Proapoptotic peptide, (KLAKLAK)2, exhibits strong anti-tumor effect with the help of cell-penetrating peptides such as Pep2, targeting TLR2 with high expression in acute myeloid leukemia (AML). However, the applications are limited due to the peptide's instability and high cost of synthesis. Recombinant PP7 bacteriophage-like particles (VLPs) can protect the peptides from degradation by proteases, based on their ability to display foreign peptides. Here, we evaluated the feasibility of PP7 VLPs carrying Pep2 and (KLAKLAK)2 (2PP7-Pep2-KLAK VLPs) expressed in E. coli. We further investigated the characteristics including size, toxicity, thermal stability, penetrating ability, anti-tumor activity, and potential anti-tumor mechanism of 2PP7-Pep2-KLAK VLPs. 2PP7-Pep2-KLAK VLPs was expressed in E. coli BL21(DE3) successfully with high yield and thermal stability. They penetrated the AML cells THP-1 rapidly after 30min of incubation. Moreover, 2PP7-Pep2-KLAK VLPs were non-replicative, non-infectious, and non-toxic against normal cells, but inhibited the proliferation of THP-1 cells by inducing cell apoptosis after 24h of exposure. This effect extends through 120h of exposure, indicating their anti-proliferation effect was superior to that of synthetic peptides. In addition to the mitochondrial apoptotic pathway, the anti-tumor activity of 2PP7-Pep2-KLAK VLPs was also correlated with down-regulation of expression of enhancer of zeste homolog 2 (EZH2) and trimethylation of histone H3K27. We identified the feasibility to prepare the stable, active Pep2-KLAK peptide by using PP7 bacteriophage as the vehicle. We revealed this peptide was an inhibitor of EZH2. 2PP7-Pep2-KLAK VLPs may have significant clinical implications in the treatment of MLL-AF9 AML as an epigenetic modulator.

Metastasis is a fundamentally physical process in which cells deform through narrow gaps and generate forces to invade surrounding tissues. While it is commonly thought that increased cell deformability is an advantage for invading cells, we previously found that more invasive pancreatic ductal adenocarcinoma (PDAC) cells are stiffer than less invasive PDAC cells. Here we investigate potential mechanisms of the simultaneous increase in PDAC cell stiffness and invasion, focusing on the contributions of myosin II, Arp2/3, and formins. We measure cell invasion using a 3D scratch wound invasion assay and cell stiffness using atomic force microscopy (AFM). To determine the effects of actin- and myosin-mediated force generation on cell stiffness and invasion, we treat cells with pharmacologic inhibitors of myosin II (blebbistatin), Arp2/3 (CK-666), and formins (SMIFH2). We find that the activity of myosin II, Arp2/3, and formins all contribute to the stiffness of PDAC cells. Interestingly, we find that the invasion of PDAC cell lines is differentially affected when the activity of myosin II, Arp2/3, or formins is inhibited, suggesting that despite having similar tissue origins, different PDAC cell lines may rely on different mechanisms for invasion. These findings deepen our knowledge of the factors that regulate cancer cell mechanotype and invasion, and incite further studies to develop therapeutics that target multiple mechanisms of invasion for improved clinical benefit.

