BALB/c and C57BL/6 (B6) mice (8-10 wk old) were purchased from the Shanghai Experimental Animal Center, Chinese Academy of Sciences (Shanghai, China). All mice were kept under pathogen-free conditions in the animal center of the Soochow University (Suzhou, China).

Gastric cancer cell line, SGC-7901, and melanoma cell line, B16, were purchased from the Shanghai Cell Biology Institutes, Chinese Academy of Sciences (Shanghai, China). Both cell lines were cultured in RPMI (Roswell Park Memorial Institute) medium 1640 (GIBCO, USA) containing 10% fetal calf serum (FCS), penicillin G (100 U/mL), and streptomycin (100 μg/mL) at 37°C in a humidified incubator supplemented with 50 mL/L CO₂.

Murine granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, tumor necrosis factor-α (TNFα), IL-2, and IL-7 were purchased from Becton Dickinson (New Jersey, USA). Phenotypic analysis, fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-labeled monoclonal antibodies (Mabs), such as MHC I (Ia), DEC-205, CD11b, CD40, CD80, CD86, and F4/80, were provided by Pharmigen (CA, USA). Mitomycin C (MMC) was purchased from Jingmei Biothe (Shenzhen, China).

Primary bone marrow-derived DCs (BM-DCs) were obtained from mouse bone marrow precursors according to a previously established protocol[10]. BALB/c mice bone marrow obtained from tibia and femurs by flushing with media. Tissue was minced in a single-cell suspension through a nylon mesh. BALB/c mice bone marrow cells were stained sequentially with biotinylated anti-c-kit MAb and Streptavidm MicroBeads (Dynal, Norway). c-kit⁺ hematopoietic progenitor cells were magnetically isolated with a MiniMACS separator (Milteyi Biotec, Auburn, CA) from the BALB/c mice bone marrow cells. The cells (6 × 10⁶) were cultured for 5 d in fresh RPMI medium 1640 containing 10% FCS, GM-CSF (4 ng/mL), and IL-4 (10 ng/mL). Then immature DCs were further cultured in GM-CSF (4 ng/mL) and TNFα (5 ng/mL) for 3 d to induce their maturation. Bone marrow-derived mature DCs were observed by light microscope (Nikon, Japan).

DCs immunofluorescence analysis was performed as previously described[11]. In brief, DCs cultured for 5 d or 8 d as described above (2 × 10⁵-4 × 10⁵ cells) were incubated with rat anti-DEC-205 MAb followed by FITC-labeled goat anti-rat IgG (Fab’)² antibodies or directly with FITC-labeled MAbs against CD40, F4/80, CD11b, or CD80 and PE-labeled MAbs against Ia, or CD86 followed by FACS analysis. The instrument compensation was set in each experiment using two-color stained samples.

The mixed leukocyte reaction (MLR) assay was performed in accordance with previously described methods[12]. Immature DCs or mature DCs derived from bone marrow c-kit⁺ cells were incubated in RPMI Medium 1640 containing 10% FCS and mitomycin C (MMC; 15 μg/mL) in six-well plates at 37°C for 3 h to arrest their proliferation. After several washes with PBS, these stimulator cells were suspended in RPMI-1640 containing 10% FCS at concentrations ranging from 1 × 10² to 5 × 10⁴ cells/mL. One hundred microliters of the above stimulator cells suspension were added to each well of 96-well plates that contained allogeneic CD4⁺ T cells (3 × 10⁵ cells/100 μL per well) that had been magnetically isolated from B6 mice using CD4 Microbeads. Five days later, T-cell proliferation was determined by using an MTT assay. Fifteen μL of MTT (5 μg/mL in PBS) was added to each well, and the plates were incubated at 37°C for an additional 4 h. The resultant absorbance at 550 nm was read by a microplate immunoreader. PBS alone was used as the negative control. Results are expressed as the mean of three wells from three individual experiments.

