Fusions of Tumor-derived Endothelial Cells with Dendritic Cells Induces Antitumor Immunity

To explore dendritic cells/tumor-derived endothelial cells (DC/EC) fusion cells are potent stimulators of T cells to impact tumor progression. ECs were isolated from mice hepatoma cell line (H22) Xenograft, and dendritic cells were isolated from bone marrow of BALB/c mice, then the isolated ECs were cultured and detected the endothelial surface expression of CD105 by flow cytometry. The endothelial characteristics of ECs were detected by tube formation assay and Dil-Ac-LDL uptake assay. After the fusion with polyethylene glycol (PEG), we used DCs, ECs, DCs mixed ECs as the control groups, DC/EC fusion cells as the experimental group, Secretion of IFN-α and IFN-γ was evaluated, T lymphocyte proliferation and cytotoxic T lymphocytes (CTL) were detected in vitro. In vivo, T lymphocyte induced by five groups was injected to detect the effect of tumor progression. Purified ECs (CD105+) took the function of endothelial cells, then successfully fused with DCs. The DC/EC fusion cells were functional in stimulating the proliferation of T cells, which produced IFN-α and IFN-γ. In vivo, T cells stimulated by DC/EC fusion cells effectively repressed tumor growth. The fusion cells, which was capable of stimulating T cells, is indispensable for antitumor immunity.


Induction of endothelial/Fusion Cells (FCs)-specific CTL responses by four types of cell preparations.
To investigate the Ag-specific CTL induction capacity by four types of cell preparations, cytotoxicity assays were performed. Incubation of the endothelial/FCs-stimulated T cells demonstrated selective lysis of the tumor endothelium, while there was no significant lysis of the DC only, EC only, DC mixed with EC (Fig. 2b). These results indicate that T cells stimulated by DC/EC fusion cells had selective lysis of tumor endothelium.

Stimulation of T cell proliferation compromised in fusion cells.
To determine the ability of fusion cells about stimulating T cell proliferation, flow cytometry (Beckman Coulter Epics XL, USA) was applied. T cells co-cultured with DC/EC fusion cells proliferated vigorously, co-cultured of these T cells with DC only, EC only, DC mixed with EC resulted in proliferation of T cells, however, at a lower level (Fig. 2c).

DC/EC fusion cells induce antitumor activity.
To assess the induction of antitumor immunity, mice were immunized with irradiated DC, EC, DC mixed with EC or DC/EC fusion cells and then challenged intravenously with 2 × 10 5 viable H22 cells. In Fig. 3a, immunization of DC/EC fusion cells significantly decreased the tumorigenicity of H22 in BALB/c mice. The tumor volume and weight of the DC/EC fusion cells group was significantly decreased compare to the PBS group ( Fig. 3b and c). These findings indicate that T cells induced by DC/EC fusion cells is effective in anti-tumor activity.
Cell proliferation, endothelial cell expression and cell apoptosis in H22 Xenograft. In Fig. 4a, the result showed that there was a significantly increase in apoptosis in DC/EC fusion groups when compared to other groups. We explored the function of activated T cells by immunization, which was confirmed by IHC staining using Ki-67 and CD105 antibody ( Fig. 4b and c). T cells induced by DC/EC fusion cells led to low level of Ki-67 protein and CD105 protein expression compared to that by mixed with DC and EC, DC only, EC only or PBS. Importantly, we found no toxicity in heart, lung, liver or kidney tissues of mice injected with T cells induced by immunization (Fig. 4d). These data indicate that T cells induced by DC/EC fusion cells can enhance antitumor immunity.

Discussion
During the development of immune system, body can produce immune tolerance to autologous antigen, so in a healthy state, the body's immune system will not produce an immune response to normal tissue. Previous study found that in situations where a tumor-associated antigen exists but remains unidentified, an approach may be needed for presentation of that antigen by a professional antigen-presenting cell (APC) 21 . Stimulus for DCs activation is in combination with Ags, mature DCs induce an effective anti-tumor immunity [22][23][24][25][26][27] . The DC/tumor fusion is an effective approach, because multiple tumor-associated Ags, including those known and unknown, are endogenously processed and presented by MHC class I and II pathways with the ability to migrate to draining lymph nodes [28][29][30][31][32][33][34][35] . In previous studies, DC-Tumor fusion have been found to have the effective treatment in carcinomas, lymphomas, and melanomas in mice 14,36-41 . These findings have recently been extended to the treatment  and long-term survival of patients 14,[42][43][44][45] . However, the fusion of DCs with tumor-derived endothelial cells and its function is largely unknown.
In this experimental setting, T cells are stimulated by four kinds of cell suspensions (DC only, EC only, DC mixed with EC, DC/EC fusion). The results demonstrate that T cells activated by DC/EC fusion cells are effective in protecting mice against tumor challenge. In this context, fusion of tumor-derived endothelial cells with DCs may result in heterokaryons that express the necessary MHC, DC/EC fusions as compared with mixed DC with EC, DC only or EC only, were sufficient to give rise to T cell proliferation, IFN-α , IFN-γ production and CTL responses. T cells treat neovascular antigens of tumor vascular endothelial cells as heterologous antigens (such as CD105). When these antigens are largely and stably presented to T cells by DCs, could activate the specific T cells, kill tumor vascular endothelial cells, but not target normal blood vessels 21 .
In conclusion, we have isolated and generated tumor-derived endothelial cells and DCs from tumor-bearing mice with success. Immunogenic cells created by DC/EC fusions have the capacity to greatly stimulate EC-specific T cells. Our findings that T cells activated by DCs fused with tumor-derived endothelial cells could induce higher levels of IFN-α and IFN-γ , as well as significantly reinforce CTL responses in vitro or in vivo, which may help in designing optimal strategies for the therapy and may improve DC/EC fusion-based vaccination strategies. Tube Formation Assay. To analyze the tube formation ability of CD105 + cells, 100 μ l/well of growth factor-reduced Matrigel (BD Bioscience) was laid into 96-well plates to solidify. Cells were seeded into 96-well plates. After 6 h, the tube formation was assessed with microscopy 20 .

