General methods

The chelating agent p-SCN-Bn-DFO was purchased from Novartis Pharma AG, France. pH value was measured using pH indicator paper (Merck Millipore). Radioactivity was measured using the CRC-25R dose calibrator (Capintec Inc.). Instant thin layer chromatography (iTLC) was performed on chromatography paper (Agilent iTLC-SG) and the bands were analyzed using the Mini-Scan/FC radio-TLC scanner (Eckert & Ziegler).

To accurately quantify the activity of 89Zr, the samples were counted for 1 min on a gamma counter (Wizard2 2480, PerkinElmer, Waltham, MA, USA) with the energy window set to 15-2000 keV for 89Zr. The radiochemical purity of the 89Zr radiolabeling were checked using thin-layer chromatography (radio-iTLC) paper and analyzed on a γ counter [29].

Preparation of 89Zr-anti-γH2AX-TATTAT synthesis

According to literature reports, the TAT sequence was selected as GRKKRRQRRRPPQGYG [28], The synthesis was carried out using solid-phase peptide synthesis, which was provided by Shenzhen BGI Group.

Anti-γH2AX-TAT coupling, p-SCN-Bn-DFO modification of anti-γH2AX-TAT

The anti-γH2AX rabbit monoclonal antibody was purchased from Cayman Chemical Company in the United States. The antibody was stored in a buffer containing 50% glycerol/PBS with 1% BSA and 0.09% sodium azide. The antibody was purified using a ultrafiltration tube (molecular weight cutoff: 50 kDa and 100 kDa), and the buffer was exchanged for a 0.2 M borate buffer to adjust the pH between 7.4 and 8.2. The antibody solution was mixed with EDC.HCl (10 eq., 10 μL) and Sulfo-NHS (10 eq., 10 μL) at room temperature for 30 min. Then, the TAT solution (1:12, 20 μL) was added to the reaction system. The unreacted small molecules were removed by ultrafiltration, and the buffer was exchanged for a 0.2 M borate buffer (pH adjusted to 7.4–8.2). The p-SCN-Bn-DFO solution (1:15, 10 μL) was added to the reaction system, and the mixture was shaken at room temperature for 5 h. The conjugate was purified by protein A column and desalted using a PD-10 gel filtration column. The buffer of the conjugate was exchanged for a 10 mM pH 7.4 PBS buffer and stored at 4 ℃.

89Zr labeling.

The 89Zr was purchased from Perkin Elmer and dissolved in a 1 M oxalic acid solution. The anti-γH2AX-TAT conjugate modified with p-SCN-Bn-DFO was radiolabeled with 89Zr using the method reported in literature [30, 31].

Cells culture

Human large cell carcinoma cell lines NCI-H460 and human lung adenocarcinoma lines A549 were kindly provided by Cobioer Life Science& Technology Co., Ltd.

The H460 cells were cultured in RPMI-1640 medium (Thermo Scientific) supplemented with 10% FBS (BI), 10 KU/mL penicillin, and 10 mg/mL streptomycin (Thermo Scientific). The A549 cells were cultured in F12K medium (BOSTER) supplemented with 10% FBS, 10 KU/mL penicillin, and 10 mg/mL streptomycin.

The cells were grown in a cell culture incubator (Thermo Scientific, Forma™ Series II 3110 Water-Jacketed CO2 Incubator) at 37 °C with 5% CO2. Sub-culturing of cells was carried out using Trypsin-EDTA solution (Thermo Fisher).

For external radiation exposure, the cells and animal models were irradiated using a Swedish Elekta image-guided radiation therapy system (Elekta Synergy; dose rate 6 MeV/min).

