SCID Mouse Model Research Articles

Application of an assay Cascade methodology for a deep preclinical characterization of polymeric nanoparticles as a treatment for gliomas

Glioblastoma multiforme (GBM) is the most devastating primary brain tumor due to its infiltrating and diffuse growth characteristics, a situation compounded by the lack of effective treatments. Currently, many efforts are being devoted to find novel formulations to treat this disease, specifically in the nanomedicine field. However, due to the lack of comprehensive characterization that leads to insufficient data on reproducibility, only a reduced number of nanomedicines have reached clinical phases. In this context, the aim of the present study was to use a cascade of assays that evaluate from physical-chemical and structural properties to biological characteristics, both in vitro and in vivo, and also to check the performance of nanoparticles for glioma therapy. An amphiphilic block copolymer, composed of polyester and poly(ethylene glycol; PEG) blocks, has been synthesized. Using a mixture of this copolymer and a polymer containing an active targeting moiety to the Blood Brain Barrier (BBB; Seq12 peptide), biocompatible and biodegradable polymeric nanoparticles have been prepared and extensively characterized. In vitro studies demonstrated that nanoparticles are safe for normal cells but cytotoxic for cancer cells. In vivo studies in mice demonstrated the ability of the Seq12 peptide to cross the BBB. Finally, in vivo efficacy studies using a human tumor model in SCID mice resulted in a significant 50% life-span increase, as compared with non-treated animals. Altogether, this assay cascade provided extensive pre-clinical characterization of our polymeric nanoparticles, now ready for clinical evaluation.

High Platelet Levels Attenuate the Efficacy of Platinum-Based Treatment in Non-Small Cell Lung Cancer

Background/Aims: The correlation between platelet levels and clinical outcomes has received increasing attention, but it is not yet clear whether and how platelet levels affect the therapeutic response in non-small cell lung cancer (NSCLC). In the current study, we aimed to explore the role of platelet levels in responsive to platinum-based chemotherapy and investigated the underlying mechanism. Methods: We evaluated the possibility of platelet level as a biomarker for response to platinum-based therapy in NSCLC by retrospective analysis of NSCLC patients. Cell proliferation was evaluated using cell counter and flow cytometry. Cell capillary-like structures of HPMEC were estimated with ECMatrix. The effect of platelets on A549, H1299, and HPMEC apoptosis was measured by flow cytometry. A A549-bearing NOD/ SCID mice model was employed to determine whether platelets could counteract cisplatin-induced apoptosis in vivo. In vivo cell proliferation and apoptosis were evaluated with Ki-67 antibody and TUNEL staining respectively. The angiogenesis of tumor was estimated by CD31 microvessel density. The protein levels of Akt, Bad and Bcl-2 were assessed by western blot. To further examine platelet-driven effects of the chemotherapeutic response, we used platelet depletion and platelet transfusion in A549-bearing NOD/SCID mice. Results: Thrombocytosis at NSCLC diagnosis was associated with lower progression-free survival and median overall survival. Platelet levels before chemotherapy in the no response group were markedly higher than in the responsive group. Platelets rescued the inhibition of cell proliferation and angiogenesis and protected against cell apoptosis induced by cisplatin, platelets rescued cisplatin-induced apoptosis via the Akt/Bad/Bcl-2 signaling pathway under endoplasmic reticulum stress. Platelet transfusion decreased the therapeutic effect of cisplatin, while it was increased by platelet depletion. Conclusion: We confirmed an important anti-apoptosis mechanism mediated by platelets and found that platelets could counteract cisplatin-induced apoptosis. Reducing platelet levels or blocking platelet-based cytoprotection may represent new methods for improving the chemotherapeutic effect.

Optimization of Large-Scale Expansion and Cryopreservation of Human Natural Killer Cells for Anti-Tumor Therapy.

