Genetically engineered mesenchymal stromal cells in cancer gene therapy.

Based on our experimental data, we aimed to emphasise the perspectives of the use of mesenchymal stromal cells (MSC) in the cancer gene therapy. On the other hand, we would like to point out factors which should be taken into consideration at their clinical use. In this review we define MSC as unique targets for targeted therapy. We proved the efficacy of experimental therapeutic approach utilising enzymatic conversion of non-toxic prodrug into chemotherapeutic by engineered MSC, and we observed significant cytotoxic effect in many preclinical models including metastatic disease. Treatment was enabled by affinity of MSC to tumour tissue and subsequent delivery of therapeutic molecule into the tumour. We also observed decreased efficacy of cell-mediated gene therapy on chemoresistant tumour cells. Moreover MSC can exert a supportive effect on tumour cells as well as to decrease the efficacy of conventional treatment. Besides obvious unique benefits connected to the use of MSC we pointed also to possible risks associated with their clinical application (Ref. 24).

Exosomes of human mesenchymal stem/stromal/medicinal signaling cells.

In this review, we intend to explore the potential therapeutic effects of exosomes released from mesenchymal stem/stromal cells (MSCs). MSCs gained credibility as a therapeutic tool due to their potential to differentiate into many cell types like osteoblasts, chondrocytes, adipocytes, muscular, endothelial, cardiovascular, and neurogenic cells. They possess potent wound healing activity due to their immunosuppressive and anti-inflammatory properties. MSCs are tested in large number of clinical trials for treatment of diseases, which do not have adequate therapy at present. MSCs engineered to express suicide genes in preclinical studies have shown promising tumor targeting therapeutic tool for malignancies difficulty treatable at present. It has been increasingly observed in many different kinds of regenerative medicine and in MSCs mediated prodrug gene therapy for cancer that the intravenously administered of MSCs did not necessarily engraft at the site of injury or tumor. The therapeutic effect was exerted mainly through a paracrine action of rich secretome released from the cells. The main biocomponent of secretome are exosomes - naturally occurring membrane nanoparticles of 30-120 nm in diameter that mediate intercellular communication by delivering biomolecules like mRNA, miRNA into recipient cells. These nanosized exosomes derived from MSCs promise to be a new and valuable therapeutic strategy in regenerative medicine and cancer therapy compared with transplanted exogenous MSCs. Advantage of nanosized exosomes compared with administration of exogenous MSCs is multiple. Exosomes are easier to preserve and be transferred, have lower immunogenicity and therefore are safer for therapeutic administration.

Dental pulp mesenchymal stem/stromal cells labeled with iron sucrose release exosomes and cells applied intra-nasally migrate to intracerebral glioblastoma.

We report on a simple iron oxide (Venofer) labeling procedure of dental pulp mesenchymal stem cells (DP-MSCs) and DP-MSCs transduced with yeast cytosinedeaminase::uracilphosphoribosyltransferase (yCD::UPRT-DP-MSCs). Venofer is a drug approved for intravenous application to treat iron deficiency anemia in patients. Venofer labeling did not affect DP-MSCs or yCD::UPRT-DP-MSCs viability and growth kinetics. Electron microscopy of labeled cells showed internalized Venofer nanoparticles in endosomes. MRI relativity measurement of Venofer labeled DP-MSCs in a phantom arrangement revealed that 100 cells per 0.1 ml were still detectable. DP-MSCs or yCD::UPRT-DP-MSCs and the corresponding Venofer labeled cells release exosomes into conditional medium (CM). CM from yCD::UPRT-DP-MSCs in the presence of a prodrug 5-fluorocytosine caused tumor cell death in a dose dependent manner. Iron labeled DP-MSCs or yCD::UPRT-DP-MSCs sustained their tumor tropism in vivo; intra-nasally applied cells migrated and specifically engrafted orthotopic glioblastoma xenografts in rats.

Stem cell based glioblastoma gene therapy.

