Safety evaluation of long-term temperature controlled whole-body thermal treatment in female Aachen minipig.

Objective: Thermal treatment (TT), defined as treatment using supra-physiological body temperatures (39-45 C), somewhat resembles fever in terms of temperature range, one of the first natural barriers for the body to fight exposure to external pathogens. Methods: Whole-body thermal treatment (WBTT) consists of heating up the complete body to a temperature range of 39 to 45 C. Despite the recognized therapeutic potential of hyperthermia, the broad clinical use of WBTT has been limited by safety issues related to medical devices and procedures used to achieve WBTT, in particular adequate control of the body temperature. To circumvent this, a sophisticated medical device was developed, allowing long-term temperature controlled WBTT (41.5 C for up to 8 h). Technical feasibility and tolerability of the WBTT procedure (including complete anesthesia) were tested using female Aachen minipig. Optical fiber temperature sensors inserted in multiple organs were used and demonstrated consistent monitoring and control of different organs temperature over an extended period of time. Results: Clinical evaluation of the animals before, during and after treatment revealed minor clinical parameter changes, but all of them were clinically acceptable. These changes were limited and reversible, and the animals remained healthy throughout the whole procedure and follow-up. In addition, histopathological analysis of selected key organs showed no thermal treatment-related changes. Conclusion: It was concluded that WBTT (41.5 C for up to 8 h) was well tolerated and safe in female Aachen minipigs. Altogether, data supports the safe clinical use of the WBTT medical device and protocol, enabling its implementation into human patients suffering from life-threatening diseases.

TGF-ß2 silencing to target biliary-derived liver diseases.

OBJECTIVE: TGF-ß2 (TGF-ß, transforming growth factor beta), the less-investigated sibling of TGF-ß1, is deregulated in rodent and human liver diseases. Former data from bile duct ligated and MDR2 knockout (KO) mouse models for human cholestatic liver disease suggested an involvement of TGF-ß2 in biliary-derived liver diseases. DESIGN: As we also found upregulated TGFB2 in liver tissue of patients with primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), we now fathomed the positive prospects of targeting TGF-ß2 in early stage biliary liver disease using the MDR2-KO mice. Specifically, the influence of TgfB2 silencing on the fibrotic and inflammatory niche was analysed on molecular, cellular and tissue levels. RESULTS: TgfB2-induced expression of fibrotic genes in cholangiocytes and hepatic stellate cellswas detected. TgfB2 expression in MDR2-KO mice was blunted using TgfB2-directed antisense oligonucleotides (AON). Upon AON treatment, reduced collagen deposition, hydroxyproline content and αSMA expression as well as induced PparG expression reflected a significant reduction of fibrogenesis without adverse effects on healthy livers. Expression analyses of fibrotic and inflammatory genes revealed AON-specific regulatory effects on Ccl3, Ccl4, Ccl5, Mki67 and Notch3 expression. Further, AON treatment of MDR2-KO mice increased tissue infiltration by F4/80-positive cells including eosinophils, whereas the number of CD45-positive inflammatory cells decreased. In line, TGFB2 and CD45 expression correlated positively in PSC/PBC patients and localised in similar areas of the diseased liver tissue. CONCLUSIONS: Taken together, our data suggest a new mechanistic explanation for amelioration of fibrogenesis by TGF-ß2 silencing and provide a direct rationale for TGF-ß2-directed drug development.

INT-1B3, an LNP formulated miR-193a-3p mimic, promotes anti-tumor immunity by enhancing T cell mediated immune responses via modulation of the tumor microenvironment and induction of immunogenic cell death.