Allogeneic natural killer (NK) cell therapy is a potential therapeutic approach for a variety of solid tumors. We established an expansion method for large-scale production of highly purified and functionally active NK cells, as well as a freezing medium for the expanded NK cells. In the present study, we assessed the effect of cryopreservation on the expanded NK cells in regards to viability, phenotype, and anti-tumor activity. NK cells were enormously expanded (about 15,000-fold expansion) with high viability and purity by stimulating CD3+ T cell-depleted peripheral blood mononuclear cells (PBMCs) with irradiated autologous PBMCs in the presence of IL-2 and OKT3 for 3 weeks. Cell viability was slightly reduced after freezing and thawing, but cytotoxicity and cytokine secretion were not significantly different. In a xenograft mouse model of hepatocellular carcinoma cells, cryopreserved NK cells had slightly lower anti-tumor efficacy than freshly expanded NK cells, but this was overcome by a 2-fold increased dose of cryopreserved NK cells. In vivo antibody-dependent cell cytotoxicity (ADCC) activity of cryopreserved NK cells was also demonstrated in a SCID mouse model injected with Raji cells with rituximab co-administration. Therefore, we demonstrated that expanded/frozen NK cells maintain viability, phenotype, and anti-tumor activity immediately after thawing, indicating that expanded/frozen NK cells can provide ‘ready-to-use’ cell therapy for cancer patients.

Mocetinostat combined with gemcitabine for the treatment of leiomyosarcoma: Preclinical correlates.

Leiomyosarcoma (LMS) is a malignant soft tissue sarcoma (STS) with a dismal prognosis following metastatic disease. Chemotherapeutic intervention has demonstrated to have modest clinical efficacy with no curative potential in LMS patients. Previously, we demonstrated pan-HDAC inhibition to have a superior effect in various complex karyotypic sarcomas. In this study, our goal is to evaluate the therapeutic efficacy of mocetinostat alone and in combination with gemcitabine in LMS. Human leiomyosarcoma (LMS) cell lines were used for in vitro and in vivo studies. Compounds tested included the class I HDAC inhibitor, mocetinostat, and nucleoside analog, gemcitabine. MTS and clonogenic assays were used to evaluate the effect of mocetinostat on LMS cell growth. Cleaved caspase 3/7 analysis was used to determine the effects of mocetinostat on apoptosis. Compusyn software was used to determine in vitro synergy studies for the combination of mocetinostat plus gemcitabine. A LMS xenograft model in SCID mice was used to test the impact of mocetinostat alone, gemcitabine alone and the combination of mocetinostat plus gemcitabine. Mocetinostat abrogated LMS cell growth and clonogenic potential, and enhanced apoptosis in LMS cell lines. The combination of mocetinostat plus gemcitabine exhibited a synergistic effect in LMS cells in vitro. Similarly, mocetinostat combined with gemcitabine resulted in superior anti-LMS effects in vivo. Mocetinostat reduced the expression of gemcitabine-resistance markers RRM1, RRM2, and increased the expression of gemcitabine-sensitivity marker, hENT1, in LMS cells. LMS are aggressive, metastatic tumors with poor prognosis where effective therapeutic interventions are wanting. Our studies demonstrate the potential utility of mocetinostat combined with gemcitabine for the treatment of LMS.

Peripheral blood vessels are a niche for blood-borne meningococci

ABSTRACT Neisseria meningitidis is the causative agent of cerebrospinal meningitis and that of a rapidly progressing fatal septic shock known as purpura fulminans. Meningococcemia is characterized by bacterial adhesion to human endothelial cells of the microvessels. Host specificity has hampered studies on the role of blood vessels colonization in N. meningitidis associated pathogenesis. In this work, using a humanized model of SCID mice allowing the study of bacterial adhesion to human cells in an in vivo context we demonstrate that meningococcal colonization of human blood vessels is a prerequisite to the establishment of sepsis and lethality. To identify the molecular pathways involved in bacterial virulence, we performed transposon insertion site sequencing (Tn-seq) in vivo. Our results demonstrate that 36% of the genes that are important for growth in the blood of mice are dispensable when bacteria colonize human blood vessels, suggesting that human endothelial cells lining the blood vessels are feeding niches for N. meningitidis in vivo. Altogether, our work proposes a new paradigm for meningococcal virulence in which colonization of blood vessels is associated with metabolic adaptation and sustained bacteremia responsible for sepsis and subsequent lethality.

