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. 2026 Feb 18;26:401. doi: 10.1186/s12885-026-15674-3

Early effects of ionizing radiation on molecular modifications and hallmarks of Immunogenic cell death in mouse tumor cells

Ursa Kesar 1,2,#, Bostjan Markelc 1,3,#, Maja Cemazar 1,4, Gregor Sersa 1,5, Tanja Jesenko 1,2,✉,#, Primoz Strojan 1,2,✉,#
PMCID: PMC13019909  PMID: 41709194

Abstract

Background

Ionizing radiation (IR) is known to activate the immune system by releasing damage-associated molecular pattern (DAMP) molecules. To combine the IR with novel immunotherapies it is important to know how different irradiation doses influence the release of DAMPs and induce other molecular changes within the initial 48 h post-irradiation, a period preceding extensive cell death. Therefore, our aim was to determine the activation of most common markers of immunogenic cell death, calreticulin (CRT), HMGB1 and ATP and the changes in the expression of MHC I, MHC II and PD-L1 molecules to evaluate early post-IR events.

Methods

We used three immunologically different mouse tumor cell lines, B16-F10, 4T1 and CT26, which form differently immunogenic tumor models in vivo. They were irradiated in vitro with doses at which 30 (IC30), 50 (IC50) or 70% (IC70) of the cells died. We determined the type of cell death, membrane exposure of CRT, the release of HMGB1 and ATP, as well as the expression of MHC I, MHC II and PD-L1 molecules at different IC doses up to 48 h after irradiation.

Results

Most of the cells were still alive at 24 to 48 h after IR, regardless of dose and cell type. The percentage of apoptotic and necrotic cells slightly increased with increasing radiation dose and with increasing time after IR. The release of HMGB1 and ATP and membrane localization of CRT after IR were increasing with radiation dose and time after IR. Only the CT26 cell line exhibited significant elevation in all three hallmarks of immunogenic cell death (HMGB1, ATP, and CRT). MHC I and PD-L1 expressions increased in all three cell lines and increased with IR dose. There was no significant change in MHC II expression following IR.

Conclusions

Our results indicate that IR induced the most potent immunological changes in CT26 cell line, which forms the most immunogenic tumor model. In general, the effect increased with the IR dose and time after IR, with the most significant changes observed 48 h post-IR. Our results highlight the onset of immunological changes in initial 48 h post-irradiation, a period preceding extensive cell death, which were cell type and IR dose dependent, indicating the importance of tumor type and its associated microenvironment in the potential systemic activation of the anti-tumor immune response after IR in vivo and in patients.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12885-026-15674-3.

Keywords: Ionizing radiation, Immune response, Immunogenic cell death, MHC i, MHC II, PD-L1, Calreticulin, HMGB1, ATP

Background

Ionizing irradiation (IR) can trigger a strong antitumor immune response by affecting almost all steps in the cycle of cancer immunity [1–3]. These effects are manifested in increased antigen presentation and release, stimulation of immune cell activation, increased density of tumor infiltrating lymphocytes, facilitated recognition of tumor cells by T cells, and enhanced antitumor effects [4–8]. However, IR represents a double-edged sword, as it can also promote immunosuppression due to extreme lymphocytes’ radiosensitivity and by increasing the presence of immunosuppressive cells in the tumor microenvironment [9–12].

The type of cell death can be crucial for the immunostimulatory effects of IR. Immunogenic cell death (ICD) is a mode of cell death that effectively stimulates the adaptive immune response against neoantigens released by dying or dead tumor cells [13]. The involved molecules are known as damage-associated molecular patterns (DAMPs), which serve as danger signals and increase the immunogenicity of dying cells by being expressed as “eat me” signals on the cell surface where they are recognized by antigen presenting cells (APCs) [14–17]. These include the exposure of calreticulin (CRT) on the cell membrane, the extracellular release of high mobility group box 1 (HMGB1) and adenosine triphosphate (ATP), as well as the expression of heat shock proteins (HSP) 70 and 90 [18].

Under homeostatic conditions, CRT is localized in the endoplasmic reticulum, where it serves as a molecular chaperone and regulates calcium homeostasis [19]. In addition, it is responsible for the loading of cellular antigens onto MHC I molecules, regulation of gene expression, cell proliferation, cell adhesion and invasion [19–21]. Under stress conditions, it is expressed on the cell membrane, where it serves as a costimulatory “eat me” signal for various immune cells, mainly APCs, but can also be released into the extracellular space [20–22].

HMGB1 is a non-histone, chromatin-associated protein that plays a key role in the pathogenesis of hematopoietic malignancies [23]. It also plays a role as an extracellular inflammatory cytokine [23]. It is normally found intracellularly, in the nucleus, where it is bound to DNA and is involved in the regulation of transcription, DNA replication and repair, telomere maintenance and nucleosome assembly [23, 24].

