Research ArticleExperimental Studies
Open Access

Beneficial Effects of Combining an Immune Checkpoint Inhibitor With Proton Radiation, OXi4503, or Hyperthermia in a Murine Solid Tumor Model

PRIYANSHU MANOJKUMAR SINHA, CHARLEMAGNE ASONGANYI FOLEFAC, MATEUSZ SITARZ, PATRICIA SWITTON NIELSEN, PERNILLE ELMING and MICHAEL ROBERT HORSMAN
Anticancer Research June 2026, 46 (6) 3005-3021; DOI: https://doi.org/10.21873/anticanres.18176

Abstract

Background/Aim: This pre-clinical study investigated the combination of various treatments with a checkpoint blocker in an immunologically unresponsive tumor.

Materials and Methods: Experiments were performed using C3H mammary carcinoma mice, where tumors of various sizes (44-467 mm3) were treated with either proton radiation (up to 20 Gy), OXi4503 (4×50 mg/kg), or hyperthermia (42.5°C for 60 min), with or without anti-CTLA-4 (4×10 mg/kg). The endpoint was the time to reach 1,000 mm3. Mechanistic studies focused on CD4/CD8 expression, assessed in histological sections up to 10 days after irradiation.

Results: The median time for untreated tumors to reach 1,000 mm3 was unaffected by anti-CTLA-4 alone. Proton radiation, OXi4503, or hyperthermia alone increased the tumor growth time at all tumor sizes when compared to untreated tumors. Combining proton radiation or OXi4503 with anti-CTLA-4 resulted in additional tumor inhibition, which was more pronounced in smaller tumors (44-131 mm3). Anti-CTLA-4 had no additional effect on tumor response to hyperthermia regardless of tumor size at treatment. Expression of CD4/CD8 levels decreased 1 day after irradiation before recovering to pre-treatment levels on day 5.

Conclusion: Overall, combining anti-CTLA-4 with either proton radiation or OXi4503, but not with hyperthermia, enhanced growth inhibition of C3H mammary carcinoma compared to that seen with each treatment alone, suggesting potential clinical relevance.

Keywords:

Introduction

Immunotherapy, using inhibitors of the immune checkpoint proteins cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed death 1 (PD-1), and programmed death 1 ligand (PD-L1), has gained significant interest as a transformative approach in cancer treatment. Treatment with inhibitors of these proteins has now shown remarkable success in certain cancer types, especially melanoma (1) and lung (2), where durable survival benefits have been reported. However, many patients are unresponsive to checkpoint inhibitor treatment (3, 4). This has led to substantial interest in finding alternative approaches that could be combined with checkpoint inhibitors to enhance response (5-7).

Conventional radiotherapy is one clinically relevant approach with the potential to enhance treatment with checkpoint inhibitors. Following irradiation of tumors, cellular DNA damage is produced, which leads to cell killing (8). This could increase the level of antigens that can then be identified by the antigen-presenting cells that prime and activate T-cells, thereby enhancing the immune response. Interestingly, radiation can also increase tumor cell death indirectly via the induction of vascular damage (9). This vascular damage also has the potential to increase cell death by increasing the extravasation of T-cells from the tumor blood supply to the tumor cells, and even trap T-cells in the tumor. Preclinical studies have shown that combining radiation with checkpoint inhibitors clearly improves tumor treatment response (10-12). However, most studies have focused on the use of x-ray photons, gamma rays, or electron irradiation for investigations in this context. The application of proton radiation might be superior to other traditionally applied radiation modalities, as it has a more precise dose delivery to the target (13). Recent studies have shown superior anti-tumor immunity over x-ray photons when combined with immune checkpoint inhibition therapy (14, 15). In fact, our recent study using a non-immunogenic C3H mammary carcinoma model, in which proton radiation and anti-CTLA-4 treatment were combined, significantly enhanced tumor growth delay and tumor control, especially at higher proton radiation doses (16).

Interestingly, the small-molecule vascular disrupting agent (VDA) combretastatin A-1 phosphate (CA1P/OXi4503) also had bifunctional activity against tumors (17). OXi4503 is an analogue of the leading VDA combretastatin A-4 phosphate (CA4P), and its principal mode of action involves disruption of the cytoskeleton of proliferating endothelial cells, causing a change in endothelial cell shape which can lead to thrombus formation and vascular collapse (18, 19). However, unlike CA4P, OXi4503 is metabolized by oxidative enzymes to form an orthoquinone that is directly cytotoxic to tumor cells (20). For radiation, cell death primarily occurs through a direct effect, with vascular damage playing a secondary role, while for OXi4503, vascular damage is the dominant mechanism and direct cell killing is secondary. In a previous study, we also combined OXi4503, and its parent compound CA4P, with checkpoint inhibitors and demonstrated a significantly enhanced anti-tumor response (21).

