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Whole-body CT scanning radiation improves the immune microenvironment of tumor tissues to enhance the antitumor effect of ICI

Abstract

Objective

The effect of frequent whole-body CT scans during immune checkpoint inhibitor (ICI) therapy on patients' anti-tumor immunity.

Methods

We conducted a retrospective clinical study aimed to investigate the correlation between the frequency of CT scans during immune checkpoint inhibitor (ICI) therapy and the duration of remission (DOR) of ICI therapy in patients with stage IV non-small cell lung cancer (NSCLC). We constructed a hormonal mouse model and administered immune checkpoint inhibitor (ICI) therapy to mice, and radiated five whole-body CT scans to mice during ICI therapy to observe whether frequent whole-body CT scans had an effect on the antitumor effect of immunotherapy in mice.

Results

The more frequent CT scans during patients' immune checkpoint inhibitor (ICI) treatment the longer the duration of remission (DOR) of ICI treatment. In a mouse model we observed that the addition of whole-body CT scanning radiation had a tendency to inhibit tumor growth in mice compared with the anti-PD-1 group alone.Frequent CT scanning radiation during the application of immune checkpoint inhibitor PD-1 increased the proportion of infiltrating CD8 + T cells in tumor tissues and significantly increased the proportion of IFNγ-secreting CD8 + T cells, and single-cell sequencing of the results also revealed that IFNγ and killing-related genes were significantly upregulated in tumor-infiltrating CD8T cells.

Conclusion

To our knowledge this is the first study on the effect of CT scan radiation on ICI.Our findings suggest that multiple CT scans during immune checkpoint inhibitor (ICI) treatment did not promote tumor progression, but instead a trend toward delayed tumor progression was observed.

Peer Review reports

Introduction

Lung cancer is one of the malignant tumors with the highest morbidity [25]. and mortality rates worldwide, and clinically most of the lung cancer patients are already in advanced stages when they present with symptoms at the clinic, and the overall 5-year survival rate of advanced lung cancer patients is only about 20% [1]. Although some new advances in immunotherapy have been made in the treatment of advanced non-small cell lung cancer (NSCLC) in recent years [15], the current treatment of advanced NSCLC with immune checkpoint inhibitors (ICIs) faces the limitation that the heterogeneity of the tumor immune microenvironment leads to different prognosis and response of patients to immunotherapy. We believe that therapeutic strategies can be improved in two ways; one strategy is to promote personalized treatment by differentiating molecular features from the tumor microenvironment [29, 30]. Another strategy is to make the heterogeneous immune microenvironment generally activated by some therapeutic means, so that it is generally effective to immune checkpoint inhibitor (ICI) therapy. Activation of the heterogeneous immune microenvironment by low-dose irradiation is an important means, and CT scan radiation, as the most common low-dose irradiation in the clinic, its effect on the tumor immune microenvironment is very worthy of study.

Computed tomography (CT) is a medical imaging technique that uses X-rays to provide detailed cross-sectional images of the internal structures of the human body, which is valuable in the diagnosis and treatment monitoring of patients with tumors. During CT scanning, patients are exposed to a certain amount of ionizing radiation due to the fact that X-rays are themselves a form of ionizing radiation, and the low-dose radiation (LDR) induced by CT scanning can have a destructive effect on immune cells; for example, it has been found that lymphocytes are highly sensitive to ionizing radiation, and that exposure to ionizing radiation leads to DNA damage in lymphocytes in the bloodstream [9, 13, 24, 26]. This DNA damage phenomenon is more pronounced in cases undergoing multiple enhanced CT scans [4, 24]. Since CT scan radiation has a detrimental effect on immune cells, do frequent whole-body CT scans during immune checkpoint inhibitor (ICI) therapy have a detrimental effect on a patient's antitumor immunity?

In the field of radiation protection and radiobiology, radiation dose is measured in milligrays (mGy), and the biological effects of radiation have a different dependence on its dose. Low Dose Radiation (LDR) is defined as a radiation dose of less than 100 mGy and is the commonly used radiation level for CT scans. Studies have shown that X-rays of 0.1 Gy or less can also kill some tumor cells, a phenomenon known as hypersensitivity to radiation therapy [12, 16] In some clinical studies, low-dose radiation in combination with chemotherapy, administered at doses to induce radiation hypersensitivity, has achieved striking rates of tumor control [2, 20, 23]. Therefore, especially in the current era of immunotherapy, the combination of low-dose radiation and immunotherapy may be a new therapeutic approach for patients.Does CT scanning, as a low-dose radiation, also enhance the tumor killing effect if performed frequently with whole-body CT scanning during the application of immune checkpoint inhibitors?