The ATP molecule is involved in various cellular metabolic functions and intracellular responses. During ICD, ATP is released from the cells via the autophagy mechanism by active exocytosis in ATP-containing vesicles [25–27]. Extracellular ATP is a potent chemoattractant and plays a role as a “find me” signal for dendritic cell precursors and macrophages, thus contributing to the immune response [22, 28]. This occurs through the binding of ATP to the purinergic P2Y2 receptor on APCs and their precursors (e.g. myeloid cells), allowing them to differentiate into mature dendritic cells [22, 29]. If no ATP is present in the microenvironment of dying tumor cells or if purinergic receptors are absent, ICD does not occur [22, 29]. Additional mechanisms of immunosuppression include the expression of immune checkpoints, the triggering of immunosuppressive immune subunits, the loss of immunogenic tumor antigens and reduced antigen presentation, making such tumors unrecognizable to the immune system [30, 31].

The process of T cell activation involves the presentation of antigens via major histocompatibility complexes (MHC) I and II on APCs to the T cell receptor (TCR) on naïve T cells [31]. Cytotoxic T cells (CD8+) recognize cancer cells via MHC I complexes that present their own (endogenous) antigens [32, 33]. After recognition, the CD8 + T cells kill the cancer cells [32, 33]. However, MHC II complexes usually present external (exogenous) antigens to CD4 + T helper cells, which play a key role in the activation of CD8 + T cells [33, 34]. Although MHC II is expressed on APCs (dendritic cells, B cells and macrophages), it can also be expressed by some tumors [35, 36]. The expression of MHC II on tumors can improve recognition by the immune system, which plays an important role in therapies, and is associated with improved prognosis in various cancers [34]. Loss of antigen presentation itself is a common and important mechanism that tumor cells use to evade the immune system [33, 37]. This can also lead to tumor cells becoming “invisible” to TCR, and consequently they cannot be killed by CD8 + T cells [33]. Reduced MHC I expression has been described in 40–90% of human tumors, and loss of effective antigen presentation via the MHC I complex is associated with poor clinical outcome in most human cancers [30, 37–39].

Other important proteins that maintain immune homeostasis are programmed cell death receptor 1 (PD-1) and programmed death-ligand (PD-L1), as their interaction prevents the hyperactivation of immune cells and thus the development of autoimmune diseases [40, 41].

Although IR has been shown to trigger ICD and other immunostimulatory changes, there is evidence that its clinical effect is a highly selective event which does not always occur [42]. The optimal doses and time frame of induced changes that effectively activate the immune system in various cancer types remain unclear. Therefore, we investigated the release of DAMPs involved in ICD (ATP, CRT, HMGB1) and immunologically important cell surface receptors (MHC I, MHC II, PD-L1) within the initial 48 h post-irradiation, a period preceding extensive cell death. We investigated the changes induced in three different murine cell lines (B16-F10, 4T1, CT26) at three different radiation doses (IC30, IC50 and IC70).

Materials and methods

Experimental design

The study was designed to evaluate the early immunological effects of irradiation at time points preceding extensive radiation-induced cell death. Three murine tumor cell lines (B16-F10, 4T1, and CT26) were selected because they are well-established and widely used preclinical cancer models that give rise to tumors with distinct immunological characteristics in vivo. The in vitro experiments were performed to characterize early radiation-induced immunological changes under controlled conditions and to provide supportive mechanistic data that could facilitate interpretation of the in vivo findings. First, the doubling times of the individual cell lines were determined, as this parameter provides an essential basis for comparing and interpreting irradiation-induced changes related to cell death (Fig. 1). Radiation-induced cell death is closely linked to cell cycle progression, as irradiated cells often undergo several rounds of division before dying, predominantly through mitotic catastrophe. Therefore, knowledge of the intrinsic proliferation rates of the different cell lines is critical for accurate interpretation of post-irradiation effects. Then, the relative irradiation doses reducing cell survival by 30% (IC30), 50% (IC50) and 70% (IC70) were determined via clonogenic assay and irradiation with 2, 4, 6–8 Gy single doses. These doses were selected because they cover the entire dose range that induces a decrease in clonogenic survival from 0% to almost 100%, thus enabling the precise evaluation of IC30, IC50 and IC70 doses in all three cell lines. These relative doses were then used for evaluation of cell death and immunological changes 24 and 48 h after irradiation (ATP and HMGB1 release and expression of calreticulin (CRT), MHC I, MHC II and PD-L1).

Fig. 1.

