We used a microfluidic chamber driven with a high-speed pressure servo to generate defined fluid shear stress to cultured astrocytes, and simultaneously measured cytoskeletal forces using a force sensitive actinin optical sensor and intracellular Ca2+. Fluid shear generated non-uniform forces in actinin that critically depended on the stimulus rise time emphasizing the presence of viscoelasticity in the activating sequence. A short (ms) shear pulse with fast rise time (2 ms) produced an immediate increase in actinin tension at the upstream end of the cell with minimal changes at the downstream end. The onset of Ca2+ rise began at highly strained areas. In contrast to stimulus steps, slow ramp stimuli produced uniform forces throughout the cells and only a small Ca2+ response. The heterogeneity of force distribution is exaggerated in cells having fewer stress fibers and lower pre-tension in actinin. Disruption of cytoskeleton with cytochalasin-D (Cyt-D) eliminated force gradients, and in those cells Ca2+ elevation started from the soma. Thus, Ca2+ influx with a mechanical stimulus depends on local stress within the cell and that is time dependent due to viscoelastic mechanics.Mechanical perturbations increase intracellular Ca The dye was dissolved in DMSO and diluted to a final concentration of 5 μM in saline.Intracellular Ca2+. The emission lights were separated using a quad-view imaging system  with filter sets, 482/25 nm, 544/25 nm and 641/75 nm for CFP, YFP and RFP channels, respectively, and a triple band dichroic mirror . The images were acquired using an EM-CCD camera  cooled to −85°C to minimize dark noise.The fluorescence signals were measured using a multichannel LED light source  with excitation filters (430–465 nm) for FRET and (540–600 nm) for CaA molecular rotor, 2-carboxy-2-cyanovinyl-julolidine farnesyl ester (FCVJ), was used to measure the tension in the lipid bilayer  was dissolved in DMSO to a 10 mM stock solution kept frozen, and diluted to a final concentration of 10 μM. Jasplakinolide  was dissolved in DMSO to 1 mM stock solution and diluted to 500 nM before experiments.Normal saline containing 1 mM CaCl2+ intensity was calculated using Relative CaF and F0 are the mean fluorescence intensities of individual cells at time t and t = 0, respectively. The mean calcium changes were an average over N selected cells from each image and from more than four experiments for each condition. FCVJ intensity was calculated using the similar method, where mean intensity changes were calculated after background subtraction. We used a new cell culture for each experiment. The data is shown as mean ± standard error of the mean (SEM). Statistical analysis used a two-sample t-test. A value of p < 0.05 was considered statistically significant.where 2, 2 ms rise time, 15 ms duration) caused a significant Ca2+ rise in astrocytes, but the response was non-uniform in time and space in single cells. To determine whether the stimulus generated heterogeneous forces in the cell, we measured the force distribution with actinin-cpst-FRET, and found that a rapid fluid shear caused non-uniform change in actinin forces . Those cells (~40% of cell population) responded to fluid shear stress with significant force gradients. The shear-induced gradient was smaller in cells that had abundant actin bundles. Using a finite element method, we modeled a viscoelastic cell body under the fluid shear stress, and the results showed a tension distribution maximal at the upstream edge and with reduced strain at the downstream edge in softer cells . It is consistent with our experimental data. Thus, an averaged impact force cannot be used to assess the activation of cell processes; the force at the high stress points can be many fold higher than the average.A narrow shear stress pulse  at the upstream edge and peaked in ~10 s in the center of cell body . This leads to a ~3 s delay of peak Ca2+ between the upstream edge and the soma . In stiffer cells having abundant actin bundles, a more uniform transient forces appeared over the entire cell . The Ca2+ increased slower than the softer cells . These data indicate that force sensitive Ca2+ influx occurred predominantly at the front edge of the cell.To correlate the Ca Figures . The Ca2 Figures . The tim Figures , and the Figures . There w2+ rise, due to viscoelasticity the distribution of the forces should be sensitive to the stimulus rise time. Slow ramps (400 ms) to the same amplitude did not produce force gradients but generated delayed and uniform tension throughout the cells . It is the local force gradients rather than the average force that is critical to the Ca2+ influx. This result explains why some research groups did and did not observe the effect of strain rate on Ca2+ rise in different TBI models  was not able to produce force gradient in actinin and actinin force was quite uniform throughout the cell  that disrupted most of stress fibers Figure . After Cl Figure . This ins Figure . There w Figures . The stas Figure . As conts Figure . The dru% Figure , becomin% Figure .2+ influx in astrocytes, and the response depends critically on the features of the stimulus  of cells, but it is above the threshold for Ca2+ response (Maneshi et al., A transient fluid shear stress generates Ca2+ influx (Figure The heterogeneous response is determined by the intrinsic mechanical properties of the cells in their resting state. Softer cells that have fewer stress fibers and lower tension in the resting state showed stress gradients, while in cells with abundant stress fibers and higher pre-tension, we observed spatially uniform stress transients Figure . Stress x Figure . This ev2+ is far delayed from the termination of the stimulus raising the question of what cellular components provide the “memory” of the earlier stimulus (Figure 2+ permeant MSCs. The highly delayed Ca2+ involves other mechanisms such as CICR that do not directly operate by local forces (Verkhratsky and Shmigol, We previously reported that at least two types of MSCs contribute to force-induced influx in astrocytes but the primary source are force sensitive NMDARs (Maneshi et al., 2+ dynamics under shear stress are governed by the viscoelastic properties of the cytoskeleton and the bilayer.The coupling of fluid shear stress to MSCs is clearly not direct evidence by the pronounced latency. Body stresses due to disrupted cytoskeletal links can be the coupling (Orr et al., MMM designed and performed the experiments and wrote the manuscript. FS interpreted data and wrote the manuscript. SZH designed the experiments, analyzed and interpreted data and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Podosomes are dynamic actin-rich cellular protrusions that facilitate cancer cell invasiveness by degrading extracellular matrix. Whether STIM1-dependent Ca2+ signaling facilitates cancer cell invasion through affecting podosome formation remains unclear. Here we show that the invasive fronts of cancer tissues overexpress STIM1, accompanied by active store-operated Ca2+ entry (SOCE). Interfering SOCE activity by SOCE inhibitors and STIM1 or Orai1 knockdown remarkably affects podosome rosettes formation. Mechanistically, STIM1-silencing significantly alters the podosome rosettes dynamics, shortens the maintenance phase of podosome rosettes and reduces cell invasiveness. The subsequently transient expression of STIM1 cDNA in STIM1-null (STIM1−/−) mouse embryo fibroblasts rescues the suppression of podosome formation, suggesting that STIM1-mediated SOCE activation directly regulates podosome formation. This study uncovers SOCE-mediated Ca2+ microdomain that is the molecular basis for Ca2+ sensitivity controlling podosome formation.The clinical significance of STIM proteins and Orai Ca Podosomes are F-actin-enriched membrane protrusions at the ventral cell surface that promote invasive motility of several types of normal cells, including macrophages3, dendritic cells4, vascular smooth muscle cells5, osteoclasts6, and endothelial cells8. Many highly invasive cancer cells display the dynamic actin-rich structures similar to podosomes, named invadopodia9, which are associated with degradation of the extracellular matrix (ECM)10. Podosome rosettes are dynamic structures that have lifespans ranging from minutes to hours11. Each podosome rosette consists of an actin core surrounded by integrins and integrin-associated proteins12. The assembled podosomes recruit matrix metalloproteinases (MMPs) and facilitate focal degradation of ECM and invasion13. Podosome dots can undergo self-organization into podosome rosettes, which are much potent than podosome dots in promoting matrix degradation11.Tumor cell migration and invasion are essential steps for cancer metastasis via disrupting the basement membrane to disseminate cancer cells from original site to distant secondary sites2+ levels provides versatile and dynamic signaling involved in multiple cellular functions, such as proliferation, migration, gene regulation, and apoptosis14. Store-operated Ca2+ entry (SOCE) is a major Ca2+ entry pathway in non-excitable cells, which involves several steps for activation, including (i) stimulation of G proteins or protein tyrosine kinases activates phospholipase C (PLC), which hydrolyzes phosphatidylinositol bisphosphate to release the second messenger inositol-1, 4, 5-trisphosphate (IP3); (ii) binding of IP3 to IP3 receptor in the endoplasmic reticulum (ER) membrane causes a rapid and transient Ca2+ release from ER lumen; (iii) the decrease of ER luminal Ca2+ activates SOCE in the plasma membrane, leading to a sustained influx of extracellular Ca2+ across the plasma membrane16. Two families of proteins, STIM  and Orai, are the molecular identities responsible for SOCE activation18. STIM proteins function as ER Ca2+ sensors that detect ER store depletion. Once ER Ca2+ is depleted, STIM proteins aggregate into multiple puncta that translocate to the close proximity of plasma membranes. Orai, an essential pore-forming component of SOCE, translocates to the same STIM-containing structures during ER Ca2+ depletion and opens to mediate Ca2+ entry.Modulation of cytosolic Ca21. Inhibition of STIM1-mediated Ca2+ entry, by a pharmacological inhibitor SKF96365 or by siRNA-mediated silencing of STIM1 or Orai1, caused the impairment of the focal adhesion turnover and invasive migrations of breast cancer cells21. Yang et al. also showed that blockade of SOCE inhibits hepatocarcinoma cell migration and invasion by decreasing focal adhesion turnover22. The STIM1/Orai1-dependent SOCE is also implicated in tumor cell migration of nasopharyngeal carcinoma and human glioblastoma24. Our previous study showed that STIM1/Orai1-dependent SOCE enhances the cell migration of cervical cancer cells through activating the Ca2+-dependent protease calpain and tyrosine kinase Pyk219. A recent study demonstrated that STIM1- and Orai1-mediated SOCE promotes melanoma invasion and ECM