BM-DCs were pulsed with freeze-thawed tumor lysate at a 1:3 DC:tumor cell ratio. SGC-7901 tumor cells (6 × 10⁶) were lysed by rapid freezing (liquid nitrogen) and thawing in a 37°C bath three times. BM-DCs (2 × 10⁶ cells/mL) were incubated in six-well plates in the presence of SGC7901 tumor lysates (6 × 10⁶ cell equivalents/mL) in RPMI Medium 1640 containing 10% FCS, GM-CSF (4 ng/mL), and IL-4 (10 ng/mL) for one day at 37°C and 50 mL/L CO₂. These SGC-7901 tumor cell lysate-pulsed (TP) DCs were used for vaccination after one day. B16 TP DCs were used as control.

To confirm that tumor-specific cytotoxic T lymphocytes (CTLs) can be generated ex vivo, splenic CD3⁺ T cells (1 × 10⁶ cells/mL) were magnetically isolated from naïve BALB/c mice using CD3 microBeads. These cells were cultured in RPMI medium 1640 containing 10% FCS, then primed ex vivo in the presence of cytokines including IL-2 and IL-7 (5 ng/mL) at day 0, 7, and 14 with SGC-7901 TP DCs or B16 TP DCs at a 1:20 stimulator to responder cell ratio. Unpulsed DCs and SGC-7901 tumor lysates were used as controls. Fresh medium containing IL-2 and IL-7 was exchanged every 4 d. The primed T cells were effector cells; SGC-7901 or B16 tumor cells were target cells. On day 21, target cell suspension was added into 96 well plates, and effector cells were titrated to dilutions of target cells by serial dilutions (E-T mix, E:T, 1:1, 5:1, 10:1, 20:1, 50:1, 100:1). Supernatant from each well was collected after 20 h and cytolytic activity against target SGC-7901 tumor cells and B16 tumor cells was measured with a Cytotoxicity Detection Kit (LDH; Boehringer Mannheim, Mannheim, Germany). IFNγ production was determined with the IFNγ ELISA kit (Endogen, Woburn, MA, USA.) at a stimulator to responder cell ratio of 1:20.

Differences were evaluated using Statistical Package for Social Science 11.0 (SPSS11.0). Statistical analysis was performed using Student’s t-test. Statistical tests were two-sided. P < 0.05 were considered to be statistically significant.

c-kit⁺ hematopoietic progenitor cells from the BALB/c mice bone marrow cells were cultured in presence of GM-CSF, IL-4, or TNFα for 8 d, then observed by light microscopy. Results show large cells with oval or irregularly shaped nuclei and many small dendrites (Figure 1).

The phenotypic profile of a representative population of bone marrow DCs was determined by FACS. BM-DCs cultured for 5 d expressed moderate levels of co-stimulatory molecule CD40, high levels of CD11b, and very low levels of Ia, DEC-205, CD80, CD86, and were negative for F4/80 (Figure 2). However, when these cells were cultured in the presence of GM-CSF, IL-4, or TNFα for 8 d, they differentiated into mature DCs that expressed high levels of Ia, DEC-205, CD11b, CD80 and CD86 antigen, moderate levels of the co-stimulatory molecule CD40, and were negative for F4/80 (Figure 2).

Allogeneic mixed-leukocyte reactions were performed using splenic T cells purified from B6 mice as responder cells. BM-DCs were treated with MMC to arrest cell proliferation and were used as stimulator cells. T cell proliferation was determined by using an MTT assay. Results show BM-derived mature DCs have the capacity to stimulate allogeneic T cells (Figure 3); however, BM-derived immature DCs and PBS did not simulate allogeneic T cells (Stimulator cells: 5 × 10⁴ cells/mL, OD 550 nm: 1.74 ± 0.15 in mature DCs group versus 0.22 ± 0.05 in immature DCs group or 0.16 ± 0.04 in PBS group, P < 0.05, Figure 3).