There is no curative therapy for glioblastoma multiforme (GBM) thus far. Combined therapies including surgery, followed by concomitant irradiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ), slightly improves patients' survival but the prognosis remains poor. The fatal nature of glioblastoma is caused by tumor-initiating glioblastoma cells. The tumor tropic ability of adult mesenchymal stem cells offers the attractive possibility to use these cells as a vehicle to deliver therapeutic agents to the site of the tumor. In preclinical studies using animal models, mesenchymal stem cells engineered to express suicide genes were shown to elicit a significant antitumor response against various tumors including glioblastoma. This review summarizes the current state of knowledge about stem cell directed glioblastoma therapy. Results obtained in a preclinical study using mesenchymal stem cells engineered to express cytosine deaminase provided evidence that stem cell based gene therapy might also attack glioblastoma stem cells and therefore be curative. In addition to stem cell directed prodrug gene therapies, other immunotherapeutic modalities using mesenchymal stem cells are discussed as well. Encouraging results of preclinical studies of stem cell based gene therapy for glioblastoma support the argument to begin clinical studies.

Genotoxic damage of human adipose-tissue derived mesenchymal stem cells triggers their terminal differentiation.

Human adipose tissue-derived mesenchymal (stromal) stem cells (AT-MSCs) and genetically modified to express cytosine deaminase:uracil phosphoribosyltransferase (CDy-AT-MSCs) were treated with hydrogen peroxide in order to induce DNA damage and subsequently evaluate their genetic stability by single cell gel electrophoresis. Both cells types (parental and transgene modified) did not differ in the sensitivity to DNA breaks induction. Potential tumorigenicity of AT-MSCs and CDy-AT-MSCs was tested by subcutaneous inoculation of cell suspension into flank of immunocompromised mice. Dose of 15x10(6) cells was not found to be tumorigenic in given experimental setup. AT-MSCs, CDy-AT-MSCs and MSCs isolated from human lipoma were treated with chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) in attempts to transform them. Surviving cells after genotoxic stress were not transformed but underwent replicative senescence. Irreparable DNA damage caused triggered adipogenic terminal differentiation, rather than apoptosis induction in all kinds of cells tested.

Modelling polymer interactions of the 'molecular Velcro' type in wood under mechanical stress.

Trees withstand wind and snow loads by synthesising wood that varies greatly in mechanical properties: flexible in twigs and in the stem of the sapling, and rigid in the outer part of the mature stem. The 'molecular Velcro' model of Keckes et al. [2003. Cell-wall recovery after irreversible deformation of wood. Nat. Mater. 2, 810-814] permits the simulation of the tensile properties of water-saturated wood as found in living trees. A basic feature of this model is the presence of non-covalent interactions between hemicellulose chains attached to adjacent cellulose microfibrils, which are disrupted above a threshold level of interfibrillar shear. However, other evidence does not confirm the importance of hemicellulose-hemicellulose association in the cohesion of the interfibrillar matrix. Here, we present an alternative model in which hemicellulose chains bridging continuously from one microfibril aggregate (macrofibril) to the next provide most of the cohesion. We show that such hemicellulose bridges exist and that the stripping of the bridging chains from the cellulose surfaces under the tensile stress component normal to the macrofibrils can provide an alternative triggering mechanism for shear deformation between one macrofibril and the next. When one macrofibril then slides past another, a domain of the wood cell wall can extend but simultaneously it twists until the spacing between macrofibrils is reduced again and contact through hemicelluloses bridges is restored. Overall deformation therefore takes place through a series of local stick-slip events involving temporary twisting of small domains within the wood cell wall. Modelled load-deformation curves for this modified 'molecular Velcro' model are similar, although not identical, to those for the original model. However, the mechanism is different and more consistent with current views of the structure of wood cell walls, providing a framework within which the developmental control of rigidity in wood synthesised in different parts of a tree may be considered.

In vitro analysis of cisplatin functionalized magnetic nanoparticles in combined cancer chemotherapy and electromagnetic hyperthermia.