microRNAs (miRNAs) are small, non-coding RNAs that regulate expression of multiple genes. MiR-193a-3p functions as a tumor suppressor in many cancer types, but its effect on inducing specific anti-tumor immune responses is unclear. Therefore, we examined the effect of our lipid nanoparticle (LNP) formulated, chemically modified, synthetic miR-193a-3p mimic (INT-1B3) on anti-tumor immunity. INT-1B3 inhibited distant tumor metastasis and significantly prolonged survival. INT-1B3-treated animals were fully protected against challenge with autologous tumor cells even in absence of treatment indicating long-term immunization. Protection against autologous tumor cell challenge was hampered upon T cell depletion and adoptive T cell transfer abrogated tumor growth. Transfection of tumor cells with our miR-193a-3p mimic (1B3) resulted in tumor cell death and apoptosis accompanied by increased expression of DAMPs. Co-culture of 1B3-transfected tumor cells and immature DC led to DC maturation and these mature DC were able to stimulate production of type 1 cytokines by CD4+ and CD8+ T cells. CD4-CD8- T cells also produced type 1 cytokines, even in response to 1B3-transfected tumor cells directly. Live cell imaging demonstrated PBMC-mediated cytotoxicity against 1B3-transfected tumor cells. These data demonstrate for the first time that miR-193a-3p induces long-term immunity against tumor development via modulation of the tumor microenvironment and induction of immunogenic cell death.

Initial testing of JNJ-26854165 (Serdemetan) by the pediatric preclinical testing program.

JNJ-26854165 was originally developed as an activator of p53 capable of inducing apoptosis in cancer cell lines. In vitro, JNJ-26854165 demonstrated cytotoxic activity. The ALL cell line panel had a significantly lower median IC(50) (0.85 µM) than the remaining cell lines. In vivo JNJ-26854165 induced significant differences in EFS distribution compared to control in 18 of 37 solid tumors and in 5 of 7 of the evaluable ALL xenografts. Objective responses were observed in 4 of 37 solid tumor xenografts, and 2 of 7 ALL xenografts achieved PR or CR. Responses were noted in xenografts with both mutant and wild-type p53.

Multi-modal effects of 1B3, a novel synthetic miR-193a-3p mimic, support strong potential for therapeutic intervention in oncology.

Compelling evidence demonstrates that miR-193a-3p is a tumor suppressor microRNA in many cancer types, and its reduced expression is linked to cancer initiation and progression, metastasis, and therapy resistance. However, its mechanism of action is not consistently described between studies, and often contradicts the pleiotropic role of a microRNA in manipulating several different mRNA targets. We therefore comprehensively investigated miRNA-193a-3p's mode of action in a panel of human cancer cell lines, with a variety of genetic backgrounds, using 1B3, a synthetic microRNA mimic. Interestingly, the exact mechanism through which 1B3 reduced cell proliferation varied between cell lines. 1B3 efficiently reduced target gene expression, leading to reduced cell proliferation/survival, cell cycle arrest, induction of apoptosis, increased cell senescence, DNA damage, and inhibition of migration. SiRNA silencing of 1B3 target mRNAs further highlighted the advantage of the pleiotropic mechanism of 1B3 action, as repression of individual targets did not achieve the same robust effect on cell proliferation in all cell lines. Importantly, a novel lipid nanoparticle-based formulation of 1B3, INT-1B3, demonstrated marked anti-tumor activity as a single agent following systemic administration in tumor-bearing mice. Together, these data strongly support the development of 1B3 as a novel therapeutic agent for treatment of human cancer.

Transcriptome-wide analysis reveals insight into tumor suppressor functions of 1B3, a novel synthetic miR-193a-3p mimic.

Emerging data show that microRNA 193a-3p (miR-193a-3p) has a suppressive role in many cancers and is often downregulated in tumors, as compared to surrounding normal tissues. Therefore, mimics of miR-193a-3p could be used as an attractive therapeutic approach in oncology. To better understand and document the molecular mechanism of action of 1B3, a novel synthetic miRNA-193a-3p mimic, RNA sequencing was performed after transfection of 1B3 in six different human tumor cell lines. Genes differentially expressed (DE) in at least three cell lines were mapped by Ingenuity Pathway Analysis (IPA), and interestingly, these results strongly indicated upregulation of the tumor-suppressive phosphatase and tensin homolog (PTEN) pathway, as well as downregulation of many oncogenic growth factor signaling pathways. Importantly, although unsurprisingly, IPA identified miR-193a-3p as a strong upstream regulator of DE genes in an unbiased manner. Furthermore, biological function analysis pointed to an extensive link of 1B3 with cancer, via expected effects on tumor cell survival, proliferation, migration, and cell death. Our data strongly suggest that miR-193a-3p/1B3 is a potent tumor suppressor agent that targets various key oncogenic pathways across cancer types. Therefore, the introduction of 1B3 into tumor cells may represent a promising strategy for cancer treatment.

