Radiation from frequent whole-body CT scans induces systemic immunosuppression and immune activation of tumor tissue

Highlights

• •Frequent CT scan radiation induces systemic immunosuppression, as evidenced by a decrease in the proportion of CD8+ T cells in the blood and spleen, down-regulation of killer-related genes, and a decrease in serum interferon levels.
• •Frequent CT scan radiation induces immune activation in tumor tissues, as evidenced by an increase in the proportion of CD8+ T cells in tumor tissues, up-regulation of killing-related genes, and an increase in interferon levels in tumor tissues.
• •Frequent CT scan radiation does not promote tumor progression.

Abstracts

Objective

This study aims to elucidate the impact of repeated whole-body computed tomography (CT) scans on systemic immunity, the tumor immune microenvironment, and tumor control. This inquiry was prompted by clinical observations indicating a decrease in the levels of IFN-β and IFN-γ in patients' blood following whole-body CT scans.

Methods

A Lewis lung carcinoma (LLC) mouse model was established and divided into two groups: a control group and a group subjected to multiple whole-body CT scanning radiation (WBCTSs). The study monitored tumor growth trends across both groups and employed a comprehensive set of analytical techniques—including enzyme-linked immunosorbent assay (ELISA), flow cytometry analysis, immunohistochemistry, RNA sequencing, and single-cell sequencing—to assess differences in cytokine profiles (IFN-β and IFN-γ), proportions of key immune cells, and gene expression variations between the groups.

Results

Repeated CT scan radiation does not promote tumor progression. In tumor tissues subjected to multiple CT scans, an increase in the proportion of CD8+ T cells, elevated interferon levels, and up-regulation of genes associated with killing in CD8+ T cells and genes associated with Ifnb in macrophages were observed. In contrast, radiation from multiple whole-body CT scans resulted in a decrease in the proportion of CD8+ T cells in the blood and spleen, a decrease in serum interferon levels, and down-regulation of killing-related genes in CD8+ T cells.

Conclusion

Our results suggest that repeated whole-body CT scanning radiation induces systemic immunosuppression and immune activation in tumor tissues. Multiple repeated CT scans do not promote tumor progression.

Introduction

Computed tomography (CT), a medical imaging technique that uses X-rays to provide detailed cross-sectional images of the body's internal structures, is valuable in the diagnosis and therapeutic monitoring of patients with tumors.Patients are exposed to a certain amount of ionizing radiation during CT scans, due to the fact that X-rays themselves are a form of ionizing radiation that can remove electrons from atoms or molecules, thereby altering the substance's chemical structure of substances and have an effect on the body's immune system. The ionizing radiation generated during CT scans is therefore of critical importance to the patient's immune system and its potential impact on tumor control.

In the field of radiation protection and radiobiology, radiation dose is measured in units of milligray (mGy) and the biological effects of radiation show different dependencies on its dose. Low-Dose Radiation (LDR), defined as a radiation dose of less than 100 mGy, is a common radiation level for CT scans. Although the radiation dose from a single CT scan is low, the cumulative radiation dose from multiple scans may pose a potential health risk. In addition, radiation doses in the range of 100–1000 mGy are considered to be moderate doses of radiation, while those exceeding 1000 mGy are considered to be high doses of radiation.

Damage to immune cells from radiation includes both deterministic and stochastic effects. Deterministic effects are those that are directly related to the radiation dose and are certain to exceed a certain threshold. Lymphocytes, one of the most radiosensitive cells in the body, can exhibit deterministic damage at relatively low radiation doses.The ICRP (International Commission on Radiological Protection) outlines dose thresholds for various tissues, and in the case of lymphocytes, doses as low as 250m Gy can have a significant effects. At doses of 500–1000m Gy, significant lymphocyte depletion can occur and the immune system begins to dysfunction. Severe lymphopenia occurs at radiation doses of 1000m Gy, severely affecting the immune response. Generally, radiation doses from CT scans do not reach radiation levels of 250m Gy, and significant effects generally do not occur, but exposure to ionizing radiation from CT scans can cause DNA damage to lymphocytes in the blood.For example, it has been found that lymphocytes are highly sensitive to ionizing radiation and that exposure to ionizing radiation causes DNA damage to lymphocytes in the blood [1]. This DNA damage phenomenon is more pronounced in cases undergoing multiple enhanced CT scans [1,2]. Stochastic effects, such as the risk of cancer and genetic mutations, have no clear threshold with respect to radiation dose and may occur even at very low doses. For example, it was found that CT radiation exposure in adolescents resulted in an increased risk of hematologic malignancies [3].

