Showing papers on "In vivo published in 2016"

In vivo gene editing in dystrophic mouse muscle and muscle stem cells

Abstract: Editing can help build stronger muscles Much of the controversy surrounding the gene-editing technology called CRISPR/Cas9 centers on the ethics of germline editing of human embryos to correct disease-causing mutations. For certain disorders such as muscular dystrophy, it may be possible to achieve therapeutic benefit by editing the faulty gene in somatic cells. In proof-of-concept studies, Long et al., Nelson et al., and Tabebordbar et al. used adeno-associated virus-9 to deliver the CRISPR/Cas9 gene-editing system to young mice with a mutation in the gene coding for dystrophin, a muscle protein deficient in patients with Duchenne muscular dystrophy. Gene editing partially restored dystrophin protein expression in skeletal and cardiac muscle and improved skeletal muscle function. Science, this issue p. 400, p. 403, p. 407 Gene editing via CRISPR-Cas9 restores dystrophin protein and improves muscle function in mouse models of muscular dystrophy. Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.

DS-8201a, A Novel HER2-Targeting ADC with a Novel DNA Topoisomerase I Inhibitor, Demonstrates a Promising Antitumor Efficacy with Differentiation from T-DM1

Abstract: Purpose: An anti-HER2 antibody–drug conjugate with a novel topoisomerase I inhibitor, DS-8201a, was generated as a new antitumor drug candidate, and its preclinical pharmacologic profile was assessed. Experimental Design:In vitro and in vivo pharmacologic activities of DS-8201a were evaluated and compared with T-DM1 in several HER2-positive cell lines and patient-derived xenograft (PDX) models. The mechanism of action for the efficacy was also evaluated. Pharmacokinetics in cynomolgus monkeys and the safety profiles in rats and cynomolgus monkeys were assessed. Results: DS-8201a exhibited a HER2 expression-dependent cell growth–inhibitory activity and induced tumor regression with a single dosing at more than 1 mg/kg in a HER2-positive gastric cancer NCI-N87 model. Binding activity to HER2 and ADCC activity of DS-8201a were comparable with unconjugated anti-HER2 antibody. DS-8201a also showed an inhibitory activity to Akt phosphorylation. DS-8201a induced phosphorylation of Chk1 and Histone H2A.X, the markers of DNA damage. Pharmacokinetics and safety profiles of DS-8201a were favorable and the highest non-severely toxic dose was 30 mg/kg in cynomolgus monkeys, supporting DS-8201a as being well tolerated in humans. DS-8201a was effective in a T-DM1–insensitive PDX model with high HER2 expression. DS-8201a, but not T-DM1, demonstrated antitumor efficacy against several breast cancer PDX models with low HER2 expression. Conclusions: DS-8201a exhibited a potent antitumor activity in a broad selection of HER2-positive models and favorable pharmacokinetics and safety profiles. The results demonstrate that DS-8201a will be a valuable therapy with a great potential to respond to T-DM1–insensitive HER2-positive cancers and low HER2–expressing cancers. Clin Cancer Res; 22(20); 5097–108. ©2016 AACR.

Real-Time Tracking and In Vivo Visualization of β-Galactosidase Activity in Colorectal Tumor with a Ratiometric Near-Infrared Fluorescent Probe.

Abstract: Development of "smart" noninvasive bioimaging probes for trapping specific enzyme activities is highly desirable for cancer therapy in vivo. Given that β-galactosidase (β-gal) is an important biomarker for cell senescence and primary ovarian cancers, we design an enzyme-activatable ratiometric near-infrared (NIR) probe (DCM-βgal) for the real-time fluorescent quantification and trapping of β-gal activity in vivo and in situ. DCM-βgal manifests significantly ratiometric and turn-on NIR fluorescent signals simultaneously in response to β-gal concentration, which makes it favorable for monitoring dynamic β-gal activity in vivo with self-calibration in fluorescent mode. We exemplify DCM-βgal for the ratiometric tracking of endogenously overexpressed β-gal distribution in living 293T cells via the lacZ gene transfection method and OVCAR-3 cells, and further realize real-time in vivo bioimaging of β-gal activity in colorectal tumor-bearing nude mice. Advantages of our system include light-up ratiometric NIR fluorescence with large Stokes shift, high photostability, and pH independency under the physiological range, allowing for the in vivo real-time evaluation of β-gal activity at the tumor site with high-resolution three-dimensional bioimaging for the first time. Our work provides a potential tool for in vivo real-time tracking enzyme activity in preclinical applications.

