No abstract

The oncolytic adenovirus (Ad) is currently being advanced as a promising antitumor remedy as it selectively replicates in tumor cells and can transfer and amplify therapeutic genes. Interleukin (IL)-12 induces a potent antitumor effect by promoting natural killer (NK) cell and cytotoxic T cell activities. IL-18 also augments cytotoxicity of NK cells and proliferation of T cells. This effect further enhances the function of IL-12 in a synergistic manner. Therefore, we investigated for the first time an effective cancer immunogene therapy of syngeneic tumors via intratumoral administration of oncolytic Ad co-expressing IL-12 and IL-18, RdB/IL-12/IL-18. Intratumoral administration of RdB/IL-12/IL-18 improved antitumor effects, as well as increased survival, in B16-F10 murine melanoma model. The ratio of T-helper type 1/2 cytokine as well as the levels of IL-12, IL-18, interferon-γ and granulocyte–macrophage colony-stimulating factor was markedly elevated in RdB/IL-12/IL-18-treated tumors. Mice injected with RdB/IL-12/IL-18 also showed enhanced cytotoxicity of tumor-specific immune cells. Consistent with these results, immense necrosis and infiltration of NK cells, as well as CD4+ and CD8+ T cells, were observed in RdB/IL-12/IL-18-treated tumor tissues. Importantly, tumors treated with RdB/IL-12/IL-18 showed an elevated number of T cells expressing IL-12Rβ2 or IL-18Rα. These results provide a new insight into therapeutic mechanisms of IL-12 plus IL-18 and provide a potential clinical cancer immunotherapeutic agent for improved antitumor immunity.

Identifying immune escape mechanisms used by tumors may define strategies to sensitize them to immunotherapies to which they are otherwise resistant. In this study, we show that the anti-apoptotic gene API5 acts as an immune escape gene in tumors by rendering them resistant to apoptosis triggered by tumor antigen-specific T cells. Its RNAi-mediated silencing in tumor cells expressing high levels of API5 restored antigen-specific immune sensitivity. Conversely, introducing API5 into API5low cells conferred immune resistance. Mechanistic investigations revealed that API5 mediated resistance by upregulating FGF2 signaling through a FGFR1/PKCδ/ERK effector pathway that triggered degradation of the pro-apoptotic molecule BIM. Blockade of FGF2, PKCδ or ERK phenocopied the effect of API5 silencing in tumor cells expressing high levels of API5, to either murine or human antigen-specific T cells. Our results identify a novel mechanism of immune escape that can be inhibited to potentiate the efficacy of targeted active immunotherapies.

No abstract

Adoptive natural killer (NK) cell therapy may offer an effective treatment regimen for cancer patients whose disease is refractory to conventional therapy. NK cells can kill a wide range of tumor cells by patterned recognition of target ligands. We hypothesized that tumor targets sensitive to NK lysis would drive vigorous expansion of NK cells from human peripheral blood mononuclear cells (PBMC). Here, we provide the basis for developing a novel ex vivo expansion process. By screening class I-negative or -mismatched tumor cell lines we identified a Jurkat Tlymphoblast subline termed KL-1, which was highly effective in specifically expanding NK cells. KL-1 addition to PBMC cultures achieved approximately 100-fold expansion of NK cells with nearly 90% purity, accompanied by reciprocal inhibition of T-cell growth. Marked elevations in expression of activation receptors, natural cytotoxicity receptors (NKp30, NKp44), and adhesion molecules (CD11a, ICAM-1) were associated with high tumor-lytic capacity, in both in vitro and in vivo models. KL-1-mediated expansion of NK cells was contact dependent and required interactions with CD16, the Fcg receptor on NK cells, with ligands that are expressed on B cells. Indeed, B-cell depletion during culture abrogated selective NK cell expansion, while addition of EBV-transformed B cells further augmented NK expansion to approximately 740-fold. Together, our studies define a novel method for efficient activation of human NK cells that employs KL-1-lysed tumor cells and cocultured B cells, which drive a robust expansion of potent antitumor effector cells that will be useful for clinical evaluation.