A third therapy that has bifunctional anti-tumor activity via direct cell killing and indirect vascular damage is hyperthermia (22, 23). However, unlike radiation or OXi4503, at high temperatures there are no apparent primary or secondary mechanisms; hyperthermia kills cells equally, whether directly or indirectly. Several pre-clinical murine tumor studies have shown the potential benefit of combining hyperthermia with checkpoint inhibitors (24-26).

This leads to several unresolved issues when combining other therapies with checkpoint inhibitors in immunologically unresponsive tumors, including whether the level of damage induced by the additional treatment influences the outcome, whether the mechanism of cell killing plays a role, and whether tumor size at the time of treatment affects the immunotherapy response. To address these points, we combined proton radiation, OXi4503, or hyperthermia with anti-CTLA-4 in our C3H mouse mammary carcinoma using a range of tumor sizes.

Materials and Methods

Animal and tumor model. The experiments were performed using 10-14-week-old male and female CDF1 mice (Janvier Labs., Le Genest-Saint-Isle, France), with the tumor model being a murine C3H mammary carcinoma subcutaneously implanted in the right rear foot of the CDF1 mice. The process of derivation and maintenance of this tumor model has been described previously (27). Tumors were routinely passaged in the flank region of mice, and experimental tumors were produced by excising these flank tumors, finely chopping them with a scalpel and scissors under sterile conditions, and inoculating this tumor material (approximately 5-10 μl per mouse) into the right rear foot of CDF1 mice. These were then kept in cages (up to four mice per cage) with food and water ad libitum, and a stable circadian rhythm was secured with a light/dark interval of 12 h. Different experiments were then initiated when the tumors reached a desired size range (44-467 mm3), the volume being determined by the product of all orthogonal diameters (length, width, and height) multiplied by π/6. Randomization of tumor-bearing mice into different groups was difficult, as tumors grew to the desirable size at different rates. Hence, some selection was inevitable to ensure that mice with tumors that reached the desired size on the same day (Day 0) were redistributed into different treatment groups. All in vivo experiments were conducted as per the animal welfare policy of Aarhus University (http://dyrefaciliteter.au.dk), and with the approval of the Danish Animal Experiments Inspectorate (Licence number: 2021-15-0201-01008) and were performed in accordance with the ARRIVE guidelines.

Proton irradiation. Proton radiation of 5-20 Gy was given as single doses on day 0 as described previously (28). Radiation to the feet of tumor-bearing mice was performed without anesthesia, and hence the mice had to be restrained in lucite jigs, with the tumor-bearing feet exposed and attached to the jig with tape. For mice bearing smaller tumors (44-131 mm3), leg fixation was performed using histoacrylic glue, where a small drop was applied to the uppermost part of the leg. The leg was then firmly compressed against the jig with tape for a 5-min period, after which the tape was loosened and left for 10 min prior to any treatment, as described previously (28, 29). Proton radiation was then performed with a Varian AC250 system (Probeam, Varian-Siemens Healthinners, Palo Alto, CA, USA) at the Danish Centre of Particle Therapy (Aarhus, Denmark) in the experimental gantry room using a fixed horizontal beam line. The whole setup consisted of a water phantom containing temperature-controlled deionized water (25°C). A plastic plate was placed on top of the water, allowing three mice to be placed simultaneously with the tumor-bearing leg submerged in temperature-controlled deionized water. The legs were positioned 7 cm from the edge of the water bath, which is the center of the proton spread-out Bragg peak (SOBP). A single fraction of pencil-scanning proton beams was delivered perpendicular to the phantom wall with a field size of (2.5 x 10 cm2) and energies of 83-107 MeV. The bodies of the mice were placed outside the planning treatment volume (PTV) and were also protected by a brass block (2 cm) placed between the mice and the water phantom’s edge. Treatment plans were created using the ECLIPSE Treatment Planning System (Varian-Siemens Healthinners) and were tested prior to the experiments using Gafchromic EBT-3 films. The field measurements confirmed uniform dose distribution within the PTV at 7 cm depth (middle of the SOBP) with less than 1% deviation outside the PTV and acceptable backscattered dose to the mouse body.

