In Vivo Imaging In the Individualized Mouse Model of Acute Lymphoblastic Leukemia Enables Highly Sensitive and Continuous Follow up of Patient-Derived Xenografts

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3259-3259
Author(s):  
Nadia Terziyska ◽  
Katarina Farkasova ◽  
Ernst Wagner ◽  
Manfred Ogris ◽  
Irmela Jeremias
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
In Vivo Imaging ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Lentiviral Transduction ◽  
Patient Derived Xenograft ◽  
Over Time

Abstract Abstract 3259 In the individualized xenograft mouse model, acute leukemia cells from patients are transplanted into severely immuno-deficient mice to serve as a preclinical animal model. The use of this mouse model for pre-clinical therapy trials is hampered by the low sensitivity of existing readouts disabling the reliable follow up of single animals. Both secretion of leukemic cells into the blood flow as well as clinical signs, if at all, occur at late states of the disease; readouts in further organs by, e.g., immunohistochemistry or flow cytometry analysis, require organ extraction and can thus be performed only once per mouse. To overcome this obstacle, we established in vivo imaging in the xenograft mouse model of acute lymphoblastic leukemia (ALL). We engrafted pediatric acute leukemia cells from patients at diagnostic bone marrow aspiration of either diagnosis or relapse. In agreement with published data, mice developed leukemia within weeks to months. Engraftment was followed by easy passaging of cells into further generations of mice. We established lentiviral transduction of xenograft cells which enabled expression of transgenes in these cells. Using lentiviral transduction, we stained patient-derived xenograft ALL-cells using luciferase as a reporter. Transgenic xenograft leukemia cells were visualized once per week by bioluminescence in vivo imaging using a charge-coupled device camera. Our first data show that in vivo imaging enabled the reliable and continuous follow up of single animals over time. The sensitivity of in vivo imaging in measuring leukemic engraftment was significantly higher compared to current readouts like examination of blood cells. In vivo imaging data support that the leukemic pattern of metastases of patient-derived xenograft ALL-cells in mice highly resembles the distribution of the disease in men. Upon intravenous injection, cells first home to the liver, where they stay alive only for a few weeks. Long-term engraftment is seen in the bone marrow of many different bones and constantly increases over time. Engraftment in spleen indicates rather late stages of disease. This kinetic of engraftment remained constant between several transgenic xenograft samples and between different mice engrafted with the same sample. Taken together, we have established molecular modulation of xenograft cells which enables expression of luciferase and in vivo bioluminescence imaging as a new sensitive and continuous in vivo readout in mice. In vivo imaging will allow realizing precise preclinical trials in the individualized mouse model in the future. Disclosures: No relevant conflicts of interest to declare.

Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1506-1506
Author(s):  
Marika Masselli ◽  
Serena Pillozzi ◽  
Massimo D'Amico ◽  
Luca Gasparoli ◽  
Olivia Crociani ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Map Kinases ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Significant Proportion ◽  
Leukemic Cells ◽  
K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

Myeloablative Chemo-Conditioning for First Hematopoietic STEM CELL Transplantation in Children with ACUTE Lymphoblastic Leukemia in First or Second Remission

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 546-546 ◽  
Author(s):  
Christina Peters ◽  
Jean-Hugues Dalle ◽  
Stelios Graphakos ◽  
Petr Sedlacek ◽  
Antonio Campos ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Lymphoblastic Leukemia ◽  
Peripheral Blood Stem Cells ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Donor Type ◽  
Blood Stem Cells

