Biological Functions And Clinical Applications Of BCR-ABL1-positive Microvesicles

Part I Characteristics and Contents of BCR-ABL1-positive microvesiclesObjective: Microvesicles are functional bubbles derived from plenty of cells. The biological functions of MVs are due to their complexity of bioactive cargo. Our aim of this section is to figure out the characteristics and content of microvesicles derived from chronic myeloid leukemia (CML) in order to investigate their biological functions.Methods: The human CML blast crisis cell line K562were purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China) and cultured in RPMI1640with10%FBS and1%FBS at37℃in5%CO2. Supernatant of K562were collected to isolate MV by gradient centrifugation. To confirm that we had isolated MVs, random samples were analyzed in a fluorescence-activated cell sorter. The morphology of the precipitate was visualized using transmission electron microscopy and confocal microscopy, stained with CFSE and PKH-26. Phalanx Human miRNA OneArray was performed to investigate the pattern of miRNA in K562and its MV when starvation. Real-time PCR was performed to measure the BCR-ABL1mRNA; and chromosome fragments were detected by fluorescence in situ hybridization. Besides, a mice model of our own was built to investigate the detectability of MV.Results:MVs form mostly by reverse budding and fission of the plasma membrane stalk detaches the cytoplasmic protrusions. The MVs measured100-1000nm, thus we could observe them under confocal microscopy when stained with a fluorescent dye. By fluorescence-activated cell sorter, we found the surfaces of most MVs are annexin V-positive. K562-MVs contained BCR-ABL1mRNA and fragments of Philadelphia chromosome. Our mice model of minimal residual disease indicated that MV could be detected by RQ-PCR earlier than the cells, acting as a previous biomarker of the hidden tumor cells. Compared with K562-MV, certain miRNAs increased in MV when starvation. Interestingly, these miRNAs decreased in K562cells when starvation, indicating that K562cells could throw out some miRNAs which were considered as not necessary via MV shedding. Other miRNAs overexpressed in K562-MV were considered onco-gene and could give implications for transformation of normal cells.Conclusion:We have defined the the characteristics and content of microvesicles derived from K562cells. MV derived from tumor cells is considered as a self-purification system that can carry special molecules including microRNA out of cells. Besides, the content of K562-MV indicated that it might give manligant transformation of normal cells. Part II BCR-ABLl-positive microvesicles malignant transform normal hematopoietic stem/progenitor cellsObjective: MVs are thought to deposit paracrine information and create paths of least resistance to transfer their cargo to adjacent or remote cells. Our previous work demonstrated that MVs derived from K562leukemia cells contained the dominant onco-mRNA of BCR-ABL1and plenty of miRNAs. It was known to all that BCR-ABL1could transform normal hematopoietic cells. As a result, the aim of this section is to investigate whether K562-MV participates in the transformation of normal hematopoietic cells via intercellular transferring of onco-materials.Methods: MV was isolated from K562, Jurakt, KG-1a cells, etc. Mononuclear cells (MNCs) were extracted from the peripheral blood mobilization; cord blood, peripheral blood and bone marrow of healthy volunteers and were cultured in StemSpan(?) SFEM. The MNCs were adjusted to4x106cells per well in six-well plate, and MVs was added to the cells three times a day for14-23days. The morphology, phenotype, cytogeneic abnormals of transformed cells were analyzed. Transformed cells were also subcutaneously injected into the right shoulders of the mice for tumor formation. mRNAs from cells on days0,7,14, and21of the transformation process were sequenced to investigate the main events of the MV-induced leukemogenesis. DNA double-strand breaks (DSBs) were assessed by agarose gel electrophoresis using genomic DNA isolated from recipient cells. Microsatellite instability was determined by loci multiplex PCR in an ABI3730x1Genetic analyzer. Intracellular reactive oxygen species (ROS) were detected using a Reactive Oxygen Species Assay Kit; and DNA methylation was performed with a MethylFlash Methylated DNA Quantification Kit. Real-time PCR and western blot were performed to investigate the expression of AICDA, DNMT3A, DNMT3B, RAD51b and RAD18. To decrease the BCR-ABL1mRNA in the MVs, K562cells were transfected with hsa-miR-203mimics. Besides, K562-MV was treated with RNase to decrease its containing RNAs. These treated K562-MVs were used to repeat the work described above.Results: After consecutive incubation with MVs for at least14days, a new group of leukemia-like cells could be observed in the MNCs derived from normal transplants. The morphology of these cells was similar to that of monoblasts and expressed myeloid (CD15, CD38) markers. Most of the