Identification of a series of substituted 2-piperazinyl-5-pyrimidylhydroxamic acids as potent histone deacetylase inhibitors.

Pursuing our efforts in designing 5-pyrimidylhydroxamic acid anti-cancer agents, we have identified a new series of potent histone deacetylase (HDAC) inhibitors. These compounds exhibit enzymatic HDAC inhibiting properties with IC(50) values in the nanomolar range and inhibit tumor cell proliferation at similar levels. Good solubility, moderate bioavailability, and promising in vivo activity in xenograft model made this series of compounds interesting starting points to design new potent HDAC inhibitors.

Therapeutic Targeting of TGFß Ligands in Glioblastoma Using Novel Antisense Oligonucleotides Reduces the Growth of Experimental Gliomas.

PURPOSE: Transforming growth factor (TGF)-ß is expressed at high levels by glioma cells and contributes to the malignant phenotype of glioblastoma. However, its therapeutic targeting remains challenging. Here, we examined an alternative therapeutic approach of TGFß inhibition using two novel phosphorothioate-locked nucleic acid (LNA)-modified antisense oligonucleotide gapmers, ISTH1047 and ISTH0047, which specifically target TGFß1 and TGFß2. EXPERIMENTAL DESIGN: We characterized the effects of ISTH1047 and ISTH0047 on TGFß1/2 expression, downstream signaling and growth of human LN-308, LN-229, and ZH-161 cells as well as murine SMA-560 glioma cells in vitro. Furthermore, we assessed their target inhibition and effects on survival in orthotopic xenogeneic and syngeneic rodent glioma models in vivo. RESULTS: Both antisense oligonucleotides specifically silenced their corresponding target and abrogated SMAD2 phosphorylation in several glioma cell lines. Moreover, inhibition of TGFß1 or TGFß2 expression by ISTH1047 or ISTH0047 reduced the migration and invasiveness of LN-308 and SMA-560 glioma cells. Systemic antisense oligonucleotide administration to glioma-bearing mice suppressed TGFß1 or TGFß2 mRNA expression as well as the expression of the downstream target PAI-1 in orthotopic gliomas. Glioma-bearing mice had significantly prolonged survival upon systemic treatment with ISTH1047 or ISTH0047, which was associated with a reduction of intratumoral SMAD2 phosphorylation and, in a fully immunocompetent model, with increased immune cell infiltration. CONCLUSIONS: Targeting TGFß expression with the novel LNA antisense oligonucleotides ISTH1047 or ISTH0047 results in strong antiglioma activity in vitro and in vivo, which may represent a promising approach to be examined in human patients with glioma.

Disruption of murine Mus81 increases genomic instability and DNA damage sensitivity but does not promote tumorigenesis.

The Mus81-Eme1 endonuclease is implicated in the efficient rescue of broken replication forks in Saccharomyces cerevisiae and Schizosaccharomyces pombe. We have used gene targeting to study the function of the Mus81-Eme1 endonuclease in mammalian cells. Mus81-deficient mice develop normally and are fertile. Surprisingly, embryonic fibroblasts from Mus81(-/-) animals fail to proliferate in vitro. This proliferation defect can be rescued by expression of the papillomavirus E6 protein that promotes degradation of p53. When grown in culture, Mus81(-/-) cells have elevated levels of DNA damage, acquire chromosomal aberrations, and are hypersensitive to agents that generate DNA cross-links. In contrast to the situation in yeast, murine Mus81 is not required for replication restart following camptothecin treatment. Mus81(-/-) mice and cells are hypersensitive to DNA cross-linking agents. Cross-link-induced double-strand break formation is normal in Mus81(-/-) cells, but the resolution of repair intermediates is not. The persistence of Rad51 foci in Mus81(-/-) cells suggests that Mus81 acts at a late step in the repair of cross-link-induced lesions. Despite these defects, Mus81(-/-) mice do not show increased predisposition to lymphoma or any other malignancy in the first year of life.

