Poly(ethylene glycol) Methyl Ether Acrylate-Grafted Chitosan-Based Micro- and Nanoparticles as a Drug Delivery System for Antibiotics.

Nanotechnology is the science of creating materials at the nanoscale by using various devices, structures, and systems that are often inspired by nature. Micro- and nanoparticles (MPs, NPs) are examples of such materials that have unique properties and can be used as carriers for delivering drugs for different biomedical applications. Chitosan (CS) is a natural polysaccharide that has been widely studied, but it has a problem with low water solubility at neutral or basic pH, which limits its processability. The goal of this work was to use a chemically modified CS with poly(ethylene glycol) methyl ether acrylate (PEGA) to prepare CS micronic and submicronic particles (MPs/NPs) that can deliver different types of antibiotics, respectively, levofloxacin (LEV) and Ciprofloxacin (CIP). The particle preparation procedure employed a double crosslinking method, ionic followed by a covalent, in a water/oil emulsion. The studied process parameters were the precursor concentration, stirring speeds, and amount of ionic crosslinking agent. MPs/NPs were characterized by FT-IR, SEM, light scattering granulometry, and Zeta potential. MPs/NPs were also tested for their water uptake capacity in acidic and neutral pH conditions, and the results showed that they had a pH-dependent behavior. The MPs/NPs were then used to encapsulate two separate drugs, LEV and CIP, and they showed excellent drug loading and release capacity. The MPs/NPs were also found to be safe for cells and blood, which demonstrated their potential as suitable drug delivery systems for biomedical applications.

Magnetic Nanoparticle Coating Decreases the Senescence and Increases the Targeting Potential of Fibroblasts and Adipose-Derived Mesenchymal Stem Cells.

Magnetic nanoparticles (MNPs) are intensely scrutinized for applications in emerging biomedical fields. Their potential use for drug delivery, tracking, and targeting agents or for cell handling is tested for regenerative medicine and tissue engineering applications. The large majority of MNPs tested for biomedical use are coated with different lipids and natural or synthetic polymers in order to decrease their degradation process and to increase the ability to transport drugs or bioactive molecules. Our previous studies highlighted the fact that the as-prepared MNP-loaded cells can display increased resistance to culture-induced senescence as well as ability to target pathological tissues; however, this effect tends to be dependent on the cell type. Here, we assessed comparatively the effect of two types of commonly used lipid coatings, oleic acid (OA) and palmitic acid (PA), on normal human dermal fibroblasts and adipose-derived mesenchymal cells with culture-induced senescence and cell motility in vitro. OA and PA coatings improved MNPs stability and dispersibility. We found good viability for cells loaded with all types of MNPs; however, a significant increase was obtained with the as-prepared MNPs and OA-MNPs. The coating decreases iron uptake in both cell types. Fibroblasts (Fb) integrate MNPs at a slower rate compared to adipose-derived mesenchymal stem cells (ADSCs). The as-prepared MNPs induced a significant decrease in beta-galactosidase (B-Gal) activity with a nonsignificant one observed for OA-MNPs and PA-MNPs in ADSCs and Fb. The as-prepared MNPs significantly decrease senescence-associated B-Gal enzymatic activity in ADSCs but not in Fb. Remarkably, a significant increase in cell mobility could be detected in ADSCs loaded with OA-MNPscompared to controls. The OA-MNPs uptake significantly increases ADSCs mobility in a wound healing model in vitro compared to nonloaded counterparts, while these observations need to be validated in vivo. The present findings provide evidence that support applications of OA-MNPs in wound healing and cell therapy involving reparative processes as well as organ and tissue targeting.

Chitosan Grafted Poly (Ethylene Glycol) Methyl Ether Acrylate Particulate Hydrogels for Drug Delivery Applications.

