Experimental study of early evaluation of radiosensitivity in mouse models of lung cancers using ⁸⁹Zr-anti-γH2AX-TAT PET imaging

Abstract

Background

Early evaluation of radiation sensitivity in lung cancer patients can facilitate the transition to personalized treatment strategies. To this end, we assessed the capability of ⁸⁹Zr-anti-γH2AX-TAT microPET imaging in determining the radiosensitivity of lung cancer xenograft models. We prepared and conducted quality control on ⁸⁹Zr-anti-γH2AX-TAT. The radiosensitivity of human non-small cell lung cancer cells (H460) and adenocarcinoma cells (A549) was analyzed through clonogenic survival experiments. Additionally, the role of γH2AX as a biomarker for radiosensitivity was validated by quantifying γH2AX foci via fluorescence staining. Subsequently, the H460 and A549 xenograft mouse models were subjected to irradiation, followed by ⁸⁹Zr-anti-γH2AX-TAT microPET imaging. Concurrently, we performed immunofluorescence staining for γH2AX in tumor tissues to establish a correlation between the uptake of ⁸⁹Zr-anti-γH2AX-TAT and γH2AX expression.

Results

The surviving fraction 2 Gy (SF2) values of H460 and A549 indicating that A549 adenocarcinoma has higher radiosensitivity. The cell immunofluorescence experiment showed that the repair of γH2AX foci in H460 cells after irradiation was significantly higher than that in A549 cells, which also confirmed that A549 has higher radiosensitivity. The microPET imaging results showed the uptake of ⁸⁹Zr-anti-γH2AX-TAT in the tumor of the A549 models after radiotherapy was higher than H460 models. The immunofluorescence staining of tumor tissue confirmed that the expression level of γH2AX was higher and the correlation with microPET imaging uptake was good.

Conclusion

⁸⁹Zr-anti-γH2AX-TAT allows PET imaging of radiosensitivity in lung cancer xenograft models, and is expected to become an early evaluation method for lung cancer radiosensitivity.

Introduction

According to the “2020 Global Cancer Burden Data” report, lung cancer is one of the most prevalent malignant tumors globally, with its incidence and mortality ranking second and first, respectively [1]. Radiotherapy (RT) is a crucial component of comprehensive treatment for lung cancer, with approximately 65–75% of patients requiring RT at various stages of their disease [2]. However, the use of a uniform prescription dose (50 ∼ 70 Gy) in lung cancer radiotherapy, without accounting for individual differences in radiation sensitivity, results in approximately 40–50% of patients experiencing local lesion failure or recurrence [3]. Therefore, early prediction of individualized radiation sensitivity prior to radiotherapy, or early evaluation during treatment, is vital for the development of personalized radiotherapy plans [4]. This approach aims to enhance the local control rate of radiotherapy [5].

Currently, the clonogenic survival assay is considered the gold standard for assessing the radiosensitivity of tumor cells in vitro. However, its slow detection speed, complex procedures, and the need for extensive technical expertise limit its suitability for routine use [6]. An alternative marker frequently utilized is the quantification of γH2AX lesions following radiotherapy. This approach is based on several key factors: DNA is the primary target of ionizing radiation, and the resulting DNA damage is a major contributor to cell death. Among the various forms of DNA damage, double-strand breaks (DSBs) are particularly critical, as they can lead to chromosomal translocations or cell death, serving as a primary mechanism for ionizing radiation-induced cell death. Research has demonstrated that the quantity and repair capacity of DSBs can influence the radiosensitivity of cells [7].

While double-strand breaks (DSBs) accurately reflect early cellular DNA damage and radiosensitivity, their low abundance limits their practical application as a detection marker. In contrast, proteins associated with DSBs, such as γH2AX, are highly phosphorylated and provide a rapid and reliable correlation with DSB numbers. Consequently, the quantification of γH2AX foci has become the preferred method for detecting in vitro DSBs [8]. Numerous studies have established γH2AX as a potential predictive factor for radiosensitivity [9–12]. Specifically, the number of residual γH2AX foci 24 h post-irradiation has been shown to correlate with the radiosensitivity of tumor cells [13–21]. Additionally, a negative correlation exists between the number of residual γH2AX foci and local tumor control [22].

Currently, γH2AX foci can only be measured in ex vivo tissue, necessitating invasive procedures such as surgery or puncture biopsy to obtain tumor cells after radiotherapy. This approach not only fails to capture the full extent of tumor heterogeneity but also raises ethical concerns due to the associated risks for patients [23]. Theoretically, the use of a targeted γH2AX radioactive drug could enable nuclear medicine imaging shortly after radiotherapy, facilitating an early, in vivo, and non-invasive assessment of tumor radiosensitivity.