degradation by increasing invadopodia formation and activity25.The functional significance of SOCE in regulating cancer cell migration, invasion, and metastasis has been addressed by the studies on breast and cervical cancer cells2+-permeable TRPV2 channel may involve in the assembly of the podosome in mouse macrophages26. Microglial podosomes are enriched in the CRAC/Orai1 channel and are closely associated with STIM127. However, the correlation between STIM1-mediated Ca2+ signaling and podosome rosettes formation remains elusive. Here we demonstrated that STIM1-mediated Ca2+ signaling is required for the formation of podosome rosettes. Blockade of STIM1-mediated Ca2+ signaling, by a pharmacological inhibitor SKF96365 or by siRNA-mediated silencing of STIM1, not only reduces the maintenance of podosome rosette, but also impairs the fate of podosome formation. Additionally, our results showed that the inhibition of STIM1-mediated Ca2+ signaling decreases podosome-mediated matrix degradation and cell invasion, supporting a role of STIM1-mediated Ca2+ signaling in malignant tumor progression. We propose that STIM1-mediated Ca2+ signaling may regulate podosome rosettes dynamics through the effect on the maintenance phase of podosome rosettes.Translocation of the CaTo study the clinical relevance of STIM1 in tumor invasion, the expression patterns of STIM1 were examined in the surgical specimens of cervical cancer Fig. . Interes28. The results implied that cervical cancer cell lines (SiHa and CaSki) presented a more active SOCE than that of normal cervical epithelial cells derived from the normal subject. To avoid bias from the clonal effect of cell lines, here we did the primary tissue cultures from 3 cervical patients with paired cancer and adjacent non-cancerous tissues. Primary cells were cultured at the same condition for 2 days before experiments. As shown in Fig. 2+ release from ER store was not significantly different between primary non-cancerous cervical cells and primary cervical cancer cells. Both primary non-cancerous cervical cells and primary cervical cancer cells displayed SOCE. However, the SOCE of primary cervical cancer cells was more active  cells, we investigated the important role of SOCE in the formation of podosome rosettes. As shown in Fig. −/−) to study the important role of STIM1-mediated SOCE in controlling podosome rosettes formation. In wild-type MEF, we defined podosome dots and rosettes altogether known as podosome-like structures. The significant decrease of podosome-like structures was noted in STIM1 knockdown groups  images of podosome rosettes. As shown in Fig. 2+ signaling during podosome rosettes formation, we monitored podosome dynamics by time-lapse recording in living cells. Cells were recorded for 2 hours for observing and analyzing the podosome dynamics. We found that inhibition of STIM1-mediated Ca2+ signaling changed the dynamics in the formation of podosome rosettes  The siRNA-mediated knockdown of STIM1 or Orai1 expression or the pharmacologic inhibition of SOCE activity significantly inhibited podosome rosette formation. (b) Blockade of STIM1-mediated Ca2+ signaling disrupted the structure of podosome rosettes and shortened the maintenance phase of podosome rosette formation. (c) Results from MEF cells lacking STIM1 confirmed that the regulation of podosome rosette formation required STIM1-dependent SOCE activation. (d) Blockade of STIM1-mediated Ca2+ signaling inhibited cancer cell migration and invasion.This study highlights that STIM1-mediated Ca2+ signaling in the regulation of cell migration and cell-substrate adhesions has been studied in different cell models. Invadopodia, also called podosomes in non-malignant cells, are actin-rich membrane protrusions mediating focal ECM degradation and invasive motility in highly invasive cancer cells. However, the exact molecular identities of Ca2+ transport proteins and the regulatory mechanisms of the Ca2+ signaling pathways involved in podosome formation have remained elusive. A recent work by Sun et al. showed that STIM1- and Orai1-mediated SOCE promotes melanoma invasion and ECM degradation by increasing invadopodia formation and activity25. Using pharmacological inhibitors, Sun et al. demonstrated that SOCE is required for invadopodia maturation and mediating focal ECM degradation25. Here we focused on the novel role of STIM1-mediated Ca2+ signaling in regulating the structural integrity and dynamics of podosome rosettes. The 3D images of podosome rosettes revealed that siRNA-mediated knockdown of STIM1 causes the structural disruption and fragmentation of podosome rosettes. Moreover, results from living-cell image recording of podosome dynamics showed that STIM1 silencing significantly decreased the duration of maintenance phase, suggesting that STIM1-mediated Ca2+ signaling affects the certain phase in podosome rosette dynamics.The importance of Ca11. The Rho-ROCK mediated signaling pathway can promote actomyosin-based cell contraction and contribute to podosome disassembly29. Our previous study proved that STIM1-dependent Ca2+ signaling controls cell migration by the regulation of actomyosin reorganization30. In this study, we demonstrated that knockdown of STIM1 shortened the maintenance phase of podosome rosettes, probably due to STIM1-mediated actomyosin distribution. Our results showed that the distribution of phospho-myosin light chain II was altered by blocking the STIM1-mediated Ca2+ signaling  cervical cancer who underwent surgery at National Cheng Kung University Hospital, Taiwan. The collection of surgical specimens was approved by the institutional review board of National Cheng Kung University Hospital. All experimental protocols and procedures were performed in accordance with relevant guidelines and regulation, and all participants provided written informed consent.v-Src-transformed MEFs and GFP-actin v-Src-transformed MEFs were grown in high-glucose DMEM (GIBCO) with 10% fetal bovine serum (GIBCO), 100 μM non-essential amino acids, 1 mM sodium pyruvate, and 55 μM 2-mercaptoethanol and cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. GFP-actin v-Src-transformed MEFs were selected using Zeocin (Invitrogen). Osteosarcoma cell line U2OS was grown in low-glucose DMEM (GIBCO) with 10% fetal bovine serum (GIBCO). Breast cancer MDA-MB-231 cells were cultured in RPMI-1640 (GIBCO) with 10% fetal bovine serum (GIBCO). Two independent pairs of different siRNAs (Sigma) and a siRNA pool of three duplexes (Santa Cruz Biotechnology) targeting STIM1 or Orai1 were used in this study.Antibodies against STIM1 (1:1000), Myosin II (1:1000) and phospho-Ser19 Myosin light chain II (1:1000) were purchased from Cell Signaling Technology. Antibody against β-actin (clone AC-15) (1:5000) was purchased from Sigma-Aldrich. Antibodies against MMP2 (1:1000) and MMP9 (1:1000) were purchased from Millipore. Antibodies against MT1-MMP (1:500) and cortactin (1:100) were purchased from Santa Cruz Biotechnology. Thapsigargin, SKF-96365, and 2-APB were from Cayman Chemical. For immunoblotting, cells were harvested with ice-cold modified radioimmune precipitation assay (RIPA) buffer containing a protease inhibitor mixture (Roche Diagnostics), 100 mM KCl, 80 mM NaF, 10 mM EGTA, 50 mM h-glycerophosphate, 10 mM p-nitrophenyl phosphate, 1 mM vanadate, 0.5% sodium deoxycholate, and 1% NP40. Protein concentrations were determined with the use of a Bio-Rad protein assay. Equal amounts of protein lysates were separated by SDS-PAGE, and then transferred to nitrocellulose blotting membranes . Immunoblots were blocked, incubated with the primary antibody, washed, and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch), and visualized by Western blotting luminol reagent (Santa Cruz Biotechnology). Bands in the immunoblots were quantified using Vision WorksLS software (UVP).To quantify STIM1 expression, we graded the surgical specimens by the distribution and intensity of immunofluorescent staining. The high grade indicates that immunofluorescent staining intensity and distribution are more than 70% of the observed area, whereas low grade indicates that the staining intensity and distribution are less than 30% of observed area. The rest of the group was graded as “intermediate”. For immunofluorescent staining, cells were seeded on glass coverslips. Cells were fixed by 4% paraformaldehyde, permeabilized by 0.1% Triton X-100 for 15 min, and blocked with commercial blocking serum (Invitrogen). These cells were incubated with primary antibody at 4 °C overnight, washed, and then incubated with AlexaFluor-conjugated secondary antibodies (Invitrogen) for 1 hour at room temperature. To detect nuclei and actin filaments, cells were stained with Hoechst 33258 and phalloidin conjugated with tetramethylrhodamine B isothiocyanate  for 1 hour at room temperature. Cells were washed and mounted, and the fluorophores were excited by laser at 405, 488, or 594 nm and detected by a scanning confocal microscope . To examine the role of STIM1 in the formation of podosome rosettes, the 3D images of F-actin were reconstructed from serial confocal Z-section scanning images using AVIZO 3D imaging and analysis software .2 atmosphere in a micro-cultivation system with temperature and CO2 control devices. The cells were monitored by a deconvolution microscopy . Images were captured every 1 min for 2 hours and analyzed by softWoRx® software. Following our previous study on podosome dynamics31, three stages of podosome formation were defined. [1] Assembly phase: Dot-shaped podosomes, which are the main structures of podosomes, are allowed to assemble into podosome rosettes. [2] Maintenance phase: Once fully assembled, the podosome itself exhibits the mechanosensory characteristics and podosome rosettes remain complete ring structures. [3] Disassembly phase: The disassembly of podosome rosettes is manifested by the collapse of the F-actin ring structure toward its center before it is completely dissolved.v-Src-transformed MEF cells that stably expressed GFP-actin were grown on glass coverslips coated with 10 µg/ml fibronectin. The cells on the microscope stage were maintained at 37 °C in a humid CO2+ was measured at 37 °C with the Fura-2 fluorescence ratio method on a single-cell fluorimeter. In brief, cells attached on glass-bottom dishes were loaded with 2 μM Fura-2/acetoxymethyl ester (Fura-2/AM) in serum-free culture medium at 37 °C for 30 min. Cells were then washed three times with PBS. The dish was then placed on the stage of an Olympus IX71 inverted microscope equipped with a xenon illumination system and an IMAGO CCD camera (TILL Photonics). The excitation wavelength was alternated between 340 nm (I340) and 380 nm (I380) using the Polychrome IV monochromator (TILL Photonics). The fluorescence intensity of excitation at 510 nm was monitored to calculate the intracellular Ca2+ levels by TILLvisION 4.0 program (Till Photonics). ER Ca2+ release was induced with 2 μM