Fig. 1

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Experimental design with procedures being performed before and after irradiation. (1) Determination of cell doubling time via proliferation assay, (2) determination of irradiation doses via clonogenic assay (IC30, IC50, IC70) and (3) Determination of immunological changes after irradiation with IC30, IC50 and IC70 single doses: 3.1 ATP determination, 3.2 HMGB1 determination and 3.3 cell death and surface marker determination. Created in BioRender

Cell lines

All cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The 4T1 murine mammary carcinoma cells and CT26 murine colorectal carcinoma cells were cultured in Advanced RPMI-1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), whereas B16-F10 murine melanoma cells were cultured in Advanced DMEM (Gibco). Media were supplemented with 5% (v/v) fetal bovine serum (FBS, Gibco), 10 mM L-glutamine (GlutaMAX, 100 x, Gibco), and penicillin-streptomycin (100 x, Sigma-Aldrich, Merck, Darmstadt, Germany). All cells were grown in a humidified incubator with 5% CO2 at 37 °C. The cells were routinely tested for mycoplasma infection by MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, Basel, Switzerland) and were mycoplasma negative.

Proliferation assay

Cells were seeded on a 96-well plates (VWR International) at density 500 cells per well in 100 µL of cell culture media. Cells were incubated at 37 °C in a 5% CO2 humidified incubator until measurements. Cell survival was measured with a Presto Blue viability assay (Thermo Fisher Scientific) 2, 24, 48 and 72 h after they were seeded. At the designated time points Presto Blue (10 µL/well) was added to the cells, and 1 h thereafter, fluorescence intensity was measured with a Cytation 1 cell imaging multimode reader (BioTek, Agilent Technologies, Inc., Santa Clara, CA, USA). The doubling time of each cell line was determined from the growth curves using the Gompertz growth model in GraphPad Prism 10 (GraphPad Software).

Clonogenic assay

Cells were seeded at different densities (Supplementary table S1) in 6-well plates (VWR International) based on the IR dose. Seeded cells were incubated for three hours at 37 °C in a 5% CO2 humidified incubator in order to attach to the bottom of the plate before IR. Each 6-well plate with seeded cells was irradiated with a single dose of 2, 4, 6–8 Gy with X-ray generator CP225 X-RAY (Gulmay, Byfleet, United Kingdom) operated at the voltage 200 kV, electrical current 9.2 mA and average dose rate 1.73 Gy/min. After irradiation cells were incubated for six days at 37 °C in a 5% CO2 humidified incubator until colony counting. After six days the colonies were stained with crystal violet solution in 99.8% methanol (Sigma-Aldrich) and colonies containing at least 50 cells were counted. Plating efficiency (PE - number of counted colonies divided by the number of seeded cells) and Surviving fraction (SF – treated group PE divided by the average PE of the Control group) were calculated and the SF curve was plotted in GraphPad Prism (GraphPad Software, San Diego, CA, USA). We used linear-quadratic model and dose-response curves to determine the α/β ratio and calculated the inhibitory doses reducing cell survival by 30% (IC30), 50% (IC50) and 70% (IC70) using the following equation:

graphic file with name d33e497.gif

where α and β are parameters describing the cell’s radiosensitivity. α represents the linear part of the survival curve, and β represents the quadratic component of cell killing [43].

Determination of extracellular ATP

For ATP determination, one day before the experiment 2 × 105 cells were seeded in T25 flasks (VWR International) in 3 mL of cell culture medium. Before IR the next day, the cell culture medium was changed with fresh cell culture medium, then the cells were irradiated with IC30 (B16-F10 and 4T1 = 2.5 Gy; CT26 = 3.4 Gy), IC50 (B16-F10 and 4T1 = 3.4 Gy; CT26 = 4.4 Gy) and IC70 (B16-F10 and 4T1 = 4.5 Gy; CT26 = 5.5 Gy) doses. Cells were then incubated at 37 °C in a 5% CO2 humidified incubator and 250 µL of cell culture medium was collected at the timepoints 0, 24 and 48 h. The samples were then centrifuged (300×g, 4 °C, 5 min) and 10 µL of the supernatant was used for ATP determination using ATP Determination Kit (Invitrogen, Thermo Fisher Scientific) assay following the manufacturer’s instructions. The resulting luminescence was measured with Cytation 1 (BioTek), and the final ATP concentration was calculated from the obtained standard curve.

Determination of HMGB1 release

For the determination of HMGB1 concentration, one day before the experiment 2 × 105 cells were seeded in T25 flasks (VWR International) in 3 mL of cell culture medium. Before IR the next day, the cell culture medium was changed with fresh cell culture medium, then the cells were irradiated with IC30 (B16-F10 and 4T1 = 2.5 Gy; CT26 = 3.4 Gy), IC50 (B16-F10 and 4T1 = 3.4 Gy; CT26 = 4.4 Gy) and IC70 (B16-F10 and 4T1 = 4.5 Gy; CT26 = 5.5 Gy) doses. Cells were then incubated at 37 °C in a 5% CO2 humidified incubator and cell culture medium was collected in Protein LoBind tubes (Eppendorf, Hamburg, Germany) at the timepoints 24 and 48 h after IR. The samples were then centrifuged (500 x g, 4 °C, 5 min), and cell supernatants were collected in new Protein LoBind tubes and frozen at −80 °C until further analysis. At every collection time point, the total volume of cell culture media was measured, and cells were counted. The HMGB1 concentration in undiluted cell culture medium was detected with an HMGB1 Detection Kit (Chondrex, Inc., Woodinville, WA, USA), following the manufacturer’s instructions. The final HMGB1 concentration was normalized to 1 × 106 cells.