In the field of tumor immunology, low-dose radiation has been found to improve the immune microenvironment within tumor tissues and promote tumor clearance by the immune system [4]. For example, low-dose radiation therapy has been shown to enhance the immune system by promoting M1-type macrophage polarization [5], affecting NK and T-cell function and activation [6], and decreasing the proportion of regulatory T cells (Tregs) in the tumor microenvironment through a number of mechanisms [7,8]. Demonstrate potential as immune amplifiers capable of reprogramming the tumor microenvironment, triggering an inflammatory response and sensitizing "cold" tumors to immune checkpoint blockade therapy [[9], [10], [11]].

Most of the above studies on tumor immunity by low-dose radiation have used low-dose irradiation of 100 mGy or more to irradiate the tumor locally, and there is also evidence that ultra-low-dose irradiation of less than 100 mGy can also induce anti-tumor immune responses [12].

Taken together, we found that low-dose radiation, on the one hand, can cause systemic immune cell damage and thus suppress systemic immunity, and on the other hand, low-dose irradiation can also cause improvement of the tumor immune microenvironment. Whole-body CT scanning is also a kind of whole-body low-dose radiation that.With regard to the radiation dose from CT scans, according to the Diagnostic Reference Levels for X-ray Computed Tomography Scans in Adults, published by the National Health and Health Commission of the People's Republic of China in 2018, the volume Computed Tomography Dose Index (CTDIvol) represents the average dose over the entire scanning area during a CT scan, with a diagnostic level of 15 mGy for chest CT, 20 mGy for abdominal CT, and 60 mGy for cranial diagnostic level.Enhanced CT scans require a plain scan, arterial and venous phases, and abdominal scans in the portal venous phase, resulting in a patient's radiation exposure that is three to four times the reference level. The maximum CTDIvol is 45mGy for chest enhancement CT, 80mGy for abdominal enhancement CT, and 180mGy for cranial enhancement CT. CT scans can include the head, chest, abdomen, and pelvis, so the radiation exposure from CT scans is much greater than that from the tumor site alone. Moreover, patients may need to undergo multiple CT scans for a short period of time in order to diagnose a tumor, so the effects of this more extensive low-dose irradiation on the patient's systemic immune system and the immune microenvironment of the tumor tissues may be quite different compared with localized low-dose irradiation.

Does whole-body CT scanning promote tumor progression through systemic immune cell damage or inhibit tumor progression by improving the immune microenvironment of tumor tissues? Previous studies on low-dose radiation have focused on a single aspect of the tumor tissue microenvironment or blood system, but the effects of low-dose radiation on the body as a whole is a complex issue, and there is no study that focuses on the effects of whole-body low-dose irradiation on the whole-body immune system of the patient and the immune microenvironment of the tumor tissue at the same time. In view of this, we conducted a case-control study to observe the effects of multiple CT scans on the blood cytokine levels of patients, but due to ethical constraints, we were unable to perform multiple CT scans on patients and obtain tumor tissues and spleens on time to observe the effects of multiple CT scans on the systemic immune system and tumor tissues of the patients, so we designed a mouse tumor model to simulate the whole-body CT scanning of tumor patients and obtained tumor tissues and spleens on time to observe the effects of multiple systemic CT scans on the systemic immune system and tumor tissues of the mice. Therefore, we designed a mouse tumor model to simulate a tumor patient undergoing whole-body CT scanning and obtaining tumor tissues and spleens on time to observe the effects of multiple whole-body CT scans on the whole-body immune system and the immune microenvironment of tumor tissues in mice.

Materials and methods

Cell lines

The LLC murine lung adenocarcinoma cell line (TCM-C742) was acquired from Suzhou Haixing Biological Technology Co., situated in Suzhou, China. The cells were cultured at a temperature of 37 °C with a CO₂ concentration of 5 %, using DMEM medium (SH30243.01, HyClone, Logan, UT, USA), supplemented with 10 % FBS (04–001–1ACS, Biological Industries, Israel) and 1 % penicillin/streptomycin biosharp BL505A.

