Fusion of HepG2 cells with mesenchymal stem cells increases cancer­associated and malignant properties: an in vivo metastasis model.

In the present study, we have tested the hypothesis that fusion between an altered cell and a mesenchymal stem cell produces a hybrid cell with enhanced characteristics associated with metastatic cancer cells, and we have developed a flexible model for investigating the mechanisms of metastasis. Human HepG2 cells with low metastatic potential were induced to fuse with rat bone marrow mesenchymal stem cells, and the progeny were compared with the parental cells for possession of enhanced in vitro and in vivo characteristics of malignant cells. Compared to the parental cells, the fused cells exhibited enhanced expression of E-cadherin, vimentin, Twist, Snail, matrix metalloproteinase 2 and 9 activities, aneuploidy and enhanced in vitro invasion and migration. In an in vivo xenograft assay, the fused cells generated increased numbers of metastatic liver and lung lesions. This model system is a flexible tool for investigation of the mechanisms of stem cell fusion in carcinogenesis and metastasis and for the discovery of new therapeutic targets to inhibit metastasis.

Chimeric antigen receptors for stem cell based immunotherapy.

The retargeting of lymphocytes is an important new strategy in immunotherapy of cancer. One can currently isolate naturally refined, high affinity specificities from antibodies and T-cell receptors (TCRs) to use in engineered applications. We have developed two new molecules that have specificity for the overexpressed tumor antigen HER2/neu. The specificity derived from an anti-HER2 antibody variable fragment was used to create a single chain Fv (scFv). A HER2 reactive TCR was also used to develop a single chain TCR (scTCR). The HER2 binding elements were linked to an intracellular signaling module, active only in the T cell signaling pathway, providing a novel molecule to retarget lymphocytes. We demonstrate here that these molecules can be expressed in several cell lines as well as in hematopoietic stem cells (HSCs). In a transplant setting, these new receptors can be expressed in multiple cells types derived from repopulating HSCs. These new chimeric receptors will be valuable tools for further research of immune function of retargeted hematopoietic cells.

Direct sequencing is more accurate and feasible in detecting single nucleotide polymorphisms than RFLP: using human vascular endothelial growth factor gene as a model.

Since the sequencing of the human genome, there has been increased interest in understanding the distribution and effects of genetic variations among individuals. Restriction fragment length polymorphism (RFLP) is a well-established and frequently used method for genotyping. This method, however, is indirect and has a number of limitations. It is thus important to reevaluate the use of RFLP in light of more contemporary methods of genotyping. The specific aims of this study are to (a) compare genotyping methods of traditional RFLP with contemporary direct sequencing for accurate identification of polymorphisms within the human vascular endothelial growth factor (VEGF) gene and (b) describe distribution of a known single nucleotide polymorphism (SNP) in the VEGF gene in a sample composed of 50 healthy volunteers. Polymerase chain reaction (PCR) was used to amplify the initial sample of DNA. Genotypes of a G-to-A substitution (GG, AG, AA) at -1154 were analyzed by RFLP and direct sequencing. RFLP was unable to discriminate among the three possible genotypes, whereas direct sequencing clearly identified genotype for all 50 samples. Observed genotype frequencies were comparable with the Hardy-Weinberg principle. This comparative study provides justification for selecting direct sequencing instead of RFLP for detecting SNPs in selected genes.

Heat-inducible amplifier vector for high-level expression of granulocyte-macrophage colony-stimulating factor.