Hyperthermia treatment. Hyperthermia treatment (42.5°C for 60 min on day 0) was achieved using a temperature-controlled circulating water bath. Non-anesthetized mice were restrained in lucite jigs, and the tumor-bearing legs were submerged in the circulating water bath (type TE 623; Heto, Birkerød, Denmark) as described above in the proton radiation section. The temperature of the water bath was adjusted 0.2°C higher than the desired tumor temperature, since this was the difference observed in previous studies (30, 31). The temperature measurements were calibrated against a certified mercury thermometer.

Drug treatments. OXi4503 was generously supplied by Mateon Pharmaceuticals (South San Francisco, CA, USA) and freshly prepared in sterile saline (0.9% NaCl) before each experiment, prior to intraperitoneal (i.p.) injection in mice at a uniform volume of 0.02 ml per gram of mouse body weight. Anti-CTLA-4 antibody [InvivoMab anti-mouse CTLA-4 (CD152), clone 9H10] was purchased from NordicBioSite/BioXCells (Kristiansand, Norway), prepared fresh in sterile phosphate-buffered saline (PBS) before each experiment and administered in a similar manner as OXi4503. The OXi4503 dose given was 50 mg/kg, which had previously been shown to have an optimal effect when administered on days 0, 3, 7, and 10 (32). For anti-CTLA-4 treatment, a standard dose of 10 mg/kg was administered four times during a two-week period, a regimen commonly used previously (10, 33) but slightly modified for consistency across different treatment groups. It was administered on days 0, 3, 7, and 10 when given alone, or on days 1, 4, 8, and 11 when combined with other treatments.

Tumor response. Once the treatments were initiated, C3H mammary carcinomas were measured until they reached a maximum permitted size of 1200 mm3 or the mice were monitored until day 90 in case of tumor growth arrest or tumor control. At either of these points, the mice were terminated by cervical dislocation. Since treatments were initiated at different tumor sizes, time to reach 1,000 mm3 was selected as the end point for the current study. Tumor sizes in the experiment ranged from 44-467 mm3 and were categorized into four groups based on a previous study (34). The subsequent group classification was the following: (i) 50 mm3 (44-73 mm3), (ii) 100 mm3 (84-131 mm3), (iii) 200 mm3 (151-257 mm3), and finally (iv) 400 mm3 (308-467 mm3). The tumor growth delay across different treatment group was assessed using the time to event Kaplan-Meier curves. In situations where tumors were controlled or tumors failed to reach the predefined endpoint of 1,000 mm3, the mice were right-censored at day 90 for the Kaplan-Meier analysis. Mouse body weight was also recorded every week to determine toxicity, and if mice became sick or lost more than 20% of their body weight, the mice were terminated for ethical reasons.

CD4 and CD8 immunohistochemistry. To determine immune cell infiltration after a single 20 Gy dose of irradiation, tumors of 59-170 mm3 were selected for analysis. Mice were sacrificed by cervical dislocation either as untreated controls or at 1, 5, or 10 days post-irradiation. The whole process of immunohistochemistry, scanning, and quantification was described previously (21). Essentially, immediately after sacrifice, the tumors were excised and placed in formalin for a maximum of 48 h and then paraffin embedded. Three serial 3-μm sections of the formalin-fixed, paraffin-embedded tissue were cut from each tumor, with at least 250 μm between each section, mounted on Superfrost Plus Slides (Thermo Fisher Scientific, Portsmouth, NH, USA) and dried at 60°C for 1 h. The expression of CD4 and CD8 in the tissue sections was then determined by automated image analysis as previously described (17).

Data and statistical analysis. All data analyses were performed using GraphPad Prism 10 (Version 3, CA, USA). Results from the tumor size studies were visualized using Kaplan-Meier analysis, while the radiation dose response and immunohistochemistry analysis were presented as box-and-whisker plots. Statistical analysis of the Kaplan-Meier curves was conducted using the Log-rank (Mantel-Cox) test, while the Wilcoxon-Mann-Whitney U-test was used for both the dose-response and immune infiltration analyses. A p-Value of <0.05 was considered statistically significant for all tests.

