Uptake of imatinib-loaded polyelectrolyte complexes by BCR-ABL(+) cells: a long-acting drug-delivery strategy for targeting oncoprotein activity.

RATIONALE & AIM: Imatinib mesylate (IM), a selective tyrosine kinase inhibitor of the oncoprotein BCR-ABL, is the 'gold standard' for patients with chronic myeloid leukemia (CML) but the drug does not eliminate CML stem cells, leading to disease relapse on drug discontinuation. At present, much effort is focused on delivery carriers that can increase the intracellular retention and antileukemic impact of IM. We previously validated IM-loaded polyelectrolyte microcapsules as effective purging agents to eradicate BCR-ABL(+) cells from CML patient autografts. The aim is to develop controlled release carriers that can increase the intracellular retention and functionality of IM in leukemia cells. MATERIALS & METHODS: Herein, novel polyelectrolyte complexes were used as model carriers for IM in a CML cell line (KU812) and CD34(+) cells freshly isolated from patients. RESULTS & DISCUSSION: Polyelectrolyte complexes promoted a long-acting BCR-ABL kinase inactivation that was necessary to promote apoptosis at approximately twofold lower intracellular IM dose compared with the microscale formulation polyelectrolyte microcapsules. CONCLUSION: IM-loaded polyelectrolyte complexes can be used as more efficient delivery devices for overcoming drug resistance of BCR-ABL(+) leukemic cells.

Physiological formation of fluorescent and conductive protein microfibers in live fibroblasts upon spontaneous uptake of biocompatible fluorophores.

We have recently reported initial results concerning an original approach to introduce additional properties into fibrillar proteins produced by live fibroblasts and extruded into the ECM. The key to such an approach was biocompatible, fluorescent and semiconducting synthetic molecules which penetrated spontaneously the cells and were progressively encompassed via non-bonding interactions during the self-assembly process of the proteins, without altering cell viability and reproducibility. In this paper we demonstrate that the intracellular secretion of fluorescent microfibers can be generalized to living primary and immortalized human/mouse fibroblasts. By means of real-time single-cell confocal microscopy we show that the fluorescent microfibers, most of which display helical morphology, are generated by intracellular coding of the synthetic molecules. We also describe co-localization experiments on the fluorescent microfibers isolated from the cell milieu demonstrating that they are mainly made of type-I collagen. Finally, we report experimental data indicating that the embedded synthetic molecules cause the proteins not only to be fluorescent but also capable of electrical conductivity.

Micropatterned polyelectrolyte nanofilms promote alignment and myogenic differentiation of C2C12 cells in standard growth media.

Alignment of skeletal myoblasts is considered a critical step during myotube formation. The C2C12 cell line is frequently used as a model of skeletal muscle differentiation that can be induced by lowering the serum concentration in standard culture flasks. In order to mimic the striated architectures of skeletal muscles in vitro, micro-patterning techniques and surface engineering have been proven as useful approaches for promoting elongation and alignment of C2C12 myoblasts, thereby enhancing the outgrowth of multi-nucleated myotubes upon switching from growth media (GM) to differentiative media (DM). Herein, a layer-by-layer (LbL) polyelectrolyte multilayer deposition was combined with a micro-molding in capillaries (MIMIC) method to simultaneously provide biochemical and geometrical instructive cues that induced the formation of tightly apposed and parallel arrays of differentiating myotubes from C2C12 cells maintained in GM media for 15 days. This study focuses on two different types of patterned/self-assembled nanofilms based on alternated layers of poly (allylamine hydrochloride) (PAH)/poly(sodium 4-styrene-sulfonate) (PSS) as biocompatible but not biodegradable polymeric structures, or poly-L-arginine sulfate salt (pARG)/dextran sulfate sodium salt (DXS) as both biocompatible and biodegradable surfaces. The influence of these microstructures as well as of the nanofilm composition on C2C12 skeletal muscle cells' differentiation and viability was evaluated and quantified, pointing to give a reference for skeletal muscle regenerative potential in culture conditions that do not promote it. At this regard, our results validate PEM microstructured devices, to a greater extent for (PAH/PSS)5-coated microgrooves, as biocompatible and innovative tools for tissue engineering applications and molecular dissection of events controlling C2C12 skeletal muscle regeneration without switching to their optimal differentiative culture media in vitro.

Modulation of alignment and differentiation of skeletal myoblasts by biomimetic materials.