Tumor models

The female C57BL/6 mice used in this study were obtained from Beijing HFK Bioscience Co, Ltd. (HFK Bioscience, Beijing, China) at the age of six weeks and weighing approximately 18 ± 2 g. The Animal Care and Use Committee of Shandong First Medical University approved all mouse experiments conducted in this research (Ethical approval number: CUTCM/2021/9/113). We strictly followed the guidelines provided by the Animal Care and Use Committee during animal husbandry and experimental procedures.

C57BL/6 mice were subcutaneously inoculated with 1 × 10⁶ LLC cells in the left hind limb. Upon attaining a tumor size of approximately 4 mm in diameter, the mice were randomly allocated into two groups: a negative control (NC) group and a group exposed to whole-body computed tomography scanning radiation (WBCTSs) (n = 7).

Treatment

When the tumor diameter was approximately 6 mm, the mice in each group were treated as follows.WBCTSs: A spiral CT scanner (Ingenuity CT, Philips Medical Systems, Eindhoven, The Netherlands) was used with an operating voltage of 30–250 mA-s at 120 kV and a rotation time of 0.5–0.75 ss. Whole-body CT was administered to the mice at the parametric dose for abdominal CT scanning once every other day for a total of 5 times;The NC group was subjected to simulated irradiation. Three repetitions of the experiment were performed 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.

ELISA assay

Mice were sacrificed 24 h after the end of the last CT scanning irradiation, and blood and tumor tissues were collected separately. Cytokine levels in the serum and supernatants of samples prepared from tumor tissue lysates were measured via ELISA using a tissue homogenizer to process the tumor tissues and lysis buffer containing a protease inhibitor (Beyotime P1045) to prepare sample supernatants. The levels of IFN-γ and IFN-β were determined via standard ELISA using specific antibody ELISA kits (J&L Biology, Shanghai, China.) according to the manufacturer's instructions(n = 5). Three replications of the experiment were performed to ensure the reliability of the test results.

Flow cytometry analysis(FCA)

Fifty microliters of peripheral blood was removed from the mice to prepare a single-cell suspension, and appropriate antibodies CD3 (FITC), CD45 (PerCP-Cy5.5), CD4 (PE-Cy), and CD8 (APC-Cy7) were added for cell surface labeling.

The tumors and spleens of the mice were removed and subsequently homogenized in 0.2 % collagenase type IV, 0.01 % hyaluronidase, and 0.002 % DNase I (all enzymes from Solarbio Science, Beijing, China) in DMEM at 37 °C for 40 min. The obtained single cell suspensions were stained with BV510 to mark cell viability, after which the harvested cells were labeled with the following antibodies:CD45(FITC), CD3(APC), CD8(percp-cy5.5), and IFN-γ(PE/APC-Cy7) (mainly analyzing T cells infiltrated by tumor tissue). (Tube 2) CD45(percpcy5.5), CD4(FITC), CD8( APC-Cy7) (Mainly analyzes lymphocytes in the blood),antibodies were used according to the manufacturer's protocol (BioLegend, USA). For IFN-γ staining, cells were stimulated in vitro with a cell stimulation cocktail (plus protein transport inhibitors) (BioLegend, USA) for 6 h. After the surface was labeled with CD45(FITC), CD3(APC), and CD8(percpcy5.5) antibodies, the cells were processed using a fixation and permeabilization kit (BioLegend, USA) and stained with the IFN-γ antibody. The stained samples were analyzed using a BD LSDFortessa flow cytometer. All flow cytometry data were analyzed with FlowJo software (version 10.0).(n = 3) Three replications of the experiment were performed to ensure the reliability of the test results.

Immunohistochemistry

Tumor tissues were fixed in 10 % neutral-buffered formalin and embedded in paraffin, and 4 μm thick sections were cut and used for immunohistochemistry (IHC). The sections were labeled with the following antibodies: CD8+ and GZMB+ according to the manufacturer's instructions (Abcam, China). Images were taken using an optical microscope (Olympus, Tokyo, Japan). For each tumor section, the total number of cells that were positive for CD8 and GZMB was counted in five randomly selected fields (original magnification×200), and the percentage of positively stained cells was calculated.(n = 3) Three replications of the experiment were performed to ensure the reliability of the test results.

RNA sequence

Tumor tissues were snap frozen in liquid nitrogen, after which total RNA was extracted. Libraries were subsequently constructed using the TruSeq Stranded mRNA LT sample preparation kit (Illumina, San Diego, CA, USA). Transcriptome sequencing and analysis were performed by Shandong Xiuyue Biotechnology Co., Ltd. (Shandong, China).