A novel platform has been developed for combined cancer chemotherapy and hyperthermia based on iron oxide magnetic nanoparticles functionalized with cis-diamminedichloroplatinum(II) (cisplatin). The capabilities of this system for heating and controlled drug release were investigated, and the system was tested in vitro by the treatment of BP6 rat sarcoma cells, where we demonstrated a synergism between the effects of cisplatin-targetMAG nanoparticles and the application of electromagnetic field.

Glioblastoma and stem cells.

This review presents compelling evidence that human glioblastoma is a heterogenous tumor composed from tumor cells and small portion of cancer stem cells -- tumor-initiating cells, which have a high tumorigenic potential and a low proliferation rate. Glioma cancer stem cells are phenotypically similar to the normal stem cells, they express CD133 gene and other genes characteristic of neural stem cells and posses the self-renewal potential. Cancer stem cells derived from glioblastoma are capable recapitulate original polyclonal tumors when xenografted to nude mice. They are chemoresistant and radioresistant and therefore responsible for tumor progression and recurrence after conventional glioblastoma therapy. Cancer stem cells contribute to glioma radioresistance by an increase of DNA repair capacity through preferential activation of the DNA damage response checkpoints. Potential therapies that modulate or target cancer stem cells are also reviewed. Mesenchymal stem cells and/or neural stem cells were shown to target brain tumors therefore these cells are considered as an effective delivery system to target and disseminate therapeutic agents to brain tumors. Stem cell-based gene therapies for glioblastoma were shown in experiments to be effective way to target brain tumors. Effects of bone morphogenetic protein (BMP4) on glioma cancer stem cells are also reviewed. BMP4 reduces effectively proliferation of CD133 positive cells in vitro and the tumor growth in vivo. BMP4 may act as a key inhibitory regulator of cancer initiation and therefore may be used in combined stem cell-based therapy as a non-cytotoxic therapeutic agent.

Presence of serum carbonic anhydrase autoantibodies in patients relapsed after autologous stem cell transplantation indicates an improved prognosis.

Here we report patients with Hodgkin's disease and multiple myeloma, who relapsed/progressed after high dose therapy and autologous stem cell transplantation. In patients who developed aplastic anemia type syndrome, spontaneous tumor regression was observed and concomitantly high titers of serum autoantibodies were found. In order to identify the antibody specificity, two-dimensional electrophoresis, blotting and immunoreactions were used to analyze the peripheral blood stem cell extract with autoantibodies containing serum. The unique protein spot visualized exclusively by serum of patients with aplastic anemia type syndrome was identified as carbonic anhydrase I (CA I, accession No. P00915 and Q7M316) by means of mass spectrometry. The specificity of autoantibodies was confirmed by reaction with commercial CAs I, II, IX and XII. Immunoreaction in Western blots with these CA isoforms differed in sera obtained from patients with various types of the disease. Sera of Hodgkin's disease patients reacted with CA I, II and XII; sera of multiple myeloma patients reacted with the CA I, II, XII and IX. Patients developing and/or possessing CA autoantibodies had a significant survival benefit over those who did not develop CA anhydrase autoantibodies. Possible relevance of the presence of CA autoantibodies and clinical outcome is discussed.

Human mesenchymal stem cell-derived iron oxide exosomes allow targeted ablation of tumor cells via magnetic hyperthermia.

Magnetic hyperthermia, or the heating of tissues using magnetic materials, is a promising approach for treating cancer. We found that human mesenchymal stem cells (MSCs) isolated from various tissues and MSCs expressing the yeast cytosine deaminase∷uracil phosphoribosyl transferase suicide fusion gene (yCD∷UPRT) can be labeled with Venofer, an iron oxide carbohydrate nanoparticle. Venofer labeling did not affect cell proliferation or the ability to home to tumors. All Venofer-labeled MSCs released exosomes that contained iron oxide. Furthermore, these exosomes were efficiently endocytosed by tumor cells. Exosomes from Venofer-labeled MSCs expressing the yCD∷UPRT gene in the presence of the prodrug 5-fluorocytosine inhibited tumor growth in a dose-dependent fashion. The treated tumor cells were also effectively ablated following induction of hyperthermia using an external alternating magnetic field. Cumulatively, we found that magnetic nanoparticles packaged into MSC exosomes are efficiently endocytosed by tumor cells, facilitating targeted tumor cell ablation via magnetically induced hyperthermia.