Chitosan (CS) crosslinking has been thoroughly investigated, but the chemical reactions leading to submicronic hydrogel formulations pose problems due to various physical/chemical interactions that limit chitosan processability. The current study employs the chemical modification of chitosan by Michael addition of poly (ethylene glycol) methyl ether acrylate (PEGA) to the amine groups to further prepare chitosan particulate hydrogels (CPH). Thus, modified CS is subjected to a double crosslinking, ionic and covalent, in water/oil emulsion. The studied process parameters are polymer concentration, stirring speed, and quantity of ionic crosslinker. The CPH were structurally and morphologically characterized through infrared spectroscopy, scanning electron microscopy, light scattering granulometry, and zeta potential, showing that modified CS allows better control of dimensional properties and morphology as compared with neat CS. Swelling properties were studied in acidic and neutral pH conditions, showing that pH-dependent behavior was maintained after grafting and double crosslinking. The applicability of the prepared materials was further tested for drug loading and in vitro delivery of levofloxacin (LEV), showing excellent capacity. CPH were found to be cyto- and hemocompatible demonstrating their potential for effective use as a controlled release system for different biomedical applications.

Solid-Phase Synthesized Copolymers for the Assembly of pH-Sensitive Micelles Suitable for Drug Delivery Applications.

Diblock copolymers of polyhistidine are known for their self-assembly into micelles and their pH-dependent disassembly due to the amphiphilic character of the copolymer and the unsaturated imidazole groups that undergo a hydrophobic-to-hydrophilic transition in an acidic pH. This property has been largely utilized for the design of drug delivery systems that target a tumor environment possessing a slightly lower extracellular pH (6.8-7.2). The main purpose of this study was to investigate the possibility of designed poly(ethylene glycol)-polyhistidine sequences synthesized using solid-phase peptide synthesis (SPPS), to self-assemble into micelles, to assess the ability of the corresponding micelles to be loaded with doxorubicin (DOX), and to investigate the drug release profile at pH values similar to a malignant extracellular environment. The designed and assembled free and DOX-loaded micelles were characterized from a physico-chemical point of view, their cytotoxicity was evaluated on a human breast cancer cell line (MDA-MB-231), while the cellular areas where micelles disassembled and released DOX were assessed using immunofluorescence. We concluded that the utilization of SPPS for the synthesis of the polyhistidine diblock copolymers yielded sequences that behaved similarly to the copolymeric sequences synthesized using ring-opening polymerization, while the advantages of SPPS may offer facile tuning of the histidine site or the attachment of a large variety of functional molecules.

Targeted Cancer Therapy via pH-Functionalized Nanoparticles: A Scoping Review of Methods and Outcomes.

(1) Background: In recent years, several studies have described various and heterogenous methods to sensitize nanoparticles (NPs) to pH changes; therefore, in this current scoping review, we aimed to map current protocols for pH functionalization of NPs and analyze the outcomes of drug-loaded pH-functionalized NPs (pH-NPs) when delivered in vivo in tumoral tissue. (2) Methods: A systematic search of the PubMed database was performed for all published studies relating to in vivo models of anti-tumor drug delivery via pH-responsive NPs. Data on the type of NPs, the pH sensitization method, the in vivo model, the tumor cell line, the type and name of drug for targeted therapy, the type of in vivo imaging, and the method of delivery and outcomes were extracted in a separate database. (3) Results: One hundred and twenty eligible manuscripts were included. Interestingly, 45.8% of studies (n = 55) used polymers to construct nanoparticles, while others used other types, i.e., mesoporous silica (n = 15), metal (n = 8), lipids (n = 12), etc. The mean acidic pH value used in the current literature is 5.7. When exposed to in vitro acidic environment, without exception, pH-NPs released drugs inversely proportional to the pH value. pH-NPs showed an increase in tumor regression compared to controls, suggesting better targeted drug release. (4) Conclusions: pH-NPs were shown to improve drug delivery and enhance antitumoral effects in various experimental malignant cell lines.

Conifer Essential Oils Reversed Amyloid Beta1-42 Action by Modulating BDNF and ARC Expression in The Rat Hippocampus.