Single-cell RNA sequene

10×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 (10x Genomics) to generate single-cell gel beads in emulsion (GEMs) by using Single Cell 3‘ Library and Gel Bead Kit V2 (10x Genomics, 120,237) and Chromium Single Cell A Chip Kit (10x Genomics ,120,236) according to the manufacturer's protocol. sequencing was performed on an Illumina Novaseq6000 with pair end 150bp (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(10x 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, (Butler et al., 2018). 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 adjusted 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 3000 genes as features. The resulting velocity estimates were projected onto the t-SNE embedding obtained in Seurat.

Patient

Patient data for whom serum cytokines were detected

We selected 16 patients with tumors who underwent enhanced CT scans of the head, chest, abdomen and pelvis at Qingdao People's Hospital Group (Jiaozhou) for a case-control study.With the approval of the Clinical Research Ethics Committee of Qingdao People's Hospital Group (Jiaozhou) and written informed consent from all participants, we collected blood samples from patients before and after CT scans (Fig. 1A\B). 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 number: thesis approval document (2023) thesis review number (002) of Jiaozhou Central Hospital.

Fig. 1.

Reduced IFN levels in the blood of patients after whole-body enhanced CT scanning. (A): We selected 16 patients for a case-control study to observe the changes in blood levels of IFN-β and IFN-γ before and after whole-body enhanced CT scanning. (B): Enrolled patients underwent enhanced CT scanning of the head and neck, chest, abdomen and pelvis. Peripheral venous blood samples were taken 24 h before and 24 h after the CT scan. (C): We observed a significant decrease in the levels of IFN-β (P < 0.05) and IFN-γ(P < 0.01) in the blood of patients after CT scanning.

The inclusion criteria for patients were patients with histologically confirmed malignant tumors, a physical status score ranging from 0–1, and complete case data, including baseline data (age, sex, clinical stage, physical status score, etc.) and treatment data (previous illnesses and treatments) (Table 1).

Table 1.

Baseline characteristics of serum cytokines in patients.

Characteristics	Total(n = 16)
Age (years)
Range	(45–76)
>65	7
≤65	9
Gender
Male	7
Female	9
ECOG PS
0–1	16
≥2	0
With / Without Immune-related diseases
Yes	0
No	16
With / Without radiotherapy
Yes	0
No	16
With / Without chemotherapy
Yes	0
No	16
With / Without ICI
Yes	0
No	16

Underlying diseases and body states that interfere with immune parameters were excluded: those with a physical status score of ≥2; those with the presence of severe liver disease; those who had received chemotherapy, radiotherapy, and immunotherapy in the last month; those who had a cardiovascular accident in the last month; those who had an acute exacerbation of a chronic infection or had an acute infection; those who had received steroid therapy in the last 3 months; those who had received a transfusion therapy; and those who had an incomplete follow-up profile were excluded (Table 1).

CT Scanning Parameters and Methods:The CT scanning method was performed with a spiral CT scanner (Ingenuity CT, Philips Medical Systems, Eindhoven, The Netherlands) operating at 30–250 mA and 120 kV with a rotation time of 0.5 to 0.75 s. The collimation (beam width) ranged from 1 to 5 mm, and the spacing (based on the total beam width) ranged from 0.5 to 1.25. Patients underwent enhanced CT scans of the head, chest, abdomen, and pelvis

Observational index: Peripheral venous blood was collected from patients before and 24 h after head, chest, abdomen and pelvis enhanced CT scans, and the levels of the cytokines IFN-γ and IFN-β were analyzed.

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's t-test was used; We indicated significance corresponding to the following: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Results

Reduced IFN levels in the blood of patients after whole-body enhanced CT scanning

We observed a significant decrease in the blood levels of IFN-β and IFN-γ in the patients after CT scanning (Fig. 1C).The mean level of IFN-β decreased from 132.6 pg/ml before CT scanning to 107.6 pg/ml after CT scanning; the mean level of IFN-γ decreased from 75.37 pg/ml before CT scanning to 61.54 pg/ml after CT scanning (Fig. 1C).

