Cell lines and reagents
The HEK293T and human ESCC cell lines KYSE150 and KYSE520 were purchased from the American Type Culture Collection (ATCC). Cells were cultured in DMEM (Gibco, USA) media supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin solution, and maintained in a 37 °C, 5% CO₂ incubator. All cell lines were routinely authenticated by short tandem repeat (STR) profiling and tested for mycoplasma to ensure cell purity. The integrin antagonist ATN-161 (MCE, USA) was dissolved in dimethyl sulfoxide (DMSO) at an appropriate concentration to prepare a stock solution, which was then stored at −80 °C until further use.
Lentivirus package and infection
The ITGA5 overexpression, knockdown, and their corresponding control plasmids were commercially synthesized by Beijing Maijin biotechnology company Ltd. For lentiviral package, packaging plasmid (psPAX2) and envelope plasmid (pMD2.G) were co-transfected into HEK-293T cells and incubated for 15–20 min. The medium was replaced with complete fresh medium 6 h later, and virus supernatants were collected at 24, 48, and 72 h. ESCC cells were pre-seeded at 30–40% confluency, and then infected with the harvested lentivirus. Stably transfected cells were selected using puromycin (Beyotime, USA).
Plasmids and small-interfering RNA transfection
Plasmids and siRNAs were obtained from Jiangsu Jikai biotechnology company Ltd. When cell confluence reached approximately 50%, transfection was performed using Lipofectamine 3000 (Thermo Scientific, USA) to deliver plasmids or siRNAs into the cells. The medium was replaced with complete medium 6hs after transfection. Transfection efficiency was assessed 48 to 72 h post-transfection. The sequences of the plasmids and siRNAs are provided in Supplementary Table 1.
Irradiation treatment
Cells and mice were irradiated using an X-ray irradiation system (RS2000-225, RAD Source Technologies, USA). In vitro assays, cells were seeded in culture dishes at an appropriate density on the day before irradiation. The day after, fresh medium was replaced, and cells were exposed to X-rays at a dose rate of 200 cGy/min with varied doses. In vivo assays, irradiation was performed at a dose of 15 Gy [35,36,37] when tumor volumes reached approximately 100 mm3.
RNA extraction and real-time PCR
Total RNA was extracted using Trizol reagent following the manufacturer’s protocol, and its concentration was measured using Nanodrop spectrophotometer before storage at −80 °C. Subsequently, cDNA synthesis was performed from total RNA using the HiScript III RT SuperMix (Vazyme, China) reverse transcription kit. Quantitative real-time PCR (qPCR) was carried out with SYBR Green Master Mix (Vazyme, China). Relative gene expression levels were calculated using the 2−ΔΔCt equation, and normalized to the expression of internal reference genes. The sequences of all primers are listed in Supplementary Table 2.
RNA-Seq analysis
RNA sequencing was performed by Novogene (Beijing, China). Total RNA was extracted from ESCC cells and quantified, with RNA integrity assessed using the Agilent 2100 system. mRNA was enriched using oligo (dT) magnetic beads, followed by fragmentation and reverse transcription into double-stranded cDNA. After end repair, A-tailing, and adapter ligation, the library was amplified by PCR, purified, and subjected to quality control. The constructed libraries were pooled and loaded onto an Illumina platform for sequencing. Finally, low quality sequences were filtered out, and clean reads were aligned to the reference genome for gene expression quantification.
Western blotting analysis
Cells were lysed using pre-chilled protein lysis buffer containing protease and phosphatase inhibitors. After sonication and centrifugation, loading buffer was added according to protein concentration and samples were boiled for denaturation. Proteins were separated by SDS-PAGE electrophoresis under constant voltage and transferred onto PVDF membranes. After blocking at room temperature for 60 min, membranes were incubated with primary antibodies at 4 °C overnight. Following TBST washes, membranes were incubated with secondary antibodies for 1 h at room temperature. Protein bands were finally visualized using enhanced chemiluminescence reagents. All antibodies used are listed in Supplementary Table 3.