Proliferation and fusion of myoblasts are needed for the generation and repair of multinucleated skeletal muscle fibers in vivo. We developed a novel cell culture technique that results in formation of myotubes, organized in parallel much like the arrangement in muscle tissue. Alignment and fusion of myoblasts into parallel arrays of multinucleated myotubes are critical in skeletal muscle tissue engineering and more micro-patterning techniques and surface engineering have been tested by switching differentiating myotubes from growth medium (GM) to differentiative media (DM). One of the goals of tissue engineering is to develop tools allowing in vitro construction and mimicking of the final tissue architectures. The fabrication of polyelectrolyte multilayers (PEMs) may represent a promising approach for recreating physiological nanometer-sized cell environments in vitro. In this study we describe a method for generating biomimetic microstructured surfaces that promote cell adhesion and differentiation of parallel arrays of mature C2C12 myotubes continuously maintained in GM for 7 days. The structure consists of a 'double-sheet' PDMS structure that provides 'compliant' or 'stiff' microdomains to guide the cell self-patterning, coupled to layer-by-layer (LbL) self-assembled multilayers of biocompatible polyelectrolytes that promote C2C12 myoblasts alignment and differentiation. Our findings have relevance to the interpretation of in vitro data as well as to the study of cellular interactions with biomaterials.

Cell self-patterning on uniform PDMS-surfaces with controlled mechanical cues.

The exploitation of cell-instructive scaffolds with uniform physical/chemical surfaces and controlled stiffness will be greatly useful in tissue engineering applications to resemble the extracellular matrix (ECM) or topographical appearance of native tissues. We herein describe a versatile and straightforward method to assemble a polydimethylsiloxane (PDMS)-composite structure in which a uniformly laminin-coated membrane is placed on top of a micropatterned substrate that applies a stiffness gradient. This 'double-sheet' structure provides soft or stiff microdomains that guide the self-patterning of different cell types [e.g. chronic myeloid leukemia (KU812), cervix carcinoma (HeLa), NIH 3T3 and BJ], thereby stimulating their cytoskeletal remodeling. More interestingly, we used these uniform PDMS surfaces with patterned rigidity for obtaining co-cultures of tumor blood cells (KU812) and adherent fibroblasts (NIH 3T3) with spatially-controlled distribution. Thus, beyond single-cell stiffening and mechanosensing, these surfaces should also be used as simple and feasible co-culture systems for mimicking and dissecting the bidirectional interactions between blood cells and specific stromal elements of their in vivo microenvironment.

Cell Uptake and Validation of Novel PECs for Biomedical Applications.

This pilot study provides the proof of principle for biomedical application of novel polyelectrolyte complexes (PECs) obtained via electrostatic interactions between dextran sulphate (DXS) and poly(allylamine hydrochloride) (PAH). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that DXS/PAH polyelectrolyte complexes were Monodispersed with regular rounded-shape features and average diameters of 250 nm at 2 : 1 weight ratios of DXS/PAH. Fluorescently labelled DXS and fluorescein-isothiocyanate- (FITC-)conjugate DXS were used to follow cell uptake efficiency of PECs and biodegradability of their enzymatically degradable DXS-layers by using confocal laser scanning microscopy (CLSM). Moreover, quantitative MTT and Trypan Blue assays were employed to validate PECs as feasible and safe nanoscaled carriers at single-cell level without adverse effects on metabolism and viability.

Lenalidomide restrains motility and overangiogenic potential of bone marrow endothelial cells in patients with active multiple myeloma.

PURPOSE: To determine the in vivo and in vitro antiangiogenic power of lenalidomide, a "lead compound" of IMiD immunomodulatory drugs in bone marrow (BM) endothelial cells (EC) of patients with multiple myeloma (MM) in active phase (MMEC). EXPERIMENTAL DESIGN: The antiangiogenic effect in vivo was studied using the chorioallantoic membrane (CAM) assay. Functional studies in vitro (angiogenesis, "wound" healing and chemotaxis, cell viability, adhesion, and apoptosis) were conducted in both primary MMECs and ECs of patients with monoclonal gammopathies (MGUS) of undetermined significance (MGEC) or healthy human umbilical vein endothelial cells (HUVEC). Real-time reverse transcriptase PCR, Western blotting, and differential proteomic analysis were used to correlate morphologic and biological EC features with the lenalidomide effects at the gene and protein levels. RESULTS: Lenalidomide exerted a relevant antiangiogenic effect in vivo at 1.75 µmol/L, a dose reached in interstitial fluids of patients treated with 25 mg/d. In vitro, lenalidomide inhibited angiogenesis and migration of MMECs, but not of MGECs or control HUVECs, and had no effect on MMEC viability, apoptosis, or fibronectin- and vitronectin-mediated adhesion. Lenalidomide-treated MMECs showed changes in VEGF/VEGFR2 signaling pathway and several proteins controlling EC motility, cytoskeleton remodeling, and energy metabolism pathways. CONCLUSIONS: This study provides information on the molecular mechanisms associated with the antimigratory and antiangiogenic effects of lenalidomide in primary MMECs, thus giving new avenues for effective endothelium-targeted therapies in MM.