Abstract Christina Peters, Petr Sedlacek, Jean Hugues Dalle, Stelios Graphakos, Antonio Campos, Akif Yesilipek, Jacek Wachowiak, Arjan Lankester, Andrea Pession, Amir Ali Hamidieh, Marianne Ifversen, Jochen Büchner, Gergely Krivan, Franca Fagioli, Arnaud Dalissier; Myriam Labopin; Peter Bader on behalf of the EBMT Pediatric Diseases Working Party Most children with acute lymphoblastic leukemia (ALL) with indication for allogeneic hematopoietic stem cell transplantation (HSCT) receive myeloablative conditioning with a total body irradiation (TBI)-containing regimen. To investigate the outcomes of patients (pts) who did not undergo TBI, we performed a retrospective registry based study on children below 18 years who received a myeloablative chemo-conditioning for a first allogeneic HSCT from different donors between 2000 and 2012. In this analysis, only chemotherapeutic regimens with more than 30 applications were included. In total, 732 pts were included: 313 pts who received bone marrow (BM) or peripheral blood stem cells (PBSC) in 1st CR, 247 pts with BM/PBSC transplantation in CR2, 85 pts and 52 pts who received umbilical cord blood (CB) in 1st or 2nd CR, respectively. The most commonly applied myeloablative chemo-combinations were: Busulfan (Bu)/Cyclophosphamide (Cy) (n=202), Bu/Cy/Etoposide (VP) (n=189), Bu/Cy/Melphalan (Mel) (n=93), Bu/AraC/Mel (n=80), Bu/Fludarabine (Flu)/Thiotepa (Thio) (n=62), Bu/Cy/Thio (n=53, Bu/Cy/Thio (n=53), and Bu/Flu (n=53). 313 pts received either BM or PBSC in CR1 with a median follow up of 26 months (1-156) and we compared Bu/Cy/VP vs the other chemo-conditioning regimens. The Bu/Cy/VP cohort had a longer follow up (med 37 vs. 20 months, p=0.002), pts were younger (med 3,6 vs. 6,5 years, p=0.003) and the median year of transplant was earlier (med 2009 vs. 2010, p=0.03). Donor type, CMV match, gender match, stem cell were comparable. In univariate analysis, conditioning with Bu/Cy/VP was better than all other combinations: relapse incidence (RI) 21% vs 32% (p=0.05), leukemia-free survival (LFS) 72 vs 54% (p=0.004), overall survival (OS) 79 vs 68% (p=0.03) and chronic GVHD (cGVHD) 9% vs 19% (p=0.014). Engraftment and incidence and severity of acute GVHD were similar and non- relapse mortality (NRM) was 7% vs 13% (p=0.10). Other significant influencing factors were: interval between diagnosis and transplantation below or beyond 208 days (NRM 6% vs 16%, p=0.015), donor sibling vs other (RI 35% vs 23%, p=0.01, NRM 5% vs 16%, p=0.001) and in vivo T cell depletion (TCD) vs no TCD (RI 35% vs. 19%, p=0.003; NRM 20% vs 4%, p=0.0001). In the cox model, conditioning type (Bu/CY/VP vs other), age, year of transplantation, interval from diagnosis to transplant, donor type, stem cell source and in vivo TCD were evaluated. For LFS only BU/CY/VP was associated with better outcome (p=0.004, HR .52), RI was lower after Bu/Cy/VP (HR .54, p=0.02), NRM was higher in pts older than 4,6 years (p=0.02, HR 2,48) and after TCD HSCT (p=0.01, HR 9,13) and OS was best after Bu/Cy/VP (p=0.03, HR 0.57). We conclude that omission of TBI is feasible for children who undergo first allogeneic HSCT in first or second complete remission. The combination of busulfan, cyclophosphamide and etoposide resulted in better LFS and OS with less NRM and RI for children who received bone marrow or peripheral blood stem cells in CR1. These observations should be the basis for prospective trials in homogenous patient groups. Disclosures No relevant conflicts of interest to declare.

scholarly journals Bioluminescence in Vivo Imaging Improves the Model of Individual Patients' AML Cells Growing in Mice for Sensitive and Reliable Preclinical Treatment Trials on Various Genetic Subgroups

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2323-2323
Author(s):  
Irmela Jeremias ◽  
Binje Vick ◽  
Maya Rothenberg ◽  
Nadine Sandhöfer ◽  
Michela Carlet ◽  
...  
Keyword(s):  
Mouse Model ◽  
In Vivo Imaging ◽  
Disease Monitoring ◽  
Treatment Approaches ◽  
Lentiviral Transduction ◽  
Treatment Trials ◽  
Novel Treatment ◽  
Preclinical Treatment ◽  
Serial Transplantation