A complicated interpretation of a therapeutic effect with humanized mice using a novel peptide platform

In the article “Systemic delivery of tumor-targeted Bax-derived membrane-active peptides for the treatment of melanoma tumors in a humanized SCID mouse model”, Karageorgis and colleagues, cyclic RGD conjugated apoptosis-inducing peptide platform strongly inhibited the growth of melanoma tumors in humanized SCID mouse model (1).

Invitro and invivo anti-primary effusion lymphoma activities of fucoidan extracted from Cladosiphon okamuranus Tokida.

Primary effusion lymphoma (PEL) caused by Kaposi's sarcoma-associated herpesvirus (KSHV) is characterized by lymphomatous effusion in body cavities and poor prognosis. There is still no effective treatment for PEL. Fucoidan, a major sulfated polysaccharide isolated from brown seaweeds, has an attractive array of bioactivities such as cancer inhibition. However, the effects of fucoidan on PEL cells remain unclear. We investigated the anti-PEL effects of fucoidan obtained from Cladosiphon okamuranus Tokida cultivated in Okinawa. Fucoidan dose-dependently inhibited the proliferation of KSHV-infected PEL cell lines, and provoked G1 cell cycle arrest, which was accompanied by CDK4 and CDK6 downregulation. Fucoidan also induced apoptosis of PEL cells through caspase-3, -8 and -9 activation; this occurred partly through the downregulation of anti-apoptotic Bcl-xL, Mcl-1 and XIAP proteins. Fucoidan also suppressed nuclear factor-κB, activator protein-1 (AP-1), and T-lymphokine-activated killer cell-originated protein kinase (TOPK) signaling pathways through inhibition of phosphorylation of IκBα and TOPK, and the expression of AP-1 family proteins, JunB and JunD. Oral administration of fucoidan effectively inhibited the development of PEL cells and ascites in a xenograft SCID mouse model, with minimal serious adverse effects. Notably, native fucoidan exhibited a more efficient anti-PEL effect than nanoparticle fucoidan. These preclinical findings highlight the anti-PEL actions of fucoidan, suggesting it could be potentially useful for the prevention and treatment of PEL.

A potent series targeting the malarial cGMP-dependent protein kinase clears infection and blocks transmission

To combat drug resistance, new chemical entities are urgently required for use in next generation anti-malarial combinations. We report here the results of a medicinal chemistry programme focused on an imidazopyridine series targeting the Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG). The most potent compound (ML10) has an IC50 of 160 pM in a PfPKG kinase assay and inhibits P. falciparum blood stage proliferation in vitro with an EC50 of 2.1 nM. Oral dosing renders blood stage parasitaemia undetectable in vivo using a P. falciparum SCID mouse model. The series targets both merozoite egress and erythrocyte invasion, but crucially, also blocks transmission of mature P. falciparum gametocytes to Anopheles stephensi mosquitoes. A co-crystal structure of PvPKG bound to ML10, reveals intimate molecular contacts that explain the high levels of potency and selectivity we have measured. The properties of this series warrant consideration for further development to produce an antimalarial drug.

Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and InVitro Efficacy Analysis