BACKGROUND: The conifer species Pinus halepensis (Pinaceae) and Tetraclinis articulata (Cupressaceae) are widely used in traditional medicine due to their beneficial health properties. OBJECTIVE: This study aimed to investigate the mechanisms by which P. halepensis and T. articulata essential oils (1% and 3%) could exhibit neuroprotective effects in an Alzheimer's disease (AD) rat model, induced by intracerebroventricular (i.c.v.) administration of amyloid beta1-42 (Aß1-42). METHODS: The essential oils were administered by inhalation to the AD rat model, once daily, for 21 days. DNA fragmentation was assessed through a Cell Death Detection ELISA kit. Brainderived neurotrophic factor (BDNF), activity-regulated cytoskeleton-associated protein (ARC), and interleukin-1ß (IL-1ß) gene expressions were determined by RT-qPCR analysis, while BDNF and ARC protein expressions were assessed using immunohistochemistry technique. RESULTS: Our data showed that both essential oils substantially attenuated memory impairments, with P. halepensis mainly stimulating ARC expression and T. articulata mostly enhancing BDNF expression. Also, the inhalation of essential oils reduced IL-1ß expression and induced positive effects against DNA fragmentation associated with Aß1-42-induced toxicity, further contributing to the cognitive improvement in the rats with the AD-like model Conclusion: Our findings provide further evidence that these essential oils and their chemical constituents could be natural agents of therapeutic interest against Aß1-42-induced neurotoxicity.

Manganese-Doped N-Hydroxyphthalimide-Derived Carbon Dots-Theranostics Applications in Experimental Breast Cancer Models.

BACKGROUND: Theranostics, a novel concept in medicine, is based on the use of an agent for simultaneous diagnosis and treatment. Nanomaterials provide promising novel approaches to theranostics. Carbon Dots have been shown to exhibit anti-tumoral properties in various cancer models. The aim of the present study is to develop gadolinium, Fe3+, and Mn2+-doped N-hydroxyphthalimide-derived Carbon Dots. The resulted doped Carbon Dots should preserve the anti-tumoral properties while gaining magnetic resonance imaging properties. METHODS: Normal and cancer cell lines have been treated with doped Carbon Dots, and the cell viability has been measured. The doped Carbon Dots that exhibited the most prominent anti-tumoral effect accompanied by the lowest toxicity have been further in vivo tested. Magnetic resonance imaging evaluates both in vitro and in vivo the possibility of using doped Carbon Dots as a contrast agent. RESULTS: According to the results obtained from both the in vitro and in vivo experimental models used in our study, Mn2+-doped Carbon Dots (Mn-CDs-NHF) exhibit anti-tumoral properties, do not significantly impair the cell viability of normal cells, and reduce lung metastasis and the volume of mammary primary tumors while allowing magnetic resonance imaging. CONCLUSIONS: Our findings prove that Mn-CDs-NHF can be used as theranostics agents in pre-clinical models.

Exploring Hyperoxia Effects in Cancer-From Perioperative Clinical Data to Potential Molecular Mechanisms.

Increased inspiratory oxygen concentration is constantly used during the perioperative period of cancer patients to prevent the potential development of hypoxemia and to provide an adequate oxygen transport to the organs, tissues and cells. Although the primary tumours are surgically removed, the effects of perioperative hyperoxia exposure on distal micro-metastases and on circulating cancer cells can potentially play a role in cancer progression or recurrence. In clinical trials, hyperoxia seems to increase the rate of postoperative complications and, by delaying postoperative recovery, it can alter the return to intended oncological treatment. The effects of supplemental oxygen on the long-term mortality of surgical cancer patients offer, at this point, conflicting results. In experimental studies, hyperoxia effects on cancer biology were explored following multiple pathways. In cancer cell cultures and animal models, hyperoxia increases the production of reactive oxygen species (ROS) and increases the oxidative stress. These can be followed by the induction of the expression of Brain-derived neurotrophic factor (BDNF) and other molecules involved in angiogenesis and by the promotion of various degrees of epithelial mesenchymal transition (EMT).

Sevoflurane Modulates AKT Isoforms in Triple Negative Breast Cancer Cells. An Experimental Study.

