristatin conjugate of the anti-FLT3 antibody (EB10-MMAF) was prepared using a dipeptide linker that allows for drug release inside the lysosomes of antigen-positive cells. The MMAF conjugates were stable in buffers and plasma. EB10-MMAF (drug/antibody raito = 8) was highly potent, and selectively inhibited the growth of FLT3-expressing leukemia cells with an IC50 of 0.19 nM and 0.08 nM for MV4;11 and BaF3-ITD cells (both positive for FLT3-ITD), 1.11 nM, 6.18 nM and 1.82 nM for REH , EOL-1, EM3 cells (all three positive for wild-type FLT3), and 135 nM for JM1 (negative for FLT3). An MMAF conjugate with a control antibody was not active in these cell lines (IC50s > 5.9 uM). Flow cytometric analysis with annexin V indicated that EB10-MMAF treatment induced apoptosis of leukemia cells in vitro. In vivo treatment with EB10-MMAF strongly inhibited leukemia growth and prolonged survival of mice in both EOL-1 and BaF3-ITD leukemia models. In summary, immunoconjugates composed of a fully human anti-FLT3 antibody and a potent auristatin drug may provide a valuable therapeutic approach for AML and other FLT3-positive leukemias.


Blood ◽  
2003 ◽  
Vol 102 (13) ◽  
pp. 4512-4519 ◽  
Author(s):  
Joya Chandra ◽  
Jennifer Hackbarth ◽  
Son Le ◽  
David Loegering ◽  
Nancy Bone ◽  
...  

Abstract Adaphostin (NSC 680410), an analog of the tyrphostin AG957, was previously shown to induce Bcr/abl down-regulation followed by loss of clonogenic survival in chronic myelogenous leukemia (CML) cell lines and clinical samples. Adaphostin demonstrated selectivity for CML myeloid progenitors in vitro and remained active in K562 cells selected for imatinib mesylate resistance. In the present study, the mechanism of action of adaphostin was investigated in greater detail in vitro. Initial studies demonstrated that adaphostin induced apoptosis in a variety of Bcr/abl- cells, including acute myelogenous leukemia (AML) blasts and cell lines as well as chronic lymphocytic leukemia (CLL) samples. Further study demonstrated that adaphostin caused intracellular peroxide production followed by DNA strand breaks and, in cells containing wild-type p53, a typical DNA damage response consisting of p53 phosphorylation and up-regulation. Importantly, the antioxidant N-acetylcysteine (NAC) blunted these events, whereas glutathione depletion with buthionine sulfoximine (BSO) augmented them. Collectively, these results not only outline a mechanism by which adaphostin can damage both myeloid and lymphoid leukemia cells, but also indicate that this novel agent might have a broader spectrum of activity than originally envisioned. (Blood. 2003;102:4512-4519)


Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1472-1476 ◽  
Author(s):  
MP Everson ◽  
CB Brown ◽  
MB Lilly

Previous studies suggest that malignant cells from some patients with myeloid leukemias produce colony-stimulating factors (CSFs) that can function as autocrine growth factors in vitro. We have examined the roles of interleukin-6 (IL-6) and granulocyte-macrophage CSF (GM-CSF) in the proliferation of myeloid leukemia cells. IL-6 activity was assessed in conditioned medium (CM) from myeloid leukemia cell cultures or cell lysates using IL-6-dependent KD83 and 7TD1 murine cell lines. Media conditioned by cells from patients with chronic myelomonocytic leukemia (CMMoL), but not by normal monocytes, chronic myelogenous leukemia (CML), or acute myelogenous leukemia (AML) cells, contained substantial levels (50 to 1,000 U/10(6) cells) of IL-6. The IL-6 content of CM correlated directly with donor peripheral blood WBC count. CM from two of five CMMoL samples also contained greater than 350 pg/mL GM-CSF. Moreover, CMMoL cells spontaneously formed colonies in semisolid medium. CMMoL colony formation could be partially inhibited by antibodies to IL-6 or GM-CSF, whereas combination of these antibodies gave additive, and nearly complete (greater than 93%), inhibition of spontaneous colony formation. Cell lysates from uncultured CMMoL cells from one patient contained abundant GM-CSF protein but no detectable IL-6. These data suggest that IL-6 and GM-CSF act in vitro as autocrine growth factors for CMMoL cells, and that CMMoL cells in vivo may represent a GM-CSF-dependent autocrine growth system.


Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2636-2636
Author(s):  
Anna Kalota ◽  
Alan M. Gewirtz

Abstract Treatment of thrombocytopenia, whether autoimmune, secondary to marrow failure, or iatrogenic, can become problematic. Stimulation of endogenous megakaryocyte (MEG) development might be an effective strategy and it was originally hoped that this could be accomplished with thrombopoietin (Tpo), the natural ligand of the thrombopoietin receptor (TpoR) and regulator of MEG development. When formulated Tpo proved immunogenic, development of small molecule TpoR agonists (TpoRA) was undertaken. In course of investigating the biological effects, and mechanism of action, of SB559457 (a nonpeptidyl, hydrazone class, TpoRA supplied by GlaxoSmithKline (GSK), Collegeville, PA) we found that the molecule robustly stimulated human megakaryocytopoiesis in vitro. In anticipation of TpoRA use in the treatment of patients with hematologic malignancies, we also evaluated SB559457’s effect on human leukemia cell (HLC) growth in vitro since it is known that HLCs may express TpoR. Specifically, we examined the effects of SB559457 on samples obtained from 21 patients with AML, and 7 with ALL. Surprisingly, SB559457 not only failed to stimulate HLC growth, it proved toxic to primary acute myelogenous leukemia (AML). In 20 of 21 primary AML samples exposed to SB559457 (5μM) a large fraction (70–90%) of cells died between days 3 and 6 of culture, while untreated control cells survived. No significant effects on cell growth or viability were observed in the ALL patient samples. To investigate the mechanism(s) of AML cell killing, we examined signaling cascades initiated by SB559457 compared to recombinant human Tpo (rhTpo), which had no apparent effect on AML cell growth. Initially, we employed N2C-Tpo cells (a Tpo dependent megakaryoblastic cell line that express TpoR) and focused on kinases known to be phosphorylated after TpoR activation; STAT5, ERK, p70S6, and ribosomal kinase S6. When N2C-Tpo cells were stimulated with rhTpo (2.8μM, 30 min) all these kinases were highly phosphorylated. In contrast, none were phosphorylated in SB559457 (5μM, 30min) stimulated cells. Normal human CD34+ marrow cells were next examined using the same experimental conditions. In contrast to N2C-Tpo cells, ERK, and p70S6 kinases were both phosphorylated after exposure to SB559457 but STAT5 remained unphosphorylated. Since AML cells may require STAT5 activation for growth, we hypothesized that perturbation of STAT5 activation might be involved in the apoptotic mechanism. The molecular consequences of differential signaling were then pursued by Affymetrix GeneChip analysis. Remarkably, in 5 separate primary AML cell samples stimulated for 6 hours with either Tpo or SB559457 we found a statistically significant difference in expression in only 2 of 22000 genes represented on the chips: GAPDH and Redd1. Both of these genes are induced in stressed cells undergoing apoptosis. In addition, stimulation of primary AML cells with SB559457 resulted in increased phosphorylation of p70S6/S6 kinases, both downstream targets of mTOR kinase. Accordingly, we hypothesize that SB559457 mediated activation of mTOR pathway leads to a stress response in primary myeloid leukemia cells as reflected by the increased expression of Redd1 and GAPDH. Further investigation of this pathway, and the leukemic cell response to SB559457 exposure may lead to development of novel strategies for the treatment of myeloid leukemias.


Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3072-3072
Author(s):  
Enzi Jiang ◽  
Eugene Park ◽  
Carlton Scharman ◽  
Yao-Te Hsieh ◽  
Asha Kadavallore ◽  
...  

Abstract Abstract 3072 Poster Board III-9 Despite advances in chemotherapeutic treatment of acute lymphoblastic leukemia (ALL), 20% of children relapse with high death rates, highlighting the need for new treatment modalities. Recent population studies have demonstrated that Survivin, a member of the inhibitor of apoptosis (IAP) family proteins, is expressed in most cancerous cells but has also been implicated in normal erythropoiesis. It is upregulated in ALL of relapsed patients but not in drug-sensitive ALL. The expression of Survivin depends on the formation of a complex between β-catenin and its co-activator CBP. Selective suppression of CBP/β-catenin signaling using the novel small-molecule inhibitor ICG-001 offers a novel mechanism to target Survivin in the sensitization of leukemia cells to conventional drug treatment. We hypothesize that inhibition of CBP/β-catenin signaling by ICG-001 in combination with conventional therapy represents a promising therapeutic principle to eradicate drug resistant ALL while sparing normal hematopoiesis. An in vivo study utilized our bioluminescent model to non-invasively monitor leukemogenesis of a primary ALL, transduced with a lentiviral construct encoding firefly luciferase prior to xenotransplantation. NOD/SCIDIL2R gamma-/- mice were sublethally irradiated prior intravenous injection of 50,000 cells per animal. Leukemic animals were treated with a combination of intraperitoneally administered VDL and ICG-001 (100mg/kg/d) (n=3), which was delivered via subcutaneous osmotic pumps to ensure stable plasma levels, with VDL only (n=4), or PBS only (n=2) as a control for 4 weeks. Bioluminescent imaging on Day 42 post-injection showed a contrast in the containment of leukemia of ICG-001+VDL mice as compared to those of the VDL control group. The animals in the PBS control group and the VDL+PBS Pump control groups had Median Survival Times (MST) of 35 days and 66.5 days post-treatment, respectively. In marked contrast, the animals treated with a combination of VDL+ICG-001 had a significant 14% extension in MST of 76 days post-treatment (p=0.016 compared to VDL group). Survivin mRNA expression was found to be downregulated after VDL+ICG treatment compared to treatment with VDL only. Analysis of peripheral blood showed no effect of ICG-001 on leukocyte or red blood cells compared to control groups. Next, we determined in vitro the ability of ICG-001 to increase sensitivity of patient-derived ALL cells and ALL celllines including BEL-1, REH, 697 and SUPB15 to chemotherapy including VDL or Imatinib. After 4 days we observed significantly increased toxicity assessed by MTT assay and AnnexinV staining as well as downregulation of Survivin confirmed by real-time PCR and Western Blot. To determine if ICG-001 is non-toxic to normal hematopoiesis, we treated normalC57BL/6 mice for 3 weeks with ICG-001 only. At end of treatment, normal blood counts including red blood cell, white blood cells and platelets, normal histology and normal weight gain indicated that ICG-001 is not detrimental to the recipient. In vitro apoptotic studies using normal white blood cells isolated from peripheral blood and co-cultured with a stromal layer confirmed further the non-toxicity of ICG-001 to normal cells. In summary, the sustained survival of the mice treated with combination of standard chemotherapy and ICG-001 is compatible with our hypothesis that ICG-001 can sensitize drug resistant leukemia cells to treatment with standard chemotherapy while sparing normal hematopoiesis and may lead to novel therapeutic options to overcome drug resistance. Disclosures No relevant conflicts of interest to declare.


Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 742-742 ◽  
Author(s):  
Noah Hornick ◽  
Jianya Huan ◽  
Natalya A Goloviznina ◽  
Amiee Potter ◽  
Peter Kurre

Abstract Small, non-coding micro RNA (miRNA) are recognized for their potent regulatory capacity. Several recent studies indicate the prognostic value of miRNA profiling in acute myelogenous leukemia (AML), although a more mechanistic understanding of the role miRNA play in AML biology is still lacking. We recently demonstrated that patient-derived AML blasts release exosomes (nanometer-size, extracellular vesicles) that traffic a non-random subset of miRNA to stromal bystander cells, eliciting changes in transcriptional activity and growth factor secretion (Huan et al., Cancer Res. 2013). Here we hypothesized that exosome miRNA provide a candidate mechanism for the adaptation of the bone marrow to a specialized leukemic niche. As oxygen levels in the bone marrow are substantially lower than those commonly used in tissue culture, we undertook a systematic study of miRNA incorporation and exosome trafficking in AML under physiological oxygen conditions. In carefully calibrated tissue culture conditions we initially observed an up to 7-fold net increase in exosome number released by Molm14 (Flt3-ITD+ AML cell line) leukemia cells at 1% O2versus 21% O2. Nanoparticle tracking analysis and RNA bioanalyzer tracings suggested that the decreased O2 did not alter vesicle composition, average RNA amount per exosome, or global RNA profiles. Further emphasizing the critical nature of appropriate compartmental oxygenation in exosome trafficking, both murine and human stromal cells demonstrated increased uptake of Molm14 exosomes under hypoxia. Low-oxygen conditions alter transcriptional profiles, phenotypic behavior and drug resistance in AML. Therefore, we next evaluated the miRNA expression of leukemic cells and their incorporation in exosomes at 1% versus 21% O2, utilizing the Affymetrix microarray platform containing >5,000 human (hsa) miRNA probesets, followed by select qRT-PCR validation. Array experiments showed broad differences between cellular and exosomal miRNA and revealed that certain miRNA were selectively regulated in an oxygen-responsive manner. For example, hematopoiesis relevant hsa-miR-124, -146a, and -155 increased an average of 4.6-, 5.5-, and 4.9-fold, respectively, in exosomes from hypoxia-conditioned cells. Intriguingly, several known, non-AML specific, hypoxia-responsive miRNA substantially increased in cells cultured at 1% O2 (e.g. miR-210 by 33-fold), but changed less than 2-fold in exosomes. Several recent reports show that leukemia cells actively convert the bone marrow microenvironment and contribute to the erosion of hematopoiesis by modulating hematopoietic-stromal interactions, in part via decrease in SDF1a, SCF, and Angpt1. We investigated the ability of AML-derived exosomes to regulate these transcripts, and found a 50% decrease in SCF and over 90% decrease in Angpt1 in murine stromal cells after in vitro exposure to leukemia exosomes, again with relatively greater differences for exosomes from hypoxia-conditioned AML cells. These experiments were complemented by observations of altered clonogenicity (CFU-C) of murine lin-negative hematopoietic cells after AML exosome exposure, whereby hypoxia conditioning prompted a decline in colony count to 46% from vesicle-free media baseline, compared with 31% decrease at 21 % O2. Exosomes equilibrate across biological fluids and can be recovered from serum. To translate our observations to an in vivo setting, we developed a xenograft model using Molm14 cells in immune-deficient NSG mice. Early after grafting animals, exosomes could be reproducibly isolated from as little as 20 microL serum and candidate miRNA (hsa-miR-146, -150, 155, 210) were amplified, allowing us to quantitatively track leukemia progression via a unique miRNA signature even before circulating leukemia cells were detectable in the peripheral blood. A comparison of leukemic animals to NSG controls bearing cord blood MNC grafts revealed that changes in circulating miRNA were disease specific and resembled those in the hypoxia setting in vitro. In sum, our work demonstrates that physiologic oxygen levels not only increase AML exosome trafficking between cells, but selectively alter the miRNA profile contained therein. These changes produce phenotypic alterations in stromal and hematopoietic bystander cells that correlate with the functional conversion of the bone marrow to a leukemic niche. Disclosures: No relevant conflicts of interest to declare.


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