Results

Anti-CTLA-4 as a single therapy agent. Figure 1 shows the effect of anti-CTLA-4 alone on the growth of this C3H mammary carcinoma when compared to untreated control tumors. The median time at which control tumors reached 1,000 mm3 naturally depended on the initial tumor size, with the time in days being 15, 9, 6.5, and 4 days for 50, 100, 200, and 400 mm3 tumors, respectively. Anti-CTLA-4, administered 4 times during a two-week period, had absolutely no effect on growth for tumors treated at 200 or 400 mm3, with the growth delay values exactly the same as for controls. For the smaller tumors, there was a slight effect, with the time to reaching 1,000 mm3 being 18 and 9.5 days for 50 and 100 mm3 tumors, respectively. However, neither of these values was significantly different from controls.

Figure 1.
Figure 1.

Kaplan-Meier response curves showing the percentage of tumors below 1,000 mm3 over 90 days. The data are based on the time taken for different-sized C3H mammary carcinomas to reach 1,000 mm3. Results are for untreated tumors (dashed line with open symbol) or after treatment with 4×10 mg/kg anti-CTLA-4 (solid line with closed symbol). The response curves are based on 8-11 mice per group. Tumor sizes at treatment are indicated in each panel as follows: (a) 50 mm3; (b) 100 mm3; (c) 200 mm3; (d) 400 mm3.

Anti-CTLA-4 combined with single dose proton radiation. The effect of proton radiation alone and in combination with anti-CTLA-4 on tumor growth is shown in Figure 2. The median times taken to reach the growth endpoint following irradiation were 42.5, 28.5, 23.25, and 15.5 days for 50, 100, 200, and 400 mm3, respectively. Combining radiation and anti-CTLA-4 had no significant effect on tumor growth in 400 mm3 tumors compared to radiation alone, the growth delay being 16.5 days. However, as tumor size decreased, the enhancement by anti-CTLA-4 increased: for 200 mm3 tumors, the delay was 27 days, though this was not significant. For 100 mm3 tumors, there was a significant increase to 41 days (p=0.0004; log-rank test; n=8). The greatest effect was seen in the smallest tumors (50 mm3), where more than 50% of mice treated with proton radiation and anti-CTLA-4 showed tumor control 90 days after treatment, and this was significantly greater than that seen with radiation alone (p=0.0079; log-rank test; n=8). Since a significant enhancement of proton radiation (20 Gy) response was first observed when 100 mm3 tumors were also treated with anti-CTLA-4, we attempted to determine the minimal radiation dose necessary for this enhancement, and the results are shown in Figure 3. Here, 100 mm3 tumor-bearing mice were treated with different radiation doses (0-20 Gy) together with anti-CTLA-4. While a modest effect was noted at the 10 Gy dose (p=0.043; Mann-Whitney U-test; n=9), no additional benefit was seen by combining radiation doses of 5 or 15 Gy with anti-CTLA-4. However, a significant improvement was observed at 20 Gy (p=0.0026; Mann-Whitney U-test; n=9).

Figure 2.
Figure 2.

Kaplan-Meier response curves showing the percentage of tumors below 1,000 mm3 over 90 days. The data are based on the time taken for different-sized C3H mammary carcinomas to reach 1,000 mm3. Results are for tumors irradiated with 20 Gy protons (dashed line with open symbol) or after treatment with protons and 4×10 mg/kg anti-CTLA-4 (solid line with closed symbol). The response curves are based on 7-9 mice per group. Tumor sizes at treatment are indicated in each panel as follows: (a) 50 mm3; (b) 100 mm3; (c) 200 mm3; (d) 400 mm3.

Figure 3.
Figure 3.

Effect of different proton radiation doses plus anti-CTLA-4 on the response of 100 mm3 C3H mammary carcinomas. Response was the time taken for tumors to reach 1,000 mm3, and the results are shown as box-and-whisker plots for proton radiation alone (open) and proton radiation with 4×10 mg/kg anti-CTLA-4 [closed (grey color)]. The response is based on 8-11 mice per group. Statistical differences are indicated by *p<0.05 and **p<0.01, following a Mann-Whitney U-test. Symbols for proton radiation alone (open circles) and proton radiation plus anti-CTLA-4 (grey circles) outside the box-and-whisker plots denote outliers in the different groups under investigation.