Mitochondrial Transfer via Tunneling Nanotubes is an Important Mechanism by Which Mesenchymal Stem Cells Enhance Macrophage Phagocytosis in the In Vitro and In Vivo Models of ARDS.

Abstract: Mesenchymal stromal cells (MSC) have been reported to improve bacterial clearance in preclinical models of Acute Respiratory Distress Syndrome (ARDS) and sepsis. The mechanism of this effect is not fully elucidated yet. The primary objective of this study was to investigate the hypothesis that the antimicrobial effect of MSC in vivo depends on their modulation of macrophage phagocytic activity which occurs through mitochondrial transfer. We established that selective depletion of alveolar macrophages (AM) with intranasal (IN) administration of liposomal clodronate resulted in complete abrogation of MSC antimicrobial effect in the in vivo model of Escherichia coli pneumonia. Furthermore, we showed that MSC administration was associated with enhanced AM phagocytosis in vivo. We showed that direct coculture of MSC with monocyte-derived macrophages enhanced their phagocytic capacity. By fluorescent imaging and flow cytometry we demonstrated extensive mitochondrial transfer from MSC to macrophages which occurred at least partially through tunneling nanotubes (TNT)-like structures. We also detected that lung macrophages readily acquire MSC mitochondria in vivo, and macrophages which are positive for MSC mitochondria display more pronounced phagocytic activity. Finally, partial inhibition of mitochondrial transfer through blockage of TNT formation by MSC resulted in failure to improve macrophage bioenergetics and complete abrogation of the MSC effect on macrophage phagocytosis in vitro and the antimicrobial effect of MSC in vivo. Collectively, this work for the first time demonstrates that mitochondrial transfer from MSC to innate immune cells leads to enhancement in phagocytic activity and reveals an important novel mechanism for the antimicrobial effect of MSC in ARDS. Stem Cells 2016;34:2210-2223.

Bystander killing effect of DS-8201a, a novel anti-human epidermal growth factor receptor 2 antibody–drug conjugate, in tumors with human epidermal growth factor receptor 2 heterogeneity

Abstract: Antibody-drug conjugates deliver anticancer agents selectively and efficiently to tumor tissue and have significant antitumor efficacy with a wide therapeutic window DS-8201a is a human epidermal growth factor receptor 2 (HER2)-targeting antibody-drug conjugate prepared using a novel linker-payload system with a potent topoisomerase I inhibitor, exatecan derivative (DX-8951 derivative, DXd) It was effective against trastuzumab emtansine (T-DM1)-insensitive patient-derived xenograft models with both high and low HER2 expression In this study, the bystander killing effect of DS-8201a was evaluated and compared with that of T-DM1 We confirmed that the payload of DS-8201a, DXd (1), was highly membrane-permeable whereas that of T-DM1, Lys-SMCC-DM1, had a low level of permeability Under a coculture condition of HER2-positive KPL-4 cells and negative MDA-MB-468 cells in vitro, DS-8201a killed both cells, whereas T-DM1 and an antibody-drug conjugate with a low permeable payload, anti-HER2-DXd (2), did not In vivo evaluation was carried out using mice inoculated with a mixture of HER2-positive NCI-N87 cells and HER2-negative MDA-MB-468-Luc cells by using an in vivo imaging system In vivo, DS-8201a reduced the luciferase signal of the mice, indicating suppression of the MDA-MB-468-Luc population; however, T-DM1 and anti-HER2-DXd (2) did not Furthermore, it was confirmed that DS-8201a was not effective against MDA-MB-468-Luc tumors inoculated at the opposite side of the NCI-N87 tumor, suggesting that the bystander killing effect of DS-8201a is observed only in cells neighboring HER2-positive cells, indicating low concern in terms of systemic toxicity These results indicated that DS-8201a has a potent bystander effect due to a highly membrane-permeable payload and is beneficial in treating tumors with HER2 heterogeneity that are unresponsive to T-DM1