Cell clone survival assay

Cell clone survival assay was performed to confirm the difference in radiosensitivity between A549 and H460 cells. Log-phase monolayer cultures of A549 and H460 cells were digested with trypsin, dispersed into single cells, and suspended in complete medium containing 10% fetal bovine serum. The cell suspension was diluted into a gradient density and seeded at a density of 300, 600, 1200, 3000, and 4500 cells per well in a 6-well plate with 2 mL pre-warmed culture medium. Three replicates were prepared for each cell density, and the plates were gently rotated to ensure even cell distribution. After incubation for 24 h, the cells were irradiated with X-rays (0, 2, 4, 6, 8 Gy) and then incubated for 2 weeks in a cell culture incubator. During the incubation period, the medium was replaced every 3 days, and the cells were observed under a microscope to monitor the cell status.

When visible cell clones appeared in the culture dish, the culture was terminated. The cells were fixed with 4% paraformaldehyde for 15 min, and then stained with Giemsa for 30 min. The stained cells were washed with deionized water and air-dried.

Using a digital camera to take pictures, Image J software is used for colony calculation. The pixel value of the clone with a low-magnification microscope cell count of more than 50 is used as the calculation threshold. The clone rate is calculated using the following formula: Clone rate = treatment group clone number/control group clone number*100%, and the average value is taken from three complex holes. The software GraphPad Prism 8.0 is used for fitting the “multi-target single-click model” dose-activity curve. The equation of the multi-target single-click model is: SF = 1–1[1-exp(-Dq/D0)]N, where SF is the cell survival rate, D is the dose (Gy) of external irradiation, D0 represents the average lethal dose, Dq represents the quasi-threshold dose, and N is the extrapolation number.

Cellular immune fluorescence confocal imaging

To verify the relationship between cell radiosensitivity and γH2AX foci, immunofluorescent confocal imaging was performed on A549 and H460 cells after irradiation.

2 × 10^4 A549 and H460 cells were seeded in 2 ml of sterile cell climbing slices in a 24-well plate containing cell growth medium. A control group and an irradiation group were established, and the cells were allowed to adhere overnight. The cells were irradiated with X-rays (1G and 4 Gy, 300 cGy/min) or simulated radiation (0 Gy). Immediately after irradiation or after 1–24 h of incubation, immunofluorescent staining was performed. The cells were fixed with 4% PFA (polyformaldehyde) for 20 min, washed with PBS to remove the PFA, and permeated with 0.5% TritonX-100 for 15 min. The TritonX-100 was removed, and the cells were washed with PBS and blocked with 4% BSA at room temperature for 1 h. The cells were stained with polyclonal rabbit anti-γH2AX antibody and goat anti-rabbit IgG labeled with Alexa Fluor 488 fluorescent dye. Olympus FV3000 laser confocal microscope was used for microscopic examination, and the number of γH2AX foci in at least 100 cells was determined for each cell.

In vivo studyEstablishment of lung cancer xenograft models

All animal procedures in the in vivo study were performed in accordance with the 1986 UK Animals (Scientific Procedures) Act and approved by the ethics committee with the ethics approval number SBQDL-2022-077. PBS (100 μL) containing 1 × 10^6 A549 and H460 lung cancer cells was injected subcutaneously into the right side of 4-week-old female BALB/c nu/nu mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) to establish A549 and H460 lung cancer xenografts. Tumor-bearing mice were divided into imaging and growth measurement groups, each with a control group.

Micro PET/CT imaging

Three to four weeks after tumor implantation, mice in the imaging group with an average tumor size of 200 μl were used for Micro PET/CT imaging. A tail vein injection of 89Zr-antiγH2AX-TAT (5 μg, 0.5 MBq) in a volume of 100 μl was given.

One hour after injection of the imaging agent, the tumor area was irradiated with X-rays (10 Gy, 300 cGy/min) to induce γH2AX generation. Mice in the control group received simulated therapy (0 Gy).

