U. Stein

Nonviral in vivo gene delivery into tumors using a novel low volume jet-injection technology

The jet-injection technology has developed as an applicable alternative to viral or liposomal gene delivery systems. In this study a novel, low-volume, ‘high-speed jet injector’ hand-held system was used for the direct gene transfer of naked DNA into tumors. Lewis-lung carcinoma bearing mice were jet-injected with the β-galactosidase (LacZ), the green fluorescence (GFP) or the human tumor necrosis factor alpha (TNF-α) gene carrying vector plasmids. The animals received five jet injections into the tumor at a pressure of 3.0 bar, delivering 3–5u2009μl plasmid DNA (1u2009μg DNA/μl in water) per single jet injection. The jet injection of DNA leads to a widespread expression pattern within tumor tissues with penetration depths of 5–10u2009mm. Analysis of tumor cryosections revealed moderate LacZ or GFP expression at 48u2009h and strong reporter gene expression 72u2009h and 96u2009h after jet injection. The simultaneous jet injection of the TNF-α and LacZ carrying vectors demonstrated efficient expression and secretion of both the cytokine, as well as LacZ expression within the tumor 24u2009h, 48u2009h, 72u2009h, 96u2009h and 120u2009h after jet injection. These studies demonstrate the applicability of jet injection for the efficient in vivo gene transfer into tumors for nonviral gene therapy of cancer using minimal amounts of naked DNA.

ASAP1 promotes tumor cell motility and invasiveness, stimulates metastasis formation in vivo, and correlates with poor survival in colorectal cancer patients.

We have previously performed an unbiased screen to identify genes whose expression is associated with the metastatic phenotype. Secondary screening of these genes using custom microarray chips identified ASAP1, a multi-domain adaptor protein with ADP-ribosylation factor-GAP activity, as being potentially involved in tumor progression. Here, we show that at least three different splice forms of ASAP1 are upregulated in rodent tumor models in a manner that correlates with metastatic potential. In human cancers, we found that ASAP1 expression is strongly upregulated in a variety of tumors in comparison with normal tissue and that this expression correlates with poor metastasis-free survival and prognosis in colorectal cancer patients. Using loss and gain of function approaches, we were able to show that ASAP1 promotes metastasis formation in vivo and stimulates tumor cell motility, invasiveness, and adhesiveness in vitro. Furthermore, we show that ASAP1 interacts with the metastasis-promoting protein h-prune and stimulates its phosphodiesterase activity. In addition, ASAP1 binds to the SH3 domains of several proteins, including SLK with which it co-immunoprecipitates. These data support the notion that ASAP1 can contribute to the dissemination of a variety of tumor types and represent a potential target for cancer therapy.

Novel Clostridium perfringens enterotoxin suicide gene therapy for selective treatment of claudin-3- and -4-overexpressing tumors

Bacterial toxins are known to be effective for cancer therapy. Clostridium perfringens enterotoxin (CPE) is produced by the bacterial Clostridium type A strain. The transmembrane proteins claudin-3 and -4, often overexpressed in numerous human epithelial tumors (for example, colon, breast, pancreas, prostate and ovarian), are the targeted receptors for CPE. CPE binding to them triggers formation of membrane pore complexes leading to rapid cell death. In this study, we aimed at selective tumor cell killing by CPE gene transfer. We generated expression vectors bearing the bacterial wild-type CPE cDNA (wtCPE) or translation-optimized CPE (optCPE) cDNA for in vitro and in vivo gene therapy of claudin-3- and -4-overexpressing tumors. The CPE expression analysis at messenger RNA and protein level revealed more efficient expression of optCPE compared with wtCPE. Expression of optCPE showed rapid cytotoxic activity, hightened by CPE release as bystander effect. Cytotoxicity of up to 100% was observed 72u2009h after gene transfer and is restricted to claudin-3-and -4-expressing tumor lines. MCF-7 and HCT116 cells with high claudin-4 expression showed dramatic sensitivity toward CPE toxicity. The claudin-negative melanoma line SKMel-5, however, was insensitive toward CPE gene transfer. The non-viral intratumoral in vivo gene transfer of optCPE led to reduced tumor growth in MCF-7 and HCT116 tumor-bearing mice compared with the vector-transfected control groups. This novel approach demonstrates that CPE gene transfer can be employed for a targeted suicide gene therapy of claudin-3- and -4-overexpressing tumors, leading to the rapid and efficient tumor cell killing in vitro and in vivo.