Generation of stem cell-derived β-cells from patients with type 1 diabetes

Abstract: We recently reported the scalable in vitro production of functional stem cell-derived β-cells (SC-β cells). Here we extend this approach to generate the first SC-β cells from type 1 diabetic patients (T1D). β-cells are destroyed during T1D disease progression, making it difficult to extensively study them in the past. These T1D SC-β cells express β-cell markers, respond to glucose both in vitro and in vivo, prevent alloxan-induced diabetes in mice and respond to anti-diabetic drugs. Furthermore, we use an in vitro disease model to demonstrate the cells respond to different forms of β-cell stress. Using these assays, we find no major differences in T1D SC-β cells compared with SC-β cells derived from non-diabetic patients. These results show that T1D SC-β cells could potentially be used for the treatment of diabetes, drug screening and the study of β-cell biology.

Correlation of patterns of anchorage-independent growth with in vivo behavior of cells from a murine fibrosarcoma (metastatic potential/in vivo growth characteristics)

Abstract: The pattern of in vitro anchorage-independent growth of tumor cells from the murine UV-2237 fibrosarcoma correlated with their ability to produce experimental metastasis in vivo. When seeded into 0.3% Noble agar semisolid medium, cells of metastatic clones developed into larger tumor colonies at a faster rate than did cells of clones with low metastatic po- tential. Furthermore, when tumor cells were plated into 0.6% Noble agar, colony development by cells of low metastatic po- tential clones was almost completely restricted. Tumor cells from the heterogeneous parent UV-2237 fibrosarcoma were plated into dishes containing 0.6% agar semisolid medium. In separate experiments, 16 colonies were isolated 2 weeks there- after and were established as individual cell lines in monolayer cultures. All of these cell lines produced experimental metastases as determined by in vivo lung colony assay. The data suggest that anchorage-independent growth of UV-2237 tumor cells in 0.6% Noble agar semisolid medium is selective and permits the isolation of metastatic subpopulations.

Targeting CD73 in the tumor microenvironment with MEDI9447

Abstract: MEDI9447 is a human monoclonal antibody that is specific for the ectoenzyme CD73 and currently undergoing Phase I clinical trials. Here we show that MEDI9447 is a potent inhibitor of CD73 ectonucleotidase activity, with wide ranging immune regulatory consequences. MEDI9447 results in relief from adenosine monophosphate (AMP)-mediated lymphocyte suppression in vitro and inhibition of mouse syngeneic tumor growth in vivo. In contrast with other cancer immunotherapy agents such as checkpoint inhibitors or T-cell agonists, MEDI9447 drives changes in both myeloid and lymphoid infiltrating leukocyte populations within the tumor microenvironment of mouse models. Changes include significant alterations in a number of tumor micro-environmental subpopulations including increases in CD8(+) effector cells and activated macrophages. Furthermore, these changes correlate directly with responder and non-responder subpopulations within animal studies using syngeneic tumors. Combination data showing additive activity between MEDI9447 and anti-PD-1 antibodies using human cells in vitro and mouse tumor models further demonstrate the potential value of relieving adenosine-mediated immunosuppression. Based on these data, a Phase I study to test the safety, tolerability, and clinical activity of MEDI9447 in cancer patients was initiated (NCT02503774).