thapsigargin for 10 minutes in the absence of extracellular Ca2+, followed by the activation of SOCE with the addition of 2 mM Ca2+.Intracellular CaAlexaFluor 488-conjugated fibronectin was prepared according to the manufacturer’s instructions (Invitrogen). Cells were seeded on glass coverslips coated with 20 μg/ml AlexaFluor 488-conjugated fibronectin for 24 hours. The matrix degraded areas were measured by MetaMorph® software.24-well transwell chambers (Corning Incorporated) with a membrane in 8 μm pore size were coated with 100 μl Matrigel . The lower chamber was loaded with 750 μl high-glucose DMEM with 10% serum. Cells were seeded into the upper chamber in 250 μl serum-free medium. After 24 hours, Cells were fixed and permeabilized by 100% methanol. Cells were stained with Giemsa stain and counted on inverted Microscopy (Olympus IX71).3. After 24 hours, stained gel with 0.1% Coomassie brilliant blue R250 for 10 minutes. Gels were washed with 10% acetic acid to destain. The activity of MMP-2 and MMP9 was estimated by computer-assisted densitometric scanning of the 62-kD and 92-kD proteolytic bands, respectively.Gelatinolytic activity in the culture media was examined by gelatin zymography. The supernatants (25 μg of protein per lane) were examined by SDS-PAGE with 0.2% gelatin. After electrophoresis, gels were washed twice in 2.5% Triton X-100 to remove the SDS, and incubated for at least 24 hours at 37 °C in 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl, 10 mM CaCl2, and 0.02% NaNAll values were reported as mean ± S.E.M. (standard error of the mean). Student’s pair t test, unpaired t test or Chi-square test was used for statistical analyses. Differences between values were considered significant when P < 0.05.Supplementary InformationSupplementary material Movie 2Supplementary material Movie 1


We therefore sought to investigate the expression and functional relevance of MS channels in human platelets. The effect of shear stress on Ca2+ entry in human platelets and Meg-01 megakaryocytic cells loaded with Fluo-3 was examined by confocal microscopy. Cells were attached to glass coverslips within flow chambers that allowed applications of physiological and pathological shear stress. Arterial shear (1002.6 s−1) induced a sustained increase in [Ca2+]i in Meg-01 cells and enhanced the frequency of repetitive Ca2+ transients by 80% in platelets. These Ca2+ increases were abrogated by the MS channel inhibitor Grammostola spatulata mechanotoxin 4 (GsMTx-4) or by chelation of extracellular Ca2+. Thrombus formation was studied on collagen-coated surfaces using DiOC6-stained platelets. In addition, [Ca2+]i and functional responses of washed platelet suspensions were studied with Fura-2 and light transmission aggregometry, respectively. Thrombus size was reduced 50% by GsMTx-4, independently of P2X1 receptors. In contrast, GsMTx-4 had no effect on collagen-induced aggregation or on Ca2+ influx via TRPC6 or Orai1 channels and caused only a minor inhibition of P2X1-dependent Ca2+ entry. The Piezo1 agonist, Yoda1, potentiated shear-dependent platelet Ca2+ transients by 170%. Piezo1 mRNA transcripts and protein were detected with quantitative RT-PCR and Western blotting, respectively, in both platelets and Meg-01 cells. We conclude that platelets and Meg-01 cells express the MS cation channel Piezo1, which may contribute to Ca2+ entry and thrombus formation under arterial shear.The role of mechanosensitive (MS) Ca Platelet activation plays a crucial role in the physiological process of hemostasis but is also the key precipitating event leading to arterial thrombosis and thus potentially life threatening pathological events such as myocardial infarction or stroke. In the circulation, shear stress exerted by laminar flow of blood is regarded as a vital environmental factor during platelet activation in both normal and pathological situations. For example, shear stress is required at the early stages of the hemostatic machinery where it unfolds von Willebrand factor to reveal the binding domains to glycoprotein Ib and thus allow attachment to collagen exposed at an injury site .2+]i is a pivotal signaling event that is essential for most major functional responses during platelet activation, including cytoskeletal rearrangements and integrin inside-out signaling : F/F0 increased from 1.0 ± 0.1 at no shear (0.0 s−1) to 1.1 ± 0.2, 1.2 ± 0.3, and 1.4 ± 0.4 at normal arterial (1002.6 s−1), stenotic (low) (2282.7 s−1), and stenotic (high) (3989.3 s−1) shear rates, respectively (C). In the absence of extracellular Ca2+  shear-dependent increases in the Fluo-3 signal were abolished except for a small, residual response (F/F0 = 1.2 ± 0.2) at the highest flow rate . Pretreating the attached Meg-01 cells with 2.5 μm GsMTx-4 abolished all increases in Fluo-3 signal in response to applied shear stress . We also compared the effect of shear flow in parallel-plate flow chambers on [Ca2+]i in human umbilical vein endothelial cells, which are known to express functional Piezo1 channels  389.3 s−1 ress see  and a moress see . In the 2+ entry observed in Meg-01 cells led us to develop a method to examine whether a similar pathway exists in human platelets. Previous measurements of Ca2+ responses under arterial shear in single platelets have used glass coverslips coated with adhesive receptor ligands such as fibrinogen and collagen or synthetic peptides mimicking their binding domains . Exposure of attached unstimulated platelets to normal arterial shear stress (1002.6 s−1) resulted in multiple transient increases in cytosolic Ca2+ after a delay of 1–2 min (B and C). Subsequent arrest of flow led to a reduction but not complete inhibition of this Ca2+ response, although a second application of arterial shear caused a further increase in the number of Ca2+ transients . The Ca2+ responses were quantified using the F/F0 integral for a total of 4 min . Prior to the application of flow, when only occasional Ca2+ transients were observed, this value was 0.4 ± 0.6; during normal arterial shear it increased significantly to 1.9 ± 1.9 . In every platelet sample, a proportion of attached platelets did not show increased Ca2+ transients in response to arterial shear. However, in the platelets that did respond, the F/F0 integrals during a first and second exposure to either arterial or stenotic shear were not significantly different (E and G). This allowed the effects of GsMTx-4 and removal of Ca2+ to be assessed during the second cycle of shear. The shear-induced Ca2+ transients were significantly reduced by either the addition of 2.5 μm GsMTx-4  or removal of extracellular Ca2+  . A higher arterial shear (3989.3 s−1), equivalent to the situation when stenosis or narrowing occurs in the arteries, induced a larger and more significant increase  above prestimulus (no flow) levels when compared with normal arterial shear  and was also inhibited by GsMTx-4 . Together, these results suggest that platelets, like Meg-01 cells, possess a MS Ca2+ influx pathway induced by physiological levels of shear. The major difference between the response in these two cell types was the longer delay between stimulus application and the [Ca2+]i increase in platelets compared with Meg-01 cells. This may result from the greater rigidity of platelet surface membranes, a consequence of the extensive cortical cytoskeleton that will resist deformation and thus activation of MS ion channels . All three aspects of thrombus dimension analyzed  were reduced compared with control conditions; mean thrombus height to 52% of control (1.2 ± 0.5 to 0.6 ± 0.4 μm), mean thrombus volume to 52% of control , and mean surface coverage to 62% of control (26.4 ± 10.2 to 16.3 ± 8.6%) (B). In contrast, GsMTx-4 had no effect on the aggregation response of platelets to 1 μg/ml collagen measured in stirred suspensions, which induce minimal levels of shear (C). This response depends upon activation of the αIIbβ3 integrin (C). Together, these data suggest that the underlying GsMTx-4-sensitive pathway is crucially dependent upon application of shear stress for its activation.Pretreating whole blood with GsMTx-4 for 30 s consistently resulted in a marked reduction in thrombus formation under arterial flow on a collagen surface A. All th ± 8.6%) B. In conof shear C. This rintegrin , as demointegrin C. Togeth2+ entry pathways are unknown. Store-operated Ca2+ entry through Orai1 channels is a major Ca2+ entry pathway evoked by multiple agonists and can be selectively activated by depletion of intracellular Ca2+ stores with the SERCA inhibitor thapsigargin . Furthermore, GsMTx-4 caused no inhibition of Ca2+ entry through transient receptor potential cation channel subfamily C member 6 (TRPC6) ion channels directly activated using the diacylglycerol analogue 1-oleoyl-2-acetyl-glycerol (OAG) (60 μm) . A third Ca2+-permeable pathway in platelets is the ATP-gated P2X1 channel, which can be selectively activated by α,β-meATP (2+ response (to 60% of control) to a supramaximal concentration of α,β-meATP  . Loss of P2X1 receptor activity did not contribute to the inhibition of platelet shear-dependent Ca2+ responses by GsMTx-4 (A). However, P2X1 receptors will be functional in the thrombus formation experiments using whole blood because of the ectonucleotidase activity of plasma and leukocytes , consistent with previous reports . Using NF449 alone, mean thrombus volume was reduced from 99,856 ± 30,184 μm3 to 62,678 ± 28,802 μm3(63% of control), whereas using GsMTx-4 alone and GsMTx-4 and NF449 combined, there were reductions to 31%  and 25%  of control in thrombus volume, respectively . Similarly, mean percentage surface coverage was reduced from 46.9 ± 7.6 to 36.1 ± 11.6% using NF449 and to 24.6 ± 4.5 and 18.4 ± 3.7% using GsMTx-4 and both GsMTx-4 and NF449, respectively . Mean thrombus height decreased from 2.4 ± 0.7 to 1.5 ± 0.7 μm using NF449 and to 0.8 ± 0.2 and 0.6 ± 0.2 μm using GsMTx-4 and both GsMTx-4 and NF449, respectively . Together, these results indicate that a pathway other than P2X1 receptors is the main target for GsMTx-4 during inhibition of thrombus formation.GsMTx-4 is widely used as an inhibitor of MS ion channels; however, its effects on well established platelet Casigargin , 4. GsMT (60 μm)   was detected at trace levels in this recent study; thus we repeated the qPCR assay using a larger sample volume. Parallel qPCR runs with primers for GYPA and CD45 were used as described previously . Meg-01 cells were found to express higher levels of FAM38A transcripts compared with platelets and to also express FAM38B (A). In contrast, TRPC6 mRNA was not detected in Meg-01 cells, although it was present in platelets as reported previously (B), and this further suggested a lower level of expression in platelets compared with Meg-01. Piezo1 protein in humans is known to be an N-linked glycoprotein . Although three alternatively spliced forms of Piezo2 protein were detected in Meg-01 cells, they