(1) Background: Triple negative breast cancer (TNBC) is a highly aggressive tumor, associated with high rates of early distant recurrence and short survival times, and treatment may require surgery, and thus anesthesia. The effects of anesthetic drugs on cancer progression are under scrutiny, but published data are controversial, and the involved mechanisms unclear. Anesthetic agents have been shown to modulate several molecular cascades, including PI3K/AKT/mTOR. AKT isoforms are frequently amplified in various malignant tumors and associated with malignant cell survival, proliferation and invasion. Their activation is often observed in human cancers and is associated with decreased survival rate. Certain anesthetics are known to affect hypoxia cell signaling mechanisms by upregulating hypoxia-inducible factors (HIFs). (2) Methods: MCF-10A and MDA-MB 231 cells were cultivated and CellTiter-Blue® Cell Viability assay, 2D and 3D matrigel assay, immunofluorescence assays and gene expressions assay were performed after exposure to different sevoflurane concentrations. (3) Results: Sevoflurane exposure of TNBC cells results in morphological and behavioral changes. Sevoflurane differently influences the AKT isoforms expression in a time-dependent manner, with an important early AKT3 upregulation. The most significant effects occur at 72 h after 2 mM sevoflurane treatment and consist in increased viability, proliferation and aggressiveness and increased vimentin and HIF expression. (4) Conclusions: Sevoflurane exposure during surgery may contribute to cancer recurrence via AKT3 induced epithelial-mesenchymal transition (EMT) and by all three AKT isoforms enhanced cancer cell survival and proliferation.

Enhancing Anti-Tumoral Potential of CD-NHF by Modulating PI3K/Akt Axis in U87 Ex Vivo Glioma Model.

BACKGROUND: In the latest years, there has been an increased interest in nanomaterials that may provide promising novel approaches to disease diagnostics and therapeutics. Our previous results demonstrated that Carbon-dots prepared from N-hydroxyphthalimide (CD-NHF) exhibited anti-tumoral activity on several cancer cell lines such as MDA-MB-231, A375, A549, and RPMI8226, while U87 glioma tumor cells were unaffected. Gliomas represent one of the most common types of human primary brain tumors and are responsible for the majority of deaths. In the present in vitro study, we expand our previous investigation on CD-NHF in the U87 cell line by adding different drug combinations. METHODS: Cell viability, migration, invasion, and immunofluorescent staining of key molecular pathways have been assessed after various treatments with CD-NHF and/or K252A and AKTVIII inhibitors in the U87 cell line. RESULTS: Association of an inhibitor strongly potentiates the anti-tumoral properties of CD-NHF identified by significant impairment of migration, invasion, and expression levels of phosphorylated Akt, p70S6Kinase, or by decreasing expression levels of Bcl-2, IL-6, STAT3, and Slug. CONCLUSIONS: Using simultaneously reduced doses of both CD-NHF and an inhibitor in order to reduce side effects, the viability and invasiveness of U87 glioma cells were significantly impaired.

AXL Is a Driver of Stemness in Normal Mammary Gland and Breast Cancer.

The receptor tyrosine kinase AXL is associated with epithelial plasticity in several solid tumors including breast cancer and AXL-targeting agents are currently in clinical trials. We hypothesized that AXL is a driver of stemness traits in cancer by co-option of a regulatory function normally reserved for stem cells. AXL-expressing cells in human mammary epithelial ducts co-expressed markers associated with multipotency, and AXL inhibition abolished colony formation and self-maintenance activities while promoting terminal differentiation in vitro. Axl-null mice did not exhibit a strong developmental phenotype, but enrichment of Axl + cells was required for mouse mammary gland reconstitution upon transplantation, and Axl-null mice had reduced incidence of Wnt1-driven mammary tumors. An AXL-dependent gene signature is a feature of transcriptomes in basal breast cancers and reduced patient survival irrespective of subtype. Our interpretation is that AXL regulates access to epithelial plasticity programs in MaSCs and, when co-opted, maintains acquired stemness in breast cancer cells.

NHF-derived carbon dots: prevalidation approach in breast cancer treatment.

Metastatic breast cancer dominates the female cancer-related mortality. Tumour-associated molecules represents a crucial for early disease detection and identification of novel therapeutic targets. Nanomaterial technologies provide promising novel approaches to disease diagnostics and therapeutics. In the present study we extend the investigations of antitumoral properties of Carbon Dots prepared from N-hydroxyphthalimide (CD-NHF) precursor. We evaluate the effect of CD-NHF on tumour cell migration and invasion in vitro and their impact on tumour progression using an in vivo model. Furthermore, we investigate the molecular mechanisms involved in CD-NHF antitumour effects. In vivo mammary tumours were induced in Balb/c female mice by injecting 4T1 cells into the mammary fat pad. Conditional treatment with CD-NHF significantly impair both migration and invasion of metastatic breast cancer cells. The presence of CD-NHF within the 3D cell cultures strongly inhibited the malignant phenotype of MDA-MB-231, 4T1 and MCF-7 cells in 3D culture, resulting in culture colonies lacking invasive projections and reduction of mammospheres formation. Importantly, breast tumour growth and metastasis dissemination was significantly reduced upon CD-NHF treatments in a syngeneic mouse model and is associated with down-regulation of Ki67 and HSP90 expression. CD-NHF nanostructures provide exciting perspective for improving treatment outcome in breast cancer.

