Cell culture and mouse models
BALB/C mouse-derived mammary carcinoma 4T1 cells were obtained from the State Key Laboratory of Biotherapy of Sichuan University (Chengdu, China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, USA), supplemented with 10% fetal bovine serum (FBS; Cellmax, Australia) and 1% penicillin–streptomycin (Sigma-Aldrich, St Louis, MO, USA). Cell cultures were incubated at 37℃ with 5% CO2 in a humidified incubator.
Female 6–8-week-old BALB/C mice weighing about 20 g were obtained from Chongqing Tengxin Biotechnology Co., LTD. (Chongqing, China). Mice were housed in separate specific pathogen-free (SPF) cages and allowed to eat and drink ad libitum. The ambient temperature was controlled between 20 and 25 °C to simulate the circadian rhythm.
4T1 mammary carcinoma cells (1.5 × 105/ml) were subcutaneously injected into the right flank of each BALB/C mouse separately. This treatment time point was designated as day 0. Tumor size was monitored every 2 days, and tumor growth or regression was recorded. Tumors were measured with a vernier caliper and volume was determined using the formula: length x width2 x 0.5 by two investigators independently. Tumors were selected for cryosurgery when they were 4–6 mm in diameter. Mice were sacrificed when tumors reached a volume of 1500 mm3.
For tumor rechallenge experiments, mice were challenged by subcutaneous injection of 4T1 cells (1.5 × 105/ml) in 0.1 ml of phosphate-buffered saline on the contralateral flank in the second experiment on day 19.
Cryotherapy and irradiation
Mice have been anesthetized with the aid of using intraperitoneal injection of chloral hydrate. Cryosurgery was performed using the direct contact liquid nitrogen method. The cotton swab was swiftly immersed in liquid nitrogen and used to contact the tumor surface, each time for about 10 s and lasted 1 min until the tumor formed an “ice ball”. Then, the cycle was repeated after the ball melts. To guarantee optimum tumor cell death, a two-cycle 60-second freeze/thaw procedure was used. Mice were placed on a heating pad after the treatment to recover from anesthesia.
The mice were secured with a rubber band in a transparent cuboid self-made radiotherapy box. The 6-MV linear accelerator (Varian Clinac 600c, USA) was used for low-dose irradiation at a dose rate of 24cGy / min, and the source–surface distance was maintained at 100 cm. Before irradiation, dose rates in the radiation field center and the middle plane were measured in the radiotherapy box, and the dose was verified by stacking thermoluminescent sheets near the tumor. L-TBI is defined in this study as whole-body irradiation at 0.1Gy at a dose rate of 24 cGy/min.
Measurement of Lung Surface Nodules
On day 26, the mice were sacrificed, and the lung tissue was collected and fixed with a 10 % formalin solution. The pulmonary metastatic nodules were counted under an anatomical microscope and their diameters were measured. Metastatic nodules were divided into four grades based on their diameters: I < 0.5 mm, II 0.5 mm–1 mm, III 1 mm–2 mm, and IV > 2 mm. The numbers of pulmonary metastatic nodules were I × 1 + II × 2 + III × 3 + IV × 4. For histopathological examination, fixed tissues were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) according to standard protocols. Microscopic analysis of all slides was performed with an optical microscope (Olympus Cor, Tokyo, Japan) linked to a computerized imaging system (Image-Pro Plus V6.0, Silver Spring, MD).
Flow cytometry analysis, ELISA measurements and immunohistochemistry
The tumor tissues were excised and homogenized in DMEM medium with 0.2% type IV collagenase, 0.01% hyaluronidase, and 0.002% DNase I (all enzymes from Solarbio Science, Beijing, China) for 40 min at 37°C. Additionally, spleen tissues were dissected, ground, and filtered into a single-cell suspension using standard protocols. The blood cell lysate kits were used for removing red blood cells (BD Biosciences, CA, USA). The resulting single-cell suspension was stained with the fixable viability stain 780 before being tagged with the following antibodies as directed: CD45-PerCP antibody, CD11b-FITC antibody, Gr1-FITC antibody, CD11c-PE antibody, CD3-PerCP-Cy5.5 antibody, CD4-FITC antibody, and CD8-PE-Cy7 antibody. Stained samples were analyzed using a Beckman Coulter Gallios flow cytometer. All antibodies were purchased from BD, and the concentrations were the same as those of the antibodies used. All data measured by flow cytometry were analyzed using FlowJo software (version 10.0).
Tumor tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin before being cut into 4 µm thick sections. Baking, dewaxing, hydration, antigen repair and peroxidase blocking, adding antibody (CD4, CD8, Ki-67, and Foxp3), coloring, and re-staining were all performed. Finally, the slices were viewed under an optical microscope, and images were collected from five randomly selected fields under a 200X microscope. CD4, CD8, Ki-67, and Foxp3 positivity rates were counted by quantitative analysis with Image-Pro Plus 6.0 software (Media Cybernetics, USA), and data are expressed as mean ± standard error (SE). The percentage of positive cells = the number of positive cells / the number of total cells in this field.
On day 26, 0.5 ml of venous blood from the eyeball was collected, left to stand for 20 min at room temperature (20–25 °C), and then centrifuged (2000r, 20 min). The supernatant was collected as serum, inhaled under sterile conditions, and kept in a refrigerator at -20 °C for subsequent studies. According to the instructions for using the ELISA kit, the concentrations of IFN-γ, TNF-α, and IL-2 in serum were measured using the standard ELISA method.
Statistical analysis
SPSS18.0 (SPSS, Inc., Chicago, IL, USA) was used for data processing, and the enumeration data were expressed as mean ± standard error (mean ± SE). An independent sample t-test was used to compare the data between the two groups. One-way ANOVA was used to compare data from three or more groups. The Kaplan–Meier method was used for survival analysis. P < 0.05 was considered statistically significant. Fisher's exact test was used to analyze re-growth rates and re-challenges. All graphs were created with GraphPad Software Prism 7.0.