A mouse-human phase 1 co-clinical trial of a protease-activated fluorescent probe for imaging cancer

Abstract: Local recurrence is a common cause of treatment failure for patients with solid tumors. Intraoperative detection of microscopic residual cancer in the tumor bed could be used to decrease the risk of a positive surgical margin, reduce rates of reexcision, and tailor adjuvant therapy. We used a protease-activated fluorescent imaging probe, LUM015, to detect cancer in vivo in a mouse model of soft tissue sarcoma (STS) and ex vivo in a first-in-human phase 1 clinical trial. In mice, intravenous injection of LUM015 labeled tumor cells, and residual fluorescence within the tumor bed predicted local recurrence. In 15 patients with STS or breast cancer, intravenous injection of LUM015 before surgery was well tolerated. Imaging of resected human tissues showed that fluorescence from tumor was significantly higher than fluorescence from normal tissues. LUM015 biodistribution, pharmacokinetic profiles, and metabolism were similar in mouse and human subjects. Tissue concentrations of LUM015 and its metabolites, including fluorescently labeled lysine, demonstrated that LUM015 is selectively distributed to tumors where it is activated by proteases. Experiments in mice with a constitutively active PEGylated fluorescent imaging probe support a model where tumor-selective probe distribution is a determinant of increased fluorescence in cancer. These co-clinical studies suggest that the tumor specificity of protease-activated imaging probes, such as LUM015, is dependent on both biodistribution and enzyme activity. Our first-in-human data support future clinical trials of LUM015 and other protease-sensitive probes.

The Role of α1-Adrenoceptor Antagonists in the Treatment of Prostate and Other Cancers.

Abstract: This review evaluates the role of α-adrenoceptor antagonists as a potential treatment of prostate cancer (PCa). Cochrane, Google Scholar and Pubmed were accessed to retrieve sixty-two articles for analysis. In vitro studies demonstrate that doxazosin, prazosin and terazosin (quinazoline α-antagonists) induce apoptosis, decrease cell growth, and proliferation in PC-3, LNCaP and DU-145 cell lines. Similarly, the piperazine based naftopidil induced cell cycle arrest and death in LNCaP-E9 cell lines. In contrast, sulphonamide based tamsulosin did not exhibit these effects. In vivo data was consistent with in vitro findings as the quinazoline based α-antagonists prevented angiogenesis and decreased tumour mass in mice models of PCa. Mechanistically the cytotoxic and antitumor effects of the α-antagonists appear largely independent of α 1-blockade. The proposed targets include: VEGF, EGFR, HER2/Neu, caspase 8/3, topoisomerase 1 and other mitochondrial apoptotic inducing factors. These cytotoxic effects could not be evaluated in human studies as prospective trial data is lacking. However, retrospective studies show a decreased incidence of PCa in males exposed to α-antagonists. As human data evaluating the use of α-antagonists as treatments are lacking; well designed, prospective clinical trials are needed to conclusively demonstrate the anticancer properties of quinazoline based α-antagonists in PCa and other cancers.

Aurora kinase inhibitor nanoparticles target tumors with favorable therapeutic index in vivo

Abstract: Efforts to apply nanotechnology in cancer have focused almost exclusively on the delivery of cytotoxic drugs to improve therapeutic index. There has been little consideration of molecularly targeted agents, in particular kinase inhibitors, which can also present considerable therapeutic index limitations. We describe the development of Accurin polymeric nanoparticles that encapsulate the clinical candidate AZD2811, an Aurora B kinase inhibitor, using an ion pairing approach. Accurins increase biodistribution to tumor sites and provide extended release of encapsulated drug payloads. AZD2811 nanoparticles containing pharmaceutically acceptable organic acids as ion pairing agents displayed continuous drug release for more than 1 week in vitro and a corresponding extended pharmacodynamic reduction of tumor phosphorylated histone H3 levels in vivo for up to 96 hours after a single administration. A specific AZD2811 nanoparticle formulation profile showed accumulation and retention in tumors with minimal impact on bone marrow pathology, and resulted in lower toxicity and increased efficacy in multiple tumor models at half the dose intensity of AZD1152, a water-soluble prodrug of AZD2811. These studies demonstrate that AZD2811 can be formulated in nanoparticles using ion pairing agents to give improved efficacy and tolerability in preclinical models with less frequent dosing. Accurins specifically, and nanotechnology in general, can increase the therapeutic index of molecularly targeted agents, including kinase inhibitors targeting cell cycle and oncogenic signal transduction pathways, which have to date proved toxic in humans.

