Fusion Of Tumor Cells Research Articles

Fusion between tumor cells enhances melanoma metastatic potential

Malignant melanoma, characterized by early distant metastasis to the lungs and brain, is a leading cause of mortality related to skin cancer. Cell fusion and the subsequent aneuploidy, commonly observed in melanoma, are associated with poor prognosis. However, the pathological consequences of cell fusion in melanoma remain unknown. Therefore, the present study aims to investigate the pathological consequences of cell fusion in melanoma and the mechanism of melanoma metastasis. Phytohemagglutinin-polyethylene glycol (PHA-PEG) fusion method was developed for the fusion of tumor cells. Melanoma cells were fused through the improved PHA-PEG fusion method and obtained by fluorescence-activated cell sorting. DNA content was analyzed through flow cytometry. Cell proliferation rate was detected by cell culture in vitro, and the cell number was counted daily. To detect the tumor growth rate in vivo, cells were injected subcutaneously and the tumor volumes were measured using a vernier caliper. To analyze the tumor metastatic potential, cells were injected intravenously, and the collected lung-metastasis samples were weighed by an electronic balance and the surface nodules were counted. We established an improved phytohemagglutinin-polyethylene glycol fusion method and successfully obtained stable melanoma tumor-tumor cell fusion hybrids. Cell size, DNA content, and chromosome numbers of the fusion hybrids were approximately twice those of the parents. The metastatic potential of the fusion hybrids was dramatically enhanced, in contrast to their proliferation rate. Their metastasis was specific to the lungs. We developed a highly efficient cell fusion method that can be applied in many fields, particularly cancer research. Our study has proven that tumor-tumor cell fusion hybrids in melanoma can acquire enhanced and specific metastatic potential. Thus, blockage of cell fusion may be a new strategy for melanoma metastasis therapy.

Spontaneous Formation of Tumorigenic Hybrids between Breast Cancer and Multipotent Stromal Cells Is a Source of Tumor Heterogeneity

Breast cancer progression involves cancer cell heterogeneity, with generation of invasive/metastatic breast cancer cells within populations of nonmetastatic cells of the primary tumor. Sequential genetic mutations, epithelial-to-mesenchymal transition, interaction with local stroma, and formation of hybrids between cancer cells and normal bone marrow-derived cells have been advocated as tumor progression mechanisms. We report herein the spontaneous in vitro formation of heterotypic hybrids between human bone marrow-derived multipotent stromal cells (MSCs) and two different breast carcinoma cell lines, MDA-MB-231 (MDA) and MA11. Hybrids showed predominantly mesenchymal morphological characteristics, mixed gene expression profiles, and increased DNA ploidy. Both MA11 and MDA hybrids were tumorigenic in immunodeficient mice, and some MDA hybrids had an increased metastatic capacity. Both in culture and as xenografts, hybrids underwent DNA ploidy reduction and morphological reversal to breast carcinoma-like morphological characteristics, while maintaining a mixed breast cancer-mesenchymal expression profile. Analysis of coding single-nucleotide polymorphisms by RNA sequencing revealed genetic contributions from both parental partners to hybrid tumors and metastasis. Because MSCs migrate and localize to breast carcinoma, our findings indicate that formation of MSC-breast cancer cell hybrids is a potential mechanism of the generation of invasive/metastatic breast cancer cells. Our findings reconcile the fusion theory of cancer progression with the common observation that breast cancer metastases are generally aneuploid, but not tetraploid, and are histopathologically similar to the primary neoplasm.

Abstract 4389: Impact of regulatory T cell depletion on dendritic cell-tumor fusion vaccine immunotherapy