Ultrasound-targeted microbubble destruction (UTMD) has been shown a promising approach for target-specific gene delivery and treatment of many diseases in the past decade. To improve the therapeutic potential of UTMD, the gene carrier of microbubbles should possess adequate DNA condensation capability and (or) specific cell or tissue selectivity. The tissue-targeted and ultrasound-targeted cationic microbubbles were developed to meet gene therapy. A tissue-targeted stearic acid-inserted cationic microbubbles (SCMBs) were prepared for ultrasound-targeted gene delivery. Branched PEI was modified with stearic acid and further mixed with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and biot-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (ammonium salt) (Biot-DSPE-PEG2000), intercellular adhesion molecule-1 (ICAM-1) antibody and plasmid DNA to prepare cationic microbubbles through ultrasonic hydration. The ICAM-1 antibody and plasmid DNA were expected to assemble to the surface of SCMBs via biotin-avidin interaction and electrostatic interaction, respectively. It was found that the SCMBs had higher zeta potential compared with neutral microbubbles (NMBs) and cationic microbubbles (CMBs). In contrast, DNA incorporated SCMBs4 showed negative potential, exhibiting good DNA-binding capacity. Confocal images showed that the HeLa cells were attached around by the SCMBs4 from the view of green fluorescence of fluorescein isothiocyanate-loaded IgG which conjugated to ICAM-1 antibody on their surface. After ultrasound treatment, HeLa cells treated with SCMBs exhibited slightly stronger red fluorescence under confocal laser scanning microscope, indicating a synergistic promotion for transfection efficiency. This tissue- and ultrasound-targeted cationic microbubble demonstrated here showed a promising strategy for improving gene therapy in the future.

In response to external stress, cells alter their morphology, metabolic activity, and functions to mechanically adapt to the dynamic, local environment through cell allostasis. To explore mechanotransduction in cellular allostasis, we applied an integrated micromechanical system that combines an 'ultrasound tweezers'-based mechanical stressor and a Förster resonance energy transfer (FRET)-based molecular force biosensor, termed "actinin-sstFRET," to monitor in situ single-cell allostasis in response to transient stimulation in real time. The ultrasound tweezers utilize 1Hz, 10-s transient ultrasound pulses to acoustically excite a lipid-encapsulated microbubble, which is bound to the cell membrane, and apply a pico- to nano-Newton range of forces to cells through an RGD-integrin linkage. The actinin-sstFRET molecular sensor, which engages the actin stress fibers in live cells, is used to map real-time actomyosin force dynamics over time. Then, the mechanosensitive behaviors were examined by profiling the dynamics in Ca2+ influx, actomyosin cytoskeleton (CSK) activity, and GTPase RhoA signaling to define a single-cell mechanical allostasis. By subjecting a 1Hz, 10-s physical stress, single vascular smooth muscle cells (VSMCs) were observed to remodeled themselves in a biphasic mechanical allostatic manner within 30min that caused them to adjust their contractility and actomyosin activities. The cellular machinery that underscores the vital role of CSK equilibrium in cellular mechanical allostasis, includes Ca2+ influx, remodeling of actomyosin CSK and contraction, and GTPase RhoA signaling. Mechanical allostasis was observed to be compromised in VSMCs from patients with type II diabetes mellitus (T2DM), which could potentiate an allostatic maladaptation. By integrating tools that simultaneously permit localized mechanical perturbation and map actomyosin forces, we revealed distinct cellular mechanical allostasis profiles in our micromechanical system. Our findings of cell mechanical allostasis and maladaptation provide the potential for mechanophenotyping cells to reveal their pathogenic contexts and their biophysical mediators that underlie multi-etiological diseases such as diabetes, hypertension, or aging.

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United States, with high associated costs. Most of the cost burden results from acute exacerbations of COPD (AE-COPD), events associated with heightened symptoms and mortality. Cellular mechanisms underlying AE-COPD are poorly understood, likely because they arise from dysregulation of complex immune networks across multiple tissue compartments. To gain systems-level insight into cellular environments relevant to exacerbation, we applied data-driven modeling approaches to measurements of immune factors (cytokines and flow cytometry) measured previously in two different human tissue environments (sputum and peripheral blood) during the stable and exacerbated state. Using partial least squares discriminant analysis, we identified a unique signature of cytokines in serum that differentiated stable and AE-COPD better than individual measurements. Furthermore, we found that models integrating data across tissue compartments (serum and sputum) trended towards being more accurate. The resulting paracrine signature defining AE-COPD events combined elevations of proteins associated with cell adhesion (sVCAM-1, sICAM-1) and increased levels of neutrophils and dendritic cells in blood with elevated chemoattractants (IP-10 and MCP-2) in sputum. Our results supported a new hypothesis that AE-COPD is driven by immune cell trafficking into the lung, which requires expression of cell adhesion molecules and raised levels of innate immune cells in blood, with parallel upregulated expression of specific chemokines in pulmonary tissue. Overall, this work serves as a proof-of-concept for using data-driven modeling approaches to generate new insights into cellular processes involved in complex pulmonary diseases.