In recent years in the field of tumor immunology, it has been found that low-dose radiation can improve the immune microenvironment within tumor tissues and promote tumor clearance by the immune system [10]. For example, low-dose radiation therapy has been shown to reduce the proportion of regulatory T cells (Tregs) in the tumor microenvironment by promoting M1-type macrophage polarization [3], affecting the function and activation of NK and T cells [14], and reducing the proportion of regulatory T cells (Tregs) in the tumor microenvironment through multiple mechanisms [11] to enhance the immune system [17]. Shows potential as an immune amplifier capable of reprogramming the tumor microenvironment, triggering an inflammatory response and sensitizing "cold" tumors to immune checkpoint blockade therapies [5, 17, 18, 22].

No studies have been reported on whether frequent whole-body CT scans during the application of immune checkpoint inhibitors have an effect on patients' antitumor immunity, and our study is the first of its kind.

We conducted a retrospective clinical study aimed at investigating the effect of the interval between CT scans during immune checkpoint inhibitor (ICI) therapy on the time to progression (TTP) survival of non-small cell lung cancer (NSCLC) patients.

We constructed a hormonal mouse model and administered immune checkpoint inhibitor (ICI) therapy to mice, and radiated five whole-body CT scans to mice during ICI therapy to observe whether frequent whole-body CT scans had an effect on the anti-tumor effect of immunotherapy in mice.

Materials and methods

Cell lines

The murine lung adenocarcinoma cell line LLC was purchased from Suzhou Haixing Biological Technology Co. (Suzhou, China; Item No: TCM-C742). The cells were cultured in DMEM (HyClone, Logan, UT, USA SH30243.01) supplemented with 10% FBS (Biological Industries, Israel Item No: 04–001-1ACS) and 1% penicillin/streptomycin biosharp BL505A and were incubated at 37 °C in 5% CO2.

Tumor models

Six-week-old female C57BL/6 mice (18 ± 2 g) were purchased from Beijing HFK Bioscience Co., Ltd. (HFK Bioscience, Beijing, China). All mouse experiments were approved by the Animal Care and Use Committee of Shandong First Medical University(Ethical approval number: CUTCM/2021/9/113). Animal husbandry and experimental procedures were carried out after strict adherence to the guidelines of the Animal Care and Use Committee.

C57BL/6 mice were injected subcutaneously with 1 × 106 LLC cells in the left hind limb. When the tumor size reached approximately 4 mm in diameter, the mice were randomly divided into 2 groups: anti-PD-1 group, whole body CT scan radiation (WBCTSs) + anti-PD-1 group.

Treatment

Multiple Whole Body CT Scanning Radiation in Mice (WBCTSs): In order to simulate as much as possible the level of CT radiation in the clinical environment, we performed whole body CT scans on mice with the parameters of abdominal CT scans commonly used in the clinic, once every other day, for a total of 5 scans. We used a spiral CT scanner (Ingenuity CT, Philips Medical Systems, Eindhoven, The Netherlands, with an operating current of 30–250 mA-s, voltage of 120 kV, and rotation time of 0.5–0.75 s).

Anti-PD-1: anti-PD-1 was administered by intraperitoneal injection of 200ug of anti-mouse PD-1 (CD279)(Bioxcell catalog #BE0146) every other day for a total of 3 injections.

When the tumor diameter was approximately 6 mm, mice in each group were treated as follows: WBCTSs + anti-PD-1 group: mice were subjected to whole-body CT scanning at the parametric dose of abdominal CT scanning every other day for a total of 5 times; anti-PD-1 was injected intraperitoneally with 200ug of anti-mouse PD-1 (CD279) every other day for a total of 3 injections.Anti-PD-1 Anti-PD-1 alone group: 200ug of anti-mouse PD-1 (CD279) (Bioxcell Catalog #BE0146) was injected intraperitoneally every other day for a total of 3 injections and simulated whole-body CT scans were performed on mice every other day for a total of 5 injections.The experiment was repeated three times to ensure the reliability of the test results.

The mice were executed by cervical dislocation when the tumors reached 15 mm in diameter. No chemicals were used in this procedure.

Flow cytometry analysis(FCA)

Tumors were taken from mice and then homogenized for 40 min at 37 °C in DMEM medium with 0.2% type IV collagenase, 0.01% hyaluronidase, and 0.002% DNase I (all enzymes were obtained from Solarbio science, Beijing, China). The resulting single-cell suspensions were stained with fixable viability BV510, and then the harvested cells were labeled with the following antibodies: CD45 + FITC, CD3 + APC, CD8 + percpcy5.5, and IFN-γ + APC-Cy7. Antibodies were used according to the manufacturer's protocol (Biolegend, USA). After antibody labeling of the cell surface, cells were treated with the Fixation and Permeabilization Kit (Biolegend, USA) and stained with antibody IFN-γ. Stained samples were analyzed with a BD LSDFortessa flow cytometer. All flow cytometry data were analyzed using FlowJo software (version 10.0).The experiment was repeated three times to ensure the reliability of the test results.