Abstract A promising branch of research in cancer immunotherapy is the development of vaccines comprised of dendritic cells hybridized to tumor cells by electrofusion. Studies in numerous murine models have shown the efficacy of treatment with a dendritic cell-tumor fusion vaccine; however, this is critically dependent on systemic administration of 3rd signals such as IL-12 (Interleukin-12). Translation of these findings into the clinic will be difficult due to toxicity, morbidity, and mortality associated with IL-12 administration. Thus, alternative strategies to enhance the efficacy of fusion vaccines are needed. Regulatory T cells (Tregs) are known to suppress anti-tumor immunity. Treg depletion has been demonstrated to improve tumor immunotherapy in murine tumor models as well as some phase I clinical studies. We therefore investigated whether Treg depletion with the anti-CD25 monoclonal antibody PC61 would significantly improve dendritic cell - tumor fusion immunotherapy in a murine melanoma model. Experiments were conducted using six groups of animals: no treatment (negative control), isotypic monoclonal antibody alone (Y13), Treg depletion alone (PC61), fusion vaccine with Y13 (Fusion + Y13), fusion vaccine with PC61 (Fusion + PC61), and fusion vaccine with IL-12 (Fusion + IL12; positive control). Pulmonary metastases were established through intravenous tail vein injection of 0.25x106 tumor cells (Day 0). Three days later, an injection of 0.6x106 fusion hybrids was delivered intranodally. 250 µg of Y13 or PC61 monoclonal antibodies were delivered one day prior to vaccine delivery. Starting on the day of vaccination in the IL-12 group, 0.2 µg of IL-12 was given daily for 4 days. Three weeks after tumor inoculation, mice were euthanized and the pulmonary metastases were enumerated. The median number of metastases and standard deviation [SD] per group were the following: no treatment= 204 [50]; Y13= 220 [59]; PC61= 211 [49]; fusion+Y13= 126 [67]; fusion + PC61= 72 [74]; fusion + IL12= 1 [11]. Using the Mann-Whitney U test, there was a statistically significant reduction in the number of tumor metastases between both the fusion vaccine + Y13 group and the fusion + PC61 group (P=.0397) as well as PC61 alone group and fusion + PC61 group (P=.002). This suggests that Treg depletion can significantly augment dendritic cell-tumor cell fusion melanoma immunotherapy. Our results support the development of novel therapeutic modalities that include Treg depletion, or modulation, to enhance the efficacy of dendritic cell - tumor fusion cancer immunotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4389. doi:1538-7445.AM2012-4389

Abstract SY41-01: Tumor-endothelial transdifferentiation

Abstract Glioblastoma (GBM) is the most malignant brain tumor and highly resistant to intensive combination therapies and anti-VEGF therapies. To assess the resistance mechanism to anti-VEGF therapy, we examined the vessels of GBMs in tumors that were induced by the transduction of p53+/− heterozygous mice with lentiviral vectors containing oncogenes and the marker GFP in the hippocampus of GFAP-Cre recombinase (Cre) mice. We were surprised to observe GFP+ vascular endothelial cells (ECs). Transplantation of mouse GBM cells revealed that the tumor-derived endothelial cells (TDECs) originated from tumor-initiating cells and did not result from cell fusion of ECs and tumor cells. An in vitro differentiation assay suggested that hypoxia is an important factor in the differentiation of tumor cells to ECs and is independent of VEGF. TDEC formation was not only resistant to an anti-VEGF receptor inhibitor in mouse GBMs but it led to an increase in their frequency. A xenograft model of human GBM spheres from clinical specimens and direct clinical samples from patients with GBM also showed the presence of TDECs. We suggest that the TDEC is an important player in the resistance to anti-VEGF therapy, and hence a potential target for GBM therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY41-01. doi:1538-7445.AM2012-SY41-01

Long-lived fusions of human haematological tumour cells and B-lymphoblastoid cells induce tumour antigen-specific cytotoxic T-cell responses in vitro

Tumour-specific cytotoxic T-cells (CTL) are important anti-cancer immune effectors. Most tumour cells, however, do not stimulate effective anti-tumour immune responses, in vivo or in vitro. To enhance tumour cell immunogenicity, we fused human tumour cells from haematological malignancies with the B-lymphoblastoid cell line (LCL), HMy2, to generate a panel of long-lived, self replicating LCL/tumour hybrid cell lines. The LCL/tumour hybrid cell lines expressed HLA class I and class II molecules, CD80 and CD86, and a range of known tumour associated antigens (TAAs). In vitro stimulation of PBLs from healthy, HLA-A2+ individuals by hybrid cell lines induced tumour antigen-specific CTLs to TAAs, including survivin, MAGE-A1, NY-ESO-1 and WT-1. Individual hybrid cell lines simultaneously induced CTL to multiple TAAs. In vitro stimulation of PBL from 2 patients with acute myeloid leukaemia by autologous LCL/tumour hybrid cell lines induced CTL capable of killing the patient's own tumour cells. Our data show, for the first time, that hybrid cell lines formed by fusion of HMy2 cells and haematological tumour cells induce tumour- and tumour antigen-specific cytotoxic T-cell responses in vitro. Hybrid cell lines such as those described may represent novel reagents for use in the immunotherapy of haematological malignancies.