Determination of cell death and cell surface markers by flow cytometry

For the determination of the type of cell death and the expression of cell surface markers MHC I, MHC II, PD-L1 and CRT after IR, one day before the experiment 5 × 105 cells (for timepoint 24 h) or 2 × 105 cells were seeded (for timepoint 48 h) in T25 flasks (VWR International) in 3 mL of cell culture medium. The cells were irradiated with IC30 (B16-F10 and 4T1 = 2.5 Gy; CT26 = 3.4 Gy), IC50 (B16-F10 and 4T1 = 3.4 Gy; CT26 = 4.4 Gy) and IC70 (B16-F10 and 4T1 = 4.5 Gy; CT26 = 5.5 Gy) doses and at the designated timepoints the cells were dissociated with Versene Solution (Gibco), centrifuged, washed once with 1x PBS, and counted to obtain 1 × 106 cells per staining. Cells were stained for above listed markers with antibodies (Supplementary table S2) and with e780 fixable viability dye (Thermo Fisher Scientific) as previously described [44]. The stained cells were analyzed with BD FACS Symphony A3 flow cytometer and the acquired data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR, USA) as described previously [44]. Cells were first gated based on forward and side scatter, followed by the exclusion of doublets. Then, the cells were gated for apoptosis (e780−, Annexin V+ population), necrosis (e780+, Annexin V+ population) and live cells (e780−, Annexin V− population) (Supplementary Fig. S1). From the live cell gate (e780−, Annexin V− population), the relative frequency (% of cells) and median fluorescence intensity (MFI) were determined for each of the following markers: CRT, MHC I, MHC II, PD-L1 (Supplementary Fig. S1). In all cases, the obtained median fluorescence intensity (MFI) values were normalized to the MFI values of the control (Ctrl) group within each independent experiment. The MFI for each measured parameter of the cells from the live cell gate (e780−, Annexin V− population) was used for the calculations.

Statistics

The values in this study represent the arithmetic mean (AM) ± standard error of the mean (SEM) as all analyzed parameters were continuous variables obtained from independent replicates with comparable variance and without pronounced skewness. Comparison of means was performed with one-way ANOVA followed by Dunnett’s multiple comparisons test. Differences were considered significant at *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Sample size (n) represents an independent biological experiment performed on a separate occasion using independently prepared cell cultures and is presented in figure legends. When technical replicates were included within a biological experiment, their values were first averaged, and the resulting mean value was used as a single data point for statistical analysis to ensure appropriate assessment of biological variability.

All graphical presentations and statistical analyses were made in GraphPad Prism 10 (GraphPad Software).

Results

Cell proliferation and survival after IR

All three cell lines had similar doubling times at around 16 to 18 h and no statistically significant differences among them were observed (Fig. 1A), therefore equal number of cell divisions could be expected at the measured time points. We showed that even though the cell line CT26 was more sensitive to IR at higher doses in comparison to B16-F10 and 4T1 cell lines, the IC30 (B16-F10 and 4T1 = 2.5 Gy; CT26 = 3.4 Gy), IC50 (B16-F10 and 4T1 = 3.4 Gy; CT26 = 4.4 Gy) and IC70 (B16-F10 and 4T1 = 4.5 Gy; CT26 = 5.5 Gy) values that were later used in this study ranged from 2.5 to 5.5 Gy where cell lines had comparable surviving fraction (Fig. 2B, Supplementary table S3). We also demonstrated that most of the cells were live after IR at both timepoints irrespective of dose and cell type (Fig. 2C-E, Supplementary Fig. S2A-F). The percentage of live cells ranged from 90 to 95% in the B16-F10 and CT26 cell lines (Fig. 2C, E), and from 60 to 85% in the 4T1 cell line (Fig. 2D). The percentage of apoptotic and necrotic cells was increasing with increasing IR dose and at longer times after IR in all three cell lines (Fig. 2C-E). The 4T1 cell line exhibited a higher sensitivity to cell manipulation, which explains the higher percentage of dead cells in the control and treatment groups (Fig. 2D).

Fig. 2.