Colony formation assay
Radiosensitivity of tumor cells was assessed using the colony formation assay. Before irradiation, a single-cell suspension of exponentially growing cells was plated on six-well dishes. Subsequently, cells were irradiated with the indicated dose and allowed to grow for additional 10–14 days. The resulting colonies were fixed with paraformaldehyde (4%) for 15 min and stained with crystal violet (5%) for 15 min. After washing three times with PBS, the colonies of more than 50 cells were counted in each group. Experiments were repeated in triplicate. Data from experimental groups were normalized to their respective control groups.
Cell apoptosis assay
Cell apoptosis was detected using the Annexin V-propidium iodide (PI) Apoptosis Detection Kit according to the manufacturer’s protocol. Briefly, cells and supernatant were collected using non-EDTA trypsin, washed with PBS, and resuspended in binding buffer. For each sample, 5 μL Annexin V and 5 μL PI were added, followed by incubation in the dark for 15 min. Samples were analyzed by flow cytometry, and the percentage of apoptotic cells was determined using FlowJo software.
Cell cycle assay
Cell cycle was analyzed using the cell cycle detection kit following the manufacturer’s instructions. Briefly, cells were seeded in 6-well plates one day prior to irradiation treatment. At designated post-irradiation time points, cells were trypsinized, washed with PBS, and fixed in pre-chilled 70% ethanol overnight at 4 °C. After fixation, cells were washed and stained with PI staining solution at 37 °C for at least 20 min. Data acquisition was performed using flow cytometry, and cell cycle distribution was analyzed using FlowJo software.
Comet assay
After exposure to either no radiation or radiation, DSBs were detected using a DNA Damage Comet Assay Kit. Cell density was adjusted to 1 × 10⁵ cells/mL, mixed with low-melting agarose (1:10 ratio), and added onto the slides. After solidification, slides were immersed in lysis buffer at 4 °C for 1–2 h. Electrophoresis was performed at 25 V for 30 minutes in a horizontal chamber with neutral buffer. Slides were then sequentially immersed in DNA neutralization buffer and 70% ethanol, and stained with SYBR Green I at room temperature for 30 min protected from light. Fluorescent images were captured using the confocal microscope in the FITC channel and analyzed with CASP software to quantify tail moment as the extent of DNA damage.
HR and NHEJ reporter assay
The repair reporter system is a tool used to detect the efficiency of DNA damage repair. The DR-GFP system is employed to monitor HR repair, while the EJ5-GFP system is used to assess NHEJ repair. I-SceI is an endonuclease that can generate DSBs at specific recognition sites. When a reporter gene containing the I-SceI recognition site is integrated into the cellular genome or constructed into a plasmid, the expression and subsequent DNA cleavage by I-SceI enzyme will trigger cellular repair mechanisms. The efficiency of HR or NHEJ can be determined based on the expression of GFP reporter gene.
Subcellular fractionation assay
Subcellular fractionation was performed using a subcellular protein fractionation kit (#78840, Thermo Fisher, USA) to detect the accumulation of DNA damage repair proteins on chromatin after radiation-induced DNA damage. After treatment with or without radiation, 2 × 106 cells were collected by centrifugation following trypsin digestion. Subcellular protein isolation and enrichment were then performed according to the manufacturer’s instructions, sequentially extracting cytoplasmic, membrane, nuclear, and chromatin proteins. All steps were carried out on ice. The subcellular fractions were stored at −80 °C, and the protein separation efficiency was verified by Western blot.
Proteomics analysis
Proteomics analysis was supported by Shanghai Houji Biotechnology Co., Ltd. using data-independent acquisition (DIA) quantitative proteomics technology, encompassing protein extraction, enzymatic digestion, mass spectrometry analysis, and data processing. First, proteins were extracted from cells using the SDT method, followed by filter-aided sample preparation (FASP) for tryptic digestion to digest proteins into peptides. Subsequently, each sample was separated via nanoflow HPLC system and analyzed using the Orbitrap Fusion Tribrid mass spectrometer. Mass spectrometry data were acquired in DIA mode to obtain extensive peptide fragment ion information. Finally, MaxQuant software was employed for database searching, matching DIA data against a spectral library to achieve protein identification and quantification.