MDR1 gene expression: evaluation of its use as a molecular marker for prognosis and chemotherapy of bone and soft tissue sarcomas.

Successful chemotherapeutic treatment of malignant tumours is often limited by the intrinsic or acquired multidrug resistance (MDR). The classical MDR phenotype is characterised by reduced drug accumulation within the cell, caused by overexpression of the MDR1 gene encoded P-glycoprotein. Some reports have been published evaluating MDR1 expression as a molecular marker for response to chemotherapy in human bone and soft tissue sarcomas. In this review, an attempt is made to summarise the accuracy of the measurement of MDR1 expression for use in prognosis, as well as in decisions on chemotherapeutic treatment of sarcomas. In addition, general problems for the performance of such studies is discussed.

The immediate early gene Ier2 promotes tumor cell motility and metastasis, and predicts poor survival of colorectal cancer patients

Here, we report unbiased screens for genes expressed in metastatic tumor cells that are associated with cell motility. These screens identified Ier2, an immediate early gene of unknown function, as potentially having a role in tumor cell motility and metastasis. Knockdown of Ier2 in 3T3 fibroblasts inhibited their motility upon relief of contact inhibition in monolayer wounding assays. Furthermore, ectopic Ier2 expression promoted the motility and invasiveness of poorly metastatic 1AS pancreatic tumor cells in vitro. Relief of contact inhibition was associated with translocation of the Ier2 protein from the cytoplasm to the nucleus in both 3T3 fibroblasts and 1AS tumor cells. Importantly, ectopic Ier2 expression in 1AS cells stimulated metastasis formation when cells were implanted into experimental animals. Furthermore, we found elevated Ier2 expression in a wide variety of human tumor types. This correlated with poor metastasis-free and overall survival in patients with colorectal adenocarcinomas. Together, these data reveal Ier2 as a new player in the regulation of tumor progression and metastasis, and suggest that Ier2 may be useful prognostically and therapeutically in the management of cancer.

SPON2, a newly identified target gene of MACC1, drives colorectal cancer metastasis in mice and is prognostic for colorectal cancer patient survival.

MACC1 (metastasis associated in colon cancer 1) is a prognostic biomarker for tumor progression, metastasis and survival of a variety of solid cancers including colorectal cancer (CRC). Here we aimed to identify the MACC1-induced transcriptome and key players mediating the MACC1-induced effects in CRC. We performed microarray analyses using CRC cells ectopically overexpressing MACC1. We identified more than 1300 genes at least twofold differentially expressed, including the gene SPON2 (Spondin 2) as 90-fold upregulated transcriptional target of MACC1. MACC1-dependent SPON2 expression regulation was validated on mRNA and protein levels in MACC1 high (endogenously or ectopically) and low (endogenously or by knockdown) expressing cells. Chromatin immunoprecipitation analysis demonstrated the binding of MACC1 to the gene promoter of SPON2. In cell culture, ectopic SPON2 overexpression induced cell viability, migration, invasion and colony formation in endogenously MACC1 and SPON2 low expressing cells, whereas SPON2 knockdown reduced proliferative, migratory and invasive abilities in CRC cells with high endogenous MACC1 and SPON2 expression. In intrasplenically transplanted NOD/SCID mice, metastasis induction was analyzed with control or SPON2-overexpressing CRC cells. Tumors with SPON2 overexpression induced liver metastasis (vs control animals without any metastases, P=0.0036). In CRC patients, SPON2 expression was determined in primary tumors (stages I–III), and survival time was analyzed by Kaplan–Meier method. CRC patients with high SPON2 expressing primary tumors demonstrated 8 months shorter metastasis-free survival (MFS) compared with patients with low SPON2 levels (P=0.053). Combining high levels of SPON2 and MACC1 improved the identification of high-risk patients with a 20-month shorter MFS vs patients with low biomarker expression. In summary, SPON2 is a transcriptional target of the metastasis gene MACC1. SPON2 induces cell motility in vitro and CRC metastasis in mice. In patients, SPON2 serves as prognostic indicator for CRC metastasis and survival, and might represent a promising target for therapeutic approaches.