Fig. 2

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Cell proliferation, cytotoxicity and percent of live, apoptotic and necrotic cells after IR (A) Doubling time of all three cell lines. n = 3. B Cytotoxicity after IR. n = 3. C Cell populations present in B16-F10 cell line samples 24 and 48 h after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses. D Cell populations present in 4T1 cell line samples 24 and 48 h after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses. E Cell populations present in CT26 cell line samples 24 and 48 h after IR with IC30 (3.4 Gy), IC50 (4.4 Gy), and IC70 (5.5 Gy) doses. The values are presented as the AM ± SEM. *p < 0.05. n = 4. Due to greater graph clarity the statistics for C-E are shown in the Supplementary Fig. S2

IR induced CRT translocation in B16-F10 and 4T1 cell lines

After IR, the increase in the percentage of CRT positive cells was observed in the B16-F10 cell line 24 h after treatment, but only at the IC70 (4.5 Gy) dose, and 48 h after treatment at all doses of IR (Fig. 3A, D). The presence of CRT on the membrane was increased relative to the control cells only 48 h post-treatment at IC50 (3.4 Gy) and IC70 (4.5 Gy) in the B16-F10 cell line. In the 4T1 cell line, CRT translocation to the cell membrane was detected at both time points, which was evident in the percentage of CRT positive cells and in CRT MFI (Fig. 3B, E). In the CT26 cell line, a significant increase in CRT translocation was observed only 48 h after IR at IC50 (3.4 Gy) and IC70 (4.5 Gy) doses (Fig. 3C, F).

Fig. 3.

Fig. 3

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CRT translocation after IR. Percentage of CRT+ cells 24 and 48 h after IR in the (A) B16-F10 cell line after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses, (B) 4T1 cell line after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses and (C) CT26 cell line after IR with IC30 (3.4 Gy), IC50 (4.4 Gy), and IC70 (5.5 Gy) doses. CRT expression 24 and 48 h after IR in the (D) B16-F10 cell line after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses, (E) 4T1 cell line after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses and (F) CT26 cell line after IR with IC30 (3.4 Gy), IC50 (4.4 Gy), and IC70 (5.5 Gy) doses. The values are presented as the AM ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 4

Release of HMGB1 was significant only in CT26 cell line 48 h after IR

To further explore the effect of IR on markers of ICD, we measured the release of HMGB1 with ELISA in cell culture medium collected at 24 and 48 h after IR. At 24 h after IR there was no statistically significant increase of extracellular HMGB1 in any of the cell lines (Fig. 4A-C). In the B16-F10 cell line, there was no increase at 48 h after IR (Fig. 4A), whereas there was a slight, but statistically non-significant increase in the 4T1 cell line (Fig. 4B). The only cell line exhibiting statistically significant increased extracellular HBGB1 was the CT26 cel line at 48 h after IR (Fig. 4C).

Fig. 4.

Fig. 4

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HMGB1 release after IR. HMGB1 release 24 h and 48 h after IR in the (A) B16-F10 cell line after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses, (B) 4T1 cell line IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses and (C) CT26 cell line after IR with IC30 (3.4 Gy), IC50 (4.4 Gy), and IC70 (5.5 Gy) doses. The values are presented as the AM ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, n = 2

Release of ATP increased with time after IR

Analysis of ATP release after IR showed that the lowest amount of ATP (compared to the control cells) was released by the B16-F10 cell line, irrespective of the radiation dose (Fig. 5A, Supplementary Fig. S3A-C). A statistically significant increase in the released ATP was detected in the 4T1 cell line at the IC70 dose (4.5 Gy) 48 h after IR (Fig. 5B, Supplementary Fig. S3D-F). The highest levels of released ATP were detected in the CT26 cell line, where it was statistically significant at the IC70 dose (5.5 Gy) 24 h after IR and at all three tested doses 48 h after IR (Fig. 5C, Supplementary Fig. S3G-I).

Fig. 5.

Fig. 5

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ATP release after IR. ATP release release 24 h and 48 h after IR in the (A) B16-F10 cell line after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses, (B) 4T1 cell line IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses and (C) CT26 cell line after IR with IC30 (3.4 Gy), IC50 (4.4 Gy), and IC70 (5.5 Gy) doses. The values are presented as the AM ± SEM. *p < 0.05, n = 3

IR increased MHC I expression in all cell lines

The MHC I expression predominantly increased after IR in all three cell lines at 24 and 48 h after IR (Fig. 6A-F). In the B16-F10 and CT26 cell lines, the increase in MHC I expression was statistically significant at both timepoint at all three IR doses (Fig. 6A, C, D, F). Differently, in the 4T1 cell line the MHC I expression was statistically increased only at the IC50 (3.4 Gy) and IC70 (4.5 Gy) doses of IR at both timepoints, but not after IC30 dose (2.5 Gy) (Fig. 6B, E). Further, in the B16-F10 cell line with low basal levels of MHC I positive cells, the increase in the MHC I expression was also readily visible also in the percentage of MHC I cells (Fig. 6D), whereas in the 4T1 and CT26 cell lines, with high basal levels of MHC I positive cells, the increase in MHC I expression was mainly evident in the increased MFI of MHC I (Fig. 6E, F).