GALV expression enhances the therapeutic efficacy of an oncolytic adenovirus by inducing cell fusion and enhancing virus distribution

The limitations of the current oncolytic adenoviruses for cancer therapy include insufficient potency and poor distribution of the virus throughout the tumor mass. To address these problems, we generated an oncolytic adenovirus expressing the hyperfusogenic form of the gibbon-ape leukemia virus (GALV) envelope glycoprotein under the control of the adenovirus major late promoter. The oncolytic properties of the new fusogenic adenovirus, ICOVIR16, were analyzed both in vitro and in vivo, and compared with that of its non-fusogenic counterpart, ICOVIR15. Our results indicate that GALV expression by ICOVIR16 induced extensive syncytia formation and enhanced tumor cell killing in a variety of tumor cell types. When injected intratumorally or intravenously into mice with large pre-established melanoma or pancreatic tumors, ICOVIR16 rapidly reduced tumor burden, and in some cases, resulted in complete eradication of the tumors. Importantly, GALV expression induced tumor cell fusion in vivo and enhanced the spreading of the virus throughout the tumor. Taken together, these results indicate that GALV expression can improve the antitumoral potency of an oncolytic adenovirus and suggest that ICOVIR16 is a promising candidate for clinical evaluation in patients with cancer.

Semiallogenic fusions of MSI+tumor cells and activated B cells induce MSI-specific T cell responses

BackgroundVarious strategies have been developed to transfer tumor-specific antigens into antigen presenting cells in order to induce cytotoxic T cell responses against tumor cells. One approach uses cellular vaccines based on fusions of autologous antigen presenting cells and allogeneic tumor cells. The fusion cells combine antigenicity of the tumor cell with optimal immunostimulatory capacity of the antigen presenting cells.Microsatellite instability caused by mutational inactivation of DNA mismatch repair genes results in translational frameshifts when affecting coding regions. It has been shown by us and others that these mutant proteins lead to the presentation of immunogenic frameshift peptides that are - in principle - recognized by a multiplicity of effector T cells.MethodsWe chose microsatellite instability-induced frameshift antigens as ideal to test for induction of tumor specific T cell responses by semiallogenic fusions of microsatellite instable carcinoma cells with CD40-activated B cells. Two fusion clones of HCT116 with activated B cells were selected for stimulation of T cells autologous to the B cell fusion partner. Outgrowing T cells were phenotyped and tested in functional assays.ResultsThe fusion clones expressed frameshift antigens as well as high amounts of MHC and costimulatory molecules. Autologous T cells stimulated with these fusions were predominantly CD4+, activated, and reacted specifically against the fusion clones and also against the tumor cell fusion partner. Interestingly, a response toward 6 frameshift-derived peptides (of 14 tested) could be observed.ConclusionCellular fusions of MSI+ carcinoma cells and activated B cells combine the antigen-presenting capacity of the B cell with the antigenic repertoire of the carcinoma cell. They present frameshift-derived peptides and can induce specific and fully functional T cells recognizing not only fusion cells but also the carcinoma cells. These hybrid cells may have great potential for cellular immunotherapy and this approach should be further analyzed in preclinical as well as clinical trials. Moreover, this is the first report on the induction of frameshift-specific T cell responses without the use of synthetic peptides.

Abstract 1477: Tumor cell-leukocyte fusion in human cancer: A melanoma brain metastasis with a donor-patient hybrid genome following allogeneic BMT

Abstract Tumor cell fusion with motile bone marrow-derived cells (BMDCs) has long been proposed as a mechanism for metastasis, but while such fusion has been demonstrated in animal models little is known of this in human cancer. Here we report the first genetic evidence for BMDC-tumor fusion and genomic hybridization in a human melanoma brain metastasis from a patient with a prior allogeneic bone marrow transplant (male-male, sibling). STR analyses of DNA from melanoma cells throughout the tumor revealed the presence of both donor and patient alleles, with common aberrant allelic patterns at multiple chromosomal loci indicating a clonal origin of the tumor. Methods. All procedures were in accordance with HIPAA regulations and approved by the Yale HIC. Donor and patient DNAs were obtained from frozen lymphocytes and from a patient buccal swab. Formalin-fixed, paraffin-embedded tumor specimens were stained by immuniohistochemistry with common leucocyte antigen (CLA) in order to avoid leucocytes during microdissection of tumor cells. LCA-negative tumor cells were isolated with an Arturus XT laser dissection microscope system. DNA was extracted and analyzed for donor and patient alleles using forensic STR analyses at 13 chromosomal loci and amelogenin for the X and Y. Tissue sections were analyzed from 14 areas throughout the tumor. Results. DNA from donor and patient lymphocytes showed balanced diploidy at all loci and the allelic patterns were consistent with the sibling relationship. On the other hand, DNA from tumor cells contained a mixture of donor and host alleles at some loci, only donor or patient alleles at others, and numerous allelic imbalances throughout–particularly for the X and Y (X:Y = 2.8:1) that pointed to a probable tumor genotype of XXXY. With few exceptions, aberrant genetic profiles seen in one tumor section were seen in all, demonstrating a clonal origin of the tumor. Conclusions. The results provide the first genetic proof of leucocyte-tumor fusion and genomic hybridization in human cancer and indicate that the tumor was generated from a single fusion event. While this report concerns only one case, we demonstrate that forensic genetic analyses of tumors arising as secondary malignancies represents a fruitful area for research in tumor cell fusion in human cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1477. doi:10.1158/1538-7445.AM2011-1477