were all absent in platelets,4 in agreement with the lack of Piezo2 mRNA (A). Piezo1 channels have been previously studied in various tissues including erythrocytes with the aid of the blocker GsMTx-4 . Most or all of this response was lost in Ca2+-free external saline , as expected if the predominant location of Piezo1 channels is on the surface membrane (B and C). The residual response to Yoda1 in platelets in Ca2+-free medium can be explained by the suggested presence of Piezo1 channels on membranes of the intracellular stores (A). Yoda1 also enhanced intracellular Ca2+ transients in platelets attached to slides via PECAM-1 (D and E). Yoda1 increased the occurrence of Ca2+ transients both in the absence of flow and upon application of shear . Under static conditions, the F/F0 integral increased more than 3-fold from a F/F0·4 min value of 0.3 ± 0.3 to 1.2 ± 0.9. Under normal arterial flow, in the absence of Yoda1 the F/F0·4 min value was 1.2 ± 0.6, which showed a 1.7-fold increase to 2.1 ± 1.1 in the presence of the Piezo1 agonist .To further assess the contribution of Piezo1 channels to platelet signaling and functional events, we used the recently characterized Piezo1 activator, Yoda1 . In Furar milieu A. Most omembrane , B and Cr stores . These er stores A. Yoda1  PECAM-1 , D and ESince their identification in 2010 by the Patapoutian group , the MS 2+ influx mechanisms that are activated by flow, and thus shear forces, and that the underlying pathway is Piezo1. First, expression of mRNA transcripts and protein for Piezo1 was detected in both platelets and Meg-01 cells -free conditions used to record shear-induced Ca2+ transients in our study 2+ influx studies . Second,e Piezo2 , 15, dir-workers , 13. The-workers  and henclatelets . Althougur study . Furtherur study –32, the agonists , 43. Ca22+ influx in platelets, the presence of such a pathway has been suggested in earlier studies    was belo2+ responses of Meg-01 cells and platelets was the more immediate nature of the cell line response compared with the delayed increase in platelets (B and 2C). A likely explanation is the very different cytoskeletal arrangement of platelets, which consists of a cortical cytoskeleton that is responsible for its discoid resting shape and also results in a less flexible plasma membrane compared with other cell types, including its precursor and related cell lines  was from Alomone Labs , and the agonist of Piezo1, Yoda1 (5586), was from Tocris Bioscience . Fura-2 AM and Fluo-3 AM were from Invitrogen. PECAM-1 antibody (WM59) (MCA1738T) for platelet attachment was purchased from AbD Serotec . Type I collagen  was from Takeda . Unless otherwise stated, all other materials were from Sigma-Aldrich.m Phe-Pro-Arg-chloromethylketone  as anticoagulant. For all other studies, blood was taken into acid-citrate-dextrose (ACD) solution  at a ratio of 6:1 (blood:ACD). For extraction of mRNA, 1.5 ml of platelet inhibitor mixture containing 0.1 μm prostaglandin E1, 2 mm EDTA, and 0.3 mm acetylsalicylic acid in ACD was added per 10 ml of whole blood or Meg-01 cell suspension. Platelet-rich plasma (PRP) was prepared by centrifugation at 150 × g for 20 min for mRNA extraction, 100 × g for 20 min for aggregometry, or 700 × g for 5 min for other experiments. For preparation of washed platelets (WP) for aggregometry, ACD was added to PRP in 1:80 (ACD:PRP) ratio and 1 μm prostaglandin E1 and then centrifuged at 700 × g for 10 min, and the platelets were resuspended in Tyrode's HEPES buffer , after which they were centrifuged at 700 × g for 10 min. WP were resuspended in a volume of Tyrode's HEPES buffer equivalent to that of the original PRP. For intracellular Ca2+ measurements, the PRP was incubated at room temperature for 15 min with 100 μm acetylsalicyclic acid and 0.32 unit/ml apyrase  and then incubated with either 2 μm Fura-2 AM  or 5 μm Fluo-3 AM . The PRP was then centrifuged at 350 × g for 20 min, and the platelets were resuspended in an equal volume of nominally Ca2+-free saline  that also contained 0.32 unit/ml apyrase for experiments when P2X1 receptor responses were being studied. Immediately prior to Fura-2 measurements in cuvettes or before introducing Fluo-3-loaded platelets to biochips for attachment, the platelet suspension was diluted 1:1 with Ca2+-free saline. For platelet lysate preparation for Western blotting, the protocol for Fura-2-loaded platelet preparation was followed without the dye addition step, and the washed platelet suspension as lysed with radioimmunoprecipitation assay buffer  with EDTA-free protease inhibitor mixture  on ice for 1 h.Blood was collected by venepuncture from informed, consenting healthy volunteers, in accordance with the Declaration of Helsinki. This study was approved by the University of Leicester Research Ethics Committee for Human Biology . In experiments where human platelets were used, the data were obtained from three to seven donors. Whole blood for thrombus formation was collected into 40 μd-lysine hydrobromide  for 15 min. The slides were then rinsed with sterile water and dried for 2 h. Meg-01 cells in medium were added to the coated slides and incubated at 37 °C for 15 min to promote attachment. Attached cells were then treated with 2 μm Fluo-3 AM at room temperature for 45 min. After washing in Hanks' balanced salt solution , slides were immediately inserted in a parallel-plate flow chamber for experimentation. Meg-01 cell lysates were prepared by washing with PBS and lysed as described for platelets. The lysates were centrifuged at 20,000 × g at 4 °C for 10 min, and the supernatant was stored at −80 °C. Meg-01 cells used in all experiments were from passage numbers 1–14.Meg-01 cells were obtained from the European Collection of Authenticated Cell Cultures and grown in RPMI 1640 supplemented with 10% FBS and 100 units/ml penicillin-streptomycin. Sterilized glass coverslips were coated with 0.1 mg/ml poly-2+]i recordings from single platelets or Meg-01 cells was carried out on an Olympus IX81 inverted confocal microscope with a FV1000 laser scanning module  using a 60×, 1.35 NA oil immersion lens (UPLSAPO). The confocal aperture was set for optimal optical slicing . Fluo-3 fluorescence images were captured at a rate of 1.74 Hz for platelets and 0.37 Hz for Meg-01 cells  attached to a parallel-plate flow chamber was used to initially introduce HEPES-buffered saline solution  to remove air bubbles. 1 ml of whole blood was then introduced into the system for a period of 5 min at the required shear level before perfusing HEPES-buffered saline to clear the components unbound to the collagen surface. The blood was applied at a flow rate of 0.235 ml/min, which equals to a normal arterial shear rate of 1002.6 s−1, calculated according to Q = flow rate (ml/s), w = microslide lumen width in cm (0.15), h = microslide lumen height in cm (0.0125), t = shear stress (Pa), and μ = viscosity of whole blood (0.001002 Pa/s). To calculate shear rate (s−1), t was divided by μ.The glass coverslips were coated with collagen (100 μg/ml) overnight in a humidified chamber at 4 °C. Whole blood was stained with 1 μZ-stack analyses of stable thrombi were carried out within 15 min of their formation using step changes (ΔZ) of 0.69 μm. The data represent averages of at least four randomly chosen fields per experiment. Analysis and 3D reconstruction of Z-stacks were performed with ImageJ 1.49 Volume Viewer 2.0 plugin . Surface coverage and thrombus volume were calculated according to the Cavalieri principle as previously described  as necessary before inserting the slides into the parallel-plate flow chamber. To apply fluid shear stress, the reservoir was filled with either HBSS, Ca2+-free HBSS containing 1 mm EGTA or HBSS containing 2.5 μm GsMTx-4 as appropriate, and the AL-1000 syringe pump (World Precision Instruments) was set to draw fluid through the system at the shear rates of 1002.6, 2282.67, and 3989.248 s−1, which represent shear conditions in normal arteries, moderately stenotic arteries, and severely stenotic arteries, respectively (F) were normalized against prestimulus fluorescence level (F0) to yield F/F0 values.Fluo-3-loaded Meg-01 cells attached to slides were treated with 2.5 μectively . The flum Fluo-3-AM for 45 min, at room temperature) and attached onto glass-bottomed Vena8 GCS biochips  that were coated with monoclonal mouse anti-human PECAM-1 (CD31) antibody by incubation at 37 °C for 1 h. Excess antibody was removed, and nonspecific sites were blocked with 2% BSA at 37 °C for 1 h to prevent glass-induced platelet activation. Immediately prior to each experiment, Fluo-3-loaded platelets were introduced into the biochip channel and incubated at 37 °C for 10 min with occasional gentle shaking. The biochip was then mounted on the microscope stage, and HBSS was introduced into the channel under gravity. Pharmacological reagents (2.5 μm GsMTx-4 or 25 μm Yoda1) were introduced at a very low shear rate (410.16 s−1) prior to the experimental recording. Captured time-series images were analyzed on ImageJ, version 1.47 . The baseline was set manually, and peaks more than 1 point above baseline were included within the calculation of a Ca2+ increase.Platelets were loaded with Fluo-3 , at 37 °C. Platelets were resuspended in nominally Ca2+ measurements were performed in stirred suspensions using a Cairn spectrofluorimeter system  at 37 °C as described elsewhere  using an extracellular calibration after digitonin permeabilization  represent the increases in [Ca2+]i above the prestimulus concentration. The materials used to study Ca2+ influx were as follows: 0.2 mm EGTA, 1 μm thapsigargin to induce store depletion , to selectively activate P2X1 receptors.Fura-2 ratiometric Calsewhere . After llization . Peak [Cepletion ; 60 μm O agonist , 60; and4 mRNA expression values relative to GAPDH were calculated as previously described , as previously described . The reaescribed .FAM38A (Piezo1) rabbit anti-human polyclonal antibody , 1:2000; P2X1 rabbit anti-human polyclonal antibody , 1:1000; and FAM38B (Piezo2) rabbit anti-human polyclonal antibody , 1:200. The specificity of the antibodies for their intended target in Western blots . One-way analysis of variance followed by Tukey's post hoc multiple comparison analyses or paired two-tailed Student's 2+ transients. A. H. G. contributed to the preparation of the manuscript, and M. P. M.-S. conceived and coordinated the study and wrote the manuscript. All authors approved the final version of the manuscript.Z. I. designed, performed, and analyzed the experiments, prepared the figures and wrote the manuscript. J. R. W. devised the mRNA extraction and quantitative-PCR protocols and generated the initial transcriptome database leading up to this project. J. M. G. and C. I. J. developed the single-platelet attachment approach using the PECAM-1 antibody that allowed the imaging of Ca