JAA-F11 is a highly specific mouse monoclonal to the Thomsen-Friedenreich Antigen (TF-Ag) which is an alpha-O-linked disaccharide antigen on the surface of ~80% of human carcinomas, including breast, lung, colon, bladder, ovarian, and prostate cancers, and is cryptic on normal cells. JAA-F11 has potential, when humanized, for cancer immunotherapy for multiple cancer types. Humanization of JAA-F11, was performed utilizing complementarity determining regions grafting on a homology framework. The objective herein is to test the specificity, affinity and biology efficacy of the humanized JAA-F11 (hJAA-F11). Using a 609 target glycan array, 2 hJAA-F11 constructs were shown to have excellent chemical specificity, binding only to TF-Ag alpha-linked structures and not to TF-Ag beta-linked structures. The relative affinity of these hJAA-F11 constructs for TF-Ag was improved over the mouse antibody, while T20 scoring predicted low clinical immunogenicity. The hJAA-F11 constructs produced antibody-dependent cellular cytotoxicity in breast and lung tumor lines shown to express TF-Ag by flow cytometry. Internalization of hJAA-F11 into cancer cells was also shown using a surface binding ELISA and confirmed by immunofluorescence microscopy. Both the naked hJAA-F11 and a maytansine-conjugated antibody (hJAA-F11-DM1) suppressed in vivo tumor progression in a human breast cancer xenograft model in SCID mice. Together, our results support the conclusion that the humanized antibody to the TF-Ag has potential as an adjunct therapy, either directly or as part of an antibody drug conjugate, to treat breast cancer, including triple negative breast cancer which currently has no targeted therapy, as well as lung cancer.

CRISPR/Cas9-Mediated Knockin Application in Cell Therapy: A Non-viral Procedure for Bystander Treatment of Glioma in Mice.

The use of non-viral procedures, together with CRISPR/Cas9 genome-editing technology, allows the insertion of single-copy therapeutic genes at pre-determined genomic sites, overcoming safety limitations resulting from random gene insertions of viral vectors with potential for genome damage. In this study, we demonstrate that combination of non-viral gene delivery and CRISPR/Cas9-mediated knockin via homology-directed repair can replace the use of viral vectors for the generation of genetically modified therapeutic cells. We custom-modified human adipose mesenchymal stem cells (hAMSCs), using electroporation as a transfection method and CRISPR/Cas9-mediated knockin for the introduction and stable expression of a 3 kb DNA fragment including the eGFP (selectable marker) and a variant of the herpes simplex virus 1 thymidine kinase genes (therapeutic gene), under the control of the human elongation factor 1 alpha promoter in exon 5 of the endogenous thymidine kinase 2 gene. Using a U87 glioma model in SCID mice, we show that the therapeutic capacity of the new CRISPR/Cas9-engineered hAMSCs is equivalent to that of therapeutic hAMSCs generated by introduction of the same therapeutic gene by transduction with a lentiviral vector previously published by our group. This strategy should be of general use to other applications requiring genetic modification of therapeutic cells.

Potent Therapeutic Activity Against Peritoneal Dissemination and Malignant Ascites by the Novel Anti-Folate Receptor Alpha Antibody KHK2805

Many ovarian cancer patients often show peritoneal metastasis with malignant ascites. However, unmet medical needs remain regarding controlling these symptoms after tumors become resistant to chemotherapies. We developed KHK2805, a novel anti-folate receptor α (FOLR1) humanized antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The primary aim of the present study was to evaluate whether the anti-tumor activity of KHK2805 was sufficient for therapeutic application against peritoneal dissemination and malignant ascites of platinum-resistant ovarian cancer in preclinical models. Here, both the ADCC and CDC of KHK2805 were evaluated in ovarian cancer cell lines and patient-derived samples. The anti-tumor activity of KHK2805 was evaluated in a SCID mouse model of platinum-resistant peritoneal dissemination. As results, KHK2805 showed specific binding to FOLR1 with high affinity at a novel epitope. KHK2805 exerted potent ADCC and CDC against ovarian cancer cell lines. Furthermore, primary platinum-resistant malignant ascites cells were susceptible to autologous ADCC with KHK2805. Patient-derived sera and malignant ascites induced CDC of KHK2805. KHK2805 significantly reduced the total tumor burden and amount of ascites in SCID mice with peritoneal dissemination and significantly prolonged their survival. In addition, the parental rat antibody strongly stained serous and clear cell-type ovarian tumors by immunohistochemistry. Overall, KHK2805 showed cytotoxicity against both ovarian cancer cell lines and patient-derived cells. These translational study findings suggest that KHK2805 may be promising as a novel therapeutic agent for platinum-resistant ovarian cancer with peritoneal dissemination and malignant ascites.