Given that γH2AX is localized in the nucleus, a targeted γH2AX radioactive drug must overcome the challenge of penetrating both the cell membrane and the nuclear membrane. This issue can be addressed by incorporating the HIV-1 TAT transcription activator (TAT) into the probe design [24]. Currently, two types of γH2AX nuclear medicine molecular probes have been synthesized: ¹¹¹In-anti-γH2AX-TAT and ⁸⁹Zr-anti-γH2AX-TAT. Multiple studies have demonstrated their capability to detect double-strand breaks (DSBs) in xenograft tumor models [13, 25–28]. However, to date, there have been no reported studies utilizing targeted γH2AX nuclear probes for in vivo evaluation of radiosensitivity.

This project aims to design and synthesize ⁸⁹Zr-anti-γH2AX-TAT tracers and perform quality control. In vitro experiments will be conducted using two different radiosensitive human lung cancer cell lines, and xenograft tumor models will be established in experimental mice. Following X-ray irradiation, ⁸⁹Zr-anti-γH2AX-TAT microPET imaging will be performed. Concurrently, tumor tissue γH2AX immunofluorescence will be observed and quantitatively assessed using standardized methods. The feasibility and potential value of ⁸⁹Zr-anti-γH2AX-TAT imaging for early evaluation of tumor radiosensitivity will be investigated.

Materials and methods

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-TAT

TAT 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 study

Establishment 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.

Results

Preparation of 89Zr-anti-γH2AX-TAT

The natural anti-γH2AX antibody, anti-γH2AX-TAT, and DFO-anti-γH2AX-TAT were subjected to mass spectrometric analysis. The S-glucosyl glycosidase cleaved the antibody glycan chain, and the mass spectrometry detection results are shown in the figure. The molecular weight was consistent with the molecular weight range of the natural anti-γH2AX antibody, anti-γH2AX-TAT, and DFO-anti-γH2AX-TAT, confirming the successful coupling of DFO-anti-γH2AX-TAT. The conventional synthesis of ⁸⁹Zr-anti-γH2AX-TAT was measured by using iTLC with 50 mM EDTA (pH 6) buffer to elute the reaction mixture, which showed a high radiochemical yield (90.04%) and high radiochemical purity (> 97%). (Fig. 2)

Fig. 2.

The chemical structure of ⁸⁹Zr-anti-γH2AX-TAT and Mass spectrometry image of the precursor and TLC image of ⁸⁹Zr-anti-γH2AX-TAT. A The chemical structure of ⁸⁹Zr-anti-γH2AX-TAT. B, C, D Mass spectrometric analysis results showed that the molecular weight was consistent with the molecular weight range of the natural anti-γH2AX antibody, anti-γH2AX-TAT, and DFO-anti-γH2AX-TAT, confirming the successful coupling of DFO-anti-γH2AX-TAT. E The iTLC results of ⁸⁹Zr-anti-γH2AX-TAT showed that a high radiochemical yield (90.04%)

Cell clone survival assay

The results showed that the D0, Dq, and SF2 values of H460 cells were higher than those of A549 cells, and the SF2 values were 0.89 and 0.47, respectively. Statistical analysis results showed that there were significant differences in SF between H460 cells and A549 cells in the same dose group (P < 0.001), indicating that A549 cells were more sensitive to radiation than H460 cells (Fig. 3; Table 1).

Fig. 3.

Dose-survival experiment and Dose-survival curves of H460 and A549 cancer cells(multi-target single-hit model) A Digital pictures of H460 cell and A549 cell clone survival assay. B Dose-survival curves of H460 and A549 cancer cells. C Cellular immune fluorescence confocal imaging of H460 cell and A549 cell showed that the number of γH2AX foci in the cell nuclei of H460 cells decreased significantly and there was a statistical difference compared to A549 cells

Table 1.

Main parameters of dose-survival curves

	D0	N	Dq	SF2
H460	1.459	8.142	3.059575	0.89
A549	1.363	2.403	1.194967	0.47

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

The cell immunofluorescence experiment showed that there was no significant difference in the number of γH2AX foci in the cell nuclei of H460 and A549 cells 1 h after 4 Gy radiation. However, 24 h later, the number of γH2AX foci in the cell nuclei of H460 cells decreased significantly and there was a statistical difference compared to A549 cells. This suggests that the double-strand DNA repair capacity of H460 cells after external irradiation is significantly stronger than that of A549 cells, confirming that A549 cells are more radiation-sensitive than H460 cells, which is consistent with previous literature results.(Fig. 3C).