Co-immunoprecipitation (Co-IP)
After digestion and centrifugation, cells were lysed on ice for 30 min in IP lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol) supplemented with protease and phosphatase inhibitors (Beyotime, China). The lysates were centrifuged at 12,000 × g for 15 min at 4 °C to remove debris. Twenty microliters of the supernatant was mixed with 5×SDS loading buffer and boiled for 10 min as the Input sample. The remaining supernatant was incubated with 2–5 μg of target antibody or control IgG overnight at 4 °C with rotation. The next day, 30 μL of Protein A/G agarose beads (Santa Cruz, USA) was added to each tube and incubated for 4–6 h at 4 °C with rotation. The beads were collected by centrifugation, washed three times with IP lysis buffer, and then boiled in 5× SDS loading buffer for 10 min to elute the bound proteins for Western blotting.
Tumor xenograft assay
This study established an ESCC cell line-derived xenograft (CDX) model by subcutaneously inoculating tumor cells into nude mice. Cells were prepared at 6 × 10⁷ cells/mL, and injected to form subcutaneous tumors. After inoculation, the mice were monitored daily for their condition and tumor growth. Tumor length (a) and width (b) were measured every 2–3 days, and the tumor volume was calculated using the formula a×b²×1/2. When the tumor volume reached 100 mm³, the mice were randomly grouped for radiotherapy or drug treatment. At the end, the mice were euthanized, tumor tissues were dissected, weighed, and fixed in 4% paraformaldehyde for histopathological analysis. For animal studies, tumor volume was measured by individuals blinded to group allocation.
Immunofluorescence
Cells were seeded in confocal dishes and cultured overnight before irradiation. After washing with PBS, cells were fixed with methanol and permeabilized with 0.2% Triton X-100 in PBS for 10 min, followed by blocking with 5% BSA for 1 h. Subsequently, cells were incubated overnight at 4 °C with primary antibodies against RAD51 (Cat# ab133534, Abcam, 1:100), 53BP1 (Cat# ab175933, Abcam, 1:100), and RAD51AP1 (Cat# ab88370, Abcam, 1:100). Next, cells were incubated with Alexa Fluor 488 (Cat# A0423, Beyotime, 1:500) or Alexa Fluor 555 (Cat# P0179, Beyotime, 1:500)-conjugated secondary antibodies at room temperature for 1 h. Finally, nuclei were stained with DAPI-containing anti-fluorescent quencher. Cells were imaged in a Zesis LSM800 confocal microscope, and images were processed with the Zeiss Zen software.
Immunohistochemistry staining (IHC)
Formalin-fixed, paraffin-embedded (FFPE) tissue sections (4 μm thick) were baked at 65 °C for 2 h, followed by deparaffinization and rehydration. Antigen retrieval was performed by microwave heating in citrate buffer. Tissue sections were blocked with normal goat serum and then incubated overnight at 4 °C with primary antibodies against ITGA5 (1:100, Abcam, ab150361), γH2AX (1:100, Abcam, ab81299), Cleaved caspase-3 (1:100, CST, #9661), and RAD51AP1 (1:100, Proteintech, 11255-1-AP). After incubation with secondary antibodies for 1 h, color was development with diaminobenzidine (DAB). Sections were counterstained with hematoxylin, dehydrated, and mounted. Quantitative analysis was calculated using a histochemistry score (H-score), which combines staining intensity and the percentage of positive tumor cells. Staining intensity was scored as follows: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The percentage of positive tumor cells was categorized as: 1 (0–25%), 2 (25–50%), 3 (50–75%), and 4 (75–100%). The final score was calculated by multiplying the intensity score by the percentage score.
Luciferase reporter assay
The dual luciferase assays were conducted using the Dual Luciferase Reporter System (Promega). Cells were seeded in 24-well plates and transfected with reporter plasmids using standard methods. After 6 h of transfection, the medium was replaced. The cells were lysed with 100 μL of PLB buffer at room temperature for 15 min, followed by centrifugation to collect the supernatant. In opaque 96-well plates, 20 μL of the lysate was mixed with 100 μL of LAR II reagent to measure Firefly luciferase activity, followed by the addition of 100 μL Stop & Glo Reagent for Renilla luciferase detection. Relative luciferase activity was calculated as (Firefly luciferase activity-blank)/(Renilla luciferase activity-blank) and normalized to the average of the negative control group.