Novel germline mutation in the transmembrane region of RET gene close to Cys634Ser mutation associated with MEN 2A syndrome.

Two mutations on the same allele of RET gene were revealed in a family with predisposition to multiple endocrine neoplasia (MEN) type 2A. The first mutation changes codon 634 from cysteine to serine. The second, a novel mutation in codon 641, changes alanine to serine in the transmembrane domain of the RET protein. Two mutations were present in close proximity in both the patients' germline and tumor DNA and were absent in DNA isolated from healthy family members and control blood donors. All MEN 2A affected family members suffered from medullary thyroid carcinoma and two of ten patients for pheochromocytoma. No parathyroid gland alterations were observed in patients with two RET gene mutations. Analysis of four genetic polymorphisms in the RET gene showed higher incidence of polymorphisms of exons 11 and 15. The observed allelic imbalance in favor of mutated allele in pheochromocytoma corresponded to higher expression of the RET gene. These observations confirm the multifactorial process leading to development of MEN 2A syndrome.

Treatment of human tumor cells by combine gene therapy harnessing plasmids expressing human tumor necrosis factor alpha and bacterial cytosine deaminase suicide gene.

We have assessed the effect of combine cancer gene therapy with exogenous human tumor necrosis factor alpha (hTNFalpha) and Escherichia coli cytosine deaminase (CD) suicide gene on two human breast adenocarcinoma cell lines MDA-MB-361 and SK-BR-3. Transfection of a plasmid containing hTNFalpha under the control of a hybrid promoter resulted in expression of hTNFalpha gene in vitro. Transduction of retroviral plasmid containing bacterial cytosine deaminase led to the expression of cytosine deaminase in adenocarcinoma cell lines as well. The significant increase in apoptotic cells and decrease of cell proliferation in both tumor cell lines was observed using combination treatment with hTNFalpha expression plus CD/5-FC suicide system. Corresponding data were generated by MTT cell proliferation assay and by flow cytometric analysis. The presence of both genes after transduction of retroviral vector containing CD and transfection of hTNFalpha plasmid was confirmed by PCR and RT-PCR. The additive cell killing effect due to the presence of bacterial CD and hTNFalpha gene after activation of non-toxic prodrug was observed. Whether the bicistronic vector containing both therapeutic genes improve the therapy need to be assessed in the future.

Combine cancer gene therapy harnessing plasmids expressing human tumor necrosis factor alpha and Herpes simplex thymidine kinase suicide gene.

We have assessed the effect of combine cancer gene therapy with exogenous human tumor necrosis factor alpha (hTNFalpha) and suicide gene therapy on three human cancer cell lines MCF-7 (breast adenocarcinoma), U-118MGand 42-MG-BA (human gliomas). Transfection of a plasmid containing hTNFalpha under the control of a hybrid promoter resulted in expression of hTNFalpha gene in vitro. Transduction of retroviral plasmid containing Herpes simplex thymidine kinase (HSVtk) led to the expression of thymidine kinase in all three cell lines. MTT cell proliferation assay and flow cytometric analysis showed a significant increase in apoptotic and necrotic cells and decrease of proliferation in all cell lines after combine therapy with hTNFalpha expression plus thymidine kinase/GCV suicide system. The presence of these two genes after transduction of retroviral vector containing thymidine kinase and hTNFalpha was confirmed by PCR. The expression of HSVtk gene was proved by Western blot analysis, and the expression of both genes was confirmed by RT-PCR. Additive cell killing effect due to presence of HSVtk and hTNFalpha therapeutic genes after activation of non-toxic prodrug was observed. Whether the bicistronic plasmid containing both genes would improve the therapeutic effect need to be assessed in the future.