Materials and Methods
Dil-Ac-LDL Uptake Assay. CD105 + cells were plated into the 6-well plates at 5 × 10 4 cells/dish. At 75% confluence, the culture medium was replaced by the serum-free DMEM for 24 h, followed by incubation with 2 μ g/ml Dil-Ac-LDL for 5 h in incubator. Then cells were washed and fixed with paraformaldehyde (4 °C, 30 min), followed by DAPI staining for 3 min. The Dil-Ac-LDL uptake was assessed with microscopy 20 .

Fusion of DCs with endothelial cells (DC/EC).
DCs were incubated with ECs for 5 min at a ratio of 10:1 in serum-free medium, which contained 50% polyethylene glycol. Then, culture medium was added to dilute the polyethylene glycol slowly. After washing, the cells were cultured in RPMI 1640 medium (10% FBS, 500 U/ml GM-CSF) for 7-14 days.

Cell proliferation assay. The effects of T cells co-cultured with DC only, EC only, DC mixed with EC, or DC/EC fusion cells on cells proliferation was examined at stimulate cells : T cells (S : T cells) ratios by flow cytometry (Beckman Coulter Epics XL, USA).
Cytotoxicity assays. T cells were stimulated and harvested, which were treated as effector cells in CTL assays 46,47 . The effects of T cells co-cultured with DC only, EC only, DC mixed with EC and DC/EC fusion cells for 5 hours, respectively. After T cells stimulation, 2 × 10 4 PKH-26 (Sigma-Aldrich) labeled-target ECs were cultured with T cells (37 °C, 5 h). Cytotoxicity assays were examined at the indicated effector-to-target cell (E:T) ratios by flow cytometry (Beckman Coulter Epics XL, USA).

ELISA.
In order to assess the production of IFN-α or IFN-γ in T cells, DC only, EC only, DC mixed with EC, or DC/EC fusion cells were washed twice and cocultured with T cells (1 × 10 5 cells) at a ratio of 1:10 in 48-well plates at 37 °C for 3 days in the absence of IL-2. T cells were purified and cultured in the low-dose of IL-2 (10 U/ ml) for 3 days. The test supernatants from these samples were collected and tested for IFN-α and IFN-γ production by ELISA (BD Pharmingen) according to manufacturer's instructions. Proliferation assay (In vivo). BALB/c mice (Four weeks old, female) were purchased from Guangxi Laboratory Animal Center (Nanning, China). All animal care and experimental procedures were according to guidelines of the Institutional Animal Care and Use Committee of Guangxi Medical University. Left flanks of ten mice were implanted subcutaneously with 1 × 10 5 H22 (mice hepatoma cell lines) suspended in 0.01 M PBS. Tumor volumes were calculated by the formula. 0.52 × a (length) × b (width) 2 in millimeters. The animals were sacrificed and the tumors were excised after 40 days.
Immunohistochemistry. In order to quench endogenous peroxidase activities, 4 μ m-thick tumor sections were made and incubated with 3% hydrogen peroxide. Heat mediation in citrate buffer (pH 6.0) was used to retrieve antigen. After blocking with 10% goat serum, the slides was incubated with primary antibody. Then, the samples were incubated with the antibodies against Ki-67 48,49 (Abcam) or CD105 9 (Abcam) overnight in a humidified container at 4 °C. 0.01 M PBS without primary antibodies was applied as the negative control. Immunohistochemical staining was performed with DAB and were counterstained with hematoxylin. Instead of adding antibody, TUNEL reaction mixture (R&D Systems, Minneapolis, MN) was then added to tumor sections, followed by incubation in a humidified chamber at 37 °C for 60 min and DAPI staining for 3 min.
Statistical analysis. Quantitative data were expressed as mean ± SD. One-way analysis of variance was used to determine significance. The difference was significant when P values were < 0.05.