cells were triploid or hypotetraploid and consistently or frequently contained several structural aberrations. Fourteen days after subcutaneous transplantation into nude mice (without cyclophosphamide), the leukemia-like cells formed tumors at the injection sites. The secondary host mice into which the tumor cells were subcutaneously transplanted presented obvious cachexia and spleen enlargement after20days. Under confocal microscopy, we found that K562-MVs labeled with CFSE were present in the cytoplasm. Besides, we found K562-MVs could deliver BCR-ABL1mRNA and P210protein into the recipient cells. Clustering analysis of sequencing data indicated that most of the significantly expressed mRNAs were involved in genomic instability. We found that there was increased DNA breakage during transformation in the vast majority of recipient cells incubated with K562-MVs, and small changes were found in the microsatellite of these cells. And the oncogenic effect of genomic instability is the result of consequent overexpression of hypermethylation, AICDA and ROS induced by K562-MVs. K562-MV with decreased BCR-ABL1did not exhibit the ability of transformation and the effect of genomic instability.Conclusion:We demonstrated that BCR-ABL1-positive MVs could initiate malignant transformation of normal hematopoietic transplants through genomic instability.These findings shed much-needed light on an unconventional and poorly understood mechanism of intercellular communication and the manner in which it may have significant consequences in the tumorigenesis of DCL and CML transformation. It also presented a unique opportunity to prospectively study the changes undergone by normal cells, leading to their malignant transformation. Part Ⅲ Clinical Applications of BCR-ABL1-positive MicrovesiclesObjective: Circulating MV harbors molecular cancer-related information, and contain a potential reservoir of prognostic and predictive biomarkers of tumor progression and response to targeted therapeutics. Multiple studies have demonstrated that tumor cells themselves may locate in some undetectable sites, but the MVs they secreted are able to circulate in the blood, transporting information of the cancer. The aim of this study was to investigate whether BCR-ABL1positive MVcould be used in the monitoring minimal residual disease (MRD) of CML and Ph positive acute lymphoblastic leukemia (ALL).Methods: Following informed consent,127Ph1and/or BCR-ABL1positive CML patients were recruited between November2010and March2011in our hospital, including68males and59females (median age35ys [10-54ys]). Median time of TKI was33months (3-82m). Of these patients,36/127was confirmed at complete molecular response (CMR),37achieved the major molecular response,9were in complete cytogeneic response and9suffered hematological response.The left24patients achieved allogeneic hematolopoietic stem cell transplantation (allo-HSCT). Similarly,15Ph1+ALL patients post-HSCT, with median year of26(19-49ys), participated in the study, following the principle of informed consent. The median monitoring time was12m (4-21m). The blood samples were collected from each patient with fixed frequency to isolated MV. Before sent for real-time PCR, MV was confirmed by electron microscopy and flow cytometry.Results:MV isolated from the peripheral blood of patients was confirmed by electron microscopy and flow cytometry as Annexin V positive (94.5%)0.8μm particles containing cell membrane. Using RQ-PCR and ABL1as a control, BCR-ABL1mRNA was detected in MV isolated from patients. Similar to those in the cells, BCR-ABL1copy numbers in MV significantly varied between CML patients in HR, CCyR, MMR and CMR (HR:106.50±38.3, CCyR:42.20±14.1, MMR:21.05±17.8, CMR:14.08±10.9, P<0.05). A positive correlation was observed between BCR-ABL1expression in MV and cells(r=0.416, P<0.05). BCR-ABLl in MV increased with patients diagnosed with CP, AP and BP (CP:577.2±11.3, AP:1109.7±131.4, BP:2783.1±462.3, P<0.05). We detected BCR-ABL1in MV from29/37CMR patients (14.08±10.9), indicating that quantification of BCR-ABL1in MV may complement cellular RQ-PCR monitoring, especially in CMR. To those achieved CMR, BCR-ABL1in MV are significantly different between imatinib and HSCT recipients (14.08±10.9vs7.3±2.1, P<0.05), while the cellular BCR-ABL1show no significantly difference. For our Ph+ALL patients,6/15suffered leukemia relapse and2/6was extramedullary relapse. In our monitoring, relapsed patients suffered durative positive BCR-ABL1copies in MV for2weeks before the definitive proof of relapse was found. Besides, durative positive BCR-ABL1copies in MV could provide much-needed implications for the extramedullary relapse.Conclusion:This is the first report of the detection of BCR-ABL1in MV isolated from peripheral blood of BCR-ABL1-positive patients. We believe that BCR-ABL1in MV could act as a supplement of normal monitoring methods to avoid the false-negative of CMR, as well as providing an early-warning signature of relapse of Ph+acute leukemia patients.