Mesenchymal stem cell (MSC) chondrogenesis is associated with increases in intracellular reactive oxygen species (ROS), which may result in oxidative stress that is detrimental to cartilage regeneration. This study evaluated the ability of the antioxidants N-acetylcysteine (NAC) or pyrrolidine dithiocarbamate (PDTC) to reduce intracellular ROS, and their effect on MSC chondrogenesis and maturation of cartilage-like extracellular matrix. Equine bone marrow MSCs were cultured in serum-supplemented chondrogenic medium with or without NAC or PDTC. ROS was quantified in monolayer after 8 and 72h of culture. MSCs were seeded into agarose, cultured for 15days, and analyzed for viable cell density, glycosaminoglycan (GAG) and hydroxyproline accumulation, and collagen gene expression. PDTC cultures were evaluated for oxidative damage by protein carbonylation, and mechanical properties via compressive testing. NAC significantly lowered levels of ROS after 8 but not 72h, and suppressed GAG accumulation (70%). In secondary experiments using serum-free medium, NAC significantly increased levels of ROS at 72h, and lowered cell viability and extracellular matrix accumulation. PDTC significantly reduced levels of ROS (~ 30%) and protein carbonylation (27%), and enhanced GAG accumulation (20%). However, the compressive modulus for PDTC-treated samples was significantly lower (40%) than controls. Gene expression was largely unaffected by the antioxidants. NAC demonstrated a limited ability to reduce intracellular ROS in chondrogenic culture, and generally suppressed accumulation of extracellular matrix. Conversely, PDTC was an effective antioxidant that enhanced GAG accumulation, although the concomitant reduction in compressive properties is a significant limitation for cartilage repair.

Landomycins are a subgroup of angucycline antibiotics that are produced by Streptomyces bacteria and possess strong antineoplastic potential. Literature data suggest that enhancement of the therapeutic activity of this drug may be achieved by means of creating specific drug delivery systems. Here we propose to adopt C60 fullerene as flexible and stable nanocarrier for landomycin delivery into tumor cells. The methods of molecular modelling, dynamic light scattering and Fourier transform infrared spectroscopy were used to study the assembly of C60 fullerene and the anticancer drug Landomycin A (LA) in aqueous solution. Cytotoxic activity of this nanocomplex was studied in vitro towards two cancer cell lines in comparison to human mesenchymal stem cells (hMSCs) using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test and a live/dead assay. The morphology of the cells incubated with fullerene-drug nanoparticles and their uptake into target cells were studied by scanning electron microscopy and fluorescence light microscopy. The viability of primary cells (hMSCs, as a model for healthy cells) and cancer cell lines (human osteosarcoma cells, MG-63, and mouse mammary cells, 4T1, as models for cancer cells) was studied after incubation with water-soluble C60 fullerenes, LA and the mixture C60 + LA. The C60 + LA nanocomplex in contrast to LA alone showed higher toxicity towards cancer cells and lower toxicity towards normal cells, whereas the water-soluble C60 fullerenes at the same concentration were not toxic for the cells. The obtained physico-chemical data indicate a complexation between the two compounds, leading to the formation of a C60 + LA nanocomposite. It was concluded that immobilization of LA on C60 fullerene enhances selectivity of action of this anticancer drug in vitro, indicating on possibility of further preclinical studies of novel C60 + LA nanocomposites on animal tumor models.