Fig. 6.

Fig. 6

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MHC I expression after IR presented as a percentage of MFI relative to Ctrl. MHC I expression in the (A) B16-F10 cell line 24 h and 48 h after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses. MHC I expression in the (B) 4T1 cell line 24 h and 48 h after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses. MHC I expression in the (C) CT26 cell line 24 h and 48 h after IR with IC30 (3.4 Gy), IC50 (4.4 Gy), and IC70 (5.5 Gy) doses. Percentage of MHC I positive cells in the (D) B16-F10 cell line 24 h and 48 h after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses. MHC I expression in the (E) 4T1 cell line 24 h and 48 h after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses. MHC I expression in the (F) CT26 cell line 24 h and 48 h after IR with IC30 (3.4 Gy), IC50 (4.4 Gy), and IC70 (5.5 Gy) doses. The values are presented as the AM ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 4

IR did not induce an increase in MHC II expression

In general, the expression of MHC II did not change in any of the three cell lines used (Fig. 7A-C). However, we observed a minor decrease in the percentage of cells expressing MHC II at IC30 dose (2.5 Gy) after 48 h in the B16-F10 cell line and at IC70 dose (4.5 Gy) 24 h after IR of 4T1 (Fig. 7D-F).

Fig. 7.

Fig. 7

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MHC II expression after IR presented as a percentage of MFI relative to Ctrl. MHC II expression in the (A) B16-F10 cell line 24 h and 48 h after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses. MHC II expression in the (B) 4T1 cell line 24 h and 48 h after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses. MHC II expression in the (C) CT26 cell line 24 h and 48 h after IR with IC30 (3.4 Gy), IC50 (4.4 Gy), and IC70 (5.5 Gy) doses. Percentage of MHC II positive cells in the (D) B16-F10 cell line 24 h and 48 h after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses. MHC II expression in the (E) 4T1 cell line 24 h and 48 h after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses. MHC II expression in the (F) CT26 cell line 24 h and 48 h after IR with IC30 (3.4 Gy), IC50 (4.4 Gy), and IC70 (5.5 Gy) doses. Percent of MHC II positive cells in the (F) CT26 cell line 24 h and 48 h after IR. The values are presented as the AM ± SEM. **p < 0.01, n = 4

IR increased PD-L1 expression in all cell lines

The expression of PD-L1 after IR was increased in all three cell lines (Fig. 8A-F). This was most evident in the B16-F10 cell line, where the PD-L1 expression was increased at all timepoints and at all doses (Fig. 8A), whereas in the 4T1 cell lines, all three doses increased PD-L1 expression only at 24 h after IR, and the IC70 dose (4.5 Gy) of IR at 48 h after IR (Fig. 8B), and in the CT26 cell line, the expression of PD-L1 was only increased at 48 h after IR after all three doses (3.4, 4.4 and 5.5 Gy) of IR used, but not at 24 h after IR (Fig. 8C). A similar trend was observed in the percentage of cells expressing PD-L1 with the only difference being that there was no statistical increase in the percentage of cells expressing PD-L1 at 48 h after the IC70 dose (4.5 Gy) of IR in the 4T1 cell line (Fig. 8D-F).

Fig. 8.

Fig. 8

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PD-L1 expression after IR presented as a percentage of MFI relative to Ctrl. PD-L1 expression in the (A) B16-F10 cell line 24 h and 48 h after IR after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses. PD-L1 expression in the (B) 4T1 cell line 24 h and 48 h after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses. PD-L1 expression in the (C) CT26 cell line 24 h and 48 h after IR with IC30 (3.4 Gy), IC50 (4.4 Gy), and IC70 (5.5 Gy) doses. Percentage of PD-L1 positive cells in the (D) B16-F10 cell line 24 h and 48 h after IR after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses. PD-L1 expression in the (E) 4T1 cell line 24 h and 48 h after IR with IC30 (2.5 Gy), IC50 (3.4 Gy), and IC70 (4.5 Gy) doses. PD-L1 expression in the (F) CT26 cell line 24 h and 48 h after IR with IC30 (3.4 Gy), IC50 (4.4 Gy), and IC70 (5.5 Gy) doses. The values are presented as the AM ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 4

Discussion

In this study, we highlighted the onset of the release of DAMPs and expression of immunologically important cell surface receptors in initial 48 h post-irradiation, a period preceding extensive cell death. We demonstrated that IR induced most potent immunological changes in CT26 cell line. In general, the effect increased with the dose and time after IR, with most of the significant changes observed 48 h post-IR. Furthermore, we demonstrate that there are intrinsic differences between the cell lines in the immunologically relevant changes after IR, which could be further potentiated in the in vivo tumor models due to more complex tumor microenvironment.