Comparison of the Superagonist Complex, ALT-803, to IL15 as Cancer Immunotherapeutics in Animal Models

Abstract: IL15, a potent stimulant of CD8(+) T cells and natural killer (NK) cells, is a promising cancer immunotherapeutic. ALT-803 is a complex of an IL15 superagonist mutant and a dimeric IL15 receptor αSu/Fc fusion protein that was found to exhibit enhanced biologic activity in vivo, with a substantially longer serum half-life than recombinant IL15. A single intravenous dose of ALT-803, but not IL15, eliminated well-established tumors and prolonged survival of mice bearing multiple myeloma. In this study, we extended these findings to demonstrate the superior antitumor activity of ALT-803 over IL15 in mice bearing subcutaneous B16F10 melanoma tumors and CT26 colon carcinoma metastases. Tissue biodistribution studies in mice also showed much greater retention of ALT-803 in the lymphoid organs compared with IL15, consistent with its highly potent immunostimulatory and antitumor activities in vivo. Weekly dosing with 1 mg/kg ALT-803 in C57BL/6 mice was well tolerated, yet capable of increasing peripheral blood lymphocyte, neutrophil, and monocyte counts by >8-fold. ALT-803 dose-dependent stimulation of immune cell infiltration into the lymphoid organs was also observed. Similarly, cynomolgus monkeys treated weekly with ALT-803 showed dose-dependent increases of peripheral blood lymphocyte counts, including NK, CD4(+), and CD8(+) memory T-cell subsets. In vitro studies demonstrated ALT-803-mediated stimulation of mouse and human immune cell proliferation and IFNγ production without inducing a broad-based release of other proinflammatory cytokines (i.e., cytokine storm). Based on these results, a weekly dosing regimen of ALT-803 has been implemented in multiple clinical studies to evaluate the dose required for effective immune cell stimulation in humans.

Polydopamine-Based Surface Modification of Novel Nanoparticle-Aptamer Bioconjugates for In Vivo Breast Cancer Targeting and Enhanced Therapeutic Effects.

Abstract: In this study, we reported a simple polydopamine (pD)-based surface modification method to prepare novel nanoparticle-aptamer bioconjugates (Apt-pD-DTX/NPs) for in vivo tumor targeting and enhanced therapeutic effects of breast cancer. With simple preparation procedures, the new functionalized Apt-pD-DTX/NPs could maximumly increase the local effective drug concentration on tumor sites, achieving enhanced treatment effectiveness and minimizing side effects. The dopamine polymerization and aptamer conjugation barely changed the characters of NPs. Both in vitro cell experiments (i.e. endocytosis of fluorescent NPs, in vitro cellular targeting and cytotoxicity assays) and in vivo animal studies (i.e. in vivo imaging, biodistribution and antitumor effects of NPs) demonstrated that the Apt-pD-DTX/NPs could achieve significantly high targeting efficiency and enhanced therapeutic effects compared with clinical Taxotere(®) and NPs without functional modification. Above all, the Apt-pD-DTX/NPs showed great potential as a promising nanoformulation for in vivo breast cancer therapy and the construction of pD-modified NP-aptamer bioconjugates could be of great value in medical use.

Human mesenchymal stromal cells reduce influenza A H5N1-associated acute lung injury in vitro and in vivo.