Furthermore, short – term treatment of the AML LSCs with these MSNs before transplant significantly delayed leukemia development in recipient mice. These data demonstrate that targeting of AML LSCs can be improved by using functionalized and antigen directed MSNs as carriers for anti-leukemic drugs.Acute leukemia is initiated and maintained by leukemia stem cells (LSCs) and therefore there is great interest to develop innovative therapeutic approaches which target LSCs. Here we show that mesoporous silica nanoparticles (MSNs) functionalized with succinic anhydride, tagged with an anti-B220 antibody and loaded with the anthracycline daunorubicin are efficiently incorporated into murine B220-positive AML LSCs and preferentially kill these cells in comparison to B220-negative AML LSCs  One of the major obstacles for the development of such therapeutic concepts is the similarity between CSCs and their normal counterparts2. Thus, many surface antigens are shared between malignant and normal tissue stem cells and there is a larger overlap in molecular programs driving growth of CSCs and normal stem cells. One of the cancers obeying the CSC model is acute myeloid leukemia (AML). The cornerstone of therapy in AML is dose intense polychemotherapy, using drugs such as anthracyclines or cytarabine. This treatment approach is largely unchanged since the 1970s and improvement in treatment outcome is virtually lacking in elderly patients suffering from this disease4. Thus, developing innovative therapeutic concepts is a major goal for this disease, from which still up to eight of ten patients are dying today. Experiments in murine models as well as in xenograft models using primary human AML samples have convincingly proven that AML is propagated by leukemic stem cells (LSCs)6. We and others have shown that LSCs share many characteristics with normal hematopoietic stem cells (HSCs) with regard to their antigen profile, but also with regard to their transcriptional profile10. Differences in the antigen profile between LSCs and HSCs would open the door to antibody – based preferential targeting of LSCs, sparing normal HSCs. We previously showed that AML can be propagated by LSCs with lymphoid characteristics in a CALM-AF10 positive AML model. These LSCs showed incomplete immunoglobulin DJ rearrangement, but also importantly expressed the lymphoid associated antigen B220 in contrast to normal HSCs. We now provide evidence that a multifunctional particle system based on zwitterionic mesoporous silica nanoparticles (MSNs) functionalized with the B220 antibody and loaded with the anti-leukemic drug daunorubicin is able to target AML LSCs in this model. This is the first study which demonstrates that functionalized mesoporous silica nanoparticles can be used for targeted drug delivery to AML stem cells.One of the major goals in developing curative treatments in cancer is to target so called cancer stem cells (CSCs), which are responsible for initiating and maintaining tumor growth in many cancer subtypes. CSC targeting treatments should be able to eradicate the CSC without harming its normal counterpart, by this generating a therapeutic window allowing to treat the patient with acceptable therapy related toxicity+ AML LSCs. Amino-functionalized MSNs were used as the starting particles11. Particle morphology and size  in B220+ murine AML LSCs in vitro was analyzed by confocal laser microscopy. Intracellular localization was proven by co-localization of the GFP protein, expressed by the retrovirally transduced CALM-AF10 positive AML LSCs and the ATTO 594 dye conjugated to the MSNs  values represented efficient data fitting for all the corresponding mean values. 24 hours treatment of anti-B220 MSN-DN on B220+ AML LSCs at lower drug concentrations achieved maximum killing in contrast to free DN, indicating better efficacy of DN loaded into functionalised MSNs  is one of the most commonly used chemotherapeutics in the treatment of AML and as an anthracycline forms the backbone of polychemotherapy together with Ara-Curs Fig. . Cell deurs Fig. . Howeverurs Fig. . The difurs Fig. . In thesurs Fig.  both the+ AML LSCs, mimicking the situation, in which the surface marker is present on both HSCs and LSCs, but at considerably different quantities. 10% of the B220 positive AML LSCs expressed CD9, whereas nearly all CD9 positive cells co-expressed B220  and loaded with DN were generated. This antibody crossreacts with murine CD9, which is expressed at low levels on the B220220 Fig. . There w220 Fig. . SignifiSCs Fig. .+ AML LSCs were treated with either untagged MSNs, anti-B220 MSNs, free DN, anti-HuCD9 MSN-DN or anti-B220 MSN-DN for 24 hours. Equivalent cell numbers were subsequently injected into lethally irradiated recipients Fig. . These r4. As AML is propagated by LSCs, there is the rationale to develop therapies, which are able to preferentially target LSCs. One approach is to use functionalized nanoparticle based approaches, which are linked to antibodies recognizing antigens expressed on LSCs. Following this concept, we developed zwitterionic MSNs, tagged with anti-B220 antibodies, thereby recognizing and binding to B220+ LSCs in a murine model of CALM-AF10 positive AML9. Nanoparticle based approaches for elimination of cancer stem cells have been investigated before, but focused so far mainly on solid cancer using largely different delivery systems16. Reports on targeted approaches for hematopoietic malignancies are scarce. Recently, nanoparticles were used carrying the selective Aurora kinase B inhibitor AZD2811 to target HL-60 and MOLM-13 AML cell lines. These nanoparticles showed improved efficacy compared AZD1152 alone, in particular when combined with Ara-C17. In another report, light-inducible polymeric retinoic acid (RA)-containing nanoparticles with the capacity to accumulate in the cytoplasm of leukemia cells were used. Upon exposure to blue/UV light they released RA accompanied by differentiation of cells18. However, both approaches did not use functionalized nanoparticles targeting LSCs. In contrast to this, Jiang et al. applied a targeted system, using FLT3 ligand-conjugated nanoparticles carrying the tumor suppressor miR-150. With this approach the authors documented selective killing of FLT3-positive AML cells, confirming the potential of targeting nanoparticle systems19. Nanoparticle based concepts using surface proteins which are internalized after ligand binding as target such as the folate or the EGF receptor were already successfully applied in preclinical models of solid cancers22.There is urgent need to develop innovative therapeutic concepts for patients with AML as still the majority of patients dies of this disease despite intensive polychemotherapy and allogeneic transplantation today+ AML LSCs compared to B220- AML LSCs, by this approach comparing cells with highly similar morphology, metabolic state and nature, but discriminated by B220 antigen expression 26. We recently identified a naturally occurring anti-CXCR4 peptide EPI-X4, which significantly impairs engraftment of patient derived AML LSCs and WM cell lines28 (data not shown). We started to tag MSNs with EPI-X4 and optimized peptide derivatives of EPI-X4 with higher activity and increased stability. The anti-leukemic efficacy of these functionalized MSNs will be tested in the human system on AML and WM cell lines, but also on primary AML patient samples, which express CXCR4 in the majority of cases29. Importantly, EPI-X4 and its derivatives crossreact to murine Cxcr4, so that the potential of these MSNs can be tested in murine AML and lymphoma models.In our approach we confirmed specificity of drug delivery by performing B220 blocking experiments and by using nanoparticles linked to an anti-CD9 antibody as control. Furthermore, we demonstrated a significantly higher toxicity of anti-B220 MSN-DN on B220in vitro and efficiently kill AML LSCs at DN concentration levels well below those needed by free DN. The efficient in vitro AML LSC killing is also evident by a strongly prolonged survival of mice into which the pre-treated LSC were injected. Further optimization of the MSNs by targeting them to antigens, which are actively incorporated after ligand binding or by tagging them with peptides, which interfere with cancer growth promoting ligands will be key steps in the future. Beside this, modifications of the nanoparticles themselves by optimization of both the surface chemistry, the size and the shape of the particles will be a prerequisite to achieve longer half – lives and prolonged blood circulation times of the particles, a key prerequisite for their successful clinical use30.In summary, our data indicate that functionalized antibody-tagged MSNs are able to target LSCs Based on nitrogen sorption measurements on a Quadrasorb SI (Quantachrome) the BET surface area and NL-DFT  pore size distribution of the particles was calculated. Particle morphology and particle size was visualized by transmission electron microscopy on a TEM 1400 (Jeol). Dynamic light scattering was used to measure the hydrodynamic diameter as well as the zeta potential of the particles in a dispersion of particles (0.1 mg/mL) in 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid (HEPES) buffer solution  from Merck KGaA  on a Zetasizer Nano-ZS ZEN 3600 .11. Briefly, cetyltrimethylammonium bromide (CTAB) was added to a solution of water (53.5 mol), caustic soda solution (4.6 mmol) and methanol (20.0 mol), followed by a mixture of tetramethoxysilane (TMOS) and 3-aminopropyltrimethoxysilane (APTMS). The reaction mixture was continuously stirred for 24 hours at room temperature. All above mentioned chemicals were purchased from Sigma-Aldrich Chemie GmbH . The particles were separated via centrifugation and surfactant was removed by three times extraction with ammonium nitrate in ethanol (6.0 g/L) for 1 hour in an ultrasonic bath. The particles were washed two times with ethanol and dried at 60 °C overnight.Particle synthesis was performed according to the literatureParticles were dried prior to functionalization with succinic anhydride in vacuum for 3 hours at 80 °C. Ethanol was dried with sodium under reflux conditions and then stored under inert atmosphere over a 3 Å molecular sieve. The dried particles were dispersed in dry ethanol (15.0 mg/mL) supplemented with succinic anhydride from Merck KGaA  (0.4 µmol/mg) and rotated for 24 hours. Afterwards the particles were washed three times with ethanol and dried overnight.For intracellular detection the particles were labelled with the fluorescent dye ATTO 594 . For this particles were dispersed in HEPES buffer solution (20.0 mg/mL)  and ATTO 594 N-hydroxysuccinimide (NHS)-ester (1.3 nmol/mg) was added to the dispersion. After 1 hour of rotation at room temperature the particles were separated, washed two times with ethanol and dried for 24 hours at 60 °C. Ammonium nitrate, methanol, ethanol, potassium chloride and sodium hydroxide were obtained from Merck KGaA .31. Daunorubicin hydrochloride (7.0 µmol) was dissolved in water (27.8 µmol) and triethylamine (8.4 µmol) was added. After 10 min of rotation, the neutralized form of daunorubicin was extracted with dichloromethane. The dichloromethane was removed in vacuum and the daunorubicin was vacuum dried for 24 hours. To remove adsorbed water from the pores of the particles, the particles were dried for 3 hours at 80 °C and afterwards dispersed in daunorubicin  (7.0 µg/mg) containing dichloromethane. Dichloromethane was dried over 4 Å molecular sieve. All other chemicals were used as received without further purification. After 24 hours of rotation at room temperature the particles were centrifuged and washed three times with dichloromethane before dried in vacuum for 24 hours at room temperature. The amount of daunorubicin on the particles was calculated based on UV/vis spectroscopic analysis on a SPECORD® 50  of the starting solution and the supernatant as well as the washing waters (λ = 499 nm) .Daunorubicin hydrochloride  was neutralized with triethylamine according to the procedure previously describedTo conjugate the antibody, the carboxyl groups of the antibody anti-human/mouse CD45R (B220) respectively anti-human CD9 (20 µg/mL) was activated with N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) (19.2 µmol/mL) and NHS (19.9 µmol/mL) in HEPES buffer solution  for 30 min at room temperature. The particles were dispersed in HEPES buffer solution (2 mg/mL), added to the solution with the activated antibody and rotated for further 90 min. Afterwards the particles were separated from the conjugation mixture, washed once with HEPES buffer solution before dispersed in DMEM supplemented with 15% FBS, 1% penicillin-streptomycin and 10 ng/ml RMIL310. The residual amount of daunorubicin on the particles was calculated based on ultra-violet (UV)/visible spectroscopic analysis of the supernatant and washing water (λ = 481 nm) .+/Mac1− cells, which were shown to have LSC characteristics, reflected by their ability to cause AML in serially transplanted mice (data not shown)9. These sorted B220+/Mac1− single cells were expanded in vitro (B220+ AML LSCs) and used for experiments. B220-negative AML LSCs, also proven to be able to cause leukemia in serial transplanted mice, were derived from the bone marrow of leukemic mice transplanted with bone marrow (BM) retrovirally engineered to overexpress the homeobox gene Cdx2 as previously published33. These cells were growing immortally as a homogenous B220-negative cell population in vitro. Before these two LSC populations were used for experiments, B220 positivity or negativity was confirmed by FACS. Cells were cultured in 15% fetal bovine serum (FBS) in 1X dulbecco’s modified eagle medium (DMEM) containing 1% penicillin-streptomycin solution and 10 ng/ml recombinant mouse interleukin3 (RMIL310).CALM-AF10 positive murine AML cells were derived from single-cell-sorted B220in vitro treatments were performed with anti-human/mouse CD45R (B220) (anti-B220) tagged MSNs  and anti-human CD9 tagged MSNs loaded with or without daunorubicin. Experiments were performed for 24 hours at 37 °C and 5% CO2. B220 antigen was blocked on the CALM-AF10 positive AML cells (B220+ murine AML LSCs) by incubation with the anti-human/mouse CD45R (B220) antibody Clone RA3-6B2, eBioscience Inc., San Diego, USA) for 4 hours. Cells were incubated with the anti-human/mouse CD45R (B220) tagged or anti-human CD9 tagged MSNs loaded with daunorubicin for 24 h. For all flow cytometry and microscopic analyses, cells were washed 1 time with 1 × dulbecco’s phosphate buffered saline (DPBS) from Thermo-Fischer Scientific  containing 0.01% Tween-20 from Sigma-Aldrich Co. LLC  thereafter with 2 consecutive 1 × DPBS washes after incubation with MSNs.All in vivo experiments, 2 × 105 cells were plated in vitro and incubated for 24 h as described above. Cells left from the 2 × 105 cells at day 0 after treatment  together were injected with 1.25 × 106 non-irradiated helper cells intravenously in lethally irradiated (1200 cGy) C57BL/6 mice. All animal experiments were performed in compliance with the ETS123  and guideline 2010/62/EU and were approved by the Regierungspräsidium Tuebingen, Germany.For +Mac1− cells were performed on a BD FACS ARIA II. Flow cytometry analysis was performed using BD FACS Diva v8.0.1 and analyzed using Microsoft Excel 2013 and GraphPad Prism 6.0.All flow cytometry experiments were performed on a BD  LSR Fortessa cell analyzer and sorting for the B220Data were analyzed using appropriate testing such as 2-tailed paired student’s t-test, Wilcoxon-mann-whitney-test and area under the curve (AUC) analysis by GraphPad Prism version 6.0 or with Microsoft Excel 2013 for linear regression fitting and trend line analysis.Supplementary Information


Using optical mapping of transmembrane potential (with RH237) and sarcoplasmic reticulum (SR) Ca2+ (with the low-affinity indicator Fluo-5N AM), the effects of 15 min of regional ischemia were assessed in isolated rabbit hearts (n = 19). The impact of β-AR inhibition on isolated hearts was assessed by pre-treatment with 100 nM propranolol (Prop) prior to ischemia (n = 7). To control for chronotropy and inotropy, hearts were continuously paced at 3.3 Hz and contraction was inhibited with 20 μM blebbistatin. Untreated ischemic hearts displayed prototypical shortening of action potential duration (APD80) in the ischemic zone (IZ) compared to the non-ischemic zone (NI) at 10 and 15 min ischemia, whereas APD shortening was prevented with Prop. Untreated ischemic hearts also displayed significant changes in SR Ca2+ handling in the IZ, including prolongation of SR Ca2+ reuptake and SR Ca2+ alternans, which were prevented with Prop pre-treatment. At 5 min ischemia, Prop pre-treated hearts also showed larger SR Ca2+ release amplitude in the IZ compared to untreated hearts. These results suggest that even when controlling for chronotropic and inotropic effects, β-AR inhibition has a favorable effect during acute regional ischemia via direct effects on APD and Ca2+ handling.β-adrenergic receptor (β-AR) blockers may be administered during acute myocardial infarction (MI), as they reduce energy demand through negative chronotropic and inotropic effects and prevent ischemia-induced arrhythmogenesis. However, the direct effects of β-AR blockers on ventricular electrophysiology and intracellular Ca Using a low-affinity Ca2+ indicator , free intra-SR Ca2+ can be directly monitored everywhere on the surface of the heart  and SR Ca2+ ATPase (SERCA) function can be directly assessed via the time constant of SR Ca2+ reuptake. Acute regional ischemia was induced by ligation of the left circumflex artery (LCA). To isolate electrophysiological effects from metabolic effects that may be secondary to negative chronotropy and inotropy induced by β-AR inhibition, hearts were continuously paced and contraction was abolished with the excitation-contraction uncoupler blebbistatin.The goal of the present study was to determine the direct impact of β-AR inhibition on ventricular electrophysiology and sarcoplasmic reticulum (SR) CaAll procedures involving animals were approved by the Animal Care and Use Committee of the University of California, Davis and adhered to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .n = 19) weighing 3–3.5 kg were anesthetized with a single intravenous injection of pentobarbital sodium (50 mg/kg) containing 1000 IU heparin. Hearts were rapidly removed and perfused as previously described  modified Tyrode's solution of the following composition (in mmol/L): NaCl 128.2, CaCl2 1.3, KCl 4.7, MgCl2 1.05, NaH2PO4 1.19, NaHCO3 20, and glucose 11.1 (pH 7.4 ± 0.05). Flow rate (25–35 mL/min) was adjusted to maintain a perfusion pressure of 60–70 mmHg. Two Ag/AgCl disc electrodes were positioned in the bath to record an electrocardiogram (ECG) analogous to a lead I configuration. A bipolar pacing electrode was positioned on the base of the right ventricular epicardium for pacing, which was performed at a pacing cycle length (PCL) of 300 ms using a 2 ms pulse at twice the diastolic threshold.Male New Zealand White rabbits (m and intra-SR free [Ca2+] ([Ca2+]SR) was performed as previously described . The emitted fluorescence signals were recorded using two CMOS cameras  with a sampling rate of 0.5–1 k Hz, and 100 × 100 pixels with a 31 × 31 mm field of view.The anterior epicardial surface was excited using LED light sources centered at 470 nm  and bandpass filtered from 475 to 495 nm . The emitted fluorescence was collected through a THT macroscope  and split with a dichroic mirror at 593 nm . The longer wavelength moiety, containing the Vn = 7) were randomly assigned to acute pre-treatment with the non-specific β-AR blocker, propranolol . A non-specific β-AR blocker was chosen because of the potentially high levels of local DA and Epi, which may activate β2- in addition to the β1-ARs activated by high local NE. Prop was added to the perfusate following the first set of baseline measurements and maintained in the perfusate throughout ischemia and reperfusion. The effect of Prop was assessed 10 min after addition and did not significantly impact either Vm or SR Ca2+ properties .Epicardial pacing at a PCL of 300 ms (3.3 Hz) was continuously maintained throughout the experiment except for brief instances at baseline, 15 min ischemia, and 15 min reperfusion, when pacing at PCL = 250 ms was performed to evoke alternans. Baseline electrophysiological parameters were measured at the beginning of the experiment and again 10 min later to assure stability of the experimental preparation. Time = 0 in subsequent plots represents the immediate pre-ischemic time point. The obtuse marginal branch of the LCA was then identified and ligated around a small piece of tubing Figure . A lead Following mapping, the tube was re-inserted through the ligature to reproduce ischemia and Evan's blue dye was injected to demarcate the ischemic region Figure . This viBV_Analyze, Brainvision, Tokyo, Japan; and Optiq, Cairn, UK). Vm and [Ca2+]SR datasets were spatially aligned and processed with a Gaussian spatial filter (radius 3 pixels). For both action potentials (APs) and SR Ca2+ transients, activation time was determined at 50% of the maximal  amplitude. APD and SR Ca2+ transient durations were calculated at 80% return to baseline. SR Ca2+ transient amplitude was defined as systolic–diastolic fluorescence and this value was normalized to the pre-ischemic amplitude since these measurements are uncalibrated and only reflect relative changes in [Ca2+]SR. SERCA function was assessed using the time constant (τ) of a single exponential fit to the recovery portion of the SR Ca2+ trace (from 5 to 90% recovery). The spectral method was used to quantify the magnitude and spatial extent of APD and SR Ca2+ alternans as previously described , data were normalized to pre-ischemic (Time = 0) values.Relative ST segment elevation on the volume-conducted ECG was quantified as the ratio of the maximal ST amplitude to the QRS amplitude. Optical mapping data were analyzed with two commercially available analysis programs  and were compared using a two-way ANOVA with Tukey's multiple comparison post-testing. 