PURPOSE: In cytokine immunotherapy of cancer it is critical to deliver sufficiently high local cytokine concentrations in order to reach the therapeutic threshold needed for clinical efficacy. Simultaneously, for optimal clinical safety adverse effects caused by high systemic cytokine levels must be minimized. One of the most promising anti-cancer therapeutic cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF), has elicited anti-tumour immune responses in animal studies and clinical trials. However, the clinical efficacy has been limited, with local GM-CSF levels being therapeutically insufficient and systemic toxicity being a limiting factor. METHODS: To address these problems we have developed a novel GM-CSF expression vector, pAD-HotAmp-GM-CSF, which can provide high levels of GM-CSF expression, and induction of cytokine expression to limited tissue areas. This expression system combines inducible and amplifying elements in a single multi-genic construct. The first transcriptional unit contains the inducible element, the heat shock protein 70B (HSP70B) promoter that regulates expression of the transcription-activating factor tat. RESULTS: Upon the binding of tat to the second promoter, the HIV2 long terminal repeat amplifies downstream gene expression of the therapeutic cytokine GM-CSF. Moderate hyperthermia at 42 degrees C for 30 min induced GM-CSF expression in pAD-HotAmp-GM-CSF that was over 2.5- and 2.8-fold higher than levels reached with HSP70B promoter alone and the prototypical human cytomegalovirus promoter. CONCLUSIONS: Thus, the inducible amplifier vector, pAD-HotAmp-GM-CSF, represents a novel system for regulated and enhanced GM-CSF expression, which enables both greater efficacy and safety in cytokine immunotherapy of cancer.

Differential gene expression profiling of CD34+ CD133+ umbilical cord blood hematopoietic stem progenitor cells.

Umbilical cord blood (CB)-derived primitive hematopoietic stem progenitor cells (HSPC) are a promising source for stem cell-based gene therapy due to the reduced incidence and severity of graftversus- host disease (GVHD) after human leukocyte antigen (HLA)-disparate CB transplantation. Cell-surface markers such as CD34 and CD133 have been used in combination to enrich primitive HSPC for research and clinical applications. To understand the molecular characteristics of the CB HSPC, we compared the global gene expression of freshly isolated CB CD34+ CD133+ cells with their progenies using a cDNA microarray containing 22,000 human cDNA clones printed on a single chip. A total of 139 genes were differentially expressed between CB HSPC and their progenies. These transcripts included a number of known genes that might play roles in key functions of CB HSPC as well as many genes of unknown function. Among the genes showing the greatest differential expression levels in HSPC were: psoriasin 1, CRHBP, HDAC3, MLLT3, HBEX2, SPINK2, c-kit, H2BFQ, CD133, HHEX, TCF4, ALDH1A1, and FHL1. These data provide more information on the molecular phenotype of CB HSPC and may lead to the identification of new genes critical to stem cell function.

Antigen epitope-expressing cytokines for DNA immunization.

Strategies to enhance the efficacy of DNA vaccination against malignancy remain to be established. In this study, a plasmid expressing a tumor antigen incorporated into the signal peptide of human IL-2 was tested as a DNA vaccine in a murine model system. Results showed that antigen-specific CTL responses were elicited by intramuscular injection of these plasmids. Importantly, compared with a minigene vector expressing the same epitope, the OVA epitope-incorporated, IL-2 expression plasmid vaccination was more effective in protecting mice from OVA-expressing tumor challenge. The improved efficacy appears to result from enhanced antigen presentation as well as the immunostimulatory activity of IL-2. This approach may provide new perspectives in designing cytokine-adjuvant DNA vaccines for clinical applications.

Immuno-gene therapy of melanoma by tumor antigen epitope modified IFN-gamma.

Cytokine-based vaccines play a major part in tumor immuno-gene therapy. However, down-regulated antigen expression on tumor cells may diminish the immuno-potentiating aspects of cellular vaccines. In this study, we coexpressed a tumor antigen epitope with IFN-gamma in the same gene by replacing the IFN-gamma signal peptide with an antigen epitope-expressing signal peptide. We then investigated the effect of the antigen epitope-incorporated IFN-gamma on the immunotherapy of murine melanoma B16 tumors. Results showed that TRP-2 epitope-expressing IFN-gamma decreased B16 tumorigenicity and enhanced its immunogenicity after gene transfer. Protective immunity against wild type B16 tumors was induced by vaccination with IFN-gamma transiently gene-modified tumor cells. These data suggest that cellular vaccines engineered to express an antigen epitope within an immunostimulatory cytokine could potentiate the immunization effect.

A stem cell fusion model of carcinogenesis.

