Reagents
The authors prepared Griess reagent kit, Dulbecco’s modified eagle medium (DMEM), and Fetal Bovine Serum from Kala zist-Iran. Nitrotetrazolium blue tetrazolium (NBT), dimethylthiazol-2, 5-diphenyl tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), dioxin, and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich (St. Louis, MO). Enzyme-linked immunosorbent assay (ELISA) kits were ordered from Qiagen (Hilden, Germany).
Bacterial Strains and Growth Conditions
Lactobacillus Casei (ATCC: 393) was purchased from Pasteur Institute of Iran. A Rogosa’s medium was applied to cultivate the bacteria. The process was performed at 37 °C for 24 hours, washed with PBS, heated at 56 °C for 60 minutes, and lyophilized.
Cell Culture
Pasteur Institute-Iran provided us with TC1 cells. The cells were cultured at 37°C humidified atmosphere, using 5% CO2 and kept in monolayer cultures in DMEM supplemented with 10% FBS (Figure 1). Also, 104 TC1 cells were exposed freeze-and-thaw (-196 °C, 30 min) and nonlethal heated shock (43 °C, 30 min), then centrifuged to be used as a killed cell extract in the treatment of tumor mice.
Experimental design, mice and Tumor Induction
C57BL/6 female mice (n=80) with an age of 6-8 weeks were purchased from Pasteur Institute in Iran. The experimental processes were in compliance with the Iran Ministry of Health regulations, and approved by the Medical Ethics Committee for Animal Studies of Islamic Azad University (IR.IAU.PS.REC.1399.265). The mice were kept under standard conditions, i.e. temperature of 22–24 °C, 12-hour light/dark cycles and standard food and water. All the mice were acclimatized to the environment for 1 week prior to the experimentations and then 1× 104 viable tumor cells in 50 µL of PBS was injected subcutaneously in their left flanks with (Figure 2). Then, rats were randomly divided into 10 equal groups (Table 1). All groups received treatment in 100 µl volume and twice a 1-week interval. One month after treatment, Sampling was started.
Table 1
The characteristics of the studied groups
Group
|
Characteristics
|
Control
|
100 µL of PBS
|
Heated TC1
|
104 heated TC1 cells
|
Heated L. casei
|
2×108 CFU/mL of Heated Lactobacillus Casei
|
α-GalCer
|
5 µg of Alpha galactosyl ceramid
|
Heated TC1+Heated L. casei
|
Combined of Heated TC1 and Heated L. casei
|
Heated TC1+α-GalCer
|
Combined of Heated TC1 and α-GalCer
|
Heated TC1+Heated L. casei+ α-GalCer
|
Combined of Heated TC1, Heated L. casei and α-GalCer
|
Gardasil
|
50 µg of Gardasil vaccine
|
The proliferation potential of lymphocytes
The MTT assay was used to check proliferation index in the splenocyte population. The splenocytes were put in 96-well flat-bottomed plates and in a DMEM medium added by 10% FBS (1×105 cells/100 μl/well) and stimulated with antigens derived from the tumor cells by freezing and thawing (20 μg/mL). After 72 hours of incubation, the cultures were pulsed with 20 μl of the MTT solution (5 mg/mL) for 4 hours at 37 °C. Then, 150 mL of DMSO was added and shaken severely to dissolve formazan crystal. The optical density (OD) at 492 nm was measured using a microplate reader (Dynatech, Denkendorf, Germany). The experiments were conducted in triplicate sets.
Lactate dehydrogenase assay
Cytotoxic activity was investigated using a lactate dehydrogenase (LDH) detection kit (Takara Company, Tehran-Iran). This assay is a simple fast colorimetric medium for cytotoxicity quantification through determining activity of LDH released from the damaged cells. LDH is a stable cytoplasmic enzyme, which is available in most cells. The splenocytes were used as effector cells, and the TC1 cell lines were applied as target cells. The effector and target cells were washed with the assay medium (RPMI-1640 with 1% bovine serum albumin) and co-cultured at a ratio of 50 effector cells to 1 target cell in 96-well round-bottomed plates for 6 hours at 37 °C. Subsequently, the plates were put in centrifuge apparatus and the supernatants were transferred to 96-well flat-bottomed plates. Then, 100 μl of the LDH detection mixture was added to each well and incubated for 30 minutes at the room temperature. The OD was measured using a microplate reader (Dynatech, Denkendorf, Germany) at 492 nm.
Measurement of nitric oxide in splenocytes population
The potential of nitric oxide production was assessed by measuring the nitrite level in the splenocyte culture supernatants and using the Griess reagent. After the splenocytes being cultured, the cell free supernatants (50 μl) were collected and intermixed with 50 μl of Griess reagent (containing 0.1% sulfanilamide, 3% phosphoric acid, and 0.1% naphthyl-ethylenediamine). The resulted mixture incubated at room temperature for 10 minutes in the dark. After the incubation, microplate reader reads the absorbance at 540 nm (Dynatech, Denkendorf, Germany). The nitrite concentration is estimated based on a standard curve.
Cytokine assay
One week after the last immunotherapy, half of the mice were euthanized to measure the cytokine assay induced in splenocytes. Splenocytes were isolated from the mice in an aseptic environment, and single-cell suspensions of the splenocytes were prepared in a DMEM medium added with 10% FBS and red blood cells (RBCs) were removed by RBC lysis buffer. Next, the cell suspensions (2×106 cells/mL) were incubated in 24-well plates and pulsed with antigens. These antigens were derived from the tumor cells by freezing and thawing (20 μg/mL). Tumor antigen was prepared as described above. The culture supernatants were collected after 72 hours. The production of IFN-γ, IL-4, and TGF-β was evaluated by the ELISA according to the manufacturer’s instructions.
Statistical Analysis
The statistical analysis was conducted using the Kruskal–Wallis test, followed by pairwise comparisons, using the Mann–Whitney U-test and with the Bonferroni adjustment. The Kaplan–Meier estimator was applied to evaluate the survival function based on the lifetime data. The results are shown as means ± SD. p<0.05 was considered statistically significant.