Abstract We introduced bioluminescence in vivo imaging as a novel, sensitive and reliable readout parameter for preclinical treatment trials in the individualized model of patients' primary AML cells growing in mice. Novel treatment approaches require preclinical in vivo evaluation. While the individualized xenograft mouse model of individual patients AML cells growing in mice inherits the advantage of mimicking the broad genetic heterogeneity of AML, disease monitoring remained challenging so far due to the lack of appropriate readout parameters. In the individualized mouse model of AML, primary patients' AML cells are xenotransplanted into immuno-compromised mice. Here, we aimed at increasing sensitivity and reliability of disease monitoring in the individualized mouse model of patient-derived AML. Towards this aim, we engrafted primary tumor cells from 16 adult patients with AML. 8/16 (50%) samples allowed serial transplantation and thereby generation of stable patient-derived xenograft (PDX) cells with constant characteristics regarding growth and immunophenotype. PDX cells were derived from genetically distinct patient samples, mimicking the known heterogeneity of AML. Targeted re-sequencing of 43 genes important for AML leukemogenesis revealed identical mutations in primary and PDX cells after initial or serial transplantation, except the loss of two minor subclones within two samples. Lentiviral transduction was established to genetically manipulate PDX cells and introduce stable expression of transgenes which was feasible in 7/8 PDX AML samples tested. Transgenic PDX cells were enriched by flow cytometry gating on a co-expressed fluorochrome. Recombinant expression of luciferase enabled bioluminescence in vivo imaging for reliable follow up of PDX cell leukemia growth in mice. Imaging was highly sensitive and detected a single PDX cell within 10,000 normal mouse bone marrow cells covering the clinically important situation of minimal disease. Furthermore, imaging facilitated reliable analysis of preclinical treatment trials, visualizing drug effects in single mice over time. Novel treatment approaches aim at eliminating AML propagating cells, and the limiting dilution transplantation assay represents the gold standard for determining frequency of AML propagating cells. Bioluminescence in vivo imaging facilitated quantifying AML propagating cells by determining engraftment as early as 5 weeks after cell transplantation. Taken together, we advanced the individualized mouse model of AML by introducing serial transplantation, lentiviral transduction and in vivo imaging. These improvements now allow sensitive and reliable preclinical treatment trials in patient-derived AML cells of various different genetic subgroups including AML propagating cells. Disclosures No relevant conflicts of interest to declare.

Xenografts of Pediatric Acute Lymphoblastic Leukemia Retain the Gene Expression Pattern From the Diagnostic Material They Originated From Over Serial Passages.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1629-1629
Author(s):  
Manon Queudeville ◽  
Elena Vendramini ◽  
Marco Giordan ◽  
Sarah M. Eckhoff ◽  
Giuseppe Basso ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Leukemia Cells ◽  
Diagnostic Samples ◽  
Xenotransplantation Model