Imatinib-loaded polyelectrolyte microcapsules for sustained targeting of BCR-ABL+ leukemia stem cells.

AIM: The lack of sensitivity of chronic myeloid leukemia (CML) stem cells to imatinib mesylate (IM) commonly leads to drug dose escalation or early disease relapses when therapy is stopped. Here, we report that packaging of IM into a biodegradable carrier based on polyelectrolyte microcapsules increases drug retention and antitumor activity in CML stem cells, also improving the ex vivo purging of malignant progenitors from patient autografts. MATERIALS & METHODS: Microparticles/capsules were obtained by layer-by-layer (LbL) self-assembly of oppositely charged polyelectrolyte multilayers on removable calcium carbonate (CaCO(3)) templates and loaded with or without IM. A leukemic cell line (KU812) and CD34(+) cells freshly isolated from healthy donors or CML patients were tested. RESULTS & DISCUSSION: Polyelectrolyte microcapsules (PMCs) with an average diameter of 3 microm, fluorescently labelled multilayers sensitive to the action of intracellular proteases and 95-99% encapsulation efficiency of IM, were prepared. Cell uptake efficiency of such biodegradable carriers was quantified in KU812, leukemic and normal CD34(+) stem cells (range: 70-85%), and empty PMCs did not impact cell viability. IM-loaded PMCs selectively targeted CML cells, by promoting apoptosis at doses that exert only cytostatic effects by IM alone. More importantly, residual CML cells from patient leukapheresis products were reduced or eliminated more efficiently by using IM-loaded PMCs compared with freely soluble IM, with a purging efficiency of several logs. No adverse effects on normal CD34(+) stem-cell survival and their clonogenic potential was noticed in long-term cultures of hematopoietic progenitors in vitro. CONCLUSION: This pilot study provides the proof-of-principle for the clinical application of biodegradable IM-loaded PMC as feasible, safe and effective ex vivo purging agents to target CML stem cells, in order to improve transplant outcome of resistant/relapsed patients or reduce IM dose escalation.

An alternative in vivo system for testing angiogenic potential of human neuroblastoma cells.

In this study we purposed an alternative method to study the angiogenic and invasive potential of neuroblastoma cell suspensions implanted on the chick embryo chorioallantoic membrane (CAM) surface. Neuroblastoma cells were seeded in Matrigel and thereafter the suspension was pipetted onto the CAM surface at day 8 of incubation inside a silicon ring previously loaded onto the CAM surface. Four days after implantation, the silicon ring was removed and the angiogenic and invasive response were studied morphologically at macroscopic and microscopic levels and by reverse transcriptase-polymerase chain reaction (RT-PCR) by using human and chicken primers for several angiogenic cytokines, namely vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), angiopoietin-1 (ANG-1), hypoxia inducible factor-2alpha (HIF-2alpha), and for an endogenous angiostatic molecule, namely endostatin. Results showed that: (1) Neuroblastoma cells induced an angiogenic response in the CAM assay comparable to that induced by FGF-2; (2) neuroblastoma cells are packed inside Matrigel or are recognizable in the CAM mesenchyme; (3) Angiogenic activity of neuroblastoma cells is associated to an high expression of the transcripts of human VEGF-A, FGF-2, ANG-1 and HIF-2alpha and to a low expression in the transcript of a human endostatin while in the control specimens there is no expression of both angiogenic and angiostatic molecules; and (4) the expression of the transcripts of the same chicken angiogenesis stimulators and inhibitor is unmodified in treated and control specimens. Overall, these data indicate that neuroblastoma cells growth on the chick CAM express characteristics of the human disease. This experimental model could be employed for further research on human tumor progression and anti-angiogenic molecules screening.

Characterization of some molecular mechanisms governing autoactivation of the catalytic domain of the anaplastic lymphoma kinase.