Critical role of the ubiquitin ligase activity of UHRF1, a nuclear RING finger protein, in tumor cell growth.

Early cellular events associated with tumorigenesis often include loss of cell cycle checkpoints or alteration in growth signaling pathways. Identification of novel genes involved in cellular proliferation may lead to new classes of cancer therapeutics. By screening a tetracycline-inducible cDNA library in A549 cells for genes that interfere with proliferation, we have identified a fragment of UHRF1 (ubiquitin-like protein containing PHD and RING domains 1), a nuclear RING finger protein, that acts as a dominant negative effector of cell growth. Reduction of UHRF1 levels using an UHRF1-specific shRNA decreased growth rates in several tumor cell lines. In addition, treatment of A549 cells with agents that activated different cell cycle checkpoints resulted in down-regulation of UHRF1. The primary sequence of UHRF1 contains a PHD and a RING motif, both of which are structural hallmarks of ubiquitin E3 ligases. We have confirmed using an in vitro autoubiquitination assay that UHRF1 displays RING-dependent E3 ligase activity. Overexpression of a GFP-fused UHRF1 RING mutant that lacks ligase activity sensitizes cells to treatment with various chemotherapeutics. Taken together, our results suggest a general requirement for UHRF1 in tumor cell proliferation and implicate the RING domain of UHRF1 as a functional determinant of growth regulation.

Real-time gene expression analysis in human xenografts for evaluation of histone deacetylase inhibitors.

Real-time analysis of gene expression in experimental tumor models represents a major tool to document disease biology and evaluate disease treatment. However, monitoring gene regulation in vivo still is an emerging field, and thus far it has not been linked to long-term tumor growth and disease outcome. In this report, we describe the development and validation of a fluorescence-based gene expression model driven by the promoter of the cyclin-dependent kinase inhibitor p21waf1,cip1. The latter is a key regulator of tumor cell proliferation and a major determinant in the response to many anticancer agents such as histone deacetylase inhibitors. In response to histone deacetylase inhibitors, induction of fluorescence in A2780 ovarian tumors could be monitored in living mice in a noninvasive real-time manner using whole-body imaging. Single p.o. administration of the histone deacetylase inhibitor MS-275 significantly induces tumor fluorescence in a time- and dose-dependent manner, which accurately predicted long-term antitumoral efficacy in individual mice following extended treatment. These findings illustrate that this technology allows monitoring of the biological response induced by treatment with histone deacetylase inhibitors. In addition to providing experimental pharmacokinetic/pharmacodynamic markers for investigational drugs, this model provides insight into the kinetics of in vivo regulation of transcription, which plays a key role in causing and maintaining the uncontrolled proliferation of tumor tissue.

Biological Role and Therapeutic Targeting of TGF-ß3 in Glioblastoma.

Transforming growth factor (TGF)-ß contributes to the malignant phenotype of glioblastoma by promoting invasiveness and angiogenesis and creating an immunosuppressive microenvironment. So far, TGF-ß1 and TGF-ß2 isoforms have been considered to act in a similar fashion without isoform-specific function in glioblastoma. A pathogenic role for TGF-ß3 in glioblastoma has not been defined yet. Here, we studied the expression and functional role of endogenous and exogenous TGF-ß3 in glioblastoma models. TGF-ß3 mRNA is expressed in human and murine long-term glioma cell lines as well as in human glioma-initiating cell cultures with expression levels lower than TGF-ß1 or TGF-ß2 in most cell lines. Inhibition of TGF-ß3 mRNA expression by ISTH2020 or ISTH2023, two different isoform-specific phosphorothioate locked nucleic acid (LNA)-modified antisense oligonucleotide gapmers, blocks downstream SMAD2 and SMAD1/5 phosphorylation in human LN-308 cells, without affecting TGF-ß1 or TGF-ß2 mRNA expression or protein levels. Moreover, inhibition of TGF-ß3 expression reduces invasiveness in vitro Interestingly, depletion of TGF-ß3 also attenuates signaling evoked by TGF-ß1 or TGF-ß2 In orthotopic syngeneic (SMA-560) and xenograft (LN-308) in vivo glioma models, expression of TGF-ß3 as well as of the downstream target, plasminogen-activator-inhibitor (PAI)-1, was reduced, while TGF-ß1 and TGF-ß2 levels were unaffected following systemic treatment with TGF-ß3 -specific antisense oligonucleotides. We conclude that TGF-ß3 might function as a gatekeeper controlling downstream signaling despite high expression of TGF-ß1 and TGF-ß2 isoforms. Targeting TGF-ß3in vivo may represent a promising strategy interfering with aberrant TGF-ß signaling in glioblastoma. Mol Cancer Ther; 16(6); 1177-86. ©2017 AACR.