Anti-CTLA-4 combined with OXi4503 or hyperthermia. Figure 4 shows the effect of treating tumors with OXi4503 plus anti-CTLA-4. As with radiation, there were clear tumor-size-dependent effects on the response to OXi4503 alone and when combined with anti-CTLA-4. The respective median times for tumors to reach 1,000 mm3 for OXi4503 and OXi4503 with anti-CTLA-4 were 16 and 16.5 days for 400 mm3 tumors, 18 and 21 days for 200 mm3 tumors (p=0.0024; log-rank test; n=17-18), and 20 and 27 days for 100 mm3 tumors (p=0.0178; log-rank test; n=9-10). For the 50 mm3 tumors, the median value for OXi4503 was 26 days, and as with radiation, the combination of OXi4503 with anti-CTLA-4 resulted in greater than 50% tumor control, thus showing a significant increase in median time (p<0.0001; log-rank test; n=9-10). The effect of hyperthermia with anti-CTLA-4 on tumor growth is summarised in Figure 5. Treating tumors at 50, 100, 200, and 400 mm3 resulted in median growth times of 18.5, 15, 10.75, and 7 days for hyperthermia alone, and 19.5, 16.5, 11, and 7.5 days for hyperthermia with anti-CTLA-4, respectively, and none of these differences were significant.

Figure 4.
Figure 4.

Kaplan-Meier response curves showing the percentage of tumors below 1,000 mm3 over 90 days. The data are based on the time taken for different-sized C3H mammary carcinomas to reach 1,000 mm3. Results are for mice receiving 4×50 mg/kg OXi4503 (dashed line with open symbol) or after treatment with OXi4503 and 4×10 mg/kg anti-CTLA-4 (solid line with closed symbol). The response curves are based on 8-18 mice per group. Tumor sizes at treatment are indicated in each panel as follows: (a) 50 mm3; (b) 100 mm3; (c) 200 mm3; (d) 400 mm3.

Figure 5.
Figure 5.

Kaplan-Meier response curves showing the percentage of tumors below 1,000 mm3 over 90 days. The data are based on the time taken for different-sized C3H mammary carcinomas to reach 1,000 mm3. Results are for tumors treated with hyperthermia at 42.5°C for 60 min (dashed line with open symbol) or after treatment with hyperthermia and 4×10 mg/kg anti-CTLA-4 (solid line with closed symbol). The response curves are based on 9-17 mice per group. Tumor sizes at treatment are indicated in each panel as follows: (a) 50 mm3; (b) 100 mm3; (c) 200 mm3; (d) 400 mm3.

Immune cell infiltration analysis after proton radiation. To investigate whether our treatments could affect the immune infiltration of CD4+ and CD8+ T-cells in the C3H mammary carcinoma, we irradiated 100 mm3 tumors with protons (20 Gy) and excised tumors for analysis at different time points (days 1, 5, and 10) after irradiation. A typical histological section showing the cell distributions shown in Figure 6a, while the counts obtained for all tumor sections and the quantification of T-cell expression on days 1, 5, and 10 are shown in Figures 6b and c. The average tumor size for control tumors was 92.4 mm3 (range=79-105 mm3; n=5), while the average sizes of the irradiated tumors at day 1, day 5, and day 10 were comparable at 104 mm3 (range=75-170 mm3; n=6), 109 mm3 (range=92-141 mm3; n=6), and 75 mm3 (range=59-84 mm3; n=6), respectively. For CD4+ T-cell infiltration, there was a significant reduction in levels 1 day after irradiation (p=0.0026; Mann-Whitney U test; n=5-6) for the whole tumor, including the core and periphery. At day 5, the levels had recovered, and although they were higher than controls, this increase was not significant. No additional changes were seen on day 10. Similar observations were made for CD8+ infiltration, with a significant reduction 1 day after irradiation (p=0.0087; Mann-Whitney U-test; n=5-6) for the whole tumor and periphery, followed by recovery on days 5 and 10 post-irradiation. However, in the core of the tumor, although CD8+ T-cells were reduced at day 1, this was not significant. On day 5, the CD8+ T-cells had returned to control levels, but by day 10 they had significantly increased (p=0.03; Mann-Whitney U-test; n=5-6).

Figure 6.
Figure 6.