211 SPON2, a Newly Identified Transcriptional Target Gene of MACC1, is Associated with Colorectal Cancer Metastasis

Background: The epithelial to mesenchymal transition (EMT) is involved in multiple aspects of cancer biology, including tumor metastasis, cancer stem cells, drug resistance, and immunosuppression. TGFb, one of the most studied regulators of EMT, is produced in a latent form that needs to be activated to enable binding to its receptor and subsequent down-stream signaling. Four of the five aVb integrin proteins have been demonstrated to induce TGFb maturation. Cross-talk between TGFb and integrin signaling can also occur downstream of initial receptor activation and regulates various cellular processes, including EMT. Materials and Methods: Lung, melanoma, and breast cancer cell lines were obtained from ATCC. Protein levels were determined by Western-blot and RNA levels were determined by q-PCR. Migration and invasion assays were carried out using trans-well system. All experiments were conducted without coating the plates with vitronectin. Results: Active TGFb1 increased integrin aV, b1, and b3 expression in lung cancer cells while integrin b5 expression was suppressed by TGFb1. This differential regulation of integrin protein expression was accompanied by a reorganization of integrin heterodimer complex between aV and b subunits. TGFb-treated cells developed a spindle-like morphology. Altered expressions of E-Cadherin, vimentin, and snail in these cells were consistent with EMT. Intetumumab (CNTO95), a monoclonal antibody that binds to aVb integrins (aVb1, aVb3, aVb5, aVb6, and aVb8), potently inhibits cellular events strongly relevant to EMT such as migration, invasion and metastasis. Co-treatment of cells with intetumumab blocked TGFb-induced EMT. Moreover, lung cancer cell lines treated with intetumumab alone underwent a mesenchymal to epithelial transition (MET), with increased expression of E-cadherin and decreased expression of vimentin and snail. The effect of intetumumab on EMT was also observed in melanoma cancer cells. Conclusions: aVb integrins are critical regulators of EMT and MET. Intetumumab activates MET and inhibits TGFb induced EMT by blocking aVb integrins, suggesting potential roles in preventing metastasis.

IN VIVO EVALUATION OF A DRUG-INDUCIBLE VECTOR SYSTEM FOR THE COMBINED GENE­ AND CHEMOTHERAPY OF CANCER*

Regulated expression of therapeutic genes in cancer gene therapy is used in a growing number of gene therapy approaches. Numerous conditionally active vectors have been tested carrying promoters which respond to certain factors or therapeutic modalities, such as irradiation, hyperthermia etc. [1].

Erste Ergebnisse einer Phase-I-Studie zur nicht-viralen Gentherapie mittels Jet-Injektion in Hautmetastasen des Mammakarzinoms sowie Intransit-Metastasen des Malignen Melanoms