2+ handling, stability of the experimental preparation was evaluated. Measurements of APD80 and SR Ca2+ transient duration (SRCaTD80) were identical at baseline (beginning of experiment) and 10 min later (pre-ischemic)   non-significantly decreased over time throughout ischemia and reperfusion in both the IZ and NI of control hearts and in the NI of Prop-treated hearts  were observed in any region or time point, regardless of treatment  was observed in the IZ of control hearts, which was reversed upon reperfusion . Under ischemic conditions, however, β-AR inhibition prevented the increase in tau observed in non-treated hearts  activation, other results are novel and suggest a multifaceted role for β-AR inhibition during ischemia.Our results indicate that even when controlling for chronotropic and inotropic effects , β-AR inhibition during the acute phase of ischemia prevents APD shortening, alters SR Ca+/H+ exchanger, leading to an increase in intracellular Na+. This Na+ overload results in intracellular Ca2+ overload via reverse-mode Na+-Ca2+ exchanger (NCX) activity. At the same time, SR Ca2+ release is dramatically reduced due to inhibition of RyR by intracellular acidification and SERCA function is inhibited due to both acidification and a reduction in cellular ATP availability  is responsible for the reuptake of extracellular NE and DA back into the neuron. NE and DA uptake by NET is coupled to the influx of Na+ and Cl− (at a ratio of 1:1:1). During ischemia, NET operates in reverse-mode and transports Na+, NE, and DA out of the cell. This non-exocytotic release mechanism can increase NE in the extracellular space up to 1000-times the normal plasma NE concentrations within 15 min of ischemia . However, due to the bidirectional coupling between Ca2+ and Vm, it is difficult to ascertain the precise contribution of intracellular Ca2+ to the observed changes in Vm   are predominating. Interestingly, Vittone et al. also reported that PLB phosphorylation was reduced with Prop pre-treatment prior to ischemia. SERCA function was not directly assessed in that study, but contractile performance was improved and ischemic contracture was reduced. The authors did not discern the precise mechanisms but speculated that β-AR inhibition may preserve cellular levels of ATP during ischemia  are added to the perfusate  were observed under any condition in the present study. Prop is a non-specific β-AR blocker. Therefore, the contribution of β1 vs. β2 receptor signaling was not assessed in the present study, but this would be an important area for future work. Prop may also have off-target membrane effects that are not fully accounted for and these effects may differ during ischemia. Finally, the present study did not fully dissect the primary effects of Prop during ischemia from the inhibition of excessive β-AR stimulation that may occur . Experiments in fully denervated or catecholamine-depleted hearts would allow for isolating any potential primary effects of Prop from ischemia-released NE.Optical mapping of free intra-SR Cae et al.  reported2+ handling characteristics during ischemia. Here we report that even when controlling heart rate and contractility, β-AR inhibition during the acute phase of ischemia prevents APD shortening, alters SR Ca2+ handling, and prevents arrhythmogenic APD and SR Ca2+ alternans. These data are consistent with improved energy metabolism with β-AR inhibition during ischemia. Investigation into the cellular and molecular mechanisms responsible for these effects remains an important area for future study.To our knowledge, this is the first study to control for chronotropic and inotropic effects and evaluate the direct impact of β-AR inhibition on action potential and CaSM, LW, and CR conceived the study, designed experiments, analyzed and interpreted data, and wrote the manuscript. ZW, PD, DL, BH, and RM analyzed and interpreted data and critically revised the manuscript. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


While supportive care has been increased in quantity and quality; substantial progress of AML therapy itself is still lacking. Primarily two predicaments challenge current therapy: first, the heterogeneity of the disease; second, the phenomenon of treatment resistance. Although many AML patients initially respond to therapy, the majority of patients relapse caused by chemoresistant clones, ultimately leading to the incurability of the disease4. On account of the poor prognosis of FLT3-ITD+ AML patients, FLT3-inhibitors are under development and in clinical testing. Nevertheless, acquired treatment resistance persists. This underlines the necessity of a greater knowledge on the origins of resistance as well as new approaches abrogating treatment resistance5.Approximately a quarter of AML patients present with FLT3-gain-of-function mutations. This usually entails a constitutive activation of FLT3 receptor downstream signaling pathways and changes gene expression patterns from healthy to malignantTherefore, we propose to deviate from the common strategy of target inhibition and focus on the reinforcement of negatively regulated downstream targets of well-known oncogenes such as FLT3-ITD.8. In accordance with this, various miRs have been shown to impact treatment resistance as well as sensitivity toward AML therapy9. First clinical trials disclosed the potential of miR-inhibitors to be a promising therapeutic option10. Nevertheless, no miR-based experimental drugs are currently available in AML.Because microRNA (miR) expression was shown to be altered by FLT3-ITD, this group of small non-coding RNAs is of interest in this work. By binding to the 3’UTRs of mRs, miRs post-transcriptionally regulate gene expression. Thus, miRs hold key regulatory functions in processes such as hematopoiesis and leukemogenesisp = 0.002) lower expressed in FLT3-ITD+ AML patients compared to healthy individuals; while among the remaining AML samples screened, miR-451a levels were lowest in FLT3-ITD patient samples  or FLT3-ITD mutations Fig. . Here, wles Fig. . In agreles Fig. . In addiion Fig. . In ordeion Fig. . Indeed,In contrast, interruption of FLT3-ITD signaling by treatment of MV4;11 cells with FLT3 inhibitors quizartinib and gilteritinib  caused an increase in miR-451a expression Fig. . As a he12, we focused on the possible underlying interaction axis.Since miR-451a correlates with an increased cancer persistence as well as recurrence12. Presently, nothing is known about the FLT3-ITD-miR-451a-MDR1 axis in AML. Hence, we conjectured that FLT3-ITD-induced repression of miR-451a and subsequent increase in MDR1 protein is in part responsible for the poor therapy response of FLT3-ITD+ AML patients. To prove this, we measured MDR1 protein amount after exogenous FLT3-ITD introduction and observed elevated MDR1 levels in FLT3-ITD transduced U937 cells by flow cytometry , an ATP dependent efflux pump mediating chemoresistance, is targeted by miR-451atry Fig.  and westtry Fig. . Upon trtry Fig. .Fig. 2MiTo corroborate our hypothesis, we determined miR-451a levels in various cell lines and measured corresponding MDR1 expression. Here, expression of MDR1 correlated inversely with miR-451a levels in the leukemic cell lines K562, MV4;11, and U937


The majority of individuals with acute myeloid leukemia (AML) respond to initial chemotherapy and achieve a complete remission, yet only a minority experience long-term survival because a large proportion of patients eventually relapse with therapy-resistant disease. Relapse therefore represents a central problem in the treatment of AML. Despite this, and in contrast to the extensive knowledge about the molecular events underlying the process of leukemogenesis, information about the mechanisms leading to therapy resistance and relapse is still limited.Recently, a number of studies have aimed to fill this gap and provided valuable information about the clonal composition and evolution of leukemic cell populations during the course of disease, and about genetic, epigenetic, and gene expression changes associated with relapse. In this review, these studies are summarized and discussed, and the data reported in them are compiled in order to provide a resource for the identification of molecular aberrations recurrently acquired at, and thus potentially contributing to, disease recurrence and the associated therapy resistance. This survey indeed uncovered genetic aberrations with known associations with therapy resistance that were newly gained at relapse in a subset of patients. Furthermore, the expression of a number of protein coding and microRNA genes was reported to change between diagnosis and relapse in a statistically significant manner.Together, these findings foster the expectation that future studies on larger and more homogeneous patient cohorts will uncover pathways that are robustly associated with relapse, thus representing potential targets for rationally designed therapies that may improve the treatment of patients with relapsed AML, or even facilitate the prevention of relapse in the first place.The online version of this article (doi:10.1186/s13045-017-0416-0) contains supplementary material, which is available to authorized users. It is cThe discrepancy between the favorable primary response rates and the substantially lower long-term survival rates in AML is due to the fact that a high proportion of patients who achieve CR eventually relapse , 5, 6. E7 cells, depending both on experimental variables and on leukemia-intrinsic factors. In agreement with LSCs representing a bastion of therapy resistance and a potential source of relapse, high LSC frequencies, as well as the presence of a stem cell expression signature, correlate with inferior outcome in AML [Similar to normal hematopoiesis, leukemic hematopoiesis is organized in a hierarchical manner. The bulk of the leukemic cell mass is derived from mostly quiescent leukemic stem cells (LSCs), which can divide either symmetrically to produce two stem cells, or asymmetrically to give rise to one stem cell and one more differentiated progenitor cell , 12. Thee in AML –13. On te in AML .Like other malignant diseases, AML is the result of somatically acquired genetic lesions, e.g., numerical and structural chromosome aberrations, copy number alterations (CNAs), uniparental isodisomies (UPDs), small insertions or deletions (indels), and single nucleotide variants (SNVs), 14–19, Recently, next generation sequencing-based investigations have yielded important novel insights into the molecular pathogenesis of AML. They have uncovered previously unknown recurrent aberrations in this disease entity , 31 and The majority of genetic and molecular studies on AML