The effect of proton radiation (20 Gy) on T-cell infiltration within the C3H mammary carcinoma. (a) Representative histological tumor section showing the distribution of T-cells (yellow) in the tumor periphery (green outline) and core (red outline). The bottom two sets of panels show quantitative assessments of (b) CD4+ and (c) CD8+ T-cell expression as a function of time after 20 Gy proton irradiation. The box-and-whisker plots represent the relative total percentage level of immune cells in the whole section (left), tumor periphery (middle), and tumor core (right). The data are based on 5-6 tumors per group. Values significantly different from controls are indicated by *p<0.05 and **p<0.01 (Mann-Whitney U-test).

Discussion

Although immunotherapy using checkpoint inhibitors has shown unprecedented success in certain types of cancer (1, 2), most solid tumors are poor candidates for immune checkpoint inhibition therapy. This can be attributed to a lack of tumor antigens due to low mutation load, the high presence of immunosuppressive cells, and the absence of infiltrating CD4+ and CD8+ T cells, or the presence of inactive ones, within the tumor microenvironment (35). Combining checkpoint inhibitors with other conventional cancer therapies that could increase the presence of damage-associated molecular patterns (DAMPs) and other tumor antigens, which would then activate infiltrating T cells to cause immune-mediated cytotoxic effects. We investigated this using three different clinically applicable therapeutic methods. These were radiation, the vascular disrupting agent OXi4503, and hyperthermia, all of which can kill cells in solid tumors via a direct cell-killing mechanism, as well as by indirect effects following the induction of vascular damage, although the primary and secondary mechanisms of cell killing differ among these three modalities. The checkpoint inhibitor selected was anti-CTLA-4, since this has been shown to strongly enhance T-cell activity and regulatory T-cell blocking function (36), as well as having previously been reported to have the best anti-tumor response when combined with proton radiation (16) and OXi4503 (21) in our C3H mammary carcinoma model. This tumor model was also considered ideal because other results obtained with it have significantly impacted clinical treatments (27, 37). It is also considered non-immunogenic when grown in CDF1 mice, because tumor take is typically 100% and tumors grow reasonably fast, reaching around 200 mm3 within 2-3 weeks after challenge.

Many tumor characteristics change as a function of tumor size, and this is especially true for the tumor microenvironment. This was clearly illustrated in our C3H mammary carcinoma, where it was shown that the necrotic fraction, partial pressure of oxygen, and hypoxic cell number all increased with tumor size up to around 400 mm3, after which they began to plateau (34). Consequently, we decided to investigate the tumor response for the different combination treatments using tumors that were 50, 100, 200, or 400 mm3 in size. When compared to untreated control tumors, anti-CTLA-4 alone had no effect on tumor response regardless of tumor size (Figure 1), and this was consistent with our previous studies with anti-CTLA-4 in this C3H mammary carcinoma using 200 mm3 tumors (16, 21). It also supports the suggestion that this tumor model is non-immunogenic.

The potential of x-ray photon radiation to enhance the immune response has been widely investigated (11, 12, 38-40), but more recent studies suggest the potential for proton radiation to also induce an immune response (14, 15, 41, 42). Because of their mass, protons have more favorable ballistic properties than x-ray photons, resulting in more precise dose delivery to the target site, compared with photon radiation (43). Furthermore, there have been suggestions that photons and protons induce different damage patterns when applied at comparable doses (13). Due to the heavier mass and charge of protons, there is a higher ionization density, resulting in clustered energy deposition and higher levels of reactive oxygen species (ROS) for the same photon radiation dose, giving rise to more complex DNA damage that is even more difficult to repair (44). These remarkable differences in damage patterns between protons and photons could result in different inflammatory and immune responses (44). In our current study, we investigated the combination of proton radiation (20 Gy) with anti-CTLA-4 (4×10 mg/kg). The tumor growth delay outcome (Figure 2) showed that this single 20 Gy dose alone caused a significant inhibitory effect at all tumor sizes, and when combined with anti-CTLA-4, a substantial enhancement was observed in smaller tumors (50 and 100 mm3), but not in larger tumors (200 and 400 mm3). The effect of combining radiation and anti-CTLA-4 in 50 mm3 tumors was actually very significant, with >50% of the mice under investigation showing tumor control at day 90. To see whether there was a radiation-dose-dependent effect, we irradiated 100 mm3 tumors with a range of proton doses (5-20 Gy) with/without anti-CTLA-4. From the results in Figure 3, a clear dose-dependent proton radiation response was observed in tumor growth inhibition. When the proton radiation was combined with anti-CTLA-4, even though a minor effect was observed at 10 Gy, likely due to an outlier in the 10 Gy proton-radiation-only group, there was no further benefit from combining anti-CTLA-4 with radiation doses below 15 Gy. A more substantial and significant enhancement was only evident at the 20 Gy dose level. This is entirely in agreement with our previous study (16), although larger 200 mm3 tumors were used. Despite our current study showing a small enhancement of radiation response by anti-CTLA-4 in 200 mm3 tumors, unlike that earlier study, the enhancement we obtained was not significant. Why this should be is unclear, but it might reflect the selection of different endpoints (time to 600 mm3 in the former study and 1,000 mm3 in our current one). Overall, our radiation studies suggest that a certain level of damage is required before an effect of anti-CTLA-4 can be seen and that tumor size plays an important role.