Single-Cell RNA Sequene

10X genomics Single-Cell RNA Sequencing

Cell capture and cDNA synthesis

Tissue collection resuspended witn PBS. cell suspensions were loaded on a Chromium Single Cell Controller (10 × Genomics) to generate single-cell gel beads in emulsion (GEMs) by using Single Cell 3 ‘ Library and Gel Bead Kit V2 (10 × Genomics, 120,237) and Chromium Single Cell A Chip Kit (10 × Genomics,120,236) according to the manufacturer’s protocol. sequencing was performed on an Illumina Novaseq6000 with pair end 150 bp (PE150) mode.

Statistical analysis

Single cell data preprocess

Raw FASTQ files were mapped to the Reference genome (human, mouse et al.) using Cell Ranger 6.0(10 × Genomics). Mouse reference (mm10)—2020-A.

t-SNE visualization and determination of the major cell types

Gene expression analysis and cell type identification was analyzed using Seurat V3.0 pipeline (http://satijalab.org/seurat/) after filtering and normalization, [6]. As the data were already normalized, they were loaded into Seurat without normalization, scaling or centring. Along with the expression data, metadata for each cell was collected, including information such as clone identity, cell cycle phase, and time point. Next, highly variable genes were identified and used as input for dimensionality reduction via principal component analysis (PCA). The resulting PCs and the correlated genes were examined to determine the number of components to include in downstream analysis. t-SNE was then performed on the first 10 principal components to visualize cells in a two-dimensional space. To identify differentially expressed genes in each cluster, the Seurat function FindAllMarkers was used. For a gene to be differentially expressed in a cluster it must be expressed by at least 10% of cells, have a log-fold change greater than 0.25, and reach statistical significance of an p < 0.05 as determined by the Wilcox test. Finally, cell clusters were annotated to known biological cell types using canonical marker.

Pseudotime Analysis

Single cell trajectory was analyzed using matrix of cells and gene expressions by Monocle 3. Differentially expressed genes or significantly variable genes among cells were identified and used for dynamic trajectory analysis which ordered cells in pseudotime. First, the expression of transcripts of each gene was determined. Genes were then ranked using the coefficient of variation versus mean metric, selecting the top 3,000 genes as features. The resulting velocity estimates were projected onto the t-SNE embedding obtained in Seurat.

Patient

Our retrospective clinical study was conducted under the conditions of approval from the Clinical Research Ethics Committee of Qingdao People's Hospital Group (Jiaozhou) and written informed consent from all participants, and all methods were performed in accordance with the relevant guidelines and regulations of the Clinical Research Ethics Committee of Qingdao People's Hospital Group (Jiaozhou). Ethics No.: Jiaozhou Central Hospital Thesis Approval Document (2023) Thesis Review No. (003).

Patient data:Twenty patients with stage IV NSCLC treated with PD-1/PD-L1 from January 1, 2019 to December 31, 2021 at Shandong Cancer Hospital and 20 patients with stage IV NSCLC treated with PD-1/PD-L1 from January 1, 2019 to December 31, 2020 at Qingdao People's Hospital Group (Jiaozhou) were retrospectively collected.The patients enrolled were those who underwent a whole-body CT scan review.

The patient inclusion criteria were as follows: all histologically confirmed NSCLC patients were stage IV; they had received treatment with at least one PD-1/PD-L1 monoclonal antibody; and the case data were complete, including baseline data (age, gender, clinical stage, physical condition score, etc.) and treatment data (previous treatment, whether combined with chemotherapy, anti-angiogenic therapy or radiotherapy). Patients were treated in accordance with the guidelines of the Chinese Society of Clinical Oncology, with complete preclinical imaging and hematological indices and follow-up data.

The exclusion criteria for patients were as follows: small cell lung cancer; severe liver disease; gastrointestinal disease or other diseases that made it difficult to eat normally; cardiovascular accident within 1 month; chronic infection or acute attack of acute infection; use of steroids in the past 3 months; blood transfusion; and incomplete follow-up data.

Observations were

Duration of remission (DOR, Duration of Response) of ICI therapy: the time between the start of the first assessment of CR or PR and the first assessment of PD (Progressive Disease) or death from any cause in patients with stage IV NSCLC treated with ICI.