Chromatin immunoprecipitation (ChIP)
Cells were fixed with 1% formaldehyde at room temperature for 10 min, and cross-linking was quenched with 250 mM glycine. After lysis in protease inhibitor-containing buffer, chromatin was sheared by sonication (5–10 cycles), and DNA fragment size was verified by electrophoresis. The lysate sonication products were divided into Input, IgG, and target antibody groups. The lysates were incubated overnight at 4 °C with 5 μg of anti-MYC antibody (ab32072, Abcam) or 1 μg IgG. Antibody-protein complexes were captured using magnetic beads for 4 h at 4 °C, eluted in elution buffer for 15 min with shaking, and reverse cross-linked overnight at 65 °C. Following RNase A and Proteinase K treatment, DNA was purified using an DNA purification kit (OMEGA, USA), and fragment size was confirmed by agarose gel electrophoresis. Finally, qPCR was performed to detect target protein-bound DNA enrichment.
Bioinformatic analysis
Differentially expressed genes and proteins were screened, clustered, and functionally annotated through bioinformatics analysis to provide a foundation for further research. Gene sets with specific biological functions were downloaded from the National Center for Biotechnology Information (NCBI) website (https://www.ncbi.nlm.nih.gov/). The Gene Set Variation Analysis (GSVA) package in R version 4.4.2 was used to score functional gene sets. Raw gene expression data were obtained from the Gene Expression Omnibus (GEO) database. Spearman correlation analysis was performed to assess the relationships between genes and pathways.
Patients and specimens
The tissue samples utilized in this study were collected retrospectively from 126 ESCC patients who underwent radical radiotherapy at Shandong Cancer Hospital between January 2013 and January 2023. The inclusion criteria comprised: pathologically confirmed ESCC by biopsy at our hospital, no prior history of any antitumor therapy, no history of previous malignancy, and complete pre- and post-radiotherapy imaging data. Exclusion criteria included: unavailable tissue samples, concurrent severe systemic diseases, interrupted radiotherapy or severe complications, unwillingness to provide informed consent, and loss to follow-up. All enrolled patients had complete clinicopathological characteristics, including sex, age, Karnofsky Performance Status (KPS) score, grade, tumor location, smoking history, alcohol history, TNM stage, and radiotherapy response. Among them, 106 were male and 20 were female. The median age was 66 years (range 45–87 years). We collected paired tissue samples before and after radiotherapy from 3 ESCC patients who received identical neoadjuvant radiotherapy protocol followed by surgery at our institution. Additionally, we retrospectively enrolled 80 ESCC patients receiving radical radiotherapy at the Affiliated Tumor Hospital of Xinjiang Medical University from January 2021 to January 2026 as the validation set. The inclusion and exclusion criteria were the same as before.
Radiotherapy efficacy evaluation
Following radiotherapy completion, the response of ESCC patients to radiotherapy was evaluated using Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, based on chest CT image. Patients with complete response (CR) or partial response (PR) were classified as radiosensitive, whereas those with stable disease (SD) or progressive disease (PD) were defined as radioresistant.
Statistics
All data were statistically analyzed and visualized using GraphPad Prism version 9.5, with results presented as mean ± standard error of mean (SEM). Comparisons between two independent groups were performed using two-sided Student’s t tests. Multiple group comparisons were conducted using one-way ANOVA. Categorical variables were analyzed by chi-square tests. Survival curves were plotted using the Kaplan-Meier method and compared by log-rank test. Optimal cutoff values were determined using X-tile software. Correlation analysis was employed by Spearman’s correlation test. All experiments were independently repeated three times. P < 0.05 was considered statistically significant.
Study approval
This study was approved by the Ethics Committee of Shandong Cancer Hospital (SDTHEC202501063). Written informed consent was obtained from all participating patients. All experimental procedures were conducted in compliance with institutional guidelines and national regulations. Additionally, the study protocol received approval from the Animal Ethics Committee of Shandong Cancer Hospital.