Abstract Abstract 1629 Poster Board I-655 Primary childhood acute lymphoblastic leukemia (ALL) samples are very difficult to culture in vitro and the currently available cell lines only poorly reflect the heterogeneous nature of the primary disease. Many groups therefore use mouse xenotransplantation models not only for in vivo testing but also as a means to amplify the number of leukemia cells to be used for various analysis. It remains unclear as to what extent the xenografted samples recapitulate their respective primary leukemia. It has been suggested for example that transplantation may result in the selection of a specific clone present only to a small amount in the primary diagnostic sample. We used a NOD/SCID xenotransplantation model and injected leukemia cells isolated from fresh primary diagnostic material of 4 pediatric ALL patients [2 pre-B-ALL, 1 pro-B-ALL (MLL/AF4}, 1 cortical T-ALL] intravenously into the lateral tail vein of unconditioned mice. As soon as the mice presented clinical signs of leukemia, leukemia cells were isolated from bone marrow and spleen. Isolated leukemia cells were retransplanted into secondary and tertiary recipients. RNA was isolated from diagnostic material and serial xenograft passages and gene expression profiles were obtained using a human whole genome array (Affymetrix U133 2.0). Simultaneously, immunophenotypic analysis via multicolor surface and cytoplasmatic staining by flow cytometry was performed for the diagnostic samples and respective serial xenograft passages. In an unsupervised clustering analysis the diagnostic sample of each patient clustered together with the 3 derived xenograft samples, although the 3 xenograft samples clustered stronger to each other than to their respective diagnostic sample. Comparison of the 4 diagnostic samples vs. all xenograft samples resulted in a gene list of 270 genes upregulated at diagnosis and 8 genes upregulated in the xenograft passages (Wilcoxon, p< .05). The high number of genes upregulated at diagnosis is most likely due to contamination of primary patient samples with normal peripheral blood and/or bone marrow cells as 15% of genes are attributed to myeloid cells, 7% to erythroid cells, 7% to lymphoid cells, 32% to bone marrow in general as well as to innate immunity, chemokines, immunoglobulins. The remaining genes can not be attributed to a specific hematopoetic cell lineage and are not known to be related to leukemia or cancer in general. Accordingly, there are no statistically significant differences between the primary, secondary and tertiary xenograft passages. The immunophenotype analysis are also in accordance with these findings, as the diagnostic blast population retains its immunophenotypic appearance during serial transplantation, whereas the contaminating CD45-positive non- leukemic cells disappear after the first xenograft passage. The few genes upregulated in xenograft samples compared to diagnosis are mainly involved in cell cycle regulation, protein translation and apoptosis resistance. Some of the identified genes have already been described in connection with cancer subtypes, their upregulation therefore being indicative of a high proliferative state in general and could argue towards a more aggressive potential of the engrafted leukemia cells but alternatively could also simply be due to the fact that the xenograft samples are pure leukemic blasts and are not contaminated with up to 15% of non-cycling healthy bone marrow cells as in the diagnostic samples. We conclude that the gene expression profiles generated from xenografted leukemias are very similar to those of their respective primary leukemia and moreover remain stable over serial retransplantation passages as we observed no statistically significant differences between the primary, secondary and tertiary xenografts. The differentially expressed genes between diagnosis and primary xenotransplant are most likely to be due to contaminating healthy cells in the diagnostic samples. Hence, the NOD/SCID-xenotransplantation model recapitulates the primary human leukemia in the mouse and is therefore an appropriate tool for in vivo and ex vivo studies of pediatric acute leukemia. Disclosures No relevant conflicts of interest to declare.

scholarly journals TPEN Selectively Eliminates Lymphoblastic B Cells from Pediatric Acute Lymphoblastic Lleukemia Patients

2021 ◽  
Author(s):  
Miguel Mendivil-Perez ◽  
Carlos Velez-Pardo ◽  
Lina Maria Quiroz-Duque ◽  
Alexandra Restrepo-Rincon ◽  
Natalia Andrea Valencia-Zuluaga ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Anticancer Agents ◽  
Pediatric Patients ◽  
De Novo ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Hematologic Disorder ◽  
Cell Counts ◽  
Proapoptotic Protein

B-acute lymphoblastic leukemia (B-ALL) is a hematologic disorder characterized by abnormal proliferation and accumulation of immature B-lymphoblast arrested at various differentiation stages. Despite some advances in treatment, there is still an important percentage of pediatric patients with precursor-B ALL who relapsed. Therefore, alternative therapies are needed to improve cure rates for pediatric patients. TPEN is a pro-oxidant agent capable of selectively inducing apoptosis in leukemia cells. Consequently, TPEN has been suggested as a potential agent for oxidative therapy. However, it is not yet known whether TPEN can selectively destroy leukemia cells in a more disease-like milieu e.g., bloodstream and bone marrow (BM) in vivo. In this investigation, we report for the first time that TPEN significantly induces apoptosis in CD34+/CD19+ cells from whole bone marrow de novo B-ALL (n=5) and refractory B-ALL (n=6) patients by oxidative stress (OS, n=8). We found that TPEN significantly increased not only positive cell counts for the oxidation of the stress sensor protein DJ-1 as a sign of the formation of H2O2, but also significantly increased positive cell counts for the proapoptotic protein TP53, PUMA, and CASPASE-3 as indicative of apoptosis in B-ALL cells irrespective of diagnostic status (de novo or refractory) and sex. Understanding the TPEN-induced cell death in leukemia cells provides insight into more effective therapeutic prooxidant-inducing anticancer agents.