Frequency of CT review during ICI maintenance therapy in patients with tumors: total number of CT scan reviews within the duration of remission (DOR) of ICI therapy/duration of remission (DOR) of ICI therapy.

Stratified grouping

The patient's overall health status and previous treatments (including chemotherapy, radiotherapy, and other treatments) are potential confounders that can affect CT scan frequency and DOR.In order to control for the effects of these confounding factors, case data will be collected with full knowledge of the presence of these confounding factors. Patients were first excluded if their overall health status had a significant impact on the observations. The exclusion criteria for patients were: small-cell lung cancer; severe liver disease; gastrointestinal disease or other conditions that make it difficult to eat normally; cardiovascular accidents within 1 month; chronic infections or acute episodes of acute infections; use of steroids in the past 3 months or not; and blood transfusions. All patients were enrolled with complete information, including baseline information reflecting overall health status (age, sex, clinical stage, physical condition score, etc.) and treatment information reflecting prior treatment (whether combined chemotherapy, antiangiogenic therapy, or radiotherapy), and then in subsequent subgroups were first stratified according to the above confounders and then randomized among the strata in order to eliminate as much as possible the and control the effects of these confounders.

Statistical analysis: According to Pearson correlation analysis, the frequency of CT review and the duration of remission (DOR) of ICI treatment in tumor patients were positively correlated (r = 0.3460, P = 0.0287).

Statistical analysis

All the statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). The results are presented as the mean ± standard error of the mean (SEM). For comparing two groups, an unpaired 2-tailed Student t test was used; We indicated significance corresponding to the following: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Results

  • 1. The more frequent CT scans during treatment of lung cancer patients ICI the longer the DOR of ICI treatment

The aim of this study was to evaluate the relationship between the frequency of CT scan review during immunocheckpoint inhibitor (ICI) therapy and the effective maintenance time of immunocheckpoint inhibitor (ICI) therapy in patients with non-small cell lung cancer (NSCLC). We conducted a retrospective study of 20 patients with stage IV NSCLC treated with PD-1/PD-L1 at Shandong Cancer Hospital from January 1, 2019, to December 31, 2021, and 20 patients with stage IV NSCLC treated with PD-1/PD-L1 at Qingdao People's Hospital Group (Jiaozhou) from January 1, 2019, to December 31, 2021, in a retrospective Study. The baseline information of the patients is detailed in Table 1.

Table 1 Baseline characteristics of patients with stage IV NSCLC

Duration of remission (DOR, Duration of Response) of ICI treatment was defined as the time from the beginning of the first assessment of CR or PR to the first assessment of PD (Progressive Disease) or death from any cause in stage IV NSCLC patients treated with ICI.

The frequency of CT review during ICI maintenance therapy in tumor patients was defined as the total number of CT scan reviews within the duration of remission (DOR) of ICI therapy/duration of remission (DOR) of ICI therapy.

According to Pearson's correlation analysis, the frequency of CT review and the duration of remission (DOR) of ICI treatment in tumor patients were positively correlated (r = 0.3460, P = 0.0287) (Fig. 1). This result suggests that the more frequent the CT scan review during immune checkpoint inhibitor (ICI) therapy, the longer the duration of remission (DOR) of ICI therapy in non-small cell lung cancer (NSCLC) stage IV patients.

Fig. 1
figure 1

Analysis of the correlation between the frequency of CT scanning during ICI treatment and the DOR of ICI treatment in lung cancer patients

  • 2. WBCTSs combined with ICI treatment inhibits tumor progression in mice

To deeply analyze the effect of multiple whole-body CT scanning radiation on ICI anti-tumor immunity, we performed in vivo validation in a mouse tumor model. Mice were divided into two groups: anti-PD-1 group, WBCTSs + anti-PD-1 group (n = 7) (Fig. 2 A). anti-PD-1 group: anti-PD-1 alone intraperitoneal injection of 200ug, 1 time every other day for a total of 3 times. WBCTSs + anti-PD-1 group: (WBCTSs) whole-body CT scanning, 1 time every other day for a total of 5 times; anti—PD-1 intraperitoneal injection of 200ug, performed 1 day after whole-body CT scan, 1 time every other day, 3 times in total.