Experimental details and acquisition parameters are as follows (Fig. 1):

Fig. 1

Schematic overview of 89Zr-anti-γH2AX-TAT PET imaging and biodistribution experiments. Xenograft models with an average tumor size of 200 μl were used for Micro PET/CT imaging. A tail vein injection of 89Zr-antiγH2AX-TAT (5 μg, 0.5 MBq) in a volume of 100 μl was given. One hour after injection of the imaging agent, the tumor area was irradiated with X-rays (10 Gy, 300 cGy/min) to induce γH2AX generation. 24, 48 and 72 h after injection of the radiopharmaceutical, mice were anesthetized with 2–4% isoflurane in air. PET/CT imaging was performed using the InliView-3000B small animal PET/CT scanner. After PET imaging, mice were euthanized, blood, tumor, and selected organs were removed, weighed, and radioactivity was measured

Twenty-four hours after injection of the radiopharmaceutical, mice were anesthetized with 2–4% isoflurane in air. PET/CT imaging was performed using the InliView-3000B small animal PET/CT scanner (Novel Medical). CT attenuation correction was performed before each PET emission scan and used as an anatomical reference. List mode data was acquired for 10 min with a gamma ray energy window of 511 keV and a coincidence time window of 0.1 ns.

Image reconstruction was performed using a filtered back projection algorithm (FBP) with a list mode and matrix size of 128 × 128. Volume of interest (VOI) analysis was performed using NMSoft-AIWS software package. The VOIs were drawn around major organs and the tumor. A VOI was drawn around the heart to measure radioactivity in the blood. Tumor to heart (T/H), tumor to muscle(T/M), tumor to bone(T/B) ratios were calculated (n = 3 per group).

After PET imaging, mice were euthanized, blood, tumor, and selected organs were removed, weighed, and radioactivity was measured.

In vitro biodistribution experiment

Another group of 16 tumor-bearing nude mice were randomly divided into 4 groups, with 4 mice in each group. After tumor formation, each mouse was injected with 0.5 MBq and 5 μg of 89Zr-anti-γH2AX-TAT via tail vein. The experimental nude mice were euthanized at 12, 24, 48, and 72 h by cervical dislocation according to groups, and selected organs, tissues, and blood were removed. The samples were immediately washed with water, dried, and transferred to pre-weighed counting tubes. After weighing the filled counting tubes, the amount of radioactivity in each counting tube was measured using a gamma counter. The counts per minute were converted to radioactive units (MBq) using a calibration curve generated from known standards. These values were decay corrected based on the injection time, and the percentage of injected dose per gram of each sample (% ID/g) was calculated.

Immunohistochemistry staining of γH2AX slices

To evaluate the difference in the number of DNA repair foci between the H460 model and A549 models after external irradiation of the tumors, 10 μm thick tumor tissue slices were prepared and stained for γH2AX foci, as described earlier. Three to six animals were assessed per group. Confocal microscopy images were acquired using an Olympus FV3000 laser confocal microscope. Relative quantification of γH2AX immunofluorescence was performed by normalizing Alexa Fluor 488 signal intensity to DAPI signal. Immunofluorescence was used to detect γH2AX-positive cells when evaluating γH2AX foci in each cell. Open-source software QuPath was used to analyze cell positivity for γH2AX in random tumor areas (n = 8 per sample). The total number of cells was determined by hematoxylin staining, while quantification of positive cells was obtained through DAB staining. Immunofluorescence analysis was performed using open-source image processing package Fiji. The mean FITC fluorescence intensity was normalized to DAPI fluorescence signal, and the number of foci/cells was counted in random tumor areas (n = 6 per sample).

Statistical analysis

Statistical analysis was conducted using GraphPad Prism 8.0 (GraphPad Software). Normality tests were performed on the data, and in appropriate cases, multiple comparisons were conducted using non-paired two-tailed Student’s t-tests or one-way ANOVA, with Dunnett’s post hoc test used to calculate the significance of differences between groups. All data was presented as mean ± SD and at least triplicate measurements were taken, unless otherwise specified. Nonlinear regression analysis was also performed using GraphPad Prism 8.0.