Identification of Gene Expression Profiles in Acute Lymphoblastic Leukemia Cells That Discriminate Intracellular Thioguanine Nucleotide Accumulation in ALL Cells after In Vivo Treatment with Mercaptopurine.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 453-453
Author(s):  
Gianluigi Zaza ◽  
Meyling Cheok ◽  
Wenjian Yang ◽  
Pei Deqing ◽  
Cheng Cheng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Post Treatment ◽  
Leukemia Cells ◽  
True Positive

Abstract Thioguanine nucleotides (TGN) are considered the principal active metabolites exerting the antileukemic effects of mercaptopurine (MP). Numerous clinical studies have reported substantial inter-patient variability in intracellular TGN concentrations during continuation therapy of acute lymphoblastic leukemia (ALL). To identify genes whose expression is related to the intracellular accumulation of TGN in leukemia cells after in vivo treatment with MP alone (MP) or in combination with MTX (MP+MTX), we used oligonucleotide microarrays (Affymetrixâ HG-U95Av2) to analyze the expression of approximately 9,670 genes in bone marrow leukemic blasts obtained at diagnosis from 82 children with ALL. TGN levels were determined in bone marrow aspirates of these patients 20 hours after mercaptopurine infusion (1 g/m2 I.V). Because, as previously reported, patients treated with MP alone achieved higher levels of intracellular TGN compared to those treated with the combination, we used Spearman’s rank correlation to identify genes associated with TGN levels separately for the 33 patients treated with MP alone and the 49 with the combination (MP: median TGN: 2.46 pmol/5x106 cells, range: 0.01–19.98; and MTX+MP: median TGN: 0.55 pmol/5x106 cells, range: 0.005–3.31). Hierarchical clustering using these selected probe sets clearly separated the 33 patients treated with MP alone into two major groups according to TGN concentration (< 2.46 and > 2.46 pmol/5x106 cells; n=60 genes) and two major branches were also found for patients treated with the combination (< 0.55 and > 0.55 pmol/5x106 cells; n=75 genes). Interestingly, there was no overlap between the two sets of genes, indicating that different genes influence the accumulation of TGN when this drug is given alone or in combination with MTX. The association between gene expression profiles and TGN levels determined by leave-one-out cross-validation using support vector machine (SVM) based on Spearman correlation, was rho=0.60 (p<0.001) for MP alone and rho=0.65 (p<0.001) for MTX+MP, with false discovery rate (FDR) computed using Storey’s q-value (MP: 50% true positive, MTX+MP: 70% true positive respectively). Genes highly associated with the post-treatment TGN level in ALL patients treated with MP alone encode transporters, enzymes involved in the MP metabolic pathway and cell proliferation. Genes associated with post-treatment levels of TGN after combined therapy have been implicated in protein and ATP biosynthesis. Together, these in vivo data provide new insights into the basis of inter-patient differences in TGN accumulation in ALL cells, revealing significant differences between treatment with MP alone or in combination with MTX.

Dynamic In Vivo Imaging Reveals That Leukemia-Induced Changes in the Bone Marrow Microenvironment Alter Mechanisms of Metastasis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2805-2805
Author(s):  
Angela Colmone ◽  
Veena Krishnamoorthy ◽  
Dorothy A. Sipkins
Keyword(s):  
Bone Marrow ◽  
Tumor Growth ◽  
Cancer Cell ◽  
In Vivo Imaging ◽  
Lymphoblastic Leukemia ◽  
Tumor Margin ◽  
Metastatic Process ◽  
Induced Changes ◽  
The Impact