Fig. 2
figure 2

WBCTSs combined with ICI treatment inhibits tumor progression in mice. A: In order to deeply analyze the effect of multiple whole-body CT scan radiation on ICI anti-tumor immunity, we performed in vivo validation in model mice. LLC was implanted into the tumor mouse model, which was divided into two groups: anti-PD-1 group and whole-body CT scan radiation group (WBCTSs) + anti-PD-1 group (n = 7). anti-PD-1 group: anti-PD-1 alone intraperitoneally injected with 200ug, once every other day, for a total of 3 times. WBCTSs + anti-PD-1 group: (WBCTSs) whole-body CT scan, once every other day, a total of 5 times; anti-PD- 1 intraperitoneal injection of 200ug, performed 1 day after whole body CT scan, 1 time every other day, a total of 3 times. B We observed a more pronounced trend of tumor growth inhibition in mice in the WBCTSs + anti-PD-1 group compared to the anti-PD-1 group (n = 7)

We observed a more pronounced trend of tumor growth inhibition in mice in the WBCTSs + anti-PD-1 group compared to the anti-PD-1 group (n = 7), (Fig. 2 B).

  • 3. WBCTSs increased CD8T cell infiltration in tumor tissues of mice

We analyzed the tumor tissues of mice 24 h after irradiation using multicolor flow cytometry. The results revealed that the average proportion of CD8 T cells to CD45 + T lymphocytes within the tumor tissues in the WBCTSs + anti-PD-1 group increased from 1.7% to 6.5% (n = 4) compared with that in the anti-PD-1 group alone (Fig. 3A), and the proportion of IFN-γ-secreting CD8 T cells in the WBCTSs + anti-PD-1 group was significantly increased, with the The average proportion increased from 10.27% to 18.4% (n = 4) (Fig. 3B).

Fig. 3
figure 3

WBCTSs increased CD8T cell infiltration in tumor tissues of mice. A: We analyzed the tumor tissues of mice 24 h after irradiation using multicolor flow cytometry, and the average proportion of CD8 T cells to CD45 + T lymphocytes within the tumor tissues of the WBCTSs + anti-PD-1 group increased from 1.7% to 6.5% compared with that of the anti-PD-1 group alone (n = 4). B: We analyzed the tumor tissues of mice 24 h after irradiation using multicolor flow cytometry, and the proportion of IFN-γ-secreting CD8 T cells was significantly increased in the WBCTSs + anti-PD-1 group compared with the anti-PD-1 group alone, with the average proportion rising from 10.27% to 18.4% (n = 4)

  • 4. WBCTSs upregulate IFNγ and killing-related genes in tumor-infiltrating CD8T cells

After Chromium™ single-cell transcriptome library construction, the project used FastQC software for data quality assessment. Then basic analysis was performed: we corrected UMI based on the results of RNA sequence unique matching and Read1's UMI (Unique Molecular Identifier) sequences, and removed PCR repeats that appeared in sequencing according to the UMI correction criteria. After de-duplication, UMI counts were performed for different genes in each cell barcodes, and the Estimated Barcode Counts of the four groups we measured were as follows: NC-spleen (9,203) WBCTs-spleen (9,283) NC-Tumor (9,062) WBCTs-Tumor (9,137).). After identifying the valid cells, Gene-barcode matrix was generated to record the expression values of the different cellular genes, Total Genes Detected for four groups: NC-spleen (20,677) WBCTs-spleen (21,202) NC-Tumor (20,426) WBCTs-Tumor (21, 154) In order to deeply analyze the heterogeneity of the samples, Cell ranger clustered the cells based on gene expression levels. The idea of clustering: firstly, UMI normalization of expression data, then PCA dimensionality reduction analysis, 10 dimensions were selected for cell clustering using Graph-based, k-means (k = 2.10) clustering algorithm, and at the same time, t-SNE dimensionality reduction analysis was performed for visualization( Fig. 4A\C). To determine the differences between the two groups, we compared to spleen and tumor separately: NC-spleen VS WBCTs-spleen; NC-Tumor VS WBCTs-Tumor, setting the following thresholds: p-value < 0.05,log2FC > 0.25,min.pct > 0.25.