Cancer associated fibroblasts (CAFs) are known to participate in anti-cancer drug resistance by upregulating desmoplasia and pro-survival mechanisms withinthe tumor microenvironment. In this regard, anti-fibrotic drugs (i.e., tranilast) have been repurposed to diminishthe elastic modulus of the stromal matrix and reduce tumor growth in presence of chemotherapeutics (i.e., doxorubicin). However, the quantitative assessment on impact of these stromal targeting drugs on matrix stiffness and tumor progression is still missing in the sole presence of CAFs. We developed a high-density 3D microengineered tumor model comprised of MDA-MB-231 (highly invasive breast cancer cells) embedded microwells, surrounded by CAFs encapsulated within collagen I hydrogel. To study the influence of tranilast and doxorubicin on fibrosis, we probed the matrix using atomic force microscopy(AFM) and assessed matrix protein deposition. We further studied the combinatorial influence of the drugs on cancer cell proliferation and invasion. Our results demonstrated that the combinatorial action of tranilast and doxorubicin significantly diminished the stiffness of the stromal matrix compared tothe control. The two drugs in synergy disrupted fibronectin assembly and reduced collagen fiber density. Furthermore, the combination of these drugs, condensed tumor growth and invasion. In this work, we utilized a 3D microengineered model to tease apart the role of tranilast and doxorubicin in the sole presence of CAFs on desmoplasia, tumor growth and invasion. Our study lay down a ground work on better understandingof the role of biomechanical properties of the matrix on anti-cancer drug efficacy in the presence of single class of stromal cells.

IntroductionPhysical and mechanical properties of ceramic-based scaffolds can be modulated by introducing hydrogel coatings on their surface. For instance, hydrogels can be used as elastic layers to overcome the brittleness of synthetic ceramic materials or to control the delivery of essential osteogenic factors. In this work, we aimed to achieve both goals by fabricating a novel cytocompatible hydrogel made of gelatin-alginate as a coating for beta-tricalcium phosphate (β-TCP) scaffolds.MethodsThe hydrogel synthesis was optimized by varying the concentration of the crosslinkers N-hydroxysuccinimide and N-Ethyl-N′-(3-dimethyl aminopropyl) carbodiimide (NHS/EDC). Swelling, degradability and mechanical studies were carried out to identify the suitable hydrogel coating formulation for the β-TCP scaffolds. The cytocompatibility of the coated ceramic was assessed in vitro by testing the proliferation and the osteogenic differentiation of human adipose stem cell (hASCs) for two weeks.ResultsThe designed hydrogel layer could withstand cyclic compression and protected the brittle internal core of the ceramic. The hydrogel coating modulated the diffusion of the model protein BSA according to the degree of crosslinking of the hydrogel layer. Additionally, the polymeric network was able to retain positively charged proteins such as lysozyme due to the strong electrostatic interactions with carboxylic groups of alginate. A higher expression of alkaline phosphates activity was found on hASCs seeded on the coated scaffolds compared to the hydrogels without any β-TCP.ConclusionOverall, the hydrogel coating characterized in this study represents a valid strategy to overcome limitations of brittle ceramic-based materials used as scaffolds for bone tissue engineering applications.

IntroductionColocalization of endothelial nitric oxide synthase (eNOS) and capacitative Ca2+ entry (CCE) channels in microdomains such as cavaeolae in endothelial cells (ECs) has been shown to significantly affect intracellular Ca2+ dynamics and NO production, but the effect has not been well quantified.MethodsWe developed a two-dimensional continuum model of an EC integrating shear stress-mediated ATP production, intracellular Ca2+ mobilization, and eNOS activation to investigate the effects of spatial colocalization of plasma membrane eNOS and CCE channels on Ca2+ dynamics and NO production in response to flow-induced shear stress. Our model examines the hypothesis that subcellular colocalization of cellular components can be critical for optimal coupling of NO production to blood flow.ResultsOur simulations predict that heterogeneity of CCE can result in formation of microdomains with significantly higher Ca2+ compared to the average cytosolic Ca2+. Ca2+ buffers with lower or no mobility further enhanced Ca2+ gradients relative to mobile buffers. Colocalization of eNOS to CCE channels significantly increased NO production.ConclusionsOur results provide quantitative understanding for the role of spatial heterogeneity and the compartmentalization of signals in regulation of shear stress-induced NO production.