Micro PET/CT imaging and tumor uptake and distribution of radiotracer

PET/CT images obtained 24 h after radiotherapy showed that the uptake of ⁸⁹Zr-anti-γH2AX-TAT in tumor of mice in A549 irradiation group was higher than that in H460 irradiation group (Fig. 4A), with statistical difference (21.7 ± 1.6%ID/ml vs. 13.9 ± 0.5%ID/ml, P < 0.001). Imaging agents were mainly concentrated in liver and tumor. There was no significant uptake of tumor tissue in A549 non-irradiated group and H460 non-irradiated group, and imaging agents mainly gathered in liver, tumor and intestinal tract.

Fig. 4.

⁸⁹Zr-anti-γH2AX-TAT PET images and biodistribution of H460 and A549 xenograft models. A The PET/CT images obtained at 24 h after radiotherapy. B The uptake of ⁸⁹Zr-anti-γH2AX-TAT obtained from the 24-hour in vitro biodistribution experiment. C Tumor to heart (T/H), tumor to muscle(T/M), tumor to bone(T/B) ratios. D Tumor %ID/g at 24,48,72 h

The %ID/g value obtained by 24-hour in vitro biological distribution experiment showed that the intake of ⁸⁹Zr-anti-γH2AX-TAT reached 13.7 ± 1.04%ID/g in the tumor of mice irradiated with A549 (Fig. 4B). The ID/g was 5.75 ± 0.25% higher than that of A549 non-irradiated mice and H460 irradiated mice (P < 0.001).

The values of T/M, T/B, and T/H obtained from the 24-hour in vitro biological distribution experiment showed that the T/M value in the tumor of mice in the A549 irradiated group was higher than that in mice in the A549 non-irradiated group and mice in the H460 irradiated group, 140.9 ± 12.2 and 192.4 ± 13.6 (P < 0.01), respectively (Fig. 4C). The T/B value of A549 irradiated mice was higher than that of A549 non-irradiated mice and H460 irradiated mice, which were 34.2 ± 3.1 and 72.6 ± 14.4 (P < 0.05). The T/H value of A549 irradiated mice was higher than that of A549 non-irradiated mice and H460 irradiated mice, 10.4 ± 1.8 and 26.9 ± 8.5 (P < 0.05), respectively.

The %ID/g value obtained by the 48-hour in vitro biological distribution experiment showed that the intake of ⁸⁹Zr-anti-γH2AX-TAT reached 3.13 ± 0.35%ID/g in the tumor of mice irradiated with A549. The ID/g was 7.43 ± 0.60% higher than that of A549 non-irradiated mice and H460 irradiated mice (P < 0.001). The %ID/g value obtained by 72-hour in vitro biological distribution experiment showed that the intake of ⁸⁹Zr-anti-γH2AX-TAT reached 3.93 ± 0.84%ID/g in the tumor of mice irradiated with A549. The ID/g was 1.86 ± 0.21% higher than that in A549 non-irradiated group and H460 irradiated group (P < 0.05). (Fig. 4D)

Immunohistochemistry staining of γH2AX slices

The IHC staining results of γH2AX slices of tumor tissue in vitro showed that the fluorescence intensity of γH2AX in A549 irradiation group was higher than that in H460 irradiation group (Fig. 5A), with statistical difference (68 ± 5 and 44 ± 7a.u.; p < 0.01). The fluorescence intensity of γH2AX in A549 irradiation group was higher than that in A549 control group (p < 0.01). The fluorescence intensity of γH2AX in H460 irradiation group was higher than that in H460 control group (p < 0.01). There was no significant difference in γH2AX fluorescence intensity between A549 control group and H460 control group (p > 0.05).(Fig. 5C)

Fig. 5.