The origin of cancer remains enigmatic. Current models of carcinogenesis based on the gene mutation hypothesis have limitations in explaining many aspects of cancer. We put forward a new model of multistage carcinogenesis and propose that cancer development involves gene mutations and cell fusions. Specifically, cancer can result from a fusion between an "altered" pre-malignant cell and a bone marrow-derived stem cell (BMDSC). "Aneuploidy", which is a hallmark of malignancy, is a direct consequence of this cell fusion. The "stem cell fusion" model explains the remarkable similarities between malignant cells and BMDSC. This model also explains why non-mutagens can be carcinogens, and why non-mutagenic processes, such as wound healing and chronic inflammation, can promote malignant transformation. This model is readily testable. Cancer has been difficult to treat because of tissue heterogeneity and gene instability. However, if the malignant characteristics of cancer cells are derived from BMDSC, new conserved targets such as homing receptors for designing novel therapies may emerge.

Transgenic TCR expression: comparison of single chain with full-length receptor constructs for T-cell function.

Genetic modification of T lymphocytes with T-cell receptor (TCR) genes provides a novel tool for adoptive immunotherapy. However, the efficiency of full-length TCR (flTCR)-transduced T cells could be limited by factors such as incorrect pairing between exogenous and endogenous TCR chains and downregulation of the CD3 complex. To overcome these hurdles, one promising strategy is to use three-domain single-chain TCRs (3D-scTCR), in which TCR Valpha and Vbeta chains are joined by a linker with signal transduction domains fused at the carboxyl termini as signal transducers and amplifiers. Our results showed that surface expression of scTCRs on T cells after retroviral transduction was affected by the origin of the transmembrane (TM) region and placement of signaling domains. scTCR-modified T cells were functional as shown by cytokine (IL-2 and IFN-gamma) release in response to antigen stimulation and cytolytic activity against specific target cells. CD8 and CD28, but not the complete CD3 complex, could enhance the scTCR-induced T cell activation. Compared with flTCR-modified T cells and native CTLs, scTCR-modified T cells require higher thresholds of antigen stimulation (approximately 10(-8) M peptide) to be functional. Despite the low efficiency of scTCRs, our data provide insight into further improvements in generating efficient scTCRs for in vivo applications.

SING: a novel strategy for identifying tumor-specific, CTL-recognized tumor antigens.

Traditional methods for identifying T cell-recognized tumor antigens (Ags) are laborious and time-consuming. In an attempt to simplify the procedure, a novel strategy, SING (SIgnal transduction molecule-mediated, NFAT-controlled, GFP expression) was established as a direct approach for cloning T cell-recognized tumor Ags. In the SING system, a mouse T cell line (BW5147) was transduced with a chimeric H-2Kb construct containing T cell-signaling domains and a green fluorescent protein (GFP) reporter gene under the transcriptional control of nuclear factor of activated T cells (NFAT). The resultant BW5147 cells were named BS cells. This cell line could "sense" TCR stimulation through the T cell-signaling domains after coculture with Ag-specific T cells and then become fluorescent (expressing green fluorescence protein, GFP+) in the presence of Ag peptides. The interaction between BS cells and Ag-specific T cells could be enhanced by addition of costimulatory signals. Currently, BS cells have been optimized to "sense" TCR stimulation after being pulsed with the relevant peptides at concentrations as low as 10(-9) M. Endogenous Ag-expressing BS cells could also become fluorescent after coculture with Ag-specific T cells. Our results provide a proof of principle for using the SING system to directly isolate Ag-expressing BS cells from BS cell repertoires, which are retrovirally transduced with tumor-derived cDNA libraries. Once tumor Ag-marked BS cells are identified, the sequences encoding tumor Ags could be easily retrieved by PCR amplification of the genomic DNA using vector-specific primers.

A novel inducible amplifier expression vector for high and controlled gene expression.