Transdifferentiation of glioblastoma cells into vascular endothelial cells

Glioblastoma (GBM) is the most malignant brain tumor and is highly resistant to intensive combination therapies and anti-VEGF therapies. To assess the resistance mechanism to anti-VEGF therapy, we examined the vessels of GBMs in tumors that were induced by the transduction of p53(+/-) heterozygous mice with lentiviral vectors containing oncogenes and the marker GFP in the hippocampus of GFAP-Cre recombinase (Cre) mice. We were surprised to observe GFP(+) vascular endothelial cells (ECs). Transplantation of mouse GBM cells revealed that the tumor-derived endothelial cells (TDECs) originated from tumor-initiating cells and did not result from cell fusion of ECs and tumor cells. An in vitro differentiation assay suggested that hypoxia is an important factor in the differentiation of tumor cells to ECs and is independent of VEGF. TDEC formation was not only resistant to an anti-VEGF receptor inhibitor in mouse GBMs but it led to an increase in their frequency. A xenograft model of human GBM spheres from clinical specimens and direct clinical samples from patients with GBM also showed the presence of TDECs. We suggest that the TDEC is an important player in the resistance to anti-VEGF therapy, and hence a potential target for GBM therapy.

Characterization of hybrid cells derived from spontaneous fusion events between breast epithelial cells exhibiting stem-like characteristics and breast cancer cells

Several data of the past years clearly indicated that the fusion of tumor cells and tumor cells or tumor cells and normal cells can give rise to hybrids cells exhibited novel properties such as an increased malignancy, drug resistance, or resistance to apoptosis. In the present study we characterized hybrid cells derived from spontaneous fusion events between the breast epithelial cell line M13SV1-EGFP-Neo and two breast cancer cell lines: HS578T-Hyg and MDA-MB-435-Hyg. Short-tandem-repeat analysis revealed an overlap of parental alleles in all hybrid cells indicating that hybrid cells originated from real cell fusion events. RealTime-PCR-array gene expression data provided evidence that each hybrid cell clone exhibited a unique gene expression pattern, resulting in a specific resistance of hybrid clones towards chemotherapeutic drugs, such as doxorubicin and paclitaxel, as well as a specific migratory behavior of hybrid clones towards EGF. For instance, M13MDA435-4 hybrids showed a marked resistance towards etoposide, doxorubicin and paclitaxel, whereas hybrid clones M13MDA-435-1 and -2 were only resistant towards etoposide. Likewise, all investigated M13MDA435 hybrids responded to EGF with an increased migratory activity, whereas the migration of parental MDA-MB-435-Hyg cells was blocked by EGF, suggesting that M13MDA435 hybrids may have acquired a new motility pathway. Similar findings have been obtained for M13HS hybrids. We conclude from our data that they further support the hypothesis that cell fusion could give rise to drug resistant and migratory active tumor (hybrid) cells in cancer.

The Establishment of Dendritic Cell-Tumor Fusion Vaccines for Hormone Refractory Prostate Cancer Cell