First-in-human phase I study of ISTH0036, an antisense oligonucleotide selectively targeting transforming growth factor beta 2 (TGF-ß2), in subjects with open-angle glaucoma undergoing glaucoma filtration surgery.

PURPOSE: To evaluate the safety and tolerability of intravitreal ISTH0036, an antisense oligonucleotide selectively targeting transforming growth factor beta 2 (TGF-ß2), in patients with primary open angle glaucoma (POAG) undergoing trabeculectomy (TE; glaucoma filtration surgery). METHODS: In this prospective phase I trial glaucoma patients scheduled for TE with mitomycin C (MMC) received a single intravitreal injection of ISTH0036 at the end of surgery in escalating total doses of 6.75 µg, 22.5 µg, 67.5 µg or 225 µg, resulting in calculated intraocular ISTH0036 concentrations in the vitreous humor of approximately 0.3 µM, 1 µM, 3 µM or 10 µM after injection, respectively. Outcomes assessed included: type and frequency of adverse events (AEs), intraocular pressure (IOP), numbers of interventions post trabeculectomy, bleb survival, visual acuity, visual field, electroretinogram (ERG), slit lamp biomicroscopy and optic disc assessment. RESULTS: In total, 12 patients were treated in the 4 dose groups. Main ocular AEs observed were corneal erosion, corneal epithelium defect, or too high or too low IOP, among others. No AE was reported to be related to ISTH0036. All other safety-related analyses did not reveal any toxicities of concern, either. The mean medicated preoperative IOP at decision time-point for surgery was 27.3 mmHg +/- 12.6 mmHg (SD). Mean IOP (±SD) for dose levels 1, 2, 3, and 4 were at Day 43 9.8 mmHg ± 1.0 mmHg, 11.3 mmHg ± 6.7 mmHg, 5.5 mmHg ± 3.0 mmHg and 7.5 mmHg ± 2.3 mmHg SD; and at Day 85 9.7 mmHg ± 3.3 mmHg, 14.2 mmHg ± 6.5 mmHg, 5.8 mmHg ± 1.8 mmHg and 7.8 mmHg ± 0.6 mmHg, respectively. In contrast to IOP values for dose levels 1 and 2, IOP values for dose levels 3 and 4 persistently remained below 10 mmHg throughout the observation period. CONCLUSION: This first-in-human trial demonstrates that intravitreal injection of ISTH0036 at the end of TE is safe. Regarding IOP control, single-dose ISTH0036 administration of 67.5 µg or 225 µg at the time of TE resulted in IOP values persistently < 10 mmHg over the three month postoperative observation period.

p53-independent regulation of p21Waf1/Cip1 expression and senescence by Chk2.

The Chk2 kinase is a tumor suppressor and key component of the DNA damage checkpoint response that encompasses cell cycle arrest, apoptosis, and DNA repair. It has also been shown to have a role in replicative senescence resulting from dysfunctional telomeres. Some of these functions are at least partially exerted through activation of the p53 transcription factor. High-level expression of virally transduced Chk2 in A549 human lung carcinoma cells led to arrested proliferation, apoptosis, and senescence. These were accompanied by various molecular events, including p21(Waf1/Cip1) (p21) transcriptional induction, consistent with p53 activation. However, Chk2-dependent senescence and p21 transcriptional induction also occurred in p53-defective SK-BR-3 (breast carcinoma) and HaCaT (immortalized keratinocyte) cells. Small interfering RNA-mediated knockdown of p21 in p53-defective cells expressing Chk2 resulted in a decrease in senescent cells. These results revealed a p53-independent role for Chk2 in p21 induction and senescence that may contribute to tumor suppression and genotoxic treatment outcome.