Safety and efficacy are both required for successful gene therapy. In this regard, our laboratory has created a novel expression system, pHi-Hot that combines inducible and amplifier strategies in one construct. In pHi-Hot, the first transcriptional unit contains an inducible heat shock protein (hsp70B) promoter controlling the expression of a transcriptional factor, Tat, which transactivates a second promoter, the HIV2 LTR, located downstream on the same construct. The second promoter drives the gene of interest. The magnitude of the amplified second gene expression can be regulated through manipulating the activity of the hsp promoter driving the Tat gene. Using the human interleukin-2 (IL-2) cytokine gene as the reporter gene, we demonstrated that moderate heat shock at 42 degrees C for 30 min, the pHi-Hot vector could achieve high gene expression levels while maintaining its inducibility. The induced IL-2 levels were 35- to 70-fold higher than achieved by using the hsp promoter alone, and 10- to 35-fold higher than achieved by using the CMV promoter. Using inducible and amplifier strategies, we can achieve high and controlled gene expression levels from a single construct. Finally, we discuss the advantages of using these strategies in developing new targeting and inducible vectors for genetic research and gene therapy.

Enhanced tumor immunogenicity through coupling cytokine expression with antigen presentation.

The density of tumor antigen in conjunction with major histocompatibility complex (MHC) class I molecules on the cell surface affects cytotoxic T cell (CTL) function in an active antitumor immune response. Thus, methods to enhance antigen expression/presentation could augment the effect of cancer immune therapy. In the present study, we investigated the feasibility of modifying a cytokine signal peptide with a tumor antigenic epitope. We inserted the genes encoding the MHC class I-restricted antigenic epitope of chicken ovalbumin and tyrosinase-related protein 2 into the signal sequence of the interleukin-2 gene, replacing part of the signal sequence at different positions. Our results showed that these modified signal peptides still functioned, as indicated by cytokine secretion. The antigenic epitope within the modified signal peptide could be processed properly and presented on tumor cell surface. Tumor cells demonstrated enhanced immunogenicity as indicated by increased susceptibility to CTL lysis in vitro and decreased tumor grow in vivo after gene modification. These data provide potential perspectives in designing therapeutic or vaccine strategies in immuno-gene therapy of cancer.

Efficient transduction of murine primary T cells requires a combination of high viral titer, preferred tropism, and proper timing of transduction.

Retroviral vectors have been used exclusively for genetic modification of primary T cells. Most T cell infection protocols have been developed for human T cells, whereas systematic investigations of the optimal conditions for transduction of murine primary T cells are limited. In this study, ecotropic and 10A1-pseudotyped retroviral vectors were compared for their efficiency in infecting murine primary T cell cells, as well as T cell lines. Various factors that affect transduction efficiency were also explored, including virus titer, times of exposure, timing of infection, low-speed centrifugation, and use of fibronectin fragment. Our results showed that up to 80% of murine primary T cells could be infected after a single exposure. Successful infection required a combination of high virus titer (>10(7) CFU/ml), proper timing of infection (within 24 h after mitogen stimulation), and preferred tropism (ecotropic vectors). These optimization results may help to establish a standard protocol for infection of murine primary T cells and provide some insight into the obstacles to retroviral infection of T cells.

Comparison of cis and trans tat gene expression in HIV LTR-based amplifier vectors.

The long terminal repeat (LTR) of the human immunodeficiency virus (HIV) drives highly efficient gene expression in the presence of the transactivator, Tat. Thus, tat-containing vectors may be very useful tools in gene therapy. However information about the optimal way of delivering the tat gene is limited. In this study, we compared the effects of cis and trans expressions of the tat gene and its effects on HIV LTR-driven gene expression in different cell lines using non-viral vectors. The human interleukin-2 (IL-2) gene was used as a reporter gene under the control of the HIV2 LTR (pHIV2-IL-2). The tat gene, driven by a cytomegalovirus (CMV) promoter, was either co-transfected separately (pCMV-Tat) or inserted downstream of the IL-2 gene (pHIV2-IL-2-neo-C-Tat). Our results showed that HIV2 LTR-Tat-based vectors were much more potent than CMV promoter-based vectors in transient expression. The co-transfection of both plasmids was comparable to a single transfection of pHIV-IL-2-neo-C-Tat in both high and low transfection efficiency cells. In conclusion, the co-placement of HIV2 LTR and tat genes on a single plasmid allows for gene expression as efficiently as a two-plasmid system, suggesting that HIV2 LTR-Tat-based vectors may be attractive tools for gene therapy.