Background: Nonviral gene delivery technologies are of increasing importance because of their easy application and the comparable low risk profile. The jet-injection technology allows efficient gene transfer of naked DNA and is based on generation of jets possessing the force to pass the skin and penetrate deeper tissues leading to transfection of the affected area. We established a phase-I-clinicaltrial to apply a LacZ-expressing reporter-plasmid by intratumoral jet-injection (DeReGe 62) to evaluate tolerance and feasibility as well as efficacy of the gene transfer. Here we present the results from the first patients. Methods: We used a jet-injector prototype (EMS Medical Systems, SA Nyon, Switzerland) and applied a pCMVβ-plasmid produced under GMP conditions. Between September 2005 and September 2006 14 patients with metastatic malignant melanoma or metastatic breast cancer were treated. All patients received 5 injections a 10μL of plasmid-DNA (1 μg/μL) into a single metastatic lesion. 72 to 96 hours after application the tumour lesion was surgically removed. In addition to the clinical and laboratory monitoring, the following molecular parameters were assessed: Detection of LacZ-expression at RNA-level by quantitative real-time PCR (qRT-PCR), and demonstration of LacZ-protein expression within the tissue (by X-Gal-staining and western blot), plasmidload within the tumour tissue by quantitative PCR (qPCR) and plasmid-clearance within the patients’ blood (30 min, 3, 6, 24, 48 and 72 hours after jet-injection). Results: All 14 patients were treated according to the protocol. No problems related to the technology and handling procedures were reported. Besides from very small bleeding signs at the injection sites, no adverse events or complications were observed during the whole treatment. The laboratory and clinical monitoring revealed no sign of systemic inflammation or any other significant findings. The treated lesions were removed and the pathologist confirmed the previous diagnoses (metastatic melanoma or breast cancer). Within all tumours plasmid-DNA (by qPCR) and LacZ-RNA (by qRT-PCR) was detected. We could prove protein expression in all tissues. The qPCR-analysis of the patients’ blood revealed a quick increase of plasmid-DNA 30 min. after jet-injection in all patients. Already 3 hours after injection, the clearance was finished and no plasmid-DNA was detected. Conclusion: The first results from this phase-I-clinical- trial clearly demonstrate the safety and feasibility of nonviral gene transfer. Within all treated tumours plasmid-DNA could be detected. This correlated well with the expression at RNA- and protein-level. The preliminary data from this trial is in accordance with previous animal experiments. For further therapeutic application increase of DNA-amount would be necessary to achieve better distribution and higher level of transgene expression. A new phase-I/II-trial for the therapeutic application of TNF-expressing-DNA has already been initiated.

Lokale nicht-virale Gentherapie in kutanen Metastasen von Mammakarzinom und Melanom: Ergebnisse einer Phase-I-Studie mittels Jet-Injektion nackter DNA

Background: This phase I clinical trial evaluated safety, feasibility and efficiency of nonviral intratumoral jet-injection reporter gene transfer in patients with skin metastases from melanoma and breast cancer. Methods: Seventeen patients were enrolled. The patients received five jet-injections of β-galactosidase (LacZ) expressing plasmid-DNA (pCMVβ) into a single cutaneous lesion. Clinical and laboratory safety monitoring were performed and systemic plasmid clearance was monitored by quantitative real-time PCR (qPCR) of blood samples throughout the study. All lesions were resected after two to six days. Intratumoral plasmid DNA-load, DNA-distribution and LacZ-expression was analyzed by qPCR, quantitative RT-PCR, Western-blot, immunohistochemistry and X-gal staining. Results: Jet-injection of plasmid-DNA was safely performed in all patients. No serious side effects were observed. Thirty minutes after jet-injection peak plasmid-DNA levels were detected in the blood followed by rapid decline and clearance. Plasmid-DNA, LacZ mRNA- and protein expression were detected in all treated lesions. Quantitative analysis revealed a correlation of plasmid-DNA load and LacZ-mRNA expression, confirmed by Western-blot. Immunohistochemistry and X-gal staining demonstrated LacZ-protein throughout the tumor. Transfected tumor areas were found close and distant to the jet-injection site with varying levels of DNA-load and transgene expression. Conclusion: Intratumoral jet-injection of plasmid-DNA led to an efficient LacZ-reporter gene expression in all patients. No side effects were experienced, supporting safety and applicability of this novel nonviral approach. Based on these results a next step with a therapeutic gene product should determine antitumor efficacy of jet-injection gene transfer.