Abstract Tissue microenvironments have been shown to critically regulate cancer cell survival and proliferation. Conversely, while tumor growth can induce neovascularization, the impact of other tumor-induced changes in the microenvironment are less well understood. Here we utilize a xenograft model of Nalm-6 pre-B acute lymphoblastic leukemia (ALL) in SCID mice to examine how tumor growth alters the bone marrow (BM) microenvironment. Using dynamic confocal and multiphoton in vivo imaging, we find that tumor growth dramatically down-regulates expression of the chemokine SDF-1 in the BM microvasculature in areas of leukemic proliferation. SDF-1 has been shown to play a key role in cancer cell metastasis for numerous cell types including Nalm-6, and directs initial Nalm-6 homing to specific SDF-1-positive vascular beds. In contrast, we found that Nalm-6 introduced in mice previously engrafted with leukemia home to SDF-1-negative vasculature, predominantly at the advancing tumor margin. Furthermore, inhibition of chemokine signaling by pertussis toxin pre-treatment of Nalm-6 cells demonstrates that Nalm-6 homing in engrafted mice is chemokine-independent. In summary, these findings suggest that leukemic growth profoundly alters the mechanisms underlying the metastatic process by inducing changes in the host vascular microenvironment. These changes may increase the efficiency of the metastatic process and/or the advancement of the tumor margin. Therapies directed at blocking tumor metastases, therefore, may need to be tailored according to tumor stage.

Functional Modulation of VLA6 in BCR-ABL1+ Pre-B Acute Lymphoblastic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2565-2565
Author(s):  
Eun Ji Gang ◽  
Yao-Te Hsieh ◽  
Huimin Geng ◽  
Jennifer Pham ◽  
Markus Muschen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Drug Resistance ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Conditional Knockout ◽  
Leukemia Cells

Abstract Abstract 2565 Chemotherapy drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem, resulting in reduced treatment efficacy and relapse. The bone marrow environment (BME) has been shown to promote resistance of leukemia cells towards chemotherapy, which has been attributed to several proteins, including integrins. Our analysis of 207 children with high-risk (BCR/ABL1−) pre-B ALL revealed that high expression of the laminin-binding integrin VLA6 (alpha6beta1) portends poor clinical outcomes in patients with minimal residual disease (MRD+) on day 29 of induction. In addition, our comparative analysis of pre-B leukemia and normal B-cells revealed that VLA6 is preferentially upregulated on BCR/ABL1+ pre-B ALL blasts. Alterations in adhesion properties have been described for BCR/ABL1+ (p210) chronic myeloid leukemia. The role of integrins and integrin VLA6 in particular for cell adhesion-mediated drug resistance (CAM-DR) in BCR/ABL1+ (p210) ALL has not been addressed. With respect to its role for normal immature hematopoietic cells, contradictory observations have been reported. Therefore, we hypothesized that VLA6-mediated adhesion of ALL cells to the bone marrow stromal niche contributes to drug resistance. We evaluated the role of VLA6 in BCR-ABL1+ leukemia using two of our established models of leukemia, a conditional knockout model of VLA6 in murine BCR-ABL1+ leukemia and a xenograft model of human BCR-ABL1+ leukemia. VLA6fl/fl cells were oncogenically transformed using BCR-ABL1 (p210) and cultured under lymphoid-skewing conditions. Induction of pre- B (B220+ CD19+) ALL was confirmed by flow cytometry. Subsequent transduction with CreERT2 or EmptyERT2 generated leukemia cells in which VLA6 ablation could be induced (CreERT2) or not (EmptyERT2) by addition of Tamoxifen. Conditional ablation of VLA6 in vitro decreased adhesion significantly compared to undeleted controls (19.7%±8.1% vs. 87.7%±6.0%; p=0.00041) and increased apoptosis of murine BCR-ABL1+ leukemia cells as determined by analysis of Annexin V−/7-AAD− viable cells by flow cytometry (VLA6 deleted vs. undeleted: 35.3%±1.1% vs. 75.1%±1.2%; p=0.0001). Moreover, VLA6 deletion sensitized murine ALL to a tyrosine kinase inhibitor (TKI), Nilotinib (p=0.022, 45.6%±2.4% vs. 73.3%±13.0%). To test the effect of VLA6 deletion on leukemic progression in vivo, VLA6 BCR/ABL1+ pre-B (B220+ CD19+) CreERT2+ or control transduced ALL cells were transferred into NOD/SCID mice. 3 days thereafter, VLA6 deletion was induced by Tamoxifen administration to all animals in 2 cycles for 5 days. In vivo deletion of VLA6 in delayed leukemia progression compared to VLA6 competent controls from a median survival time (MST) of 30 days post-leukemia injection to a MST of 43 days post-leukemia injection (p=0.008 Log-rank test). In vivo deletion of VLA6 in combination with Nilotinib treatment delayed leukemia progression compared to VLA6 competent, as Nilotinib-treated control animals have uniformly died of leukemia with a MST of 39.5 days, however the Nilotinib treated VLA6 deleted group is completely alive and is still being monitored (p=0.0025). When VLA6 was ablated before transfer and recipients were observed for leukemia progression, the recipients of VLA6–deficient murine leukemia cells also showed attenuated leukemia progression compared to recipients of VLA6 competent cells. Moreover, we show that VLA6 blockade de-adheres primary ALL cells from their cognate counter receptor laminin in vitro, and sensitizes primary ALL cells to TKI Taken together, modulating the function of VLA6 in ALL offers a new approach to overcome drug resistance in ALL. Given that VLA6 is probably largely redundant for normal immature hematopoiesis, this approach may be preferable over targeting of other integrins in BCR/ABL1+ leukemias on which VLA6 is expressed. Disclosures: No relevant conflicts of interest to declare.