Modified poly(propylene imine) dendrimers as effective transfection agents for catalytic DNA enzymes (DNAzymes).

The major bottleneck in gene therapy remains the issue of delivery. In this work, various modified poly(propylene imine) (PPI) dendrimers are introduced as gene transfection agents. Commercially available PPI-dendrimers have been modified (i) at the exterior primary amines with acetyl groups or glycol gallate (PEG-like) groups, and (ii) at the interior tertiary amines with methyl iodide (MeI) or MeCl to produce multiple quaternized cationic sites in the core of the dendrimer. The prepared materials have been tested with respect to their binding capabilities to DNA, their toxicity in cell cultures, their in vitro transfection efficiency and their in vivo delivery possibilities. In all cases, a 33-mer oligonucleotide (DNAzyme) was used. Polyacrylamide gel electrophoresis (PAGE) studies have demonstrated strong but reversible binding, where the quarternized and higher generation dendrimer species have shown more potent binding. Typically, for the modified fourth PPI-dendrimers, binding is observed at a concentration of about 4 microM DNA and a dendrimer-DNA charge ratio of around 2:1-1:1. All the tested PPI-dendrimers display a low cellular toxicity, especially when higher serum contents are used in the culture medium. For example, most of the prepared fourth generation PPI-dendrimers are not or hardly toxic up to at least 20 microM in 20% serum. An in vitro characterization has revealed a high dendrimer-mediated intracellular uptake of the DNAzyme: all the tested fourth generation PPI-dendrimers display transfection efficiencies close to or exceeding 80%, even when the concentration of serum in the medium is increased from 10 to 40%. Finally, the potential of using modified PPI-dendrimers for in vivo gene therapy experiments is demonstrated. Injecting a G4-PEG(MeI)-ssDNA complex intravenously into Nude mice has resulted in a high nuclear uptake as confirmed by co-localization studies.

TGF-ß1 and TGF-ß2 abundance in liver diseases of mice and men.

TGF-ß1 is a major player in chronic liver diseases promoting fibrogenesis and tumorigenesis through various mechanisms. The expression and function of TGF-ß2 have not been investigated thoroughly in liver disease to date. In this paper, we provide evidence that TGF-ß2 expression correlates with fibrogenesis and liver cancer development.Using quantitative realtime PCR and ELISA, we show that TGF-ß2 mRNA expression and secretion increased in murine HSCs and hepatocytes over time in culture and were found in the human-derived HSC cell line LX-2. TGF-ß2 stimulation of the LX-2 cells led to upregulation of the TGF-ß receptors 1, 2, and 3, whereas TGF-ß1 treatment did not alter or decrease their expression. In liver regeneration and fibrosis upon CCl4 challenge, the transient increase of TGF-ß2 expression was accompanied by TGF-ß1 and collagen expression. In bile duct ligation-induced fibrosis, TGF-ß2 upregulation correlated with fibrotic markers and was more prominent than TGF-ß1 expression. Accordingly, MDR2-KO mice showed significant TGF-ß2 upregulation within 3 to 15 months but minor TGF-ß1 expression changes. In 5 of 8 hepatocellular carcinoma (HCC)/hepatoblastoma cell lines, relatively high TGF-ß2 expression and secretion were observed, with some cell lines even secreting more TGF-ß2 than TGF-ß1. TGF-ß2 was also upregulated in tumors of TGFα/cMyc and DEN-treated mice. The analysis of publically available microarray data of 13 human HCC collectives revealed considerable upregulation of TGF-ß2 as compared to normal liver.Our study demonstrates upregulation of TGF-ß2 in liver disease and suggests TGF-ß2 as a promising therapeutic target for tackling fibrosis and HCC.

Discovery of pyrimidyl-5-hydroxamic acids as new potent histone deacetylase inhibitors.

A series of pyrimidyl-5-hydroxamic acids was prepared for evaluation as inhibitors of histone deacetylase (HDAC). Amino-2-pyrimidinyl can be used as a linker to provide HDAC inhibitors of good enzymatic potency.