PurposeDendritic cell (DC)-based tumor vaccine is an attractive modality for the treatment of hormone-refractory prostate cancer (HRPC) because it has some efficacy and few side effects in patients with poor general conditions. The aim of this study was to establish which is the most effective DC vaccine for the treatment of HRPC. We compared DC vaccine sensitized with tumor lysate and a fusion vaccine of DCs and tumor cells.Materials and MethodsThe DU145 cancer cell line was purchased from the American Type Culture Collection. DCs were cultured from peripheral blood monocytes. Peripheral blood monocytes were cultured in RPMI 1640 medium supplemented with interleukin-4 (IL-4), granulocyte-macrophage colony-stimulating factor, and 10% fetal calf serum. Tumor necrosis factor-alpha was added on day 7 to support maturation. Functional activity was measured in three groups: the DC single-culture group, the DC culture group with DC vaccine sensitized with tumor lysates, and the DC culture group prepared with tumor fusion vaccine made from irradiated tumor cells and monocyte-derived DCs by the polyethylene glycol method.ResultsBy FACS analysis, the rate of DC-tumor fusion vaccine was 20.3±3%. The IL-12 level produced by the DC-tumor fusion vaccine was significantly higher than that of DCs pulsed with tumor lysate (p<0.05). Also, the generation of interferon-γ by tumor-specific T cells in the DC-tumor fusion vaccine group was superior to that of DCs pulsed with tumor lysate (p<0.05). In addition, the T cells of the tumor lysate-pulsed DCs and tumor fusion vaccine had 1.6 and 2.5 times the functional activity, respectively, of the DC single-culture group in killing tumor cells in the cytotoxicity assay.ConclusionsThe DC-tumor fusion vaccine seems to be more effective than DC single-culture or DC-tumor lysate vaccine in the treatment of HRPC.

Dendritic and tumor cell fusions transduced with adenovirus encoding CD40L eradicate B-cell lymphoma and induce a Th17-type response

Fusion of dendritic cells and tumor cells (FCs) constitutes a promising tool for generating an antitumor response because of their capacity to present tumor antigens and provide appropriate costimulatory signals. CD40-CD40L interaction has an important role in the maturation and survival of dendritic cells and provides critical help for T-cell priming. In this study, we sought to improve the effectiveness of FC vaccines in a murine model of B-cell lymphoma by engineering FCs to express CD40L by means of an adenovirus encoding CD40L (Adv-CD40L). Before transduction with Adv-CD40L, no CD40L expression was detected in FCs, DCs or tumor cells. The surface expression of CD40L in FC transduced with Adv-CD40L (FC-CD40L) ranged between 50 and 60%. FC-CD40L showed an enhanced expression of CD80, CD86, CD54 and MHC class II molecules and elicited a strong in vitro immune response in a syngeneic mixed lymphocyte reaction. Furthermore, FC-CD40L showed enhanced migration to secondary lymphoid organs. Splenocytes from mice treated with FC-CD40L had a dramatic increase in the production of IL-17, IL-6 and IFN-gamma, compared with controls. Treatment with the FC-CD40L vaccine induced regression of established tumors and increased survival. Our data demonstrate that FC transduced with Adv-CD40L enhances the antitumor effect of FC vaccines in a murine lymphoma model and this is associated with an increased Th17-type immune response.

Dendritic Cell Tumor Fusion Vaccination in Conjunction with Autologous Transplantation for Multiple Myeloma.

Abstract 783▪▪This icon denotes an abstract that is clinically relevant.Autologous stem cell transplantation (ASCT) for multiple myeloma (MM) offers a unique setting to explore the role of immunotherapeutic strategies in eradicating malignancy. Patients achieve tumor cytoreduction following ASCT, however ultimately experience disease relapse from a persistent reservoir of chemotherapy resistant disease. Cancer vaccines that educate host immunity to target myeloma cells can be used to eradicate residual disease following ASCT. Our group has developed a cancer vaccine whereby dendritic cells (DCs) are fused with autologous tumor cells. DC/MM fusion cells present a broad array of tumor antigens in the context of DC derived costimulatory molecules. We are conducting a clinical trial in which patients with MM undergo ASCT followed by post-transplant vaccination with 3 doses of DC/MM fusions (cohort 1). A second cohort of patients receive an additional vaccination prior to stem cell collection in order to induce the expansion of tumor specific lymphocytes that are collected in the stem cell product (cohort 2). The infusion of educated lymphocytes provides a platform for subsequent post-transplant vaccination. To date, 26 patients have been enrolled in cohort 1 and 9 patient have been enrolled in cohort 2. Adherent mononuclear cells were isolated from leukapheresis collections and cultured with GM-CSF and IL-4 for 5-7 days, then exposed to TNFα for 48-72 hours to generate mature DCs. DCs expressed co-stimulatory (mean CD86 70%) and maturation markers (mean CD83 55%). MM cells were isolated from bone marrow and were identified by their expression of CD38 or CD138. DC and MM cells were co-cultured with PEG and fusion cells were quantified by determining the percentage of cells that co-express unique DC and myeloma antigens. Mean yield of the DC and myeloma preparations was 1.72 × 108 and 6.6 × 107 cells, respectively. Mean fusion efficiency was 38% and the mean cell dose generated was 3.6 × 106 fusion cells. Mean viability of the DC, myeloma, and fusion preparations was 87%, 87%, and 78%, respectively. As a measure of their potency as antigen presenting cells, fusion cells potently stimulated allogeneic T cell proliferation in vitro. Mean stimulation indexes were 13, 60, and 32 for T cells stimulated by myeloma cells, DCs, and fusion cells at an APC: T cell ratio of 1:10. Adverse events judged to be potentially vaccine related were mild, and included injection site reactions, pruritis, myalgias, fever, chills, and tachycardia. ASCT was associated with suppression of measures of cellular immunity. Circulating CD4 cells were depressed in the post-transplant period and CD4:CD8 ratios remained inverted for greater than 10 months. Similarly, 65% of patients had a positive DTH response to candida antigen prior to transplant while only 21% demonstrated a positive response in the early post-transplant period. T cell response to PHA mitogen was transiently depressed post-transplant with mean stimulation indexes of 79, 10, 26, 36, and 63 prior to transplant, 1, 2, 3, and 6 months post-transplant, respectively. Consistent with these findings, in vitro T cell responses to tetanus toxoid were blunted in the post-transplant period. In contrast, a significant increase in circulating tumor reactive lymphocytes was noted, as determined by T cell expression of IFN by CD4 and CD8 cells following ex vivo coculture with autologous myeloma cell lysate (Mean percentage of tumor reactive CD8 cells was 1 and 7.7 pre and post-transplant, respectively; mean percentage of CD4 cells was 0.9 and 3.2). A further amplification of tumor reactive lymphocytes was seen with vaccination in a subset of patients (mean percentage of CD4 and CD8 tumor reactive T cells was 6.4 and 13.4, respectively). In the post-transplant period, regulatory T cells fell to minimal levels. To date, 23 patients have completed follow up and were evaluable for clinical response. 3 patients achieved CR at 1 month following ASCT. Of note, an additional 7 patients obtained a CR following completion of vaccinations, suggesting a role for post-transplant immunotherapy in mediating elimination of disease. In summary, fusion cell vaccination in conjunction with ASCT was well tolerated, stimulated anti-tumor immunity and was associated with the induction of post-transplant complete response. Disclosures:Richardson:Millenium (Research Funding and Advisory Board), Celgene, Keryx, BMS, Merck, Johnson and Johnson (All Advisory Board): Membership on an entity's Board of Directors or advisory committees, Research Funding. Anderson:Millenium (Research Funding and Advisory Board: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Keryx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Johnson and Johnson: Membership on an entity's Board of Directors or advisory committees.

Toll‐like receptor agonists as third signals for dendritic cell–tumor fusion vaccines

The aim of the present study was to evaluate the therapeutic efficacy of dendritic cell (DC)-tumor fusion hybrids with Toll-like receptor (TLR) agonists. DC-tumor fusion hybrids were generated by electrofusion and injected into the inguinal lymph nodes of C57BL/6 mice with 3-day established pulmonary metastases. Paired TLR agonists polyinosine:polycytadilic acid [poly(I:C)] and cytosine-phosphate-guanine (CpG) were then injected intraperitoneally. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate interleukin (IL)-12 production from the DC-tumor fusion hybrids in vitro. Fusion + TLR agonists (60 metastases) had significantly fewer metastases than did the untreated control (262 metastases, p = .0001) and fusion alone (150 metastases, p = .02). ELISA showed that the DC-tumor fusion hybrids yielded 90 pg of IL-12 after TLR stimulation compared with 1610 pg from dendritic cells alone. CpG and poly(I:C) administered as a third signal with fusion hybrids as described significantly reduce melanoma metastasis compared with fusion hybrids alone. Fusion hybrids do not appear to be a significant source for IL-12 secretion.

Dendritic cell-tumor cell fusion vaccine prevents growth of subcutaneous transplanted esophageal carcinomas

Our previous studies have shown that dendritic cell (DC)-tumor cell fusion vaccine can induce specific antitumor response against esophageal carcinoma cells. This study was to investigate the inhibitory effect of intratumor injection of the antigen-specific cytotoxic T lymphocytes (CTLs) induced by DC-tumor cell fusion vaccine against subcutaneously transplanted esophageal carcinoma cells in nude mice, and to analyze the influence of DC/tumor cell fusion vaccine on proliferation and apoptosis of esophageal carcinoma cells. Fusion cell vaccine of mature DCs with EC109 cells were generated by the polyethylene glycol (PEG) protocol and the antigen-specific CTLs were induced. The models of transplanted human esophageal carcinoma in nude mouse were established using EC-109 cell line. Thirty-three nude mice with subcutaneous tumors were randomly divided into three groups. Subcutaneous tumors of group A (n=11), group B (n=11) and group C (n=11) were intratumorally injected with the CTLs induced by DC/tumor fusion vaccine, T lymphocytes and RPMI 1,640 medium respectively once a week. After four weeks of intratumor injection, the nude mice were killed and the nodules were anatomized. The mean volume and weight of tumors of each group were measured, and the tumor inhibitory rates of the Group A and the Group B were calculated and compared. The expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry (S-P method). The mean PCNA-label index (LI) of three groups was compared. The cell cycle and cell apoptosis of the xenograft tumor cells were analyzed by flow cytometry. The mean S-phase fraction (SPF) and the mean rate of cell apoptosis of three groups was compared respectively. Both the mean volume and the mean weight of xenograft tumors in group A (881.45+/-31.14 mm3 and 0.88+/-0.04 g) were significantly smaller than those of group B (1493.37+/-51.67 mm3 and 1.38+/-0.07 g) and group C (2065.77+/-87.55 mm3 and 2.04+/-0.11 g). The tumor inhibitory rates of Group A was significantly higher than that of group B (56.86% vs. 32.35%, F=1218.08, P=0.001). The mean PCNA-LI of xenograft tumors was less in the group A (26.83+/-0.95)% than in the group B (51.82+/-1.51)% and group C (68.93+/-2.40)% (F=1528.39, P=0.000). The mean SPF of xenograft tumors was less in the group A (12.46+/-0.36)% than in the group B (29.39+/-0.96)% and the group C (42.25+/-1.43)% (P<0.05). The mean apoptotic rate of xenograft tumors was less in the group A (38.03+/-1.21)% than in the group B (17.75+/-0.56)% and the group C (6.59+/-0.22)% (P<0.05). The model of subcutaneous xenograft tumors in nude mice using human esophageal carcinoma cell line EC-109 has been successfully established. CTLs induced by DC/tumor fusion vaccine has specific antitumor immunity efficacy in vivo. CTLs can inhibit the proliferation of tumor cells and induce apoptosis of tumor cells in local tumors.

Efficient CTL productivity of modified fusion cells by increase of heat shock protein 70

In various loading methods of whole tumor cells or their derivatives, cells fusion of dendritic cells (DCs) and tumor cells have some advantages for antigen presentation, and could up-regulate cytotoxic T cells (CTL) for multiple tumor antigens, including unknown antigens. However, the mechanisms of strong CTL productivity of fusion cells (FCs) are still unknown. Heat shock proteins (HSPs) are molecular chaperones that cross-present chaperoned antigenic peptides with MHC class I molecules. Herein, we focused on clarifying the potential of FCs for CTL production from the comparison of DCs pulsed with two kinds of tumor cell lysates, such as soluble tumor cell lysates or freeze-thawed tumor cell lysates. DCs, CD8+ T cells and tumor cells were prepared from ten patients with gastric cancer, and paired autologous tumor cells were used in all experiments. FCs of OK432-treated DCs and heat-stressed tumor cells (modified FCs) showed significant up-regulation of tumor-associated CEA and HER-2 antigen, and DC-related HLA-DR and co-stimulatory molecules (CD83 and CD86). FCs showed significantly higher IFN-gamma and CTL productivity of CD8+ T cells than DCs pulsed with soluble or freeze-thawed tumor cell lysates. IFN-gamma and CTL productivity of FCs was significantly increased by the heat stress of tumor cells. HSP70 mRNA levels and production of HSP70 protein of modified FCs increased significantly as compared with those of DCs pulsed with soluble or freeze-thawed tumor cell lysates. DCs pulsed with HSP70.PC extracted from modified FCs could enhance CTL productivity significantly more than that of DCs pulsed with HSP70.PC from soluble or freeze-thawed tumor cell lysate pulsed-DCs. The significant up-regulation of HSP70 mRNA and protein levels of modified FCs was related to the potential of CTL productivity. These results suggest that modified FCs possess stronger ability for MHC-restricted CTL production than DCs loaded with soluble or freeze-thawed tumor cell lysates.

Bone Marrow-Derived Cells Are Not the Origin of the Cancer Stem Cells in Ultraviolet-Induced Skin Cancer

Several lines of evidence have demonstrated that various cancers are derived from cancer stem cells (CSCs), which are thought to originate from either tissue stem or progenitor cells. However, recent studies have suggested that the origin of CSCs could be bone marrow-derived cells (BMDCs); for example, gastric cancer, which follows persistent gastric inflammation, appears to originate from BMDCs. Although our previous research showed the capability of BMDCs to differentiate into epidermal keratinocytes, it has yet to be determined whether skin CSCs originate from BMDCs. To assess the possibility that BMDCs could be the origin of CSCs in skin squamous cell carcinoma (SCC), we used a mouse model of UVB-induced skin SCC. We detected a low percentage of BMDCs in the lesions of epidermal dysplasia (0.59%), SCC in situ (0.15%), and SCC (0.03%). Furthermore, we could not find any evidence of clonal BMDC expansion. In SCC lesions, we also found that most of the BMDCs were tumor-infiltrating hematopoietic cells. In addition, BMDCs in the SCC lesions lacked characteristics of epidermal stem cells, including expression of stem cell markers (CD34, high alpha6 integrin) and the potential retention of BrdU label. These results indicate that BMDCs are not a major source of malignant keratinocytes in UVB-induced SCC. Therefore, we conclude that BMDCs are not the origin of CSCs in UVB-induced SCC.

Involvement of bone marrow‐derived stromal cells in gastrointestinal cancer development and metastasis

The involvement of bone marrow (BM) in tumor-stroma reactions or tumor development has not been examined in a cancer allograft, which has otherwise been appropriate for assessing therapeutic modalities. We investigated the fate of BM-derived cells in colon cancer allografts and liver metastases in mice. C57BL/6 mice were irradiated and rescued by BM transplantation from green fluorescent protein (GFP)-transgenic mice. MC38 colon cancer cells were stably transfected with the pDsRed gene in order to identify tumor cells by fluorescence. These were inoculated into the mice to generate subcutaneous allografted tumors or liver metastases. The tumors were observed under confocal microscopy and fluorescent immunohistochemistry to determine the fate of tumor versus BM-derived cells. GFP-positive (GFP(+)) cells were consistently identified as vimentin(+), alpha-smooth muscle actin (alphaSMA)(+), spindle-shaped stromal cells in both the subcutaneous tumors and the liver metastases. GFP(+) cells of leukocyte lineage also infiltrated the tumors. Neither GFP(+) CD31(+) endothelial cells nor GFP(+) DsRed(+) cells were detected in the tumor. BM-derived cells frequently and consistently infiltrated the tumor allografts and metastases as interstitial cells and leukocytes. Cells derived from the fusion of BM cells and tumor cells were not observed. This model may be appropriate for the clarification of the effects of anticancer therapies and the study of BM-derived cells in tumor-host interactions.

Study progress about dendritic/tumor fusion cell-based vaccine in tumor immunotherapy

Dendritic cells (DCs) are potent professional antigen-presenting cells which are able to in-duce primary immune responses. DC can capture and process antigens into small-molecule peptides and present them to T cells and B cells through MHC class I and II molecules. An alternative approach to the induction of antitumor immunity is the use of fusion of DC and tumor cells. In this approach, a broad spectrum of known and unknown tumor-associated antigens(TAA) are presented by MHC class I and II molecules. In animal and clini-cal studies, dendritic/tumor fusion cell-based vaccine has made much progress and become a focus of the tumor immunotherapy. Key words: Dendritic cells ; Cancer vaccines ; Antigens,tumor-associated ; T-Lymphocytes,cytotoxic ;

Cell fusion: from hybridoma to dendritic cell-based vaccine

The deployment of dendritic cell (DC) and tumor cell fusions is increasing in tumor immunotherapy. In animal and human studies, fusion cell vaccines have been shown to possess the elements essential for processing and presenting tumor antigens to host immune cells, for inducing effective immune response and for breaking T-cell tolerance to tumor-associated antigens. Moreover, fusion cell vaccines provide protection against challenge with tumor cells and mediate regression of established tumors. Despite these unique features of fusion cell vaccines and the observation of tumor eradication in animal studies, limited success has occurred in clinical trials. This article reviews the methods used for optimizing the preparation and selection of DC–tumor fusion cells and analyzes factors influencing the success or failure of fusion cell-mediated immunotherapy. In addition, we discuss the challenges facing effective fusion cell vaccine production, including factors in preparation, selection and quality control of fusion cell vaccines, as well as approaches for enhancing anti-tumor immunity.