Differential effects of WBCTS on IFN levels in blood and tumor tissue

To deeply analyze the effect of multiple whole-body CT scanning radiation on anti-tumor immunity, we performed in vivo validation on model mice.LLC implanted tumor mouse model, divided into 2 groups: control, whole-body CT scanning radiation (WBCTSs), (Fig. 2A). We showed from the tumor volume change curves of the two groups that multiple whole-body CT scanning radiation did not promote tumor progression, and the tumors of the mice in the WBCTs group grew more slowly, but there was no statistically significant difference between the WBCTs group and the control group (n = 7), (Fig. 2B).

Fig. 2.

Different effects of WBCTSs on IFN levels in blood and tumor tissues of mice. (A): LLC implanted tumor mouse model, divided into two groups: NC group, WBCTSs group: whole-body CT scanning, every other day for 5 times. (B): Compared with the control group, we observed that WBCTSs did not promote tumor progression in mice (n = 7). (C): The levels of IFN-β and IFN-γ in the blood of mice were significantly decreased (P < 0.01) after the use of WBCTS (n = 5). (D): Elevation of IFN-β and IFN-γ levels in tumor tissues of mice after WBCTS (P < 0.05) (n = 5). (E): We performed RNAseq analysis on the tumor tissues of mice in both groups. The results showed that IFN generation-related genes and monocyte-macrophage phagocytosis-related genes were significantly upregulated after WBCTSs.

We observed a significant decrease in the levels of IFN-β and IFN-γ in the blood of mice after CT scanning, with the average level of IFN-β decreasing from 9.8 pg/ml before CT scanning to 5.5 pg/ml after CT scanning; and the average level of IFN-γ decreasing from 14.2 pg/ml before CT scanning to 10.6 pg/ml after CT scanning, which was the same as what we observed in patients , mice only (n = 5) (Fig. 2C). Unlike mouse blood, the levels of IFN-β and IFN-γ in mouse tumor tissues did not decrease, with the mean level of IFN-γ increasing from 21.5pg/g before CT scan to 33.00 pg/g after CT scan; the mean level of IFN-β in mouse tumor tissues increased from 46.07 pg/g before CT scan to 67.87 pg /g (n = 5) (Fig. 2D). We performed RNAseq analysis of tumor tissues after radiation from multiple whole-body CT scans. The results showed that genes associated with monocyte-macrophage phagocytosis and IFN production were significantly upregulated after CT irradiation (Fig. 2E).

WBCTSs enhances immune cells and IFN genes in tumor tissues and suppresses the expression of related genes in the spleen

In order to better understand the mechanism by which whole-body CT scan radiation affects IFN levels, we selected 1 mouse from each of the WBCTSs and NC groups, and took mouse tumor tissues and spleens for single-cell sequencing analysis, respectively.

Grouping and number of mice: 1 mouse from the WBCTSs group and 1 mouse from the NC group, number of samples: each mouse was sequenced from mouse tumor tissues and spleens respectively, with a total of 4 samples (Fig. 3AC), and the total number of cells measured Estimated Number of Cells 36,685; Fraction Reads in Cells 90.6 %; Median Genes per Cell 2352; Median UMI Counts per Cell 8661.

Fig. 3.

WBCTSs enhanced the expression of immune cells and IFN genes in tumor tissues and inhibited the expression of related genes in spleen. (A/C): We selected 1 mouse in the WBCTSs group and 1 mouse in the NC group, and took the tumor tissues and spleens of the mice for single-cell sequencing analysis, respectively. A total of 4 samples were collected from each mouse, and the tumor tissue and spleen were sequenced separately. The total number of cells measured was estimated to be 36,685. (B): The proportion of CD3+ T cells, CD4+ T cells and CD8+ T cells among CD45+ T lymphocytes in tumor tissues increased after WBCTS. (D): After WBCTS, there was a decrease in the proportion of CD3+, CD4+ and CD8+ T cells to CD45+ cells and an increase in the proportion of CD45+ cells to CD8+ cells in the spleen. (E): The results showed that CD8+ T cells and NK cells were the main expressers of Ifng, while the expression of Ifnb1 was mainly from the macrophage population. (F): CD8+ T cell population identified by CD8+ T cell signature genes (CD3g CD3e CD3d Cd8a Cd28 Gzmk Ifng Klrg1). (G): Successful identification of macrophage population by macrophage signature genes (Maf, Mafeb, C1qa, C1qb, C1qc, Mgl2, Lyz2, Mrc1). (H): In tumor tissues, WBCTSs up-regulated the expression of genes associated with killing (Klrd1, Gzmf, Tnfrsf9, Tnfrsf4) in tumor-infiltrating CD8+ T cells, and in spleen WBCTSs down-regulated genes associated with killing (Ifng, Klrd1) in CD8+ T cells. (I): In tumor tissues, WBCTSs led to upregulation of Ifnb1-related genes (Ifit1, Oals1, Ifnb1, Isg15, Cxcl9) in macrophages, and in spleens WBCTSs did not lead to upregulation of Ifnb1-related genes in macrophages. (J): Signal exchange between CD8+ T cells and other cells was significantly enhanced after WBCTS in tumor tissues. (K): Significantly enhanced afferent signal intensity of CD8+ T cells after WBCTS. (L): WBCTS enhanced functional signaling pathways associated with T cell receptors, cytokines and antigen presentation. (M): WBCTS enhanced signaling pathways associated with the initiation of natural immune responses in macrophages, such as cytokine production, Toll receptor signaling pathway and other functional signaling pathways.