Abstract: Influenza can cause acute lung injury. Because immune responses often play a role, antivirals may not ensure a successful outcome. To identify pathogenic mechanisms and potential adjunctive therapeutic options, we compared the extent to which avian influenza A/H5N1 virus and seasonal influenza A/H1N1 virus impair alveolar fluid clearance and protein permeability in an in vitro model of acute lung injury, defined the role of virus-induced soluble mediators in these injury effects, and demonstrated that the effects are prevented or reduced by bone marrow-derived multipotent mesenchymal stromal cells. We verified the in vivo relevance of these findings in mice experimentally infected with influenza A/H5N1. We found that, in vitro, the alveolar epithelium's protein permeability and fluid clearance were dysregulated by soluble immune mediators released upon infection with avian (A/Hong Kong/483/97, H5N1) but not seasonal (A/Hong Kong/54/98, H1N1) influenza virus. The reduced alveolar fluid transport associated with down-regulation of sodium and chloride transporters was prevented or reduced by coculture with mesenchymal stromal cells. In vivo, treatment of aged H5N1-infected mice with mesenchymal stromal cells increased their likelihood of survival. We conclude that mesenchymal stromal cells significantly reduce the impairment of alveolar fluid clearance induced by A/H5N1 infection in vitro and prevent or reduce A/H5N1-associated acute lung injury in vivo. This potential adjunctive therapy for severe influenza-induced lung disease warrants rapid clinical investigation.

Probe for the measurement of cell surface pH in vivo and ex vivo

Abstract: We have developed a way to measure cell surface pH by positioning a pH-sensitive fluorescent dye, seminaphtharhodafluor (SNARF), conjugated to the pH low insertion peptide (pHLIP). It has been observed that many diseased tissues are acidic and that tumors are especially so. A combination of effects acidifies tumor cell interiors, and cells pump out lactic acid and protons to maintain intracellular pH, acidifying the extracellular space. Overexpression of carbonic anhydrases on cell surfaces further contributes to acidification. Thus, the pH near tumor cell surfaces is expected to be low and to increase with distance from the membrane, so bulk pH measurements will not report surface acidity. Our new surface pH-measurement tool was validated in cancer cells grown in spheroids, in mouse tumor models in vivo, and in excised tumors. We found that the surface pH is sensitive to cell glycolytic activity: the pH decreases in high glucose and increases if glucose is replaced with nonmetabolized deoxyglucose. For highly metastatic cancer cells, the pH measured at the surface was 6.7-6.8, when the surrounding external pH was 7.4. The approach is sensitive enough to detect 0.2-0.3 pH unit changes in vivo in tumors induced by i.p. injection of glucose. The pH at the surfaces of highly metastatic cells within tumors was found to be about 6.1-6.4, whereas in nonmetastatic tumors, it was 6.7-6.9, possibly creating a way to distinguish more aggressive from less aggressive tumors. Other biological roles of surface acidity may be found, now that targeted measurements are possible.

Bioinspired Alkenyl Amino Alcohol Ionizable Lipid Materials for Highly Potent In Vivo mRNA Delivery.

Abstract: Thousands of human diseases could be treated by selectively controlling the expression of specific proteins in vivo. A new series of alkenyl amino alcohol (AAA) ionizable lipid nanoparticles (LNPs) capable of delivering human mRNA with unprecedented levels of in vivo efficacy is demonstrated. This study highlights the importance of utilizing synthesis tools in tandem with biological inspiration to understand and improve nucleic acid delivery in vivo.

Nanoemulsion gel-based topical delivery of an antifungal drug: in vitro activity and in vivo evaluation

Abstract: Objective: In this study, attempt has been focused to prepare a nanoemulsion (NE) gel for topical delivery of amphotericin B (AmB) for enhanced as well as sustained skin permeation, in vitro antifungal activity and in vivo toxicity assessment.Materials and methods: A series of NE were prepared using sefsol-218 oil, Tween 80 and Transcutol-P by slow spontaneous titration method. Carbopol gel (0.5% w/w) was prepared containing 0.1% w/w AmB. Furthermore, NE gel (AmB-NE gel) was characterized for size, charge, pH, rheological behavior, drug release profile, skin permeability, hemolytic studies and ex vivo rat skin interaction with rat skin using differential scanning calorimeter. The drug permeability and skin irritation ability were examined with confocal laser scanning microscopy and Draize test, respectively. The in vitro antifungal activity was investigated against three fungal strains using the well agar diffusion method. Histopathological assessment was performed in rats to investigate their tox...