Immunohistochemistry staining of γH2AX slices and %positive cells, Normalized fluorescence intensity and Foci per cell of IHC in xenograft models

The IHC staining of γH2AX sections of tumor tissue in vitro showed that the percentage of positive cells of γH2AX lesions in the A549 IR group was higher than that in the H460 irradiated group, with statistical difference (49.2 ± 3.6% and 19.7 ± 1.5%, respectively; p < 0.0001). (Fig. 5B)The percentage of positive cells of γH2AX lesions in A549 irradiation group was higher than that in A549 control group, and the difference was statistically significant (p < 0.01). The percentage of positive cells of γγH2AX lesions in H460 irradiation group was higher than that in H460 control group, and the difference was statistically significant (p < 0.01). There was no significant difference between A549 control group and H460 control group in the percentage of γH2AX lesion positive cells (p > 0.05). (Fig. 5D)

Discussion

This study distinguishes itself from others by conducting mass spectrometry analysis at every step of the synthesis process to ensure the quality of the final product [26, 32, 33]. Additionally, we utilized ⁸⁹Zr-anti-γH2AX-TAT for the first time to evaluate in vivo radiosensitivity. Our findings revealed that A549 cells exhibited higher in vitro radiosensitivity compared to H460 cells. After irradiation, the residual γH2AX foci in the A549 group were significantly higher than in the H460 group. In vivo PET imaging demonstrated that tumor uptake was significantly elevated in both the A549 and H460 irradiation groups compared to the control group, with the A549 irradiation group showing notably higher tumor uptake than the H460 group, which was statistically significant. Tissue immunofluorescence further indicated that the A549 irradiation group had a greater quantity and intensity of γH2AX fluorescent foci in the tumor tissue than the H460 irradiation group. The tumor growth assay confirmed that A549 cells possess higher in vivo radiosensitivity than H460 cells. Overall, ⁸⁹Zr-anti-γH2AX-TAT effectively detects the upregulation of γH2AX during radiotherapy regimens, demonstrating significant changes earlier than those observed in relative tumor volume.

The cell-penetrating peptide TAT (GRKKRRQRRRPPQGYG) is derived from the HIV-1 transactivator of transcription and contains a nuclear localization signal domain, granting it cell-penetrating properties [24]. The physical half-life of the positron-emitting nuclide ⁸⁹Zr is 78.4 h, which aligns well with the in vivo metabolism of antibodies that have similar biological half-lives, making it suitable for PET imaging of antibody labeling. This allows for real-time monitoring of target protein expression in vivo and facilitates the quantification of tumor uptake of ⁸⁹Zr-labeled monoclonal antibodies [34]. Furthermore, ⁸⁹Zr PET imaging is anticipated to enhance sensitivity compared to the limitations encountered with ¹¹¹In SPECT imaging [35]。.

This study has certain limitations. In the Micro PET/CT imaging section, the use of a single dose (10 Gy) restricts our ability to evaluate the method’s efficacy across a range of radiation doses and treatment responses. Without this assessment, the method’s potential to predict varied responses to different radiation protocols remains uncertain. Additionally, we were unable to identify cell lines with varying radiosensitivities within the same type of cells, which led us to compare two different lung cancer cell lines; this may have influenced the interpretation of our results. In future studies, we plan to incorporate multiple dosage groups and to screen and cultivate both radiosensitive and radioresistant groups of the same lung cancer cells, followed by comparative experiments between these cell lines. This approach is expected to yield more robust and convincing results.

In summary, we conducted in vitro experiments and in vivo ⁸⁹Zr PET imaging on two different lung cancer cell lines exhibiting varying radiosensitivities. The results indicated that A549 cells demonstrated greater in vitro radiosensitivity compared to H460 cells, with the A549 irradiation group exhibiting higher tumor uptake of the imaging agent than the H460 irradiation group. These findings suggest that ⁸⁹Zr-anti-γH2AX-TAT PET imaging has the potential to serve as a powerful non-invasive imaging tool for the in vivo evaluation of lung cancer radiosensitivity in clinical practice.

Conclusion

The PET imaging agent ⁸⁹Zr-anti-γH2AX-TAT can be utilized to identify different radiosensitive lung cancer mouse models at an early stage, making it a promising method for the early evaluation of lung cancer radiosensitivity. In comparison to the gold-standard clonogenic survival assay, ⁸⁹Zr-anti-γH2AX-TAT offers an early, in vivo, non-invasive, sensitive, and direct approach for monitoring tumor radiosensitivity. Moving forward, we can explore the combination of ⁸⁹Zr-anti-γH2AX-TAT with other PET imaging agents to enhance the development, efficacy prediction, and evaluation of radiation therapy plans for lung cancer patients.

Author contributions

XL, JG, JL, JS and SL conceived and designed this experiment. XL, JG obtained, analyzed and interpreted the data. JG, JL, JS provided technical and material support. XL, JG wrote the original manuscript. All authors read and approved the final manuscript.

Funding

This research was supported by Fundamental Research Program of Shanxi Province (no.202203021212086) in the design of the study and collection, analysis of data.

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

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.

Consent for publication

Not applicable.

Competing interests

No potential conflicts of interest were disclosed.