Fig. 4
figure 4

WBCTSs upregulate IFNγ and killing-related genes in tumor-infiltrating CD8T cells. A/C We selected 1 mouse from each of the WBCTSs group and NC group, and took mouse tumor tissue and spleen for single-cell sequencing analysis, respectively. A total of 4 samples were collected from each mouse for sequencing of mouse tumor tissue and spleen, respectively. The total number of cells measured was estimated to be 36,685. B CD3T cells, CD4T cells, and CD8T cells as a percentage of CD45 + T lymphocytes were increased in tumor tissues after WBCTS. D After WBCTSs, there was a significant decrease in the proportion of CD3, CD4 and CD8 T cells to CD45 + cells and a significant increase in the proportion of CD45 + T lymphocytes to CD45 + cells in the spleen. E CD8T cell population identified by CD8T cell signature gene (CD3g CD3e CD3d Cd8a Cd28 Gzmk Ifng Klrg1). F In tumor tissues, WBCTSs upregulated the expression of IFN-γ and killing-related genes (IFNg, KLRD1, Gzmf, Tnfrsf9, Tnfrsf4) in tumor-infiltrating CD8T cells. While in spleen WBCTSs downregulated (IFNg, KLRD1) genes. G Radiation from multiple CT scans resulted in up-regulation of CD8 T cell exhaustion-related genes (including Tox, LAG-3, CTLA-4 and PDCD1) in tumor tissues. H The afferent signal intensity of CD8 T cells was significantly enhanced after WBCTS in tumor tissues. I WBCTS in tumor tissues enhanced functional signaling pathways related to t-cell receptors, cytokines and antigen presentation in CD8T cells, and also up-regulated the PD-L1/PD-L2 signaling pathwa

In order to better understand the effect of radiation from frequent whole-body CT scans on CD8 + T cells in the whole body and tumor tissues, we performed WBCTSs on mice, 1 in the WBCTSs group and 1 in the NC group, and single-cell sequencing was performed on the tumor tissues and spleens of each mouse, respectively, for a total of 4 samples (Fig. 4A\C), with a total of 4 samples (Fig. 4A\C), with a total of 4 samples (Fig. 4A\C), and the total number of cells measured Estimated Number of Cells 36,685; Fraction Reads in Cells 90.6%; Median Genes per Cell 2,352; Median UMI Counts per Cell 8,661.

The CD8T cell population was identified by the characteristic gene of CD8T cells (CD3g CD3e CD3d Cd8a Cd28 Gzmk Ifng Klrg1) (Fig. 4E), and the focus of our study was to detect the changes of CD8T cells before and after exposure to radiation from whole-body CT scans in tumor tissues and in the spleen.

It was found that in the spleen, the percentage of CD3, CD4 and CD8 T cells to CD45 + cells in the spleen decreased after radiation, where the percentage of CD8 T cells to CD45 + cells in the spleen decreased from 6.53% before radiation exposure from whole-body CT scanning to 4.51% after radiation exposure (Fig. 4D). Further gene expression analysis showed that low-dose irradiation downregulated the expression of IFNγ and killing-related genes (IFNg, KLRD1) in splenic CD8T cells (Fig. 4F).

In tumor tissues, the proportions of CD3T cells, CD4T cells and CD8T cells among CD45 + T lymphocytes were increased after multiple CT scanning radiation, in which the proportion of CD8T cells in tumor tissues as a percentage of CD45 + cells was increased from 4.49% before whole-body CT scanning radiation to 7.15% after radiation (Fig. 4B); moreover, multiple CT scanning radiation up-regulated tumor expression of IFNγ and killing-related genes (IFNg, KLRD1, Gzmf, Tnfrsf9, Tnfrsf4) in infiltrating CD8T cells (Fig. 4F),and the signal communication between CD8T cells and other cells was significantly enhanced after radiation (Fig. 4H), specifically, the afferent signal intensity of CD8T cells was significantly enhanced (Fig. 4H). We also noted that radiation enhanced functional signaling pathways related to t-cell receptors, cytokines, and antigen presentation in CD8T cells (Fig. 4I), and also upregulated the PD-L1/PD-L2 signaling pathway (Fig. 4I). In addition, radiation from multiple CT scans resulted in upregulation of CD8 T cell exhaustion-related genes (including Tox, LAG-3, CTLA-4, and PDCD1) in tumor tissues (Fig. 4G).

Discussion

The emergence of immune checkpoint inhibitors (ICIs) has ushered in a new era of cancer treatment, marking the emergence of immunotherapy as a mainstream approach. In this study, we aimed to investigate whether frequent whole-body CT scans during immune checkpoint inhibitor (ICI) treatment had an effect on the antitumor therapeutic efficacy of ICI through a retrospective clinical study and a study in a mouse model, respectively. Our results showed that multiple CT scans during immune checkpoint inhibitor (ICI) treatment did not promote tumor progression; instead, a trend toward delayed tumor progression was observed. Previous studies have shown that low-dose radiation enhances immune activation, thereby increasing the sensitivity of tumor cells to ICI [11, 22, 27, 28]. The results observed in our study are consistent with the results of these previous similar studies.