A bioengineered niche promotes in vivo engraftment and maturation of pluripotent stem cell derived human lung organoids

Abstract: Human pluripotent stem cell (hPSC) derived tissues often remain developmentally immature in vitro, and become more adult-like in their structure, cellular diversity and function following transplantation into immunocompromised mice. Previously we have demonstrated that hPSC-derived human lung organoids (HLOs) resembled human fetal lung tissue in vitro (Dye et al., 2015). Here we show that HLOs required a bioartificial microporous poly(lactide-co-glycolide) (PLG) scaffold niche for successful engraftment, long-term survival, and maturation of lung epithelium in vivo. Analysis of scaffold-grown transplanted tissue showed airway-like tissue with enhanced epithelial structure and organization compared to HLOs grown in vitro. By further comparing in vitro and in vivo grown HLOs with fetal and adult human lung tissue, we found that in vivo transplanted HLOs had improved cellular differentiation of secretory lineages that is reflective of differences between fetal and adult tissue, resulting in airway-like structures that were remarkably similar to the native adult human lung.

Combination Therapy using Co-encapsulated Resveratrol and Paclitaxel in Liposomes for Drug Resistance Reversal in Breast Cancer Cells in vivo

Abstract: Multidrug resistance (MDR) is a major impediment to cancer treatment. A promising strategy for treating MDR is the joint delivery of combined anticancer agents to tumor cells in a single nanocarrier. Here, for the first time, Resveratrol (Res) was co-encapsulated with paclitaxel (PTX) in a PEGylated liposome to construct a carrier-delivered form of combination therapy for drug-resistant tumors. The composite liposome had an average diameter of 50 nm with encapsulated efficiencies of above 50%. The studies demonstrated that the composite liposome could generate potent cytotoxicity against the drug-resistant MCF-7/Adr tumor cells in vitro and enhance the bioavailability and the tumor-retention of the drugs in vivo. Moreover, systemic therapy with the composite liposome effectively inhibited drug-resistant tumor in mice (p < 0.01), without any notable increase in the toxicity. These results suggested that the co-delivery of Res and a cytotoxic agent in a nanocarrier may potentially improve the treatment of drug-resistant tumors.

HIF-1α Is an Essential Mediator of IFN-γ–Dependent Immunity to Mycobacterium tuberculosis

Abstract: The cytokine IFN-γ coordinates macrophage activation and is essential for control of pathogens, including Mycobacterium tuberculosis . However, the mechanisms by which IFN-γ controls M. tuberculosis infection are only partially understood. In this study, we show that the transcription factor hypoxia-inducible factor-1α (HIF-1α) is an essential mediator of IFN-γ–dependent control of M. tuberculosis infection both in vitro and in vivo. M. tuberculosis infection of IFN-γ–activated macrophages results in a synergistic increase in HIF-1α protein levels. This increase in HIF-1α levels is functionally important, as macrophages lacking HIF-1α are defective for IFN-γ–dependent control of infection. RNA-sequencing demonstrates that HIF-1α regulates nearly one-half of all IFN-γ–inducible genes during infection of macrophages. In particular, HIF-1α regulates production of important immune effectors, including inflammatory cytokines and chemokines, eicosanoids, and NO. In addition, we find that during infection HIF-1α coordinates a metabolic shift to aerobic glycolysis in IFN-γ–activated macrophages. We find that this enhanced glycolytic flux is crucial for IFN-γ–dependent control of infection in macrophages. Furthermore, we identify a positive feedback loop between HIF-1α and aerobic glycolysis that amplifies macrophage activation. Finally, we demonstrate that HIF-1α is crucial for control of infection in vivo as mice lacking HIF-1α in the myeloid lineage are strikingly susceptible to infection and exhibit defective production of inflammatory cytokines and microbicidal effectors. In conclusion, we have identified HIF-1α as a novel regulator of IFN-γ–dependent immunity that coordinates an immunometabolic program essential for control of M. tuberculosis infection in vitro and in vivo.

HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Antiangiogenesis Is Produced by Nontoxic Doses of Vinblastine

Abstract: The effects of vinblastine (VBL) on endothelial cell functions involved in angiogenesis, namely proliferation, chemotaxis, spreading on fibronectin (FN), secretion of matrix-metalloproteinase-2 (MMP-2) and MMP-9, and morphogenesis on Matrigel were tested in vitro, whereas its effects on angiogenesis were studied in vivo by using the chick embryo chorioallantoic membrane (CAM) model. In vitro, at noncytotoxic doses (0.1, 0.25, 0.5, 0.75, and 1 pmol/L), VBL impacted all these functions, except secretion of MMPs, in a dose-dependent fashion. By contrast, proliferation of other primary cells such as fibroblasts and lymphoid tumor cells was not impacted. In vivo, VBL at 0.5, 0.75, and 1 pmol/L again displayed a dose-dependent antiangiogenic activity. Lack of cytotoxicity in vitro and in vivo was shown both morphologically, and also because the antiangiogenic effects were rapidly abolished when VBL was removed. Apoptosis was not induced. At the ultrastructural level, impairment of cell functions in vitro was associated with thin disturbance of the cytoskeleton, in the form of slight depolymerization and accumulation of microfilaments, which was equally reversible. Results suggest that VBL has an antiangiogenic component at very low, noncytotoxic doses, and that antiangiogenesis by VBL could be used to treat a wide spectrum of angiogenesisdependent diseases, including certain chronic inflammatory diseases, Kaposi’s sarcoma, and cancer. r 1999 by The American Society of Hematology.

A Glutathione (GSH)-Responsive Near-Infrared (NIR) Theranostic Prodrug for Cancer Therapy and Imaging

Abstract: To reduce the side effects of chemotherapy, nontoxic prodrugs activated by the tumor microenvironment are urgently required for use in cancer treatment. In this work, we developed prodrug 4 for tumor-targeting treatment and imaging of the anticancer drug release in vivo. Taking advantage of the high glutathione (GSH) concentration in cancer cells, the disulfide bond in prodrug 4 was cleaved, resulting in the release of an active anticancer drug and a near-infrared (NIR) fluorescence dye turn-on. Furthermore, contrast to the free anticancer drug, the prodrug exhibited higher cytotoxicity to hepatoma cells than that to normal HL-7702 cells. Thus, prodrug 4 is a promising platform for specific tumor-activatable drug delivery system, because of its favorable features of in situ and in vivo monitoring of the drug release and therapeutic efficacy.

In Vitro and in Vivo Mechanism of Bone Tumor Inhibition by Selenium-Doped Bone Mineral Nanoparticles.

Abstract: Biocompatible tissue-borne crystalline nanoparticles releasing anticancer therapeutic inorganic elements are intriguing therapeutics holding the promise for both tissue repair and cancer therapy. However, how the therapeutic inorganic elements released from the lattice of such nanoparticles induce tumor inhibition remains unclear. Here we use selenium-doped hydroxyapatite nanoparticles (Se-HANs), which could potentially fill the bone defect generated from bone tumor removal while killing residual tumor cells, as an example to study the mechanism by which selenium released from the lattice of Se-HANs induces apoptosis of bone cancer cells in vitro and inhibits the growth of bone tumors in vivo. We found that Se-HANs induced apoptosis of tumor cells by an inherent caspase-dependent apoptosis pathway synergistically orchestrated with the generation of reactive oxygen species. Such mechanism was further validated by in vivo animal evaluation in which Se-HANs tremendously induced tumor apoptosis to inhibit tum...