The CD8+T cell population was identified by CD8+T cell signature genes (CD3g CD3e CD3d Cd8a Cd28 Gzmk Ifng Klrg1) (Fig. 3F), and the macrophage population was successfully identified by macrophage signature genes (Maf, Mafeb, C1qa, C1qb, C1qc, Mgl2, Lyz2, Mrc1) (Fig. 3G). The results showed that CD8+T cells and NK cells were the main expressers of Ifng, whereas the expression of Ifnb1 was mainly from the macrophage population (Fig. 3E). Therefore, the focus of our study was to examine the changes of CD8+T cells and macrophages in tumor tissues and in the spleen before and after exposure to radiation from whole-body CT scans.

In tumor tissues, the proportion of CD8+T cells in tumor tissues as a percentage of CD45+ cells increased from 4.49 % before whole-body CT scanning radiation exposure to 7.15 % after radiation exposure (Fig. 3B), and furthermore, multiple CT scanning radiation up-regulated tumor expression of killing-related genes (Klrd1, Gzmf, Tnfrsf9, Tnfrsf4) in the infiltrating CD8+T cells (Fig. 3H),which may be responsible for the elevated levels of IFN-γ in the tumor tissues (Fig. 2D). We also noted that radiation enhanced functional signaling pathways associated with t-cell receptors, cytokines and antigen presentation (Fig. 3L). Signal communication between CD8+T cells and other cells was significantly enhanced after radiation (Fig. 3J), specifically, the afferent signal strength of CD8+T cells was significantly enhanced (Fig. 3K). In tumor tissues, radiation from multiple CT scans resulted in up-regulation of the expression of Ifnb-related genes (Ifit1, Oals1, Ifnb1, Isg15, Cxcl9) in macrophages (Fig. 3I), which may be responsible for the elevated levels of IFN-β in tumor tissues. We also noted that radiation enhanced signaling pathways associated with the initiation of the natural immune response in macrophages, such as cytokine production, Toll receptor signaling pathway, and other functional signaling pathways (Fig. 3M).

In the spleen, the proportion of CD8+ T cells to CD45+ cells in the spleen was reduced from 6.53 % before radiation of whole-body CT scan to 4.51 % after radiation (Fig. 3D). Further gene expression analysis showed that low-dose irradiation down-regulated the expression of Ifng and killing-related genes (Ifng, Klrd1) in splenic CD8+T cells (Fig. 3H). Unlike tumor tissue, radiation from multiple CT scans in the spleen does not result in upregulation of Ifnb-related gene (Ifit1, Oals1, Ifnb1, Isg15, Cxcl9) expression in macrophages (Fig. 3I).