Myosin-IIa Is Required for Leukemia Cell Extravasation and Its Inhibition Reduces Leukemia Dissemination and Prolongs Survival in a Mouse Model of Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 967-967
Author(s):  
Jordan Jacobelli ◽  
Eric Wigton ◽  
Scott B Thompson
Keyword(s):  
Cell Migration ◽  
Acute Lymphoblastic Leukemia ◽  
Mouse Model ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Transfer Model ◽  
Endothelial Migration ◽  
Myosin Iia

Abstract Background: Leukemia affects approximately 45,000 people each year in the USA with more than 20,000 fatalities. Many leukemia patients experience initial remission but often relapse, with the relapsing leukemia affecting organs such as the central nervous system (CNS). The CNS acts as a sanctuary site allowing leukemia cells to escape treatments such as tyrosine kinase inhibitors (TKIs) and chemotherapy. Leukemia dissemination is a complex process requiring leukemia cells to exit the blood circulation by extravasation and invade target tissues. To extravasate, leukemia cells cross through vascular endothelial walls in a process called trans-endothelial migration, which requires cytoskeletal remodeling. However, the specific cytoskeletal effectors of leukemia extravasation are not fully known. Notably, leukemia dissemination correlates negatively with survival rates. Goal: Our goal was to validate Myosin-IIA, a class II myosin motor protein, as a molecular target to inhibit lymphoid leukemia cell extravasation and hinder leukemia dissemination, particularly infiltration into the CNS. Myosin-IIA has been shown to play a role in cell migration. We recently showed that this myosin is more profoundly required for activated lymphocyte entry into the CNS than for homeostatic entry into secondary lymphoid organs. This suggests that Myosin-IIA may be a promising candidate to prevent leukemia infiltration into the CNS without completely inhibiting homeostatic lymphocyte trafficking. Results: For this study, we used a mouse model of Bcr-Abl driven B cell acute lymphoblastic leukemia (B-ALL), which closely recapitulates Philadelphia chromosome positive human leukemias. We used shRNA interference to inhibit Myosin-IIA protein expression in the leukemia cells. Myosin-IIA depletion did not affect baseline apoptosis of the leukemia cells but did result in a small but significant reduction in their growth rate. Myosin-IIA was key in promoting leukemia cell migration in response to the chemokine CXCL12. Expression of Myosin-IIA was also critical for leukemia cells to complete trans-endothelial migration through brain-derived endothelial cells in an in vitro model of extravasation. In addition, our data suggested that inhibition of Myosin-IIA reduces the ability of leukemia cells to disseminate in vivo, including reducing leukemia infiltration into the CNS. Finally, compared to control leukemia cells, inhibition of Myosin-IIA significantly prolonged survival in an in vivo leukemia transfer model. Conclusion: Our data suggest that Myosin-IIA regulates leukemia migration thus making this myosin a promising target to inhibit leukemia dissemination in vivo, particularly into the CNS. Given the serious side-effects of cranial irradiation and (intrathecal or systemic) chemotherapy, the ability to inhibit CNS infiltration of leukemia cells can be a valuable therapeutic tool to improve the efficacy of current therapies by reducing the relapse frequency following TKI and chemotherapy treatments. Disclosures No relevant conflicts of interest to declare.