We first determined IC values for each cell line, which were used for all further experiments, and we chose three IC values: IC30 (B16-F10 and 4T1 = 2.5 Gy; CT26 = 3.4 Gy), IC50 (B16-F10 and 4T1 = 3.4 Gy; CT26 = 4.4 Gy), and IC70 (B16-F10 and 4T1 = 4.5 Gy; CT26 = 5.5 Gy), to determine the response of cells at low doses of IR as well as at high doses. Using the determined IC values, we then investigated how IR affected cell death in early time points by determining the occurrence of apoptosis and necrosis, which are among the most frequently described types of cell death after radiotherapy (RT) [45–47]. The majority of cells were alive after IR at both time points, regardless of dose and cell type. The predominant type of cell death after IR was necrosis, which occurred at 24 and 48 h after IR in all three cell lines. In all three cell lines, the number of dead cells increased with increasing radiation dose and prolongation of the time interval after IR. With these experiments, we confirmed that the majority of cells were still alive in the time interval of our experiment, therefore the measured changes were induced prior to massive cell death after IR. The similar doubling times and expected number of cell divisions for all three cell lines also enabled more direct comparisons of the effects after IR. We identified the 4T1 as the cell line that was most sensitive to manipulation, as the percentage of dead cells was higher than in the other two cell lines at both time points and in the control groups.

Next, we investigated whether the features of ICD appear at early time points after IR. We focused on the determination of CRT, HMGB1 and ATP, which are among the most important DAMP molecules expressed or released in case of ICD. First, we determined the translocation of CRT from the ER. We observed that CRT was translocated to the cell membrane in all three cell lines and at all doses, with the exception of the IC30 dose (3.4 Gy) in the CT26 cell line. These results are consistent with those of Gameiro et al. [48], who demonstrated the same in carcinomas using higher doses (10 Gy) and longer monitoring of translocation (72 h). In contrast to CRT, statistically significant HMGB1 release after IR was only observed in the CT26 cell line 48 h after IR with higher doses. The role of HMGB1 in RT is mixed: doses of radiation that are not cytotoxic leave the cell membrane intact, and HMGB1 that remains in the cell promotes tumor cell survival and radioresistance by repairing damaged DNA and inducing autophagy [49, 50]. Radiation doses, which are cytotoxic, cause fragmentation of the cell membrane, which releases HMGB1 from the cell and contributes to a complex immune response. This is supported by the studies of Gameiro et al. [48] which showed that HMGB1 is released both at high doses (100 Gy), which are too high to be clinically relevant, and at lower, clinically relevant doses (10 Gy). Xu et al. and Li et al. [51, 52] concluded from their results that elevated levels of nuclear and cytoplasmic HMGB1 could predict a worse prognosis in the early stages of cancer. Thus, in our study, we showed that, HMGB1 is not released at lower IR doses in the first 48 h after IR, as these time points precede the delayed massive cell death via mitotic catastrophe. This release became significant at higher doses at 48 h only in CT26 cell line. Our results point to the importance of dose selection in order to induce ICD in the tumor cells and prevent the accumulation of HMGB1 in the nucleus and cytoplasm that results in continuous proliferation instead of cell death and HMGB1 release.

ATP release also varied depending on the type of cell line. After IR, the B16-F10 cell line released the least ATP, followed by the 4T1 and CT26 cell lines, with the latter releasing a significant amount of ATP at 48 h after IR. The fact that higher doses cause a greater release of ATP than lower doses has already been shown by Gameiro et al. [48].

Although it is known that higher doses cause a higher release of DAMP molecules, it is not known exactly which doses and irradiation regimens activate the immune system most effectively in different cancer types [53–56]. In this study, we found that IR induces the release of ICD-inducing DAMPs at higher doses, i.e. IC70: it appears that only this dose is high enough to induce the amount of damage sufficient to induce markers of immunogenic cell death at early time points in the first 48 h after IR. All three ICD-inducing DAMPs were significantly released only in the CT26 cell line. This cell line forms tumors with the hottest immunological status in mice among the three tested in our study (CT26, B16-F10 and 4T1) [57, 58]. Interestingly, our results show that this cell line also released the most ICD-inducing DAMPs as soon as 48 h after IR among the three tested cell lines.