WBCTSs increased CD8+T cell infiltration in tumor tissues of mice

To further validate the findings of single-cell sequencing, we conducted an experiment involving three mice from both the WBCTSs group and the NC group (n = 3). The mice in the WBCTSs group were exposed to whole-body CT scans, and tumor tissues were collected 24 h after irradiation. We employed multicolor flow cytometry and immunohistochemistry techniques to investigate any alterations in key immune cells within the tumor tissues. The results obtained from multicolor flow cytometry revealed an increase in the proportions of CD3+ and CD8+ T cells among CD45+ T lymphocytes following multiple whole-body CT scanning radiation. Specifically, there was a rise in the proportion of CD8+ T cells within CD45+ T cells from 8.6 % to 14 %, as well as an increase in their proportion among all cells present in the tumor tissue from 1.9 % to 3.9 % (Fig. 4A). Furthermore, stimulation led to an elevation in intracellular IFN-γ+ CD8+ T cell population from 4.9 % to 11.9 % (Fig. 4B). These observed trends align with those identified through single-cell sequencing analysis, thereby providing additional validation for our findings.To further support these observations, we utilized immunohistochemistry analysis to assess changes in the number of CD8+, GZMB+, and FOXP3+ cells within tumor tissues derived from both irradiated and non-irradiated mice subjected to multiple whole-body CT scans at a time interval of 24 h post-irradiation.Our results demonstrated significant increases (P < 0.01) in proportions of both CD8+ cells (Fig. 4C) and GZMB+ cells (Fig. 4D) within tumor tissues subsequent to multiple whole-body CT scanning radiation.

Fig. 4.

WBCTSs increased CD8+T cell infiltration in tumor tissues of mice. (A): We selected 3 mice (n = 3) from each of the WBCTSs and NC groups, and tumor tissues were taken from mice 24 h after WBCTSs. Multi-color flow cytometric analysis (FCA) showed an increase in the proportion of both CD3 and CD8+ T cells among CD45+ T lymphocytes (P < 0.01). (B): FCA in mouse tumor tissues showed a significant increase in the proportion of IFN-γ+ CD8+ T cells after WBCTSs (P < 0.05). (C): Immunohistochemistry in mouse tumor tissues after WBCTSs showed a significant increase in the proportion of CD8+ cells (P < 0.01). (D): Immunohistochemistry after WBCTSs in mouse tumor tissues showed a significant increase in the proportion of GZMB+ cells (P < 0.01).

WBCTSs reduces the proportion of CD8+T cells in the spleen and blood of mice

To further observe the changes of lymphocytes in blood and spleen after whole-body CT scanning radiation, we selected three mice (n = 3) each from the WBCTSs group and the NC group, and administered whole-body CT scanning radiation to the mice in the WBCTSs group, and we took the blood and spleens of the mice 24 h after irradiation. Multicolor flow cytometry was used to detect alterations of key immune cells in blood and spleen.

Multicolor flow cytometry analysis of the blood showed that the proportion of CD3+ and CD8+ T lymphocytes to CD45+ cells in the blood of mice decreased after CT scan irradiation, with the proportion of CD8+ T cells to CD45+ T cells in the blood decreasing from 11.8 % to 9.5 %, (P < 0.01) (Fig. 5A).

Fig. 5.

WBCTSs reduces the proportion of CD8+T cells in the spleen and blood of mice. (A): Blood drawn from mice 24 h after WBCTSs Blood measured blood analyzed by FCA showed a decrease in the percentage of CD3, CD4 and CD8+ T lymphocytes to CD45+ cells in the blood of mice after CT scanning irradiation (P < 0.01). (B): Given that a large number of immune cells accumulate mainly in secondary lymphoid tissues such as the spleen, by FCA, we found that the proportion of CD3, CD4 and CD8+ T cells to CD45+ cells in the spleens of mice also decreased after WBCTSs (P < 0.05).

Given that a large number of immune cells mainly accumulated in secondary lymphoid tissues such as the spleen, by multicolor flow cytometry, we found that the ratio of CD3+, CD4+, and CD8+ T cells to CD45+ cells in the spleens of the mice also decreased (P < 0.05) after CT scan irradiation (Fig. 5B), in which the percentage of CD8+ T cells to CD45+ T cells in the spleens decreased from 11.6 % to 9.6 %, (P < 0.05).

Discussion

In this study, we explored the effects of multiple whole-body computed tomography (CT) scans on a mouse tumor model and further analyzed the specific effects of radiation on the systemic immune system and the tumor immune microenvironment. In line with previous studies [13]. Consistent with this, our results showed that multiple CT scans did not promote tumor progression; instead, a trend of delayed tumor progression was observed.

To gain a deeper understanding of the mechanisms underlying this phenomenon, we used ELISA, flow cytometry, immunohistochemistry, RNA sequencing, and single-cell sequencing to comprehensively analyze the changes in cytokines (IFN-γ and IFN-β), major immune cell ratios, and gene expression. The results showed that after multiple CT scans, the proportion of CD8+ T cells in tumor tissues increased, IFN levels were elevated, and killing-related genes in CD8+ T cells and Ifnb-related genes in macrophages were upregulated. There are several previous studies on the effects of low-dose radiation on tumors, and these studies are consistent with our findings. All of them found that low-dose radiation can improve the immune microenvironment within tumor tissues and promote the immune system to clear tumors [14]. The present study focuses more on the response of multiple whole-body CT scanning radiation to the tumor than the others. Due to ethical constraints, it is difficult to obtain tumor tissues before and after whole-body enhanced CT scans of patients; therefore, changes in the proportion of CD8+ T, cytokines, and genes in patients' tumor tissues cannot be directly detected. We used multiple whole-body CT scanning radiation on mice to observe the changes in the proportion of CD8+T, cytokines and genes in the tumor tissues of mice, but the responses induced by multiple whole-body CT scanning radiation doses in animals may differ from those in humans, which requires us to continue to study in the future.

In addition, previous studies have focused on the effects of low-dose irradiation on the immunity of tumor tissues, while neglecting the potential effects of whole-body low-dose irradiation (e.g., whole-body CT scanning) on the entire immune system. It has been shown that CT scanning radiation induces DNA damage in blood lymphocytes [1,2]. In our study, we found that radiation from multiple whole-body CT scans resulted in a decrease in the proportion of CD8+ T cells in the blood and spleen, a decrease in serum interferon levels, and down-regulation of killing-related genes in CD8+ T cells. These findings are consistent with the results of other studies, for example, other studies have also found that whole-body low-dose radiotherapy can treat inflammatory diseases by inhibiting the release of inflammatory factors [15,16]. Multiple research teams during the COVID-19 pandemic found that low-dose irradiation (LDR) exerted its anti-inflammatory effects by inhibiting the production of inflammatory factors and causing a decrease in lymphocytes in the blood [17,18]. Thus, radiation from multiple whole-body CT scans exerts a suppressive effect on the immune system throughout the body.

The paradox that multiple whole-body CT scans lead to systemic immunosuppression and immune activation in tumor tissues 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 the immunity by initiating damage-associated molecular patterns (DAMPs) [19]. Radiation-induced DNA damage in tumor cells can promote IFN production and activate immunity [[20], [21], [22]]. Thus, immune activation is induced within the tumor tissue, counteracting the immunosuppressive effects caused by radiation damage to immune cells (Fig. 6).

Fig. 6.

Schematic illustration of radiation-activated antitumor immunity from frequent WBCTSs.

We used CT radiation to activate innate immunity by causing damage-associated molecular patterns (DAMP) in tumor cells. We believe that the cell types activated by this activation process are chronologically sequential, with macrophages and DC cells being the first cells activated by radiation, thus producing type I IFN earlier, and then activating CD8T cells and producing type II IFN slightly later, after the antigen is raised by macrophages and DC cells. The activation of CD8T cells and the production of type II IFN after macrophages and DC cells have been raised to antigen is a little later. The process of activation of the immune system by CT radiation in tumor tissues is continuously changing, therefore, the detection of different immune cells and cytokines at different points in time may be very different, and it is also an important direction of research in the future to tell this story clearly and comprehensively in terms of the time, place, and characters.

We speculate that there may be a radiation dose threshold above which the immune activation induced by tumor cell damage fails to counteract the immunosuppressive effects of the radiation itself, leading to systemic immunosuppression and immunosuppression of the tumor tissue. In contrast, when the radiation dose was below this threshold, the immune activation induced by tumor cell injury was sufficient to counteract the immunosuppressive effects of radiation, resulting in systemic immunosuppression and immune activation of tumor tissue observed. We speculate that this radiation dose threshold correlates with the radiosensitivity of the tumor cells themselves; for example, several studies have found, many years ago, that patients with radiosensitive tumors have extremely high remissions after receiving whole-body low-dose radiotherapy [23]. At a later stage, we will conduct further relevant studies to provide a scientific basis for optimizing radiation therapy for tumor patients.

Funding resource

This study was supported by the Special Fund for the Key Laboratory of the Affiliated Tumor Hospital of the First Medical University of Shandong Province, China.

CRediT authorship contribution statement

Jigang Dong: Writing – original draft, Software, Methodology, Investigation, Data curation, Conceptualization. Chengrui Fu: Software, Data curation. Minghao Li: Writing – review & editing, Data curation. Zhongtang Wang: Funding acquisition. Baosheng Li: Writing – review & editing, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.