Design and Selection of Antisense Oligonucleotides Targeting Transforming Growth Factor Beta (TGF-ß) Isoform mRNAs for the Treatment of Solid Tumors.

Transforming growth factor beta isoforms (TGF-ß1, -ß2, and -ß3) are cytokines associated with a wide range of biological processes in oncology including tumor cell invasion and migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Hence, blocking the TGF-ß signaling pathways may have a multifold therapeutic benefit for the treatment of solid tumors. Here, we describe the identification and selection processes for the development of highly potent and selective chemically modified antisense oligodeoxynucleotides (fully phosphorothioate locked nucleic acid gapmers) allowing effective and selective suppression of TGF-ß isoform expression in cell-based assays and in vivo preclinical models.

TGF-ß isoforms in cancer: Immunohistochemical expression and Smad-pathway-activity-analysis in thirteen major tumor types with a critical appraisal of antibody specificity and immunohistochemistry assay validity.

The literature on TGF-ß in cancer including data on the expression or activation of TGF-ß pathway components in specific tumors types is steadily growing. However, no systematic and uniform analysis exists reporting expression levels of the main TGF-ß pathway components across the most frequent tumor types. We used a standardized immunohistochemical assay investigating TGF-ß isoform expression and pathway activation across 13 different tumor types and corresponding non-neoplastic tissues. The study was performed on tissue microarrays allowing for the parallel analysis of a total of 1638 human tumor samples. TGF-ß1, TGF-ß2 and p-Smad2/3 were substantially expressed in multiple cancers widening the options for TGF-ß isoform directed therapies. Of note, TGF-ß antigens appear to be expressed in an individual manner pointing towards a need for patient preselection for TGF-ß isoform specific treatment. Yet, a thorough investigation of antibody specificity and assay validity revealed that immunohistochemistry did not correlate with other detection methods on mRNA or protein level in all instances. As such, with the currently available means (i.e. antibodies tested) a stratification of patients within clinical trials for TGF-ß directed antisense therapies based upon TGF-ß immunohistochemistry alone has to be interpreted with caution and should be carefully evaluated in combination with other parameters.

Targeted delivery of RNA-cleaving DNA enzyme (DNAzyme) to tumor tissue by transferrin-modified, cyclodextrin-based particles.

Short nucleic acid sequences specific to oncogene targets such as bcl-2, bcr-abl, and c-myc have been shown to exhibit specific anti-cancer activity in vitro through antigene or antisense activity. Efficient in vivo delivery of oligonucleotides remains a major limitation for the therapeutic application of these molecules. We report herein on the preparation of transferrin-modified nanoparticles containing DNAzymes (short catalytic single-stranded DNA molecules) for tumor targeting as well as their biodistribution using various methods of administration in the mouse. Linear, beta-cyclodextrin-based polymers are complexed with DNAyzme molecules to form sub-50 nm particles termed "polyplexes". The surface properties of the cyclodextrin-containing polyplexes are modified by exploiting the ability of the beta-cyclodextrin substructure and adamantane to form inclusion complexes. Accordingly, conjugates of adamantane with poly(ethylene glycol) (PEG) are prepared and combined with the polyplexes. The adamantane form inclusion complexes with the surface cyclodextrins of the polyplexes to provide a sterically stabilizing layer of PEG. The stabilized polyplexes are also modified with transferrin for increasing targeting to tumor cells expressing transferrin receptors. The preparation, characterization, and in vitro application of these nanoparticles are discussed. The transferrin-polyplexes containing fluorescently-labeled DNAzyme molecules are administered to tumor-bearing nude mice and their biodistribution and clearance kinetics are monitored using a fluorescence imaging system. Four methods of administration are studied: intraperitoneal bolus and infusion, intravenous bolus, and subcutaneous injection. DNAzymes packaged in polyplex formulations are concentrated and retained in tumor tissue and other organs, whereas unformulated DNAzyme is eliminated from the body within 24 hours post-injection. Intravenous and intraperitoneal bolus injections result in the highest fluorescent signal (DNAzyme) at the tumor site. Tumor cell uptake is observed with intravenous bolus injection only, and intracellular delivery requires transferrin targeting.