Animal Model and Tumor Induction
In this study, we designed an experiment where female C57BL/6 mice, aged six weeks, were divided into four distinct groups(N=7): NC group(N=7):a control set, L group(N=7): subjected to low-dose radiation(0.1 Gy radiation every other day for five times), CTLA-4 group(N=7) :treated solely with anti- CTLA-4(injections of 200 μg of anti- CTLA-4 once every other day for three times), L+CTLA-4 group(N=7): combination of low-dose radiation and anti-CTLA-4 treatment.
Irradiation Geometry:
In the conducted experiments, irradiation was precisely delivered at a rate of 10 cGy/min, encompassing a total of five cycles. The irradiation was performed using a linear accelerator (LINAC) as the radiation source. The LINAC emitted X-rays with a mean energy of 6 MV (megavolts), ensuring consistent and controlled irradiation conditions throughout the experimental cycles.
Cell lines
Dr. Xiaoyang Yi kindly provided the LLC cell line utilized in our research. We cultivated this cell line in DMEM with high glucose content (sourced from Gibco, USA), enriched with 10% fetal bovine serum and a 1% mixture of penicillin-streptomycin (acquired from Biosharp, China). These cells were consistently incubated at a stable temperature of 37°C, in an atmosphere containing 5% CO2, and under conditions of elevated humidity. Upon reaching near 80% confluence, the cells were transferred to new Corning 100mm culture dishes (originating from Corning, USA) for further growth and passaging.
Tumor models
We acquired six-week-old female C57BL/6 mice, each weighing around 18±2 grams, from HFK Bioscience in Beijing, China, ensuring they were kept in an environment free from specific pathogens. The Shandong First Medical University's Institutional Animal Care and Use Committee granted approval for the mouse studies. We administered a subcutaneous injection of 1×10^6 LLC cells into the left posterior limb of the mice. Subsequent to the tumors reaching an estimated volume of 100 mm^3, we allocated the mice into four distinct experimental groups, which are elaborated upon in the following section.
Flow Cytometry Analysis
In this study, the mice were allocated into four separate cohorts. The excised tumors were subsequently subjected to homogenization using a mixture containing 0.2% collagenase type IV, 0.01% hyaluronidase, and 0.002% DNase I, all sourced from Solarbio Science in Beijing, China. This process was performed in a DMEM medium and maintained at a temperature of 37°C for a duration of 40 minutes. The resulting single-cell suspension was stained using fixable viability dye BV510. The cells obtained were then marked with a set of antibodies for analysis: (Tube 1) contained antibodies such as CD45+ FITC, CD3+ APC, CD8+ percpcy5.5, and IFN-γ+ PE/APC-Cy7 to primarily assess the T cells infiltrating the tumor tissue; (Tube 2) was used with CD45+ percpcy5.5, CD4+ FITC, CD25 PE, and foxp3 APC to primarily evaluate Treg cells within the tumor tissues, following the guidelines provided by Biolegend, USA. For the detection of INF-γ, cells underwent in vitro stimulation using a cell stimulation cocktail, which included protein transport inhibitors from Biolegend, USA, for a period of 6 hours. Post-stimulation, cells were labeled on the surface with antibodies CD45+ FITC, CD3+ APC, CD8+ percpcy5.5, followed by processing with a fixation and permeabilization kit from Biolegend, USA, and subsequent staining with IFN-γ antibody at a dilution of 1:1000. Flow cytometry analysis was performed on the stained cells using a BD LSDFortessa system. The data from the flow cytometry were processed and analyzed using FlowJo software, version 10.0
Immunohistochemistry
The experimental mice were evenly distributed into four distinct groups. Following this, the tumor specimens were preserved in 10% neutral-buffered formalin, then embedded within paraffin blocks. From these blocks, sections with a thickness of 4 μm were prepared for subsequent immunohistochemical analysis (IHC). These sections underwent staining using specific antibodies: CD8, FOXP3, GZMB with the procedure aligned with the guidelines provided by abcam, China. Image capturing was conducted with the aid of an optical microscope provided by Olympus, Tokyo, Japan. For the quantification of immunoreactivity, in each tumor specimen, cells that exhibited positive staining for CD8, FOXP3, GZMB were tallied across five fields selected at random under a magnification of 200x. From these counts, the proportion of cells showing positive staining was determined.
ELISA Measurements
The tumor specimens were homogenized, and the resulting supernatants were collected after being treated with a lysis buffer that included protease inhibitors provided by beyotime (P1045). The concentrations of cytokines and chemokines, specifically IFN-γ, IFN-α, IFN-β, CXCL9, CXCL10, and CXCL11, were quantified using the ELISA technique. These measurements were performed with specific antibody-based ELISA kits, following the protocols recommended by J&L Biological, based in Shanghai, China. More specifically, the mice were euthanized 24 hours subsequent to the final session of low-dose radiation, and their tumor tissues were excised for analysis.
Western blot
LLC cell proteins and those from related samples were isolated using RIPA buffer supplied by bioss (C5029-100 ml) with the addition of a cocktail that inhibits proteases from beyotime (P1045). The BCA technique, utilizing beyotime's kit (P0010S), was employed to ascertain the protein levels. Each sample contributed 50 μg of protein, which was then separated on a 10% SDS-PAGE gel under a steady 120 V. Proteins were then transferred to a pre-activated PVDF membrane (Millipore, Cat # IPVH00010) at a current of 220 mA and temperature of 4 °C throughout the night. The membranes were incubated with primary antibodies targeting Interferon alpha 2 (ab193055 at a dilution of 1:1,000), and GAPDH (Proteintech 10494-1-AP, also at a dilution of 1:1,000) in cold conditions overnight. Subsequent to triple washes, the blots were exposed to a secondary antibody specific to rabbit IgG (Abcam ab150077; at a dilution of 1:5,000) at ambient temperature for half an hour. Protein bands were visualized using a chemiluminescence method (Millipore WBKLS0100 ECL), and images were captured with an Azure c600 imager from Azure Biosystems. Bands corresponding to molecular weights of 25, 37, and 55 kDa were pinpointed and annotated on the film prior to gel scanning. The unaltered scans of the western blots are presented in the supplementary materials The ratio of Interferon alpha 2 to GAPDH for each specimen was calculated for relative quantification using the ImageJ software.
RNA sequencing analysis
Specimens of tumor tissue were immediately preserved by freezing in liquid nitrogen, and from these specimens, total RNA was isolated. Following this, the construction of libraries was carried out with the aid of the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, based in San Diego, CA, USA). Shandong Xiuyue Biotechnology Co., Ltd., located in Shandong, China, was responsible for conducting the transcriptome sequencing as well as the subsequent data analysis.
Statistical analysis
GraphPad Prism 8.0 (GraphPad Software, based in La Jolla, CA, USA) was utilized for all statistical evaluations. The results are depicted as the mean ± SEM (standard error of the mean). The analysis in this research included the use of Two-Way ANOVA for analyzing the influence of varying treatments and time intervals on the proliferation of tumors. Comparisons between two sets were conducted using Unpaired 2-Tailed Student's t-Tests, and for comparing multiple groups, One-Way ANOVA followed by Bonferroni adjustments was applied. The levels of statistical significance were indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). These statistical methods were carefully chosen to provide a thorough assessment of the effects of treatments on tumor progression, immune reactions, and other pertinent factors within the research.