Cytokine-Based Log-Scale Expansion of Functional Murine Dendritic Cells

BACKGROUND: Limitations of the clinical efficacy of dendritic cell (DC)-based immunotherapy, as well as difficulties in their industrial production, are largely related to the limited number of autologous DCs from each patient. We here established a possible breakthrough, a simple and cytokine-based culture method to realize a log-scale order of functional murine DCs (>1,000-fold), which cells were used as a model before moving to human studies. METHODOLOGY/PRINCIPAL FINDINGS: Floating cultivation of lineage-negative hematopoietic progenitors from bone marrow in an optimized cytokine cocktail (FLT3-L, IL-3, IL-6, and SCF) led to a stable log-scale proliferation of these cells, and a subsequent differentiation study using IL-4/GM-CSF revealed that 3-weeks of expansion was optimal to produce CD11b+/CD11c+ DC-like cells. The expanded DCs had typical features of conventional myeloid DCs in vitro and in vivo, including identical efficacy as tumor vaccines. CONCLUSIONS/SIGNIFICANCE: The concept of DC expansion should make a significant contribution to the progress of DC-based immunotherapy

Nucleotide Sequence of the Proton ATPase Beta-Subunit Homologue of the Sea Urchin Hemicentrotus pulcherrimus

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Membrane-Type 5 Matrix Metalloproteinase Is Expressed in Differentiated Neurons and Regulates Axonal Growth 1

Expression of membrane-type (MT) 5 matrix metalloproteinase (MMP) in the mouse brain was examined. MT5-MMP was expressed in the cerebrum in embryos, but it declined after birth. In contrast, expression in the cerebellum started to increase postnatally and continued thereafter. The cells expressing MT5-MMP were postmitotic neurons that showed gelatinolytic activities. Specific expression of MT5-MMP was observed in the neurons but not in the glial cells when embryonal mouse carcinoma P19 cells were differentiated in vitro by retinoic acid treatment. Neurons isolated from dorsal root ganglia also expressed MT5-MMP, and it was localized at the edge of growth cone. Proteoglycans inhibit neurite extension and regulate synaptogenesis. The inhibitory effect of the proteoglycans on neurite extension of dorsal root ganglia neurons was effectively eliminated by recombinant MT5-MMP. Thus, MT5-MMP expressed in neurons may play a role in axonal growth that contributes to the regulation of neural network formation

In Vitro Blood-Brain Barrier Permeability and Cytotoxicity of an Atorvastatin-Loaded Nanoformulation Against Glioblastoma in 2D and 3D Models

Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of the family of statins have been suggested as therapeutic options in various tumors. Atorvastatin is a statin with the potential to cross the blood-brain barrier; however, the concentrations necessary for a cytotoxic effect against cancer cells exceed the concentrations achievable via oral administration, which made the development of a novel atorvastatin formulation necessary. We characterized the drug loading and basic physicochemical characteristics of micellar atorvastatin formulations and tested their cytotoxicity against a panel of different glioblastoma cell lines. In addition, activity against tumor spheroids formed from mouse glioma and mouse cancer stem cells, respectively, was evaluated. Our results show good activity of atorvastatin against all tested cell lines. Interestingly, in the three-dimensional (3D) models, growth inhibition was more pronounced for the micellar formulation compared to free atorvastatin. Finally, atorvastatin penetration across a blood-brain barrier model obtained from human induced-pluripotent stem cells was evaluated. Our results suggest that the presented micelles may enable much higher serum concentrations than possible by oral administration; however, if transport across the blood-brain barrier is sufficient to reach the therapeutic atorvastatin concentration for the treatment of glioblastoma via intravenous administration remains unclear.Peer reviewe

Single-Stranded DNA-Packaged Polyplex Micelle as Adeno-Associated-Virus-Inspired Compact Vector to Systemically Target Stroma-Rich Pancreatic Cancer.

Despite the rigidity of double-stranded DNA (dsDNA), its packaging is used to construct nonviral gene carriers due to its availability and the importance of its double-helix to elicit transcription. However, there is an increasing demand for more compact-sized carriers to facilitate tissue penetration, which may be easily fulfilled by using the more flexible single-stranded DNA (ssDNA) as an alternative template. Inspired by the adeno-associated virus (AAV) as a prime example of a transcriptionally active ssDNA system, we considered a methodology that can capture unpaired ssDNA within the polyplex micelle system (PM), an assembly of DNA and poly(ethylene glycol)--poly(l-lysine) (PEG-PLys). A micellar assembly retaining unpaired ssDNA was prepared by unpairing linearized pDNA with heat and performing polyion complexation on site with PEG-PLys. The PM thus formed had a compact and spherical shape, which was distinguishable from the rod-shaped PM formed from dsDNA, and still retained its ability to activate gene expression. Furthermore, we demonstrated that its capacity to encapsulate DNA was much higher than AAV, thereby potentially allowing the delivery of a larger variety of protein-encoding DNA. These features permit the ssDNA-loaded PM to easily penetrate the size-restricting stromal barrier after systemic application. Further, they can elicit gene expression in tumor cell nests of an intractable pancreatic cancer mouse model to achieve antitumor effects through suicide gene therapy. Thus, single-stranded DNA-packaged PM is appealing as a potential gene vector to tackle intractable diseases, particularly those with target delivery issues due to size-restriction barriers

RIG-I Helicase-Independent Pathway in Sendai Virus-Activated Dendritic Cells Is Critical for Preventing Lung Metastasis of AT6.3 Prostate Cancer123

We recently demonstrated highly efficient antitumor immunity against dermal tumors of B16F10 murine melanoma with the use of dendritic cells (DCs) activated by replication-competent, as well as nontransmissible-type, recombinant Sendai viruses (rSeV), and proposed a new concept, “immunostimulatory virotherapy,” for cancer immunotherapy. However, there has been little information on the efficacies of thismethod: 1) inmore clinically relevant situations including metastatic diseases, 2) on other tumor types and other animal species, and 3) on the related molecular/cellular mechanisms. In this study, therefore, we investigated the efficacy of vaccinating DCs activated by fusion gene-deleted nontransmissible rSeV on a rat model of lung metastasis using a highly malignant subline of Dunning R-3327 prostate cancer, AT6.3. rSeV/dF-green fluorescent protein (GFP)-activated bone marrow-derived DCs (rSeV/dF-GFP-DC), consistent with results previously observed in murine DCs. Vaccination of rSeV/dF-GFP-DC was highly effective at preventing lung metastasis after intravenous loading of R-3327 tumor cells, compared with the effects observed with immature DCs or lipopolysaccharide-activated DCs. Interestingly, neither CTL activity nor DC trafficking showed any apparent difference among groups. Notably, rSeV/dF-DCs expressing a dominant-negative mutant of retinoic acid-inducible gene I (RIG-I) (rSeV/dF-RIGIC-DC), an RNA helicase that recognizes the rSeV genome for inducing type I interferons, largely lost the expression of proinflammatory cytokines without any impairment of antitumor activity. These results indicate the essential role of RIG-I-independent signaling on antimetastatic effect induced by rSeV-activated DCs and may provide important insights to DC-based immunotherapy for advanced malignancies