Long-Term Deleterious Effects of Short-term Hyperoxia on Cancer Progression-Is Brain-Derived Neurotrophic Factor an Important Mediator? An Experimental Study.

Perioperative factors promoting cancer recurrence and metastasis are under scrutiny. While oxygen toxicity is documented in several acute circumstances, its implication in tumor evolution is poorly understood. We investigated hyperoxia long-term effects on cancer progression and some underlying mechanisms using both in vitro and in vivo models of triple negative breast cancer (TNBC). We hypothesized that high oxygen exposure, even of short duration, may have long-term effects on cancer growth. Considering that hyperoxic exposure results in reactive oxygen species (ROS) formation, increased oxidative stress and increased Brain-Derived Neurotrophic Factor (BDNF) expression, BDNF may mediate hyperoxia effects offering cancer cells a survival advantage by increased angiogenesis and epithelial mesenchymal transition (EMT). Human breast epithelial MCF10A, human MDA-MB-231 and murine 4T1 TNBC were investigated in 2D in vitro system. Cells were exposed to normoxia or hyperoxia (40%, 60%, 80% O2) for 6 h. We evaluated ROS levels, cell viability and the expression of BDNF, HIF-1α, VEGF-R2, Vimentin and E-Cadherin by immunofluorescence. The in vivo model consisted of 4T1 inoculation in Balb/c mice and tumor resection 2 weeks after and 6 h exposure to normoxia or hyperoxia (40%, 80% O2). We measured lung metastases and the same molecular markers, immediately and 4 weeks after surgery. The in vitro study showed that short-term hyperoxia exposure (80% O2) of TNBC cells increases ROS, increases BDNF expression and that promotes EMT and angiogenesis. The in vivo data indicates that perioperative hyperoxia enhances metastatic disease and this effect could be BDNF mediated.

Entrapment of N-Hydroxyphthalimide Carbon Dots in Different Topical Gel Formulations: New Composites with Anticancer Activity.

In the present study, the antitumoral potential of three gel formulations loaded with carbon dots prepared from N-hydroxyphthalimide (CD-NHF) was examined and the influence of the gels on two types of skin melanoma cell lines and two types of breast cancer cell lines in 2D (cultured cells in normal plastic plates) and 3D (Matrigel) models was investigated. Antitumoral gels based on sodium alginate (AS), carboxymethyl cellulose (CMC), and the carbomer Ultrez 10 (CARB) loaded with CD-NHF were developed according to an adapted method reported by Hellerbach. Viscoelastic properties of CD-NHF-loaded gels were analyzed by rheological analysis. Also, for both CD-NHF and CD-NHF-loaded gels, the fluorescence properties were analyzed. Cell proliferation, apoptosis, and mitochondrial activity were analyzed according to basic methods used to evaluate modulatory activities of putative anticancer agents, which include reference cancer cell line culture assays in both classic 2D and 3D cultures. Using the rheological measurements, the mechanical properties of gel formulations were analyzed; all samples presented gel-like rheological characteristics. The presence of CD-NHF within the gels induces a slight decrease of the dynamic moduli, indicating a flexible gel structure. The fluorescence investigations showed that for the gel-loaded CD-NHF, the most intense emission peak was located at 370 nm (upon excitation at 330 nm). 3D cell cultures displayed visibly larger structure of tumor cells with less active phenotype appearance. The in vitro results for tested CD-NHF-loaded gel formulations revealed that the new composites are able to affect the number, size, and cellular organization of spheroids and impact individual tumor cell ability to proliferate and aggregate in spheroids.

Association of intracellular lipid accumulation in subcutaneous adipocyte precursors and plasma adipokines in bariatric surgery candidates.