Abstract 4788: Abraxane eliminates cancer stem cells in triple-negative breast cancers

Abstract Triple-negative breast cancer (TNBC) represents about 20 percent of all breast cancers, with a poorer survival rate and being associated with high-grade tumors and the least favorable prognosis. Due to the absence of hormone receptors and Her2 receptor, no targeted therapy is available and chemotherapy is the standard treatment for TNBC patient. Paclitaxel is a widely used chemotherapy drug for various cancers with severe side effects due to its solvent. Abraxane is an albumin-bound nanoparticle of paclitaxel with less severe side effects. Here we investigated whether cancer stem cells (CSCs) in TNBC response differentially to abraxane and paclitaxel both in cell culture and xenograft mouse models. Using a series of human TNBC cell lines, we showed that there is no difference between solvent-based paclitaxel and abraxane in vitro in term of efficacy and eliminating cancer stem cells using MTS assay, mammosphere formation assay, and flow analysis of cancer stem cells. Interestingly, we demonstrated that abraxane shows superior efficacy than solvent-based paclitaxel in human SUM149 xenograft NOD SCID mouse model in adjuvant treatment. More important, we demonstrated that abraxane eliminates cancer stem cells in xenograft NOD SCID mouse model in advanced treatment, while solvent-based paclitaxel increases cancer stem cells. Cell uptake assay indicated that TNBC cells take more abraxane than solvent-based paclitaxel, which partially attributes to its superior efficacy in xenograft mouse models. Taken together, our data confirmed that abraxane is superior to solvent-based paclitaxel and may be a better option for TNBC patients in adjuvant or metastatic treatment. Citation Format: Hebao Yuan, Hongwei Guo, Feng Li, Joseph Burnett, Miao He, Nathan Truchan, Ila Myers, Duxin Sun. Abraxane eliminates cancer stem cells in triple-negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4788. doi:10.1158/1538-7445.AM2017-4788

Disease-Specific Effects of Matrix and Growth Factors on Adhesion and Migration of Rheumatoid Synovial Fibroblasts.

In rheumatoid arthritis (RA), cartilage and bone matrix are degraded, and extracellular matrix (ECM) proteins, acting as cellular activators, are liberated. Similar to ECM proteins, matrix-bound chemokines, cytokines, and growth factors (GFs) influence functional properties of key cells in RA, especially synovial fibroblasts. The role of these molecules on attachment, migration, and proinflammatory and prodestructive activation of RASFs was analyzed. Adhesion/migration of RASFs were examined under GF-enriched (GF+) or -reduced (GF-) conditions with or without addition of matrix-associated GFs, TGF-β, and platelet-derived GF to GF- or culture supernatants. Fibroblast adhesion and alterations in proinflammatory/prodestructive properties (e.g., IL-6/matrix metalloproteinase 3-release) in response to matrix-associated molecules were compared. Effects of GF+, GF-, and other ECM components on human RASF-mediated cartilage invasion were examined in the SCID mouse model. RASF adhesion under GF- conditions was significantly lower compared with GF+ conditions (6.8- versus 8.3-fold). This effect was specific for RA because control cells showed opposite effects (e.g., osteoarthritis synovial fibroblasts [SF]; GF- versus GF+: 10.7- versus 8-fold). Addition of TGF-β to GF- increased RASF attachment (12.7-fold) compared with other matrices and components. RASF adhesion to GF+ matrix resulted in the strongest IL-6 and matrix metalloproteinase-3 release, and was even more pronounced compared with supplementation of single GFs. In vivo, GF- matrix decreased RASF-mediated cartilage invasion compared with GF+ matrix. ECM components and especially GFs when bound within ECM actively enhance RASF attraction and cartilage adhesion. This observation was specific for RASFs as a reverse behavior was observed for controls.

Impact of adipose tissue or umbilical cord derived mesenchymal stem cells on the immunogenicity of human cord blood derived endothelial progenitor cells.

The application of autologous endothelial progenitor cell (EPC) transplantation is a promising approach in therapeutic cardiovascular diseases and ischemic diseases. In this study, we compared the immunogenicity of EPCs, adipose tissue (AD)-derived mesenchymal stem cells (MSCs) and umbilical cord (UC)-derived MSCs by flow cytometry and the mixed lymphocyte reaction. The impact of AD-MSCs and UC-MSCs on the immunogenicity of EPCs was analyzed by the mixed lymphocyte reaction and cytokine secretion in vitro and was further tested by allogenic peripheral blood mononuclear cell (PBMC) induced immuno-rejection on a cell/matrigel graft in an SCID mouse model. EPCs and AD-MSCs express higher levels of MHC class I than UC-MSCs. All three kinds of cells are negative for MHC class II. UC-MSCs also express lower levels of IFN-γ receptor mRNA when compared with EPCs and AD-MSCs. EPCs can stimulate higher rates of proliferation of lymphocytes than AD-MSCs and UC-MSCs. Furthermore, AD-MSCs and UC-MSCs can modulate immune response and inhibit lymphocyte proliferation induced by EPCs, mainly through inhibition of the proliferation of CD8+ T cells. Compared with UC-MSCs, AD-MSCs can significantly improve vessel formation and maintain the integrity of neovascular structure in an EPC+MSC/matrigel graft in SCID mice, especially under allo-PBMC induced immuno-rejection. In conclusion, our study shows that AD-MSC is a powerful candidate to minimize immunological rejection and improve vessel formation in EPC transplantation treatment.

Characterization of Immunodeficient Mouse Models

Leakiness in SCID mouse models refers to the occasionally productive VDJ rearrangement leading to clonal expansion of these limited B and T cell clones. The rate and extent of leakiness in immunodeficient models is a factor of age as well as housing environment and the background strain of the model. In the current study, serum IgG was used to determine the extent of leakiness in various immunodeficient mouse models; survival and lymphoma incidence was also analyzed. Male and female (n=23–26) C.B‐17‐SCID, NOD SCID, SCID Beige, SHrN®, Beige Nude XID and Nude mice were housed in flexible film isolators. Blood was collected and analyzed using dried blood spot technology at 12, 16, 20 and 24 weeks of age for IgG concentration analysis using multiplex fluorescent immunoassay. The leakiness threshold was set at 5 μg/mL of IgG for analysis. At 24 weeks of age, necropsies were performed to determine incidence of lymphoma. At 24 wk of age, the SHrN® model had the smallest percentage of leaky animals followed closely by the NOD SCID. Beige Nude XID mice display an age associated increase in serum IgG levels as well as an increasing percentage of animals above the 5 μg/mL threshold from 12–24 weeks of age. Nude mice also show increases in IgG from 12 to 24 weeks of age. The NOD SCID and SHrN® mice had the lowest survival rate at 24 weeks; however, survivability in all models was greater than or equal to 79%. There is a positive correlation between the percentage of leaky animals and survival in males and females across all models. The C.B17‐SCID shows low incidence of lymphoma at 24 weeks of age whereas the NOD SCID model has the highest incidence of lymphoma but when the hairless mutation is crossed onto the NOD SCID mouse to create the SHrN®, incidence of lymphoma is decreased. This is the first study reporting IgG concentration in multiple immunodeficient models to examine leakiness as well as 24 week survival and lymphoma incidence. Additional studies measuring immune cell activity and function will be useful to further characterize immunodeficient models; however, these data will be valuable information in determining the best model for xenograft research.Support or Funding InformationThis research was privately funded by Envigo.

Abstract B89: The chimeric antibody NZ-12 or CAR T cells targeting human podoplanin possesses antitumor activity against orthotopic xenograft models

Abstract Podoplanin (PDPN) is a type I transmembrane mucin-like glycoprotein, which is expressed in lymphatic endothelium. PDPN is also highly expressed in many cancers such as malignant pleural mesothelioma (MPM), lung cancer, and malignant brain tumors including glioblastoma (GBM). We previously developed a rat anti-PDPN monoclonal antibody NZ-1 (rat IgG2a, lambda), and a rat-human chimeric anti-PDPN antibody NZ-8 (human IgG1, kappa), which induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against PDPN-positive cancer cells. Firstly, we demonstrated the antitumor effect of not only NZ-1/NZ-8 but also NZ-12 (human IgG1, lambda), a novel rat-human chimeric anti-PDPN antibody derived from NZ-1, in an MPM orthotopic xenograft SCID mouse model. Treatment with NZ-1 and rat NK (CD161a+) cells inhibited the growth of tumors and the production of pleural effusion in NCI-H290/PDPN or NCI-H226 orthotopic xenograft mouse models. NZ-8 and human NK (CD56+) cells also inhibited tumor growth and pleural effusion in MPM orthotopic xenograft mice. Moreover, NZ-12 induced potent ADCC mediated by human MNC, compared with NZ-1/NZ-8. Antitumor effects were observed following treatment with NZ-12 and human NK (CD56+) cells in MPM orthotopic xenograft mice. Secondly, we constructed a lentiviral vector expressing chimeric antigen receptor (CAR) comprising NZ-1-based single chain variable fragment (scFv) with CD28, 4-1BB, and CD3zeta intracellular domains. CAR-transduced peripheral blood monocytes (PBMCs) were immunologically evaluated by calcein-mediated cytotoxic assay, ELISA, tumor size, and overall survival. The generated CAR T cells were specific and effective against PDPN-positive GBM cells in vitro. Systemic injection of the CAR T cells into an immunodeficient mouse model inhibited the growth of intracranial glioma xenografts in vivo. Taken together, PDPN-targeting antibody therapy or PDPN-targeting CAR T cell therapy might provide an efficacious therapeutic strategy for the treatment of MPM or GBM. Citation Format: Yukinari Kato, Satoshi Shiina, Atsushi Natsume, Shinji Abe, Yasuhiko Nishioka, Satoshi Ogasawara, Mika K. Kaneko. The chimeric antibody NZ-12 or CAR T cells targeting human podoplanin possesses antitumor activity against orthotopic xenograft models. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B89.

Correction: Influence of Extracellular RNAs, Released by Rheumatoid Arthritis Synovial Fibroblasts, on Their Adhesive and Invasive Properties.

Extracellular RNA (exRNA) has been characterized as a molecular alarm signal upon cellular stress or tissue injury and to exert biological functions as a proinflammatory, prothrombotic, and vessel permeability-regulating factor. In this study, we investigated the contribution of exRNA and its antagonist RNase1 in a chronic inflammatory joint disease, rheumatoid arthritis (RA). Upon immunohistochemical inspection of RA, osteoarthritis (OA), and psoriatic arthritis synovium, exRNA was detectable only in the RA synovial lining layer, whereas extracellular DNA was detectable in various areas of synovial tissue. In vitro, exRNA (150-5000 nt) was released by RA synovial fibroblasts (RASF) under hypoxic conditions but not under normoxia or TNF-α treatment. RNase activity was increased in synovial fluid from RA and OA patients compared with psoriatic arthritis patients, whereas RNase activity of RASF and OASF cultures was not altered by hypoxia. Reduction of exRNA by RNase1 treatment decreased adhesion of RASF to cartilage, but it had no influence on their cell proliferation or adhesion to endothelial cells. In vivo, treatment with RNase1 reduced RASF invasion into coimplanted cartilage in the SCID mouse model of RA. We also analyzed the expression of neuropilins in synovial tissue and SF, as they may interact with vascular endothelial growth factor signaling and exRNA. The data support the concepts that the exRNA/RNase1 system participates in RA pathophysiology and that RASF are influenced by exRNA in a prodestructive manner.

Systemic Delivery of Tumor-Targeted Bax-Derived Membrane-Active Peptides for the Treatment of Melanoma Tumors in a Humanized SCID Mouse Model

Melanoma is a highly metastatic and deadly form of cancer. Invasive melanoma cells overexpress integrin αvβ3, which is awell-known target for Arg-Gly-Asp-based (RGD) peptides. We developed a sophisticated method to synthetize milligram amounts of a targeted vector that allows the RGD-mediated targeting, internalization, and release of a mitochondria-disruptive peptide derived from the pro-apoptotic Bax protein. We found that 2.5μM Bax[109-127] was sufficient to destabilize the mitochondria in ten different tumor cell lines, even in the presence of the anti-apoptotic Bcl2 protein, which is often involved in tumor resistance. This pore-forming peptide displayed antitumor activity when it was covalently linked by a disulfide bridge to the tetrameric RAFT-c[RGD]4-platform and after intravenous injection in a human melanoma tumor model established in humanized immuno-competent mice. In addition to its direct toxic effect, treatment with this combination induced the release of the immuno-stimulating factor monocyte chimoattractant protein 1 (MCP1) in the blood and a decrease in the level of the pro-angiogenic factor FGF2. Our novel multifunctional, apoptosis-inducing agent could be further customized and assayed for potential use in tumor-targeted therapy.

Metformin Inhibits Tumorigenesis and Tumor Growth of Breast Cancer Cells by Upregulating miR-200c but Downregulating AKT2 Expression.

Background: Metformin has been reported to inhibit the growth of various types of cancers, including breast cancer. Yet the mechanisms underlying the anticancer effects of metformin are not fully understood. Growing evidence suggests that metformin's anticancer effects are mediated at least in part by modulating microRNAs, including miR-200c, which has a tumor suppressive role in breast cancer. We hypothesized that miR-200c has a role in the antitumorigenic effects of metformin on breast cancer cells.Methods: To delineate the role of miR-200c in the effects of metformin on breast cancer, plasmids containing pre-miR-200c or miR-200c inhibitor were transfected into breast cancer cell lines. The MDA-MB-231, BT549, MCF-7, and T-47-D cells' proliferation, apoptosis, migration, and invasion were assessed. The antitumor role of metformin in vivo was investigated in a MDA-MB-231 xenograft tumor model in SCID mice.Results: Metformin significantly inhibited the growth, migration, and invasion of breast cancer cells, and induced their apoptosis; these effects were dependent on both dose and time. Metformin also suppressed MDA-MB-231 tumor growth in SCID mice in vivo. Metformin treatment was associated with increased miR-200c expression and decreased c-Myc and AKT2 protein expression in both breast cancer cells and tumor tissues. Overexpression of miR-200c exhibited effects on breast cancer cells similar to those of metformin treatment. In contrast, inhibiting the expression of miR-200c increased the growth, migration, and invasion of MCF-7 and MDA-MB-231 cells.Conclusion: Metformin inhibits the growth and invasiveness of breast cancer cells by upregulation of miR-200c expression by targeting AKT2. These findings provide novel insight into the molecular functions of metformin that suggest its potential as an anticancer agent.

Kaempferol Inhibits the Invasion and Migration of Renal Cancer Cells through the Downregulation of AKT and FAK Pathways.

Kaempferol, which is isolated from several natural plants, is a polyphenol belonging to the subgroup of flavonoids. Kaempferol exhibits various pharmacological activities, including anti-inflammatory, antioxidant, antimicrobial, and anticancer activities. In this study, kaempferol can significantly inhibit the invasion and migration of 786-O renal cell carcinoma (RCC) without cytotoxicity. We examined the potential mechanisms underlying its anti-invasive activities on 786-O RCC cells. Western blot was performed, and the results showed that kaempferol attenuates the manifestation of metalloproteinase-2 (MMP-2) protein and activity. The inhibitive effect of kaempferol on MMP-2 may be attributed to the downregulation of phosphorylation of Akt and focal adhesion kinase (FAK). By examining the SCID mice model, we found that kaempferol can safely inhibit the metastasis of the 786-O RCC cells into the lungs by about 87.4% as compared to vehicle treated control animals. In addition, the lung tumor masses of mice pretreated with 2-10 mg/kg kaempferol were reduced about twofold to fourfold. These data suggested that kaempferol can play a promising role in tumor prevention and cancer metastasis inhibition.