We also investigated the combination of the vascular disrupting agent OXi4503 (four intraperitoneal injections of 50 mg/kg over two weeks) with anti-CTLA-4 (4×10 mg/kg also over a two-week period). OXi4503, also called combretastatin A-1 phosphate (CA1P), is a structural analogue of the initial combretastatin compound, combretastatin A-4 phosphate (CA4P). Both OXi4503 and CA4P induce vascular damage by targeting dividing endothelial cells (18, 19), but unlike CA4P, OXi4503 also undergoes oxidative activation to an orthoquinone metabolite that has a direct cytotoxic effect (20), thus giving it greater anti-tumor effects (17, 32). Previously, this combination had been shown to enhance growth inhibition in a C3H mammary carcinoma tumor, but only one tumor size (200 mm3) was used (21). OXi4503 inhibited tumor growth at all tumor sizes used in the current study. When combined with anti-CTLA-4, there was no enhanced effect in 400 mm3 tumors; however, a small but significant effect was observed in 200 mm3 tumors. In 50 mm3 and 100 mm3 tumors, the enhancement was even more pronounced, with 56% of 50 mm3 and 22% of 100 mm3 tumors being controlled at day 90. Our previous study with OXi4503 and checkpoint inhibitors, using a tumor size of 200 mm3 and time to 1,000 mm3 as the endpoint, reported a similar enhancement by anti-CTLA-4 when using the same 50 mg/kg OXi4503 treatment. However, no improvement was seen when lower OXi4503 doses (5-25 mg/kg) were applied (21), again strongly suggesting that a certain level of damage is needed before the anti-CTLA-4 treatment can work.

The final therapy that we combined with anti-CTLA-4 was hyperthermia. Here, we heated tumors once at 42.5°C for 60 min, followed by the usual 4×10 mg/kg injections of anti-CTLA-4. Hyperthermia has both direct and indirect effects on tumors, although the applied temperature plays a role in determining which mechanism predominates. The direct effects include denaturation of cellular proteins, resulting in direct cell cytotoxicity, while the indirect effects include disruption of blood flow and inhibition of DNA repair mechanisms, resulting in indirect cytotoxic effects (23, 45). We selected 42.5°C, as it is a clinically relevant threshold temperature for hyperthermia that has been shown to induce both direct and indirect effects in tumors (45). In our study, there was no significant difference between the hyperthermia alone and hyperthermia plus anti-CTLA-4 combination groups for any of the tumor sizes under investigation (Figure 5). Two other pre-clinical studies investigated the combination of hyperthermia with anti-CTLA-4 in murine tumors (26, 46). Ando et al. reported a similar outcome to that in our study, where no significant tumor growth delay was seen when hyperthermia (42°C for 60 min) was combined with anti-CTLA-4 using an EL-4 lymphoma tumor model (46). However, significant primary tumor growth inhibition was reported by Ibuki et al. when treating a 4T1 breast tumor model with anti-CTLA-4 and 42.5°C for 20 min (26). In the former study, the heating was applied using a temperature-controlled water bath and the tumors at treatment were approximately 100 mm3 in size, while in the latter study, treatment was initiated when tumors reached 65 mm3 and radiofrequency was used to heat the primary tumor. Just like our C3H mammary carcinoma, the 4T1 tumor model is also considered to be an immune non-responder (47), so the discrepancy in the results between our investigation and that by Ibuki et al. could be attributed to the heating method, with radiofrequency perhaps also inducing an additional non-thermal effect, although what this could be is unclear. Nevertheless, the fact that radiofrequency heating had an effect is a positive finding because it is a more clinically relevant method for effectively delivering hyperthermia.