In addition to the release of DAMPs, several other molecules are important for enhancing antigenicity. The most common receptors include MHC I, MHC II and PD-L1 [30, 34, 35, 59]. The effect of IR on these molecules is better understood, but more research is needed in this area to determine which irradiation scenarios induce the most beneficial changes in these molecules. In our study, we have shown that IR increases the expression of MHC I molecules in all three cell lines and at all doses of radiation, except in the 4T1 cell line at the IC30 dose. This confirms greater dependence of the expression of MHC I molecules in the cell lines on irradiation dose than reported in previous studies [2, 36, 60]. In contrast to MHC I, no significant changes were observed in MHC II expression within the groups, except for minor decrease in B16-F10 at IC30 (2.5 Gy) 48 after IR and in 4T1 at IC70 (4.5 Gy) 24 h after IR. These results are not in line with those of Lhuillier et al. [61]. They demonstrated that IR could expose neoantigens via the MHC II complex and effectively activate the antitumor immune response by activating cytotoxic T cells and T helper cells in a poorly immunogenic 4T1 model in vivo [61]. In general, little is known about the effect of IR on MHC II molecules. Published reports suggest there are different responses of MHC II molecules to IR, which is also the case in our study. Furthermore, we have shown that IR induces an increase in the expression of PD-L1 molecules in all cell lines and doses. This is consistent with the results of previous studies [62–64] indicating on the immunosuppressive action of IR.

Study limitations

In our study, we examined the behavior of several damage-associated molecular patterns (DAMPs) and cell surface receptors following irradiation (IR) at different time points, but only up to 48 h after treatment. This is a limitation of the study, as we observed that a second wave of cell death begins toward the end of the observation period. Therefore, it is possible that additional or more pronounced changes in the analyzed markers occur beyond 48 h post-irradiation. Although we demonstrated that IR induces features of immunogenic cell death (ICD) in vitro, these findings do not address whether similar effects occur in vivo. Further studies in appropriate in vivo models are required to confirm whether IR induces ICD and to define the relevant dose ranges. Additionally, as this work was limited to in vitro experiments, we cannot determine whether IR-induced ICD is sufficient to elicit a functional anti-tumor immune response. Our results also indicate that the ICD-inducing potential of IR is cell line dependent; therefore, experiments using relevant human cell lines should be performed to validate the translational potential of our findings. Nevertheless, this study provides important new insights into the early induction of ICD-related signals following irradiation and lays the foundation for future in vivo validation in immunocompetent mouse tumor models.

Conclusion

Our results highlight the onset of immunological changes in the first 48 h after irradiation, a period that precedes extensive cell death. In general, the effect increased with IR dose and time after IR, with the most pronounced changes observed 48 h after IR. Thus, we can conclude that higher doses are needed for the induction of ICD at early timepoints after IR. Irradiation induced the most profound immunological changes in the CT26 cell line among the three tested ones. Thus, we demonstrated the intrinsic differences in the immunological changes between the cell lines following IR, which could be further potentiated in the in vivo tumor models due to the more complex tumor microenvironment. Therefore, further studies in mice are needed to confirm our observations on the immunological changes and the induction of an immune response in the tumors.

Supplementary Information

Supplementary Material 1. (765.3KB, docx)

Acknowledgements

We would like to thank Teja Valant for her technical support in managing the laboratory’s material supply.

Generative AI statement

The author(s) declare that no Generative AI was used in the creation of this manuscript.

Abbreviations

AM

Arithmetic mean

ANXA1

Annexin A1

APC

Antigen presenting cell

ATP

Adenosine triphosphate

CD8+

Cytotoxic T cells

CD4+

Helper T cells

CRT

Calreticulin

DAMP

Damage-associated molecular pattern

ER

Endoplasmic reticulum

HMGB1

High mobility group box 1

HSP

Heat shock protein

IC

Inhibitory concentration

IR

Ionizing radiation

MDSC

Myeloid-derived suppressor cells

MFI

Median fluorescence intensity

MHC I

Major histocompatibility complex I

MHC II

Major histocompatibility complex II

PD-1

Programmed cell death protein 1

PD-L1

Programmed cell death ligand 1

RO(N)S

Reactive oxygen (and nitrogen) species

RT

Radiotherapy

SEM

Standard error of the mean

TCR

T cell receptor

TIL

Tumor-infiltrating lymphocytes

Authors’ contributions

Conceptualization: G.S., U.K., P.S., T.J., B.M.; data curation: U.K., B.M.; formal analysis: T.J., B.M., U.K.; methodology: T.J., B.M.; investigation: U.K., B.M.; visualization: U.K., B.M., T.J.; supervision: G.S., M.C., T.J. P.S.; writing – original draft: U.K.; writing – review and editing: T.J., B.M., G.S., M.C., P.S. All authors read and approved the final manuscript.

Funding

This research was funded by the Slovenian Research and Innovation Agency (ARIS) under the postgraduate program, and programs P3-0003 and P3-0307.

Data availability

All relevant data supporting the findings of this study are available in the article and its additional files.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Ursa Kesar and Bostjan Markelc contributed equally to this work and share first authorship.

Tanja Jesenko and Primoz Strojan share senior and last authorship.

Contributor Information

Tanja Jesenko, Email: tjesenko@onko-i.si.

Primoz Strojan, Email: pstrojan@onko-i.si.

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Associated Data

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Supplementary Materials

Supplementary Material 1. (765.3KB, docx)

Data Availability Statement

All relevant data supporting the findings of this study are available in the article and its additional files.

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