have focussed on the characterization of alterations present at the time of diagnosis, yet, as outlined above, a large proportion of AML patients with primarily responsive disease ultimately die due to relapse with refractory leukemia. The survival of stem cells, whose regrowth leads to disease recurrence, is assumed to be due in part to protective effects of the microenvironment –50 and iCytogenetics yielded the first insights into leukemia genetics, and cytogenetic analyses were the first to compare leukemic samples from the times of presentation and recurrence. During progression to relapse, karyotypes developed following five major patterns: no change (stability), acquisition of additional alterations (progression or evolution), loss of alterations (regression or devolution), progression combined with regression, and the emergence at recurrence of karyotypes that were unrelated to those found at presentation. Studies including 45–168 patients with AML observed a stable karyotype in 39-62% of them –56. AmonAberrations newly acquired at relapse in a recurrent manner are summarized in Fig. Some studies also investigated possible associations of karyotypic changes between diagnosis and relapse, or of chromosome aberrations present at relapse, with various outcome parameters. Two independent studies, including 67 and 56 patients, respectively, reported that the duration of first remission (CR1), or the time from diagnosis to first relapse (TTR), did not differ significantly between patients with a normal karyotype at both diagnosis and relapse and patients who progressed from a normal to an abnormal karyotype , 56. Forn = 11–53) at presentation and recurrence. aCNAs/UPDs were rather infrequent in AML, with an average of <1–~5 such events per sample, but their number increased significantly from diagnosis to relapse [Several studies employed single nucleotide polymorphism (SNP) arrays to compare acquired CNAs , and copy neutral losses of heterozygosity  between samples collected from AML patients  that had existed in a heterozygous state at diagnosis to homozygosity [TP53 in chromosome band 17p13 was frequent in cytogenetically complex diagnostic samples and independently predicted poor survival on the background of both complex and non-complex karyotypes [Some aCNAs/UPDs affecting specific chromosomal regions were acquired at relapse in a recurrent manner  and for mutations in ASXL1, DNMT3A, and RUNX1 [FLT3-ITD and FLT3 tyrosine kinase domain (FLT3-TKD) mutations, as well as RAS, TP53, WT1, and IDH1 mutations were recurrently gained at relapse of AML [FLT3-ITD/wild-type ratio, which represents an additional prognostic factor, was increased at relapse in several patients [TP53, were also recurrently lost at relapse  and core-binding factor AML (n = 10), respectively, found numbers of exonic mutations comparable to those in the earlier investigation. Again, the majority of these mutations persisted during disease progression, while some were specific to either diagnosis or relapse [Subsequent reports employing whole exome sequencing (WES)  and/or targeted resequencing confirmed and extended these findings. Two WES studies, restricted to adult patients with  relapse , 68. The relapse , 68. As  relapse , 38, 69. relapse .Three studies applied WES to pediatric AML, two including each 4 , 92, andPML and RARA were repeatedly observed in patients treated with arsenic trioxide and all-trans retinoic acid, respectively [PML-RARA fusion persisted between presentation and recurrence [Due to its different biology and treatment modalities , 9, 93, ectively . WES on currence , suggestOR2T12 and NPM1, gain of a synonymous mutation in VTN, and a moderate increase in the VAF of a splicing mutation in TMEM63C). Secondly, a clone containing the DNMT3A-R882H mutation of the founding clone, along with five additional variants, expanded strongly during CRs two to four to reach VAFs of up to 50%  and decreased, but was not eliminated, during subsequent relapses [In an approach somewhat different from the above discussed studies, Kim et al. used WES to track the course of disease of a patient presenting with cytogenetically normal AML at the age of 36 over 9 years, during which he experienced four CRs and four relapses . Two finrelapses . Similarrelapses . The cliCEBPA. In a cohort comprising ten pedigrees with this condition, patients presented with AML at a median age of 24.5 years [CEBPA allele, as well as additional SNVs identified by WES, were discordant between diagnosis and recurrence, suggesting that in this condition, recurrences more commonly represent new leukemic episodes rather than relapses of the original disease, in agreement with the clinical observation that they frequently retain therapy sensitivity [A unique subgroup among patients with AML are those with familial disease due to predisposing germ line mutations, e.g., in .5 years . WES on .5 years , comparasitivity .ASXL1, SETBP1, and ZRSR2 were recurrently gained but not recurrently lost at relapse , where activating mutations in the gene encoding the drug inactivating 5′-nucleotidase NT5C2 were acquired in almost one fifth of patients at disease recurrence , 52, no Even though it remains possible that application of unbiased methods like WES or WGS to larger, more homogeneous patient cohorts will lead to the identification of  candidate driver mutations of relapse, a universal role of SNVs or indels—at least in their “classical” mode of action—in the evolution of therapy resistance is also questioned by observations that disease can recur in a refractory manner without the acquisition of additional non-synonymous mutations , 92, 95.Alterations in DNA methylation are common in myeloid malignancies, and demethylating agents play a role in their clinical management . To explCDH13, NPM2, PGRA, HIN1, SLC26A4, and CDKN2B genes [CDKN2B CpG island also increased significantly from 48/77 (62%) cases at presentation to 30/36 (83%) at relapse of APL [Kröger et al. focussed on nine genes whose CpG islands had previously been shown to be hypermethylated in primary and cultured malignant hematopoietic cells, and probed them by bisulfite pyrosequencing in paired diagnostic and relapse samples from 30 patients with cytogenetically heterogeneous AML . The med2B genes . Methylae of APL .TGM2, CDK1, miR-331-5p, and miR-27a, were significantly deregulated between diagnosis and relapse of AML [Altered expression of certain genes contributes to leukemogenesis, and the mRNAs levels of single genes as well as the presence of specific multi-gene expression signatures at diagnosis are predictive of outcome in AML , 103–105e of AML –108. Othe of AML , 109–111e of AML . Supporte of AML , 22, 113e of AML . Consiste of AML .MLL rearrangements uncovered 53 microRNAs that were up-, but none that were down-, regulated at relapse at an FDR of <10%, among them the miR-106b~25 cluster. The expression pattern of about half of the tested microRNAs could be confirmed by qRT-PCR in the original, plus eight additional, sample pairs [In addition to protein-coding genes, microRNA genes may be differentially expressed between diagnosis and relapse of AML: a Taqman low-density array screen on six paired samples from pediatric patients with le pairs .CADM1 was upregulated upon exposure to cytosine arabinoside, daunorubicin, or etoposide, and its experimental expression in U937 cells led to an increase in the proportion of apoptotic cells [CADM1 was upregulated in response to in vitro exposure to cytosine arabinoside in 3/3 primary AML samples from the time of diagnosis, but in none of the matched relapse samples [While all of the abovementioned studies measured gene expression in a static manner, expression changes elicited by chemotherapeutic drugs may also play a role in the response to them. Therefore, Fisser et al. searched for genes that were induced by cytostatics in the human myeloid cell line U937. ic cells . Corrobo samples .MSH2 and MLH1 genes in unpaired diagnostic, relapsed, and refractory samples from 53 pediatric and adult patients with AML, suggested that aberrations affecting mismatch repair genes were significantly more frequent in refractory/relapsed samples than in diagnostic ones (12/25 versus 6/28) [The following chapter briefly summarizes several studies that compared molecular parameters not readily attributable to any of the above categories between diagnosis and relapse of AML. In the first of these, Shlush et al. inferred the number of cell divisions that had occurred in a cell population from single cell microsatellite data and concluded that in two investigated patients with AML, proliferative activity was higher at diagnosis than at relapse . Anotherus 6/28) . Zhou etus 6/28) . Finallyus 6/28) .Based on mutational profiles and clonal dynamics observed in patients with AML, three major pathways to relapse have been proposed , 44, 69 In some patients with apparent relapse-specific mutations, these could retrospectively be demonstrated to have been present at very low levels also in the diagnostic sample, while in others, they were undetectable at this time point even with highly sensitive methods , 46, 47,TP53 gene in chromosome band 17p13 or point mutations in ASXL1. However, these lesions were present only in a low single-digit percentage of the investigated patients. In contrast, in patients selected for the presence of specific genetic features at diagnosis, the expression of certain mRNAs or miRNAs changed between presentation and recurrence in a statistically significant manner, implying that these alterations occurred in a large proportion of cases. Furthermore, the mRNA expression profile associated with relapse of normal karyotype AML was enriched for gene signatures associated with LSCs and with a poor prognosis, suggesting that some of its member genes may contribute to therapy resistance at relapse. However, it remains to be shown whether these gene expression changes are sufficiently uniform among single leukemic cells and sufficiently stable to represent useful therapeutic targets.Indeed, the studies summarized above have identified some aberrations that were newly gained at relapse in a recurrent manner and may plausibly contribute to therapy resistance at this stage, like deletions of the In summary, even though so far only a limited number of studies has addressed the molecular alterations specifically acquired at relapse of AML, important progress has been made in understanding the genetics and molecular biology associated with this largely therapy-resistant disease stage. Despite of these advances, many open questions remain. Among these are the extent to which genetic alterations present at diagnosis and variations in treatment determine which additional lesions are able to allow leukemic cells to survive therapy and regrow at relapse. The possible functional role of mutations without predicted translational consequences warrants further exploration . The gen