Our further study in a mouse tumor model revealed that frequent CT scan radiation during the application of the immune checkpoint inhibitor PD-1 increased the proportion of infiltrating CD8 + T cells in tumor tissues and significantly increased the number of IFN-γ-secreting CD8 + T cells. Thus, frequent whole-body CT scanning combined with immunotherapy inhibits tumor growth in mice. Consistent with our findings, Nowosielska et al. reported that whole-body low-dose radiation significantly inhibited LLC cell growth. In addition, the combination therapy of CTLA-4 + PD-1 + LDR showed significant effects both in vivo (lung and subcutaneous tumor growth) and in vitro (clonogenic potential of tumor cells) [21].

Ionizing radiation directly or indirectly causes DNA damage, including single-strand breaks, double-strand breaks and base damage. Lymphocytes are extremely sensitive to DNA damage, and accumulation of damage leads to apoptosis or loss of function, resulting in a decrease in the number of lymphocytes. It has been demonstrated that radiation from multiple whole-body CT scans has a damaging effect on immune cells in both spleen and tumor tissues [9, 13, 24, 26]. Our study also observed that radiation from multiple whole-body CT scans resulted in a decrease in the proportion of splenic CD8 + T cells and downregulation of IFN-γ-related gene expression in splenic CD8 + T cells. However, an increased proportion of CD8 + T cells in tumor tissues upregulated IFNγ and killing-related genes in tumor-infiltrating CD8T cells.These findings are consistent with other studies [10, 11, 26]. Consistent with this, low-dose radiation improves the immune microenvironment within the tumor tissue and promotes tumor clearance by the immune system.

This paradox is explained by the fact that radiation from multiple whole-body CT scans has an injurious effect on immune cells in both spleen and tumor tissues, but tumor cells in tumor tissues are more sensitive to radiation and are able to activate immunity by initiating damage-associated molecular patterns (DAMPs) [8]. Radiation-induced DNA damage in tumor cells promotes IFN production and activates immunity [7, 19, 31]. As a result, immune activation is induced in the tumor tissue, counteracting the immunosuppressive effects of radiation damage on the immune cells.

However, CD8 + T cells may be in a dynamic balance of activation and exhaustion after CT scan radiation. The initial phase is dominated by immune activation and therefore shows upregulation of (IFNg, KLRD1, Gzmf, Tnfrsf9, Tnfrsf4)) genes, but prolonged and sustained antigenic stimulation and inflammatory environment leads to T cell exhaustion, which is manifested by upregulation of (Tox, LAG-3, CTLA-4 and PDCD1) genes. Therefore, our collection of CD8 T cells included both cells in the immunologically activated state and CD8 + T cells in the exhausted state. We used the therapeutic strategy of CT scanning radiation combined with immune checkpoint inhibitors, which can activate a part of CD8 + T cells by CT scanning radiation, and then, by immune checkpoint inhibitors, can rejuvenate another part of CD8 + T cells in the depleted state, so that our use of the combination therapy can enhance the sustained anti-tumor ability of CD8 + T cells, which can be used to further improve anti-tumor effect.

The potential mechanism for the upregulation of genes associated with antigen presentation and immune signaling in tumor-infiltrating CD8 + T cells after CT scan radiation is that CT scan radiation induces damage-associated molecular patterns (DAMPs) in tumor cells and causes the release of tumor antigens that lead to the release of tumor antigens.The DAMPs activate dendritic cells (DCs) to promote the uptake, processing, and presentation of tumor antigens and activation of CD8 + T cells. In addition, CT scanning radiation-induced DNA damage in tumor cells activates damage response pathways (e.g., STING pathway), promotes secretion of type I interferons (e.g., IFN-α/β), and enhances antigen presentation and activation of DCs and CD8 + T cells. This part of the study has been elaborated in our other articles.

Up-regulation of genes related to antigen presentation and immune signaling in tumor-infiltrating CD8 + T cells after CT scan radiation provides a more favorable immune microenvironment for the application of immune checkpoint inhibitors. On the one hand, CT scan radiation-induced damage-associated molecular patterns (DAMPs) and DNA damage response in tumor cells increased the immunogenicity of tumor cells, making them more easily recognized and attacked by the immune system. This complements the mechanism of action of ICIs. On the other hand, CT scanning radiation promotes the activation and proliferation of CD8 + T cells through the upregulation of antigen presentation and co-stimulatory signals, increasing the number of effector T cells, and this state of immune activation provides more favorable conditions for the action of ICIs.

The commonly used radiation level for CT scanning is about 0.1 Gy or lower, and our findings suggest that CT scanning, as a low-dose radiation, is enhanced by frequent whole-body CT scanning during the application of immune checkpoint inhibitors to kill tumors. Several previous studies have shown that X-rays of 0.1 Gy or lower can also kill some tumor cells, a phenomenon known as radiotherapy hypersensitivity [12, 16]. In some clinical studies, low-dose radiation in combination with chemotherapy, administered at doses to induce radiation hypersensitivity, has achieved striking rates of tumor control [2, 20, 23]. Therefore, in the current era of immunotherapy, the combination of low-dose radiation and immunotherapy may be a new treatment for patients.