Suppression of the avian sarcoma virus genome in 8-azaquanine-resistant, transformed, hamster cells.

The avian sarcoma virus genome (Schmidt-Ruppin strain) in transformed hamster cells resistant to 8-azaquanine [Ha(SR)AG-50] was strongly suppressed. The suppression was genetically stable and could not be overcome by attempts at induction with 5-iodo-2'-deoxyuridine. Fusion of hamster cells, which had suppressed virus genome, with chicken Rous-associated virus (RAV-1)-preinfected cells easily rescued the sarcoma virus. The rescued virus had envelope properties of RAV-1, as determined by viral interference, virus neutralization, and plating on genetically resistant chicken cells. By repeatedly cloning the rescued virus, we determined that virus recombined in the rescue experiment and that the recombinant virus had the envelope properties of helper virus used for its rescue. Cells with suppressed avian sarcoma virus genome were suitable for preparation of different recombinant viruses.

Pathogenicity for rats of a strain of chicken sarcoma virus.

The RBI rat tumor, induced with cell suspensions from chicken sarcoma B77, is pathogenic for chicks as well as for rats. Cell suspensions from RBI tumors induced sarcomas in 100 percent of inoculated chicks. Cell suspensions of this chicken sarcoma induced early RBA sarcomas in 50 percent of 171 rats. Most of these rats died within 4 weeks after inoculation. The early tumors regressed in 15 of the 85 tumor-bearing rats, and the animals died of cystic hemorrhagic disease. The sarcoma was induced in 9 animals within 50 to 70 days after inoculation, and cystic hemorrhagic disease developed in 117. None of the 171 rats remained free from either tumor or cyst and only 12 survived for 3 months or longer. The tumors induced in rats were transplantable into rats and after transplantation of early-appearing tumors, tumors and cysts developed. Virus strongly infective in chicks was demonstrated by cell-free filtrates and virus preparations from RBI and RBA rat sarcomas induced by chicken sarcoma cells. Cell suspensions from the wall of small cysts induced tumors in chicks and cystic hemorrhagic disease and tumors in rats.

Cancer stem cells.

There is an increasing evidence supporting the cancer stem cell hypothesis. Normal stem cells in the adult organism are responsible for tissue renewal and repair of aged or damaged tissue. A substantial characteristic of stem cells is their ability for self-renewal without loss of proliferation capacity with each cell division. The stem cells are immortal, and rather resistant to action of drugs. They are able to differentiate and form specific types of tissue due to the influence of microenvironmental and some other factors. Stem cells divide asymmetrically producing two daughter cells -- one is a new stem cell and the second is progenitor cell, which has the ability for differentiation and proliferation, but not the capability for self-renewal. Cancer stem cells are in many aspects similar to the stem cells. It has been proven that tumor cells are heterogeneous comprising rare tumor initiating cells and abundant non-tumor initiating cells. Tumor initiating cells -- cancer stem cells have the ability of self-renewal and proliferation, are resistant to drugs, and express typical markers of stem cells. It is not clear whether cancer stem cells originate from normal stem cells in consequence of genetic and epigenetic changes and/or by redifferentiation from somatic tumor cells to the stem-like cells. Probably both mechanisms are involved in the origin of cancer stem cells. Dysregulation of stem cell self-renewal is a likely requirement for the development of cancer. Isolation and identification of cancer stem cells in human tumors and in tumor cell lines has been successful. To date, the existence of cancer stem cells has been proven in acute and chronic myeloid leukemia, in breast cancer, in brain tumors, in lung cancer and gastrointestinal tumors. Cancer stem cell model is also consistent with some clinical observations. Although standard chemotherapy kills most cells in a tumor, cancer stem cells remain viable. Despite the small number of such cells, they might be the cause of tumor recurrence, sometimes many years after the "successful" treatment of primary tumor. Growth of metastases in distinct areas of body and their cellular heterogeneity might be consequence of cancer stem cell differentiation and/or dedifferentiation and asymmetric division of cancer stem cells. Further characterization of cancer stem cells is needed in order to find ways to destroy them, which might contribute significantly to the therapeutic management of malignant tumors.

Tumor targeted gene therapy with plasmid expressing human tumor necrosis factor alpha in vitro and in vivo.

We have assessed the effect of exogenous human tumor necrosis factor alpha (hTNFalpha) in three human cancer cell lines; MDA-MB-361 (breast adenocarcinoma), HCT 116 (colon carcinoma) and 8-MG-BA (glioma). In vitro transfection of a plasmid containing hTNFalpha under the control of a hybrid promoter resulted in expression of hTNFalpha gene in all three cell lines and secretion into the culture medium was seen with MDA-MB-361 cells. Flow cytometric analysis showed a significant increase in apoptotic and necrotic cells in MDA-MB-361 and to a lesser extent in HCT 116 cells. Increased apoptosis was confirmed by an increase in pro-caspase 3 activation. No effects of hTNFalpha expression were seen in 8-MG-BA cells. Intratumoral delivery of the hTNFalpha expression plasmid into MDA-MB-361 tumor xenografts grown in nude mice caused hemorrhagic tumor necrosis. This strategy may be a simple and promising gene therapy approach to the treatment of some human tumors.

Induction of immune deficiency syndrome in rabbits by bovine leukaemia virus.

Newborn rabbits were inoculated with bovine leukaemia virus (BLV). The majority of infected rabbits produced antiviral antibodies. All the seroconverted animals developed symptoms resembling AIDS and died several months after inoculation. The course of experimental infection of rabbits with BLV resembled in many respects the broad spectrum of clinical disorders associated with AIDS induced by HIV. Antibody response to virus proteins was followed by immune deficiency and signs of neuropathy, and the animals subsequently died of opportunistic infections. Virus transmission from infected babies to the mothers by contact was also observed. In some cases the virus was salvaged from lymphocytes of rabbits with the immune deficiency syndrome. The virus-specific sequences were found to be integrated at random in the DNA of haematopoietic cells and of some organs. Slight expression of viral RNAs in lymphocytes was found. Experimental infection of rabbits with BLV can be used in experiments to understand AIDS induction.

Modifications of hybridoma technology which improve the yield of monoclonal antibody producing cells.

Several modifications at various stages of the standard hybridoma technique were found to increase the yield of monoclonal antibody-producing cells. Lymphocytes obtained from draining lymph nodes of mice immunized over a 10 day period with antigen injected into the foot pads were used for cell fusion. Preincubation of myeloma cells with lymphocytes in the presence of 0.25% polyethylene glycol at 37 degrees C for 90 min increased the yield of antibody-secreting hybrid colonies ten times. The use of conditioned medium from cultivated rat thymocytes ('lymphokines') as a supplement to cultivation medium made it unnecessary to use feeder cells, and increased the growth rate of the hybridomas. No change of the culture fluid was needed during the time which was necessary to grow up the cells to be tested for monoclonal antibody production. By a combination of the described procedures, the time required from the start of immunization to the screening for positive hybridomas was shortened to 23 days.

Two immunodominant regions revealed by monoclonal antibodies on the main structural protein p24 of bovine leukemia virus.

Eleven different monoclonal antibodies (Mabs) directed against the main structural protein p24 of bovine leukemia virus (BLV) were prepared. All Mabs reacted with p24 in Western blot and in radioimmunoprecipitation. Competition antibody binding assays with the prepared Mabs distinguished three independent groups of Mabs. Two immunodominant regions (IDRs) of p24 BLV were defined by these Mabs. The Mabs were induced preferentially against two immunodominant regions on the native form of p24 BLV (BLVp24 IDR-1 and BLVp24 IDR-2). Mab of the third group was directed against a different immunogenic epitope of p24 BLV. A model of the IDRs based on the differences in the fine epitope specificity of Mabs defining these immunodominant regions is proposed.