BACKGROUND: The adipocyte expansion is a critical process with implications in the pathogenesis of obesity associated metabolic syndrome. Impaired adipogenesis leads to dysfunctional, hypertrophic adipocytes, local inflammation and peripheric insulin resistance. METHODS: We assessed the relationship between the adipogenic differentiation capacity of the subcutaneous adipose derived stem cells (ASCs), evaluated by total lipid accumulation, and the metabolic and hormonal profile in a group of obese female patients proposed for bariatric surgery (N = 20) versus normal weight female controls (N = 7). RESULTS: The lipid accumulation (measured as optical density at 492 nm) of ASCs during their differentiation to adipocytes was significantly lower in ASCs isolated from obese patients as compared to ASCs isolated from normal weight patients (0.49 ± 0.1 vs. 0.71 ± 0.1, p < 0.001). Significant negative correlations between lipid accumulation in adipogenic differentiated ASCs and plasma concentrations of triglycerides (p < 0.01), insulin (p < 0.001), HOMA-IR (p < 0.01), adiponectin (p < 0.05) and leptin/adiponectin ratio (p < 0.05) were found in obese group. CONCLUSIONS: In severely obese female patients, the abnormal adipogenesis is related to insulin resistance and leptin/adiponectin ratio. The abnormal lipid accumulation in the mature adipocyte derived from obese ASCs could possible predict the further development of type 2 diabetes mellitus in severely obese patients and influence the selection of patients for bariatric surgery.

Microenvironment-Induced Non-sporadic Expression of the AXL and cKIT Receptors Are Related to Epithelial Plasticity and Drug Resistance.

The existence of rare cancer cells that sporadically acquire drug-tolerance through epigenetic mechanisms is proposed as one mechanism that drives cancer therapy failure. Here we provide evidence that specific microenvironments impose non-sporadic expression of proteins related to epithelial plasticity and drug resistance. Microarrays of robotically printed combinatorial microenvironments of known composition were used to make cell-based functional associations between microenvironments, which were design-inspired by normal and tumor-burdened breast tissues, and cell phenotypes. We hypothesized that specific combinations of microenvironment constituents non-sporadically impose the induction of the AXL and cKIT receptor tyrosine kinase proteins, which are known to be involved in epithelial plasticity and drug-tolerance, in an isogenic human mammary epithelial cell (HMEC) malignant progression series. Dimension reduction analysis reveals type I collagen as a dominant feature, inducing expression of both markers in pre-stasis finite lifespan HMECs, and transformed non-malignant and malignant immortal cell lines. Basement membrane-associated matrix proteins, laminin-111 and type IV collagen, suppress AXL and cKIT expression in pre-stasis and non-malignant cells. However, AXL and cKIT are not suppressed by laminin-111 in malignant cells. General linear models identified key factors, osteopontin, IL-8, and type VIα3 collagen, which significantly upregulated AXL and cKIT, as well as a plasticity-related gene expression program that is often observed in stem cells and in epithelial-to-mesenchymal-transition. These factors are co-located with AXL-expressing cells in situ in normal and breast cancer tissues, and associated with resistance to paclitaxel. A greater diversity of microenvironments induced AXL and cKIT expression consistent with plasticity and drug-tolerant phenotypes in tumorigenic cells compared to normal or immortal cells, suggesting a reduced perception of microenvironment specificity in malignant cells. Microenvironment-imposed reprogramming could explain why resistant cells are seemingly persistent and rapidly adaptable to multiple classes of drugs. These results support the notion that specific microenvironments drive drug-tolerant cellular phenotypes and suggest a novel interventional avenue for preventing acquired therapy resistance.

Flow cytometry-based functional selection of RNA interference triggers for efficient epi-allelic analysis of therapeutic targets.