To study the potential of in anti-tumor immunity in vitro, BM-derived DCs were prepared by pulsing BM-DCs with tumor lysates after 5 d of culture in the presence of GM-CSF and IL-4. Naïve mouse splenic T cells were primed in vitro with SGC-7901 TP DCs or B16 TP DCs in the presence of IL-2 and IL-7 to elicit cytolytic reactivity against tumor cells. The results show that T cells in vitro primed with SGC-7901 TP DCs were able to lyse efficiently and specifically parental SGC-7901 tumor cells, but not B16 tumor cells, and T cells primed with B16 TP DCs, SGC-7901 tumor lysates or unpulsed DCs did not induce specific CTL against SGC-7901 tumor cells (E:T = 100:1, 69.55% ± 6.05% specific lysis in the SGC-7901 TP DCs primed T cell/SGC-7901 group versus 15.72% ± 2.9% specific lysis in the SGC-7901 TP DCs primed T cell/B16 group, 13.75% ± 3.14% specific lysis in the B16 TP DCs primed T cell/SGC-7901 group, 10.6% ± 3.01% specific lysis in the SGC-7901 tumor lysates primed T cell/SGC-7901 group, or 8.75% ± 2.15% specific lysis in the unpulsed DCs primed T cell/SGC-7901 group, P < 0.05, Figure 4).

Splenic CD3⁺ T cells primed with SGC-7901 TP DCs produced higher levels of IFNγin vitro when stimulated with SGC-7901 tumor cells; however, they did not produce higher levels of IFNγ when stimulated with B16 tumor cells (1575.31 ± 60.25 pg/mL in the SGC-7901 group versus 164.11 ± 18.52 pg/mL in the B16 group, P < 0.01, Figure 5).

Dendritic cells (DCs) are professional antigen presenting cells (APCs) that both initiate and modulate the immune response. DCs are cells in the pathway of antigen capture and presentation to T cells, with the unique ability to directly prime naïve CD4⁺ and CD8⁺ T cells. Gastric cancer is one of the most common cancers. Although gastric cancer therapy has made great progress, it is still difficult to treat advanced gastric cancer, as it has spread to the lymph glands and metastasized. Currently, tumor immunotherapy for gastric cancer has potential. DCs are believed to be essential for stimulating tumor-specific cytotoxic T lymphocyte (CTL) and inducing the protective and therapeutic anti-tumor immunity.

Currently, no defined tumor specific antigen is available for many tumors, including gastric cancer. Now, DCs can be pulsed with synthetic peptides or proteins derived from known tumor associated antigens (TAA) such as MUC1, Her-2/neu, tyrosinase, CEA or Melan-A/MART. Some studies have shown that it is feasible that DCs-based tumor vaccines will become a new effective immunoadjuvant therapy for gastric cancer, which can decrease the incidence and recurrence rate after operation for gastric cancer. Primary clinical studies were performed on melanoma patients using DCs pulsed with peptides or loaded with tumor cell lysates. Kono et al[24] report that tumor vaccination therapy with DCs pulsed with HER-2/neu-peptides may be a potential candidate for the novel treatment of gastric cancer patients.

c-kit+ hematopoietic progenitor cells from mice bone marrow cells cultured with GM-CSF, IL-4 or TNFα for 8 d showed the character of typical mature DCs. Gastric cancer cell lysates were obtained by rapid freezing and thawing, and were regarded as tumor specific antigens. Vaccination with bone marrow-derived dendritic cells pulsed with tumor cell lysates induced anti-tumor immunity specific to gastric cancer ex vivo.

Vaccination with bone marrow-derived dendritic cells pulsed with tumor cell lysates induced anti-tumor immunity specific to gastric cancer ex vivo. These results suggest that an evaluation of BM-DCs pulsed with tumor lysates against gastric cancer is an important next step in vivo. A trial evaluating this approach in mice is currently in preparation.

DCs means dendritic cells, BM-DCs indicates bone marrow DCs, APCs stands for antigen presenting cells, MHC is major histocompatibility complex, CTL is cytotoxic T lymphocyte, IFNγ means interferon gamma.

Using an in vitro system, the authors suggest a specific anti-tumor effect based on immunological mechanisms. The fatal prognosis of cancer requires the development of novel therapeutic concepts. Unfortunately, various promising strategies have failed in the clinical practice. Therefore, suitable animal models are strongly needed to investigate the possible effectiveness of the shown ex vivo principle for an anti-cancer treatment.