scholarly journals Cathepsin B Mediated Lysosomal Degradation in Macrophages Controls the Pharmacokinetics of the Therapeutic Protein Asparaginase

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3768-3768
Author(s):  
Laurens T Van Der Meer ◽  
Samantha YA Terry ◽  
Dorette van Ingen Schenau ◽  
Kiki Andree ◽  
Gerben M Franssen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Vivo Imaging ◽  
Cathepsin B ◽  
Rational Design ◽  
Lymphoblastic Leukemia ◽  
Therapeutic Protein ◽  
Bone Marrow Niche ◽  
Lysosomal Protease

Abstract Asparaginase (ASNase) is one of the cornerstones of the multi-drug treatment protocol that is used to treat acute lymphoblastic leukemia (ALL) in pediatric and adult patients. Despite the fact that ASNase has been used in ALL treatment protocols for decades, little is known about the biodistribution and the mechanism of ASNase turnover in vivo. A large inter-individual variation in ASNase pharmacokinetics is observed in patients. While elevated ASNase levels are associated with an increase in adverse events, underexposure, frequently caused by antibody mediated clearance, seriously reduces therapeutic efficacy. To date, it is not possible to predict pharmacokinetics of ASNase in individual patients and therefore current therapeutic protocols are supported by frequent monitoring of ASNase levels and adjustments of administration schemes. We used an in vivo imaging approach to study ASNase biodistribution and pharmacodynamics in a mouse model and provide in vitro and in vivo evidence that identifies the endo-lysosomal protease Cathepsin B in macrophages as a critical component of ASNase degradation. Results/Discussion Mice were injected with 111Indium-labeled ASNase and biodistribution was monitored by quantitative microSPECT/CT scans and ex vivo analysis of organs using a gamma counter. Over time, ASNase accumulated in the liver and particularly the spleen and the bone marrow. We hypothesized that macrophages in these organs, efficiently take up the ASNase, thereby rapidly clearing the active enzyme from the blood. Immunohistochemical analysis confirmed the presence of ASNase in cells positive for the murine macrophage marker F4/80. To confirm the importance of macrophage populations in ASNase clearance, we depleted mice from phagocytic cells by injection of clodronate liposomes, and studied ASNase biodistribution and kinetics. Indeed, clodronate pretreatment significantly diminished the accumulation of ASNase in the liver, spleen and the bone marrow while doubling the circulatory half-life of serum ASNase activity. We conclude from these experiments that macrophages determine the pharmacokinetics of asparaginase, which raises the question whether rapid clearance of the drug by bone marrow resident macrophages will negatively affect the depletion of asparagine in the bone marrow niche. We previously linked a germline mutation in the gene encoding endosomal protease Cathepsin B to strongly diminished asparaginase degradation in a pediatric ALL patient. To connect the macrophage mediated clearance to the proposed role of Cathepsin B in ASNase degradation, we studied the contribution of this protease in macrophage-mediated degradation of asparaginase. We used cell lines to show that Cathepsin B expression is induced during differentiation from monocytes towards macrophages. This is consistent with our finding that macrophages, but not monocytes, are capable of degrading ASNase. Furthermore, we used both chemical inhibition and RNAi mediated knockdown of Cathepsin B to show that this protease is required for ASNase degradation in these macrophages. Finally, by comparing Cathepsin B knockout mice with wildtype littermates, we demonstrated that loss of Cathepsin B activity significantly delayed clearance of serum asparaginase, consistent with a prominent role for this lysosomal protease in ASNase turnover. In conclusion, by using in vivo imaging we showed that asparaginase is efficiently cleared from the circulation by macrophages. In particular, bone marrow resident macrophages may provide a protective environment for leukemic cells by effectively removing the therapeutic protein from the bone marrow niche. However, both the prominent role of macrophages and the importance of the lysosomal protease Cathepsin B in asparaginase clearance, may allow the rational design of a next generation asparaginase. Disclosures Metselaar: Enceladus Pharmaceuticals: Employment, Equity Ownership.

Sign in / Sign up

Export Citation Format

Share Document