A wide variety of drug delivery systems have been developed for the delivery of anticancer agents. One of the most frequently used natural biomaterials in drug delivery systems is polysaccharides; however, they are difficult to digest and to eliminate from the body after systemic administration due to their high molecular weight natures and the absence of degrading enzymes. Therefore, the development of degradable and eliminable natural biomaterials is critical for successful in vivo applications. In the present study, we report the development of self-assembled biodegradable nanoparticles based on recombinant human gelatin (rHG) modified with alpha-tocopheryl succinate (TOS). The rHG-TOS nanoparticles efficiently encapsulated 17-AAG (17-allylamino-17-demethoxygeldanamycin), a small molecular anticancer drug targeting heat shock protein 90. The formation of 17-AAG-loaded nanoparticles was confirmed using TEM and dynamic light scattering analysis and found to be within the size of 90-220 nm. The loading efficiency, sustained release pattern, and stability of 17-AAG from the rHG-TOS nanoparticles were determined using HPLC. Furthermore, the passive targeting of rHG-TOS nanoparticles to the tumor area via enhanced permeability and retention effect was examined by noninvasive live animal imaging in a tumor mouse model. Finally, the 17-AAG-loaded nanoparticles were nonimmunogenic and more efficient than free 17-AAG in manifesting an anticancer effect in the tumor model. Overall, our data demonstrate rHG-TOS as a promising tool for the delivery of 17-AAG featuring therapeutic efficacy and biocompatibility.

To the best of our knowledge, no reports have directly compared synovial fluid (SF)- and synovial membrane (SM)-derived mesenchymal stem cells (MSCs) from primary knee osteoarthritis patients in terms of MSC proportion, either immediately after isolation or during culture. Any possible correlation between SM- and SF-MSC purity and osteoarthritis severity, also remains unclear. We therefore assessed quantitative and phenotypic differences in MSCs isolated from SF and SM. We also evaluated the correlation between sample MSC purity, and disease severity, in patients with osteoarthritis. The main result of the current study was that the mean SF-MSC proportion at passage 0 was negatively correlated with Kellgren-Lawrence (KL) grade (r = -0.565, P = 0.002). In addition, KL grade was a only significant independent negative predictor of SF-MSC proportion at passage 0 (β = -0.356, P = 0.039). Conclusively, the proportion of SF-MSCs in fresh samples, evaluated at the single cell level, was inversely correlated with osteoarthritis severity.

Synovium-derived mesenchymal stem cells (MSCs) offer a promising therapeutic option for cartilage regeneration. The conventional method of MSC isolation involves single-cell suspensions using collagenases. Recently, a nonenzymatic explant technique was developed to isolate MSCs. We compared these techniques in the isolation of functional MSCs. MSCs were isolated from human fibrous and adipose synovium of osteoarthritic patients using explants or enzymatic methods. Total cell number, percentage of MSCs, and surface marker expression of MSCs were measured following expansion. Multipotentiality was determined using a MSC functional identification kit. MSCs isolated from fibrous or adipose synovium using these two techniques expressed similar levels of the surface markers CD44, CD90, and CD105, and displayed similar multipotentiality in generating adipocytes, osteoblasts, and chondrocytes. Total cell number and number of CD34(-)CD44(+)CD90(+) MSCs after 10-day expansion were similar in each culture, regardless of the source and method used, although the percentage of MSCs was slightly higher in explant cultures. There were no correlations between MSC yield and patient age, Hospital for Special Surgery score, and degree of deformity under all culture conditions. Both the enzymatic and explant techniques yielded similar yields of MSCs with similar characteristics. Because the explant technique is simpler and less invasive, it may be preferred over enzymatic techniques for isolating MSCs from the synovium of osteoarthritic patients for cartilage regeneration.