In our study, we aimed to investigate whether frequent whole-body CT scans during immune checkpoint inhibitor (ICI) therapy have an impact on the antitumor therapeutic efficacy of ICI through retrospective clinical studies and mouse model studies. Although the use of mouse models has enhanced the study, there are significant differences between mice and humans in terms of DNA repair capacity, cell cycle regulation, immune cell composition, cytokine networks, immune memory, inflammatory response, and immunosuppression, and thus there are still significant limitations in translating the results of the mouse studies to humans, especially in terms of radiosensitivity and immune response mechanisms. Although our study found that radiation from frequent whole-body CT scans promotes immune activation and thus increases tumor cell sensitivity to ICI, these need to be followed up with more in-depth clinical studies.

Data availability

Sequence data that support the findings of this study have been deposited in the National Genomics Data Center with the primary accession code PRJCA026556.

Abbreviations

WBCTs:

Whole-body CT scans

ICI:

Immune checkpoint inhibitor

DOR:

Duration of remission

NSCLC:

Non-small cell lung cancer

LDR:

Low Dose Radiation

Tregs:

Regulatory T cells

TTP:

Therapy on the time to progression

LLC:

Lewis Lung Carcinoma

DMEM:

Dulbecco's Modified Eagle Medium

DAMP:

Damage-associated molecular patterns

DCs:

Dendritic cells

IFN-α:

Interferon-α

IFN-β:

Interferon-β

IFN-γ:

Interferon-γ

TNF-α:

Tumor necrosis factor-α

ELISA:

Enzyme-linked immunosorbent assay

FCA:

Flow cytometry analysis

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Acknowledgements

We express our gratitude to the Key Laboratory of Radiation Oncology in Shandong Province for providing the research platform, which includes Molecular Biology Laboratory, Cell Biology Laboratory, Small Animal Radiation Research Platform, Flow Cytometry in the Animal Experiment Center. We also appreciate all the laboratory members for their valuable discussions and technical support. This work was funded by the Academic Enhancement Program of Shandong First Medical University .This study was supported by a grant from the Key Laboratory of Marine Drugs, Chinese Ministry of Education. This study was also supported by the Qingdao Municipal Medical and Health Research Guidance Program.This study was also supported by a grant from the Qingdao Clinical Key Specialty of Comprehensive Treatment of Digestive Tract Tumors.

Funding

This work was supported by a grant from the Academic Enhancement Program of Shandong First Medical University (2019LJ004). This study was also supported by a grant from the Qingdao Clinical Key Specialty for Comprehensive Treatment of Gastrointestinal Tumors.This study was supported by a grant from the Key Laboratory of Marine Drugs, Chinese Ministry of Education. This study was also supported by the Qingdao Municipal Medical and Health Research Guidance Program.

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Authors and Affiliations

Contributions

Jigang Dong (First Author):Conceptualization, Methodology, Software, Investigation, Formal Analysis, Writing—Original Draft; Ying qi and Xiao Xu: Visualization, Investigation;(Jigang Dong and Ying qi contributed equally to this work) Sha sha: Data Curation, Writing—Original Draft; Chengrui Fu:Software, Validation Xiao Xu, Software, Investigation Baosheng Li(Corresponding Author):Funding Acquisition, Resources, Supervision,

Corresponding authors

Correspondence to Xiao Xu or Baosheng Li.

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Ethics approval and consent to participate

All mouse experiments in this study were approved by the Animal Care and Use Committee of Shandong First Medical University (Ethical Approval No. CUTCM/2021/9/113).All animal housing premises and conditions, animal care and monitoring details and experimental conditions were in accordance with ARRIVE guidelines.All clinical studies were conducted in accordance with the relevant guidelines and regulations of the Clinical Research Ethics Committee of Qingdao People's Hospital Group (Jiaozhou). Ethics number: thesis approval document (2023) thesis review number (002) of Jiaozhou Central Hospital.

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This study does not include any identifiable patient data or images requiring consent for publication. Therefore, this section is not applicable.

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The authors declare no competing interests.

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Dong, J., Qi, Y., Sha, S. et al. Whole-body CT scanning radiation improves the immune microenvironment of tumor tissues to enhance the antitumor effect of ICI. BMC Cancer 25, 824 (2025). https://doi.org/10.1186/s12885-025-14119-7

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