BACKGROUND: The dose-response relationship is a fundamental pharmacological parameter necessary to determine therapeutic thresholds. Epi-allelic hypomorphic analysis using RNA interference (RNAi) can similarly correlate target gene dosage with cellular phenotypes. This however requires a set of RNAi triggers empirically determined to attenuate target gene expression to different levels. RESULTS: In order to improve our ability to incorporate epi-allelic analysis into target validation studies, we developed a novel flow cytometry-based functional screening approach (CellSelectRNAi) to achieve unbiased selection of shRNAs from high-coverage libraries that knockdown target gene expression to predetermined levels. Employing a Gaussian probability model we calculated that knockdown efficiency is inferred from shRNA sequence frequency profiles derived from sorted hypomorphic cell populations. We used this approach to generate a hypomorphic epi-allelic cell series of shRNAs to reveal a functional threshold for the tumor suppressor p53 in normal and transformed cells. CONCLUSION: The unbiased CellSelectRNAi flow cytometry-based functional screening approach readily provides an epi-allelic series of shRNAs for graded reduction of target gene expression and improved phenotypic validation.

Akt1 activity regulates vessel maturation in a tissue engineering model of angiogenesis.

Akt kinase is a central signal transduction node that integrates extracellular cues that regulate cell migratory, proliferative, and morphological functions during angiogenesis. However, how Akt activity is modulated and contributes to subsequent vessel maturation is unclear. In this study we investigated the role of Akt1 in vessel maturation using human dermal microvascular endothelial cells (HDMVECs) expressing constitutively active and hemiphosphorylated Akt1 epi-alleles with graded kinase activity. HDMVECs expressing Akt1 epi-alleles were analyzed in vivo in a tissue engineering setting using a model of angiogenesis comprising cell-seeded poly-L-lactic acid scaffolds implanted subcutaneously into NOD/SCID murine hosts. The resultant intraimplant microvasculature was quantified for vascular parameters, including vessel diameter, perfusion, vascular density, and pericyte coverage. We found that constitutive Akt1 kinase activity in implanted HDMVECs correlated with loss of neovasculature function. Further, we found that the presence of coimplanted vascular smooth muscle cells (vSMCs) in the implants failed to promote blood vessel growth and maturation in a graded, Akt1 kinase activity-dependent manner. These results indicate that constitutive Akt1 activity disrupts the normal blood vessel growth and maturation. Therefore, we suggest that a downregulation of Akt1 activity is necessary for vSMC-induced maturation of newly formed blood vessels to occur.

Efficient in vivo vascularization of tissue-engineering scaffolds.

The success of tissue engineering depends on the rapid and efficient formation of a functional blood vasculature. Adult blood vessels comprise endothelial cells and perivascular mural cells that assemble into patent tubules ensheathed by a basement membrane during angiogenesis. Using individual vessel components, we characterized intra-scaffold microvessel self-assembly efficiency in a physiological in vivo tissue engineering implant context. Primary human microvascular endothelial and vascular smooth muscle cells were seeded at different ratios in poly-L-lactic acid (PLLA) scaffolds enriched with basement membrane proteins (Matrigel) and implanted subcutaneously into immunocompromised mice. Temporal intra-scaffold microvessel formation, anastomosis and perfusion were monitored by immunohistochemical, flow cytometric and in vivo multiphoton fluorescence microscopy analysis. Vascularization in the tissue-engineering context was strongly enhanced in implants seeded with a complete complement of blood vessel components: human microvascular endothelial and vascular smooth muscle cells in vivo assembled a patent microvasculature within Matrigel-enriched PLLA scaffolds that anastomosed with the host circulation during the first week of implantation. Multiphoton fluorescence angiographic analysis of the intra-scaffold microcirculation showed a uniform, branched microvascular network. 3D image reconstruction analysis of human pulmonary artery smooth muscle cell (hPASMC) distribution within vascularized implants was non-random and displayed a preferential perivascular localization. Hence, efficient microvessel self-assembly, anastomosis and establishment of a functional microvasculture in the native hypoxic in vivo tissue engineering context is promoted by providing a complete set of vascular components.

Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival.

Metastasis underlies the majority of cancer-related deaths. Thus, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelial-to-mesenchymal transitions (EMTs) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in primary breast cancers independently predicts strongly reduced overall patient survival, and that matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by EMT in immortalized mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Epiallelic RNA interference analysis in metastatic breast cancer cells delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness and formation of tumors in foreign and tissue-engineered microenvironments in vivo. Importantly, in two different optical imaging-based experimental breast cancer models, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs and increased overall survival. These findings suggest that Axl represents a downstream effector of the tumor cell EMT that is required for breast cancer metastasis. Thus, the detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer.