A biophysical approach to the optimisation of dendritic-tumour cell electrofusion

Electrofusion of tumour and dendritic cells (DCs) is a promising approach for production of DC-based anti-tumour vaccines. Although human DCs are well characterised immunologically, little is known about their biophysical properties, including dielectric and osmotic parameters, both of which are essential for the development of efficient electrofusion protocols. In the present study, human DCs from the peripheral blood along with a tumour cell line used as a model fusion partner were examined by means of time-resolved cell volumetry and electrorotation. Based on the biophysical cell data, the electrofusion protocol could be rapidly optimised with respect to the sugar composition of the fusion medium, duration of hypotonic treatment, frequency range for stable cell alignment, and field strengths of breakdown pulses triggering membrane fusion. The hypotonic electrofusion consistently gave a tumour-DC hybrid rate of up to 19%, as determined by counting dually labelled fluorescent hybrids in a microscope. This fusion rate is nearly twice as high as that usually reported in the literature for isotonic media. The experimental findings and biophysical approach presented here are generally useful for the development of efficient electrofusion protocols, especially for rare and valuable human cells.     

Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device

The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro.     

Electrofusion of a weakly immunogenic neuroblastoma with dendritic cells produces a tumor vaccine.

The absence of surface costimulatory molecules explains in part the lack of an effective anti-tumor immune response in tumor-bearing animals, even though unique tumor antigens may be presented by class I MHC. We determined that the immunogenicity of a murine neuroblastoma, Neuro-2a, which lacks surface costimulatory molecules, could be increased by electrically induced fusion with dendritic cells. Electrofusion induced a higher level of cell fusion than polyethylene glycol, and tumor/dendritic cell heterokaryons expressed high levels of costimulatory molecules. While Neuro-2a was unable to induce the proliferation of syngeneic or allogeneic T cells in vitro, fused cells were able to induce T cell responses both in vitro and in vivo. When fused dendritic tumor cells were used as a cancer vaccine, immunized mice were significantly protected from challenge with Neuro-2a. We propose that electrofusion with patient-derived tumor and dendritic cells may provide a rapid means to produce patient-specific tumor vaccines.     

Generation of dendritic cell–tumor cell hybrids by electrofusion for clinical vaccine application

Vaccination with hybrids comprising fused dendritic cells (DCs) and tumor cells is a novel cancer immunotherapy approach designed to combine tumor antigenicity with the antigen-presenting and immune-stimulatory capacities of DCs. For clinical purposes, we have incorporated a large-scale process for the generation of clinical-grade DCs together with novel electrofusion technology. The electrofusion system provides for ease and standardization of method, efficient DC–tumor cell hybrid formation, and large-quantity production of hybrids in a high-volume (6-ml) electrofusion chamber. In addition, we have evaluated DC electrofusion with a variety of allogeneic human tumor cell lines with the rationale that these tumor cell partners would prove a ready, suitable source for the generation of DC–tumor cell hybrid vaccines. The DC production process can generate 6×10⁸ to 2×10⁹ DCs from a single leukapheresis product (~180 ml). As determined by FACS analysis, electrofusion of 6×10⁷ total cells (1:1 ratio of DC and tumor cells) resulted in a consistent average of 8–10% DC–tumor cell hybrids, irrespective of the tumor type used. Hybrids were retained in the population for 48 h postfusion and following freezing and thawing. Upon pre-irradiation of the tumor cell partner for vaccine purposes, the overall fusion efficiency was not altered at doses up to 200 Gy. Evaluation of DC–tumor cell hybrid populations for their ability to stimulate T-cell responses demonstrated that electrofused populations are superior to mixed populations of DCs and tumor cells in generating a primary T-cell response, as indicated by IFN- release. Moreover, hybrids comprising HLA-A^*0201 DCs and allogeneic melanoma tumor cells (Colo 829 cell line) stimulated IFN- secretion by antigen-specific CD8⁺ T cells, which are restricted for recognition of a melanoma gp100 peptide antigen (gp100_209–217) within the context of the DC HLA haplotype. Maturation of the DC-Colo 829 cell hybrid population served to further improve this T-cell gp100-specific response. Overall, our results are promising for the large-scale generation of electrofused hybrids comprising DCs and allogeneic tumor cells, that may prove useful in human vaccine trials.     

Separation of apoptotic cells using a microfluidic device

A highly sensitive microfluidic device has been developed to separate apoptotic cells. Apoptotic Jurkat cells were selectively labeled with magnetic beads (0.8 μm diam) using the C2A protein which recognizes phosphatidylserine. The cell mixture was flowed through a microfluidic channel and apoptotic cells were separated by a 0.3 T permanent magnet. Separations using our device showed 96% agreement with those of a commercial flow cytometer, indicating our device can be used to sort apoptotic cells in a miniaturized system.     

A new genetic method to generate and isolate small,short-lived but highly potent dendritic cell-tumor cell hybrid vaccines

Fusion of tumor cells with antigen-presenting cells (APCs) has been proposed for the preparation of cancer vaccines. However, generation of these hybrids, using physical or chemical methods such as electrofusion or polyethylene glycol (PEG), has been difficult to standardize. Characterization of cell fusion has also been problematic because of difficulties in differentiating fusion from cell aggregation, leakage of cellular dyes and dendritic-cell (DC) phagocytosis of tumor material. In this report, we describe a new method to generate hybrid cell vaccines, based on gene transfer of a viral fusogenic membrane glycoprotein (FMG) into tumor cells, and incorporate a genetic method by which true hybrid formation can be unambiguously detected. We describe a new class of tumor cell-DC hybrid that can be rapidly isolated after cell fusion. These hybrids are highly potent in in vitro antigen presentation assays, target lymph nodes in vivo and are powerful immunogens against established metastatic disease.     

Therapeutic immune response induced by electrofusion of dendritic and tumor cells

To elicit a therapeutic antitumor immune response, dendritic cells (DCs) have been employed as a cellular adjuvant. Among various DC-based approaches, fusion of DCs and tumor cells potentially confers not only DC functionality, but also a continuous source of unaltered tumor antigens. We have recently demonstrated successful generation of fusion hybrids by a large-scale electrofusion technique. The immunogenicity and therapeutic potential of fusion hybrids were further analyzed in a model system of a murine melanoma cell line expressing beta-galactosidase (beta-gal) as a surrogate tumor antigen. A single vaccination with fusion hybrids plus IL-12 induced a therapeutic immune response against 3-day established pulmonary metastases. This immunotherapy was beta-gal specific and involved both CD4 and CD8 T cells. In vitro, fusion hybrids stimulated specific IFN-gamma secretion from both CD4 and CD8 immune T cells. They also nonspecifically induced IL-10 secretion from CD4 but not CD8 T cells. Compared to other DC loadings, our results demonstrate the superior immunogenicity of fusion. The current technique of electrofusion is adequately developed for clinical use in cancer immunotherapy.     

In vivo cell electrofusion

In vitro electrofusion of cells brought into contact and exposed to electric pulses is an established procedure. Here we report for the first time the occurrence of fusion of cells within a tissue exposed in vivo to permeabilizing electric pulses. The dependence of electrofusion on the ratio of applied voltage to distance between the electrodes, and thus on the achievement of in vivo cell electropermeabilization (electroporation) is demonstrated in the metastasizing B16 melanoma tumor model. The kinetics of the morphological changes induced by cell electrofusion (appearance of syncytial areas or formation of giant cells) are also described, as well as the kinetics of mitosis and cell death occurrence. Finally, tissue dependence of in vivo cell electrofusion is reported and discussed, since electrofusion has been observed neither in liver nor in another tumor type. Particular microenvironmental conditions, such as the existence of reduced extracellular matrices, could be necessary for electrofusion achievement. Since biomedical applications of in vivo cell electropermeabilization are rapidly developing, we also discuss the influence of cell electrofusion on the efficacy of DNA electrotransfer for gene therapy and of antitumor electrochemotherapy, in which electrofusion could be an interesting advantage to treat metastasizing tumors.     

Human tumour and dendritic cell hybrids generated by electrofusion: potential for cancer vaccines

Hybrid cells created by fusion of antigen presenting and tumour cells have been shown to induce potent protective and curative anti-tumour immunity in rodent cancer models. The application of hybrid cell vaccines for human tumour therapy and the timely intervention in disease control are limited by the requirement to derive sufficient autologous cells to preserve homologous tumour antigen presentation. In this study, the efficiency of various methods of electrofusion in generating hybrid human cells have been investigated with a variety of human haemopoietic, breast and prostate cell lines. Cell fusion using an electrical pulse is enhanced by a variety of stimuli to align cells electrically or bring cells into contact. Centrifugation of cells after an exponential pulse from a Gene Pulser electroporation apparatus provided the highest yield of mixed cell hybrids by FACS analysis. An extensive fusogenic condition generated in human cells after an electrical pulse contradicts the presumption that prior cell contact is necessary for cell fusion. Alignment of cells in a concurrent direct current charge and osmotic expansion of cells in polyethylene glycol also generated high levels of cell fusion. Waxing of one electrode of the electroporation cuvette served to polarize the fusion chamber and increase cell fusion 5-fold. Optimisation of a direct current charge in combination with a fusogenic pulse in which fusion of a range of human cells approached or exceeded 30% of the total pulsed cells. The yield of hybrid prostate and breast cancer cells with dendritic cells was similar to the homologous cell fusion efficiencies indicating that dendritic cells were highly amenable to fusion with human tumour cells under similar electrical parameters. Elimination of unfused cells by density gradient and culture is possible to further increase the quantity of hybrid cells. The generation and purification of quantities of hybrid cells sufficient for human vaccination raises the possibility of rapid, autologous tumour antigen presenting vaccines for trial with common human tumours.     

Resistance to the proapoptotic effects of interferon-gamma on melanoma cells used in patient-specific dendritic cell immunotherapy is associated with improved overall survival

The use of whole cell tumor vaccines and various means of loading antigen onto dendritic cells have been under investigation for over a decade. Induction of apoptosis and the exposure of immune-stimulating proteins are thought to be beneficial for the use in immunotherapy protocols, but conclusive evidence in the clinical setting has been lacking. Incubation of melanoma cell lines with interferon-gamma (IFN-γ) increased phosphatidylserine and calreticulin exposure, but not in the IFN-γ-resistant cell line Lu-1205. Short-term autologous melanoma cell lines used for loading dendritic cells for immunotherapy showed differential response to the pro-apoptotic effects of IFN-γ. These IFN-γ-treated tumor cells (TCs) were irradiated and used for loading antigen for dendritic cell therapy. A log-rank comparison of survival for patients whose TCs were found to be either sensitive (upregulated phosphatidylserine and calreticulin) or insensitive to IFN-γ revealed a strongly significant correlation to progression-free (p = 0.003) and overall survival (p = 0.002) favorably in those patients whose cell lines were resistant to the proapoptotic effect of IFN-γ. These results suggest that the use of IFN-γ in anti-melanoma dendritic cell-based immunotherapy may only be beneficial when the cells do not undergo apoptosis in response to IFN-γ and support the contention that the use of some apoptotic cells in vaccines may be detrimental.     

Distinct roles in phagocytosis of the early and late increases of cell surface calreticulin induced by oxaliplatin

Calreticulin (CRT), a chaperone typically located in the endoplasmic reticulum (ER), is known to translocate to the cell surface in response to anticancer drugs. Cell surface CRT (ecto-CRT) on apoptotic or pre-apoptotic cells serves as an “eat me” signal that can promote phagocytosis. In this study, we observed the biphasic (early transient and late sustained) increase of ecto-CRT on HT-29 cells after treatment with oxaliplatin (L-OHP). To investigate the role of ecto-CRT that accumulates in the early and late phases as “eat me” signals, we examined the phagocytosis of HT-29 cells by macrophage-like cells and dendritic cell (DC) -like cells prepared from THP-1 cells. The results indicated that the early ecto-CRT-expressed cells were phagocytosed by immature DC-like cells, and the late ecto-CRT-expressed cells were phagocytosed primarily by macrophage-like cells, while mature DC-like cells did not respond to the either class of ecto-CRT-expressed cells. Both types of phagocytotic events were inhibited by CRT Blocking Peptide, suggesting that such events depended on the ecto-CRT. Our results suggested that the early increase of ecto-CRT is related to phagocytosis as part of immunogenic cell death (ICD), while the late increase of ecto-CRT is related to the removal of apoptotic cells by macrophages.     

Cancer Cell Analyses at the Single Cell-Level Using Electroactive Microwell Array Device

Circulating tumor cells (CTCs), shed from primary tumors and disseminated into peripheral blood, are playing a major role in metastasis. Even after isolation of CTCs from blood, the target cells are mixed with a population of other cell types. Here, we propose a new method for analyses of cell mixture at the single-cell level using a microfluidic device that contains arrayed electroactive microwells. Dielectrophoretic (DEP) force, induced by the electrodes patterned on the bottom surface of the microwells, allows efficient trapping and stable positioning of single cells for high-throughput biochemical analyses. We demonstrated that various on-chip analyses including immunostaining, viability/apoptosis assay and fluorescent in situ hybridization (FISH) at the single-cell level could be conducted just by applying specific reagents for each assay. Our simple method should greatly help discrimination and analysis of rare cancer cells among a population of blood cells.     

Fused Late Endocytic Compartments and Immunostimulatory Capacity of Dendritic–Tumor Cell Hybridomas

Late endocytic compartments, containing MHC class II molecules in antigen presenting cells, fuse to each other in order to deliver antigens to these molecules. We have shown previously that fusion of late endocytic compartments takes place also in hybridomas. Therefore, we investigate here whether the level of fused late endocytic compartments affects the immunostimulatory capacity of hybridomas obtained by the electrofusion of dendritic and tumor cells. The level of fused late endocytic compartments in a single hybridoma cell was assessed and samples of electrofused cells were then cocultured with autologous T cells, resulting in the priming of naïve T cells. To test the immunostimulatory capacity of hybridoma cells, T-cell-induced cytotoxicity of tumor cells was assayed. The results demonstrate that in vitro cytotoxic T cell responses are enhanced if a higher percentage of fused late endocytic compartments is present in the cell population of electrofused hybridoma cells.     

Generation of cell hybrids via a fusogenic cell line

BACKGROUND: Hybrids obtained by fusion between tumour cells (TC) and dendritic cells (DC) have been proposed as anti-tumour vaccines because of their potential to combine the expression of tumour-associated antigens with efficient antigen presentation. The classical methods used for fusion, polyethylene glycol (PEG) and electrofusion, are cytotoxic and generate cell debris that can be taken up by DC rendering the identification of true hybrids difficult. METHODS: We have established a stable cell line expressing a viral fusogenic membrane glycoprotein (FMG) that is not itself susceptible to fusion. This cell line has been used to generate hybrids and to evaluate the relevance of tools used for hybrid detection. RESULTS: This FMG-expressing cell line promotes fusion between autologous or allogeneic TC and DC in any combination, generating 'tri-parental hybrids'. At least 20% of TC are found to be integrated into hybrids. CONCLUSIONS: It is speculated that this tri-parental hybrid approach offers new possibilities to further modulate the anti-tumour effect of the DC/TC hybrids since it allows the expression of relevant immunostimulatory molecules by appropriate engineering of the fusogenic cell line.     

Nuclear membrane fusion in electrofused mammalian cells

Fusion of nuclei was studied in electrofused cells using staining procedures and DNA flow cytometry. Homogeneous and heterogeneous electrofusion of Ehrlich ascites tumor cells. Muntjac cells and V79-S181 cells were performed in balanced-salt solutions at low temperature. Incubation of the cells subjected to electrofusion in fusion media for about 2 h was required to complete cell fusion and, in particular, nuclear membrane fusion. Under optimum electrofusion conditions it was found that fusion of nuclei is a very frequent event. Half of the fused cells (about 30 to 50% of the field-exposed cells) underwent nuclear membrane fusion. It is shown that the high frequency of nuclear membrane fusion in electrofused, unsynchronised cells resulted from intracellular dielectrophoresis occurring during cell alignment. In accordance with theory, maximum nuclear membrane fusion was observed using alignment fields of between 1 and 4 MHz (depending on the cell species), that is above the frequencies at which the plasmalemma capacity no longer shielded the cell interior from participation in the conduction process. In this frequency range a potential difference can be built up across the nuclear membrane leading to repositioning of the nuclei into the contact zone of the plasmalemmas of two attached cells. This intracellular dielectrophoresis apparently facilitated fusion of nuclei once intermingling of the plasma membranes had occurred. It was further demonstrated that exponentially growing cells showed higher cell fusion rates than cells taken from the unfed plateau phase. One, but not the only reason, might be the higher ATP content of exponentially growing cells compared to cells of the plateau phase. Addition of external ATP to plateau phase cells during electrofusion resulted, in accordance with this assumption, in an increase of fusion frequency, whereas ATP had apparently no effect on the fusion yield of exponentially growing cells. G1 cells obtained by mitotic selection after nocodazole-induced blockage in metaphase also showed higher cellular and nuclear membrane fusion yields than exponentially growing cells. Most importantly, it could be demonstrated both experimentally and theoretically that electrofusion of cells in a dielectrophoretically aligned chain is controlled by a simple law of probability resulting predominantly in fusion of two cells independent of the number of cells in the chain. The likelihood of fusion of various numbers of cells in a chain is given by the appropriate power of the probability of two-cell fusion.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Systematic Study of the Electroporation and Electrofusion of B16-F1 and CHO Cells in Isotonic and Hypotonic Buffer

The fusogenic state of the cell membrane can be induced by external electric field. When two fusogenic membranes are in close contact, cell fusion takes place. An appropriate hypotonic treatment of cells before the application of electric pulses significantly improves electrofusion efficiency. How hypotonic treatment improves electrofusion is still not known in detail. Our results indicate that at given induced transmembrane potential electroporation was not affected by buffer osmolarity. In contrast to electroporation, cells’ response to hypotonic treatment significantly affects their electrofusion. High fusion yield was observed when B16-F1 cells were used; this cell line in hypotonic buffer resulted in 41?±?9?% yield, while in isotonic buffer 32?±?11?% yield was observed. Based on our knowledge, these fusion yields determined in situ by dual-color fluorescence microscopy are among the highest in electrofusion research field. The use of hypotonic buffer was more crucial for electrofusion of CHO cells; the fusion yield increased from below 1?% in isotonic buffer to 10?±?4?% in hypotonic buffer. Since the same degree of cell permeabilization was achieved in both buffers, these results indicate that hypotonic treatment significantly improves fusion yield. The effect could be attributed to improved physical contact of cell membranes or to enhanced fusogenic state of the cell membrane itself.     

Hybrid-cell vaccines for cancer immune therapy

Hybrid cells generated by fusing allogenic-presenting cells, such as dendritic cells, with tumor cells are a new tool in cancer immunotherapy which are designed to enhance the immunogenicity of antigenic tumors by presenting the whole spectrum of tumor-associated antigens, by providing the co-stimulatory molecules required for T-cell activation, and by the expression of allogenic MHC molecules for recruitment and activation of T-cell help. This approach has been successfully tested in animal models as well as in clinical phaseI/II trials with various tumors. Besides clinical repsonses, induction of tumor-specific cytolytic T-cells were observed. The electrofusion protocol described here has the advantage of high fusion efficiency, high hybrid-cell viability, as well as high reproducibility, and can be used for various tumor cell types after minor adjustments are made to the instrument settings in order to process large numbers of dendritic cells with consistent efficiencies.     

Fabrication of a microfluidic device for probiotic drug's dosage screening: Precision Medicine for Breast Cancer Treatment

Breast cancer is the most common cancer in women; it has been affecting the lives of millions each year globally and microfluidic devices seem to be a promising method for the future advancements in this field. This research uses a dynamic cell culture condition in a microfluidic concentration gradient device, helping us to assess breast anticancer activities of probiotic strains against MCF-7 cells. It has been shown that MCF-7 cells could grow and proliferate for at least 24 h; however, a specific concentration of probiotic supernatant could induce more cell death signaling population after 48 h. One of our key findings was that our evaluated optimum dose (7.8 mg/L) was less than the conventional static cell culture treatment dose (12 mg/L). To determine the most effective dose over time and the percentage of apoptosis versus necrosis, flowcytometric assessment was performed. Exposing the MCF-7 cells to probiotic supernatant after 6, 24 and 48 h, confirmed that the apoptotic and necrotic cell death signaling were concentration and time dependent. We have shown a case that these types of microfluidics platforms performing dynamic cell culture could be beneficial in personalized medicine and cancer therapy.     

Prevention of spontaneous breast carcinoma by prophylactic vaccination with dendritic/tumor fusion cells

Genetically modified mice with spontaneous development of mammary carcinoma provide a powerful tool to study the efficacy of tumor vaccines, since they mimic breast cancer development in humans. We used a transgenic murine model expressing polyomavirus middle T oncogene and mucin 1 tumor-associated Ag to determine the preventive effect of a dendritic/tumor fusion cell vaccine. The MMT (a transgenic murine model) mice developed mammary carcinoma between the ages of 65-108 days with 100% penetrance. No spontaneous CTL were detected. However, prophylactic vaccination of MMT mice with dendritic/tumor fusion cells induced polyclonal CTL activity against spontaneous mammary carcinoma cells and rendered 57-61% of the mice free of the disease at the end of experiment (180 days). Furthermore, the level of CTL activity was maintained with multiple vaccinations. The antitumor immunity induced by vaccination with dendritic/tumor fusion cells reacted differently to injected tumor cells and autochthonous tumor. Whereas the injected tumor cells were rejected, the autochthonous tumor evaded the attack and was allowed to grow. Collectively these results indicate that prophylactic vaccination with dendritic/tumor fusion cells confers sufficient antitumor immunity to counter the tumorigenesis of potent oncogenic products. The findings in the present study are highly relevant to cancers in humans.