NPM/ALK is an oncogenic fusion protein expressed in approximately 50% of anaplastic large cell lymphoma cases. It derives from the t(2;5)(p23;q35) chromosomal translocation that fuses the catalytic domain of the tyrosine kinase, anaplastic lymphoma kinase (ALK), with the dimerization domain of the ubiquitously expressed nucleophosmin (NPM) protein. Dimerization of the ALK kinase domain leads to its autophosphorylation and constitutive activation. Activated NPM/ALK stimulates downstream survival and proliferation signaling pathways leading to malignant transformation. Herein, we investigated the molecular mechanisms of autoactivation of the catalytic domain of ALK. Because kinases are typically regulated by autophosphorylation of their activation loops, we systematically mutated (Tyr --> Phe) three potential autophosphorylation sites contained in the "YXXXYY" motif of the ALK activation loop, and determined the effect of these mutations on the catalytic activity and biological function of NPM/ALK. We observed that mutation of both the second and third tyrosine residues (YFF mutant) did not affect the kinase activity or transforming ability of NPM/ALK. In contrast, mutation of the first and second (FFY), first and third (FYF), or all three (FFF) tyrosine residues impaired both kinase activity and transforming ability of NPM/ALK. Furthermore, a DFF mutant, in which the aspartic residue introduces a negative charge similar to a phosphorylated tyrosine, possessed catalytic activity similar to the YFF mutant. Together, our findings indicate that phosphorylation of the first tyrosine of the YXXXYY motif is necessary for the autoactivation of the ALK kinase domain and the transforming activity of NPM/ALK.

Unique substrate specificity of anaplastic lymphoma kinase (ALK): development of phosphoacceptor peptides for the assay of ALK activity.

The anaplastic lymphoma kinase (ALK), whose constitutively active fusion proteins are responsible for 5-10% of non-Hodgkin's lymphomas, shares with the other members of the insulin receptor kinase (IRK) subfamily an activation loop (A-loop) with the triple tyrosine motif Y-x-x-x-Y-Y. However, the amino acid sequence of the ALK A-loop differs significantly from the sequences of both the IRK A-loop and the consensus A-loop for this kinase subfamily. A major difference is the presence of a unique "RAS" triplet between the first and second tyrosines of the ALK A-loop, which in IRK is replaced by "ETD". Here we show that a peptide reproducing the A-loop of ALK is readily phosphorylated by ALK, while a homologous IRK A-loop peptide is not unless its "ETD" triplet is substituted by "RAS". Phosphorylation occurs almost exclusively at the first tyrosine of the Y-x-x-x-Y-Y motif, as judged by Edman analysis of the phosphoradiolabeled product. Consequently, a peptide in which the first tyrosine had been replaced by phenylalanine (FYY) was almost unaffected by ALK. In contrast, a peptide in which the second and third tyrosines had been replaced by phenylalanine (YFF) was phosphorylated more rapidly than the parent peptide (YYY). A number of substitutions in the YFF peptide outlined the importance of Ile and Arg at positions n - 1 and n + 6 in addition to the central triplet, to ensure efficient phosphorylation by ALK. Such a peculiar substrate specificity allows the specific monitoring of ALK activity in crude extracts of NPM-ALK positive cells, using the YFF peptide, which is only marginally phosphorylated by a number of other tyrosine kinases.

ALK as a novel lymphoma-associated tumor antigen: identification of 2 HLA-A2.1-restricted CD8+ T-cell epitopes.

Oncogenic anaplastic lymphoma kinase (ALK) fusion proteins (NPM/ALK and associated variants) are expressed in about 60% of anaplastic large cell lymphomas (ALCLs) but are absent in normal tissues. In this study, we investigated whether ALK, which is expressed at high levels in lymphoma cells, could be a target for antigen-specific cell-mediated immunotherapy. A panel of ALK-derived peptides was tested for their binding affinity to HLA-A*0201 molecules. Binding peptides were assessed for their capacity to elicit a specific immune response mediated by cytotoxic T lymphocytes (CTLs) both in vivo, in HLA-A*0201 transgenic mice, and in vitro in the peripheral blood lymphocytes (PBLs) from healthy donors. Two HLA-A*0201-restricted CTL epitopes, p280-89 (SLAMLDLLHV) and p375-86 (GVLLWEIFSL), both located in the ALK kinase domain were identified. The p280-89- and p375-86-induced peptide-specific CTL lines were able to specifically release interferon-gamma (IFN-gamma) on stimulation with ALK peptide-pulsed autologous Epstein-Barr virus-transformed B cells (LCLs) or T2 cells. Anti-ALK CTLs lysed HLA-matched ALCL and neuroblastoma cell lines endogenously expressing ALK proteins. CTL activity was inhibited by anti-HLA-A2 monoclonal antibody CR11.351, consistent with a class I-restricted mechanism of cytotoxicity. These results show the existence of functional anti-ALK CTL precursors within the peripheral T-cell repertoire of healthy donors, clearly indicating ALK as a tumor antigen and ALK-derived peptides, p280-89 and p375-86, as suitable epitopes for the development of vaccination strategies.