To shed some light on the possible underlying mechanisms for tumor responses, we performed histological assessment of immune cell infiltration, as the presence of immune cells within the tumor microenvironment strongly determines tumor responses to checkpoint inhibition therapy in combination with other treatment modalities (48). For this, we assessed the effect of proton radiation (20 Gy) on the infiltration pattern of CD4 and CD8 cells in approximately 100 mm3 tumors. In general, the patterns of CD4+ and CD8+ T-cell infiltration following irradiation were similar, with a significant reduction 1 day after treating tumors with 20 Gy and a return to control levels by days 5-10. There was a suggestion that the levels were higher than those found in controls at these later time points, but this increase was not significant except for CD8+ cells in the tumor core at day 10 after radiation.

Among the immune cell population, T-lymphocytes are known to be the most radiosensitive (49). Therefore, direct cytotoxic effects of radiation on these infiltrating immune cells could possibly explain the decline in T-cell expression on day 1 post-irradiation. In fact, transitory lymphopenia, a temporary decrease in immune cells, is one of the common immunological side effects of radiotherapy (50, 51). The later increase in immune cell infiltration may also be the consequence of damaged tumor cells generating tumor-associated antigens, pro-inflammatory cytokines, and other danger signals, facilitating T-cell infiltration into tumors (38, 52). Strong immune responses, especially CD8+ T-cell responses from higher-dose irradiation, are not an uncommon phenomenon (53-56). One study using B16-F10 melanoma tumors compared a single 15 Gy dose to fractionated doses of 5×3 Gy (57). They showed that the former led to increased antigen-presenting cells and CD8+ T cells in tumor-draining lymph nodes, as well as higher IFN-γ production and CD8+ T cell-mediated lysis (57). Another study found that tumors resistant to fractionated radiation treatments could be made responsive with a single high dose of 20 Gy, provided CD8+ T cells are present (38). These results are consistent with the trends in our study, where CD4+ and CD8+ T-cell infiltration after 20 Gy irradiation initially dropped, but then recovered and rose over a period of 10 days. This suggests that high-dose radiation promotes the recruitment and activity of these immune cells. In our previous study using the same tumor and animal model, the vascular disrupting agent OXi4503 also showed a rapid decrease in T-cell populations within the tumor post-treatment, which was followed by a partial recovery and then another decline (21). Generally, this delayed but robust infiltration of immune cells supports the theory that treatments that induce substantial tumor damage, especially high-dose radiation, induce a potent and prolonged immune response, most likely through mechanisms involving improved antigen presentation and cytokine production (53).

Conclusion

In conclusion, our current study with the C3H mammary carcinoma found that combining anti-CTLA-4 with proton radiation or the vascular disrupting agent OXi4503 resulted in enhanced anti-tumor effects, and that this was largely a dose- and tumor-size-dependent phenomenon. Since only one tumor model was used for our investigations, there are still uncertainties as to whether these findings are consistent across other solid and metastatic tumor models. Furthermore, there is also uncertainty as to whether we would see additional enhancements if anti-PD-1 and anti-PD-L1 were also used in addition to anti-CTLA-4, even though there were suggestions of a benefit from both preclinical and clinical studies (25, 58, 59). Additional studies with more models and possible trimodal treatments are clearly warranted.

Acknowledgements

The Authors thank Dorthe Grand, Maria Arnoldus Bech, Marianne Kristiansen, Jeanette Bæhr Georgsen, and Mogens Jøns Johannsen for their excellent help in animal care, handling, and other technical support.

Footnotes

  • Authors’ Contributions

    Conceptualization: P.M.S., C.A.F., and M.R.H. Data acquisition: P.M.S., C.A.F., P.B.E., and P.S.N. Data analysis and interpretation: P.M.S. and C.A.F. Statistical analysis and drafting of the manuscript: P.M.S., and C.A.F. Administrative, technical, and material support: M.K.S., P.S.N., and M.R.H. All authors contributed to the concept and design of the experiments and to critical revision of the manuscript for important intellectual content.

  • Conflicts of Interest

    The Authors declare no conflicts of interest.

  • Funding

    This study has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 955625 (Hyperboost; https://hyperboost.eu/) and the Danish Cancer Society (Grant number R40-A2022-11-S2).

  • Artificial Intelligence (AI) Disclosure

    No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

  • Received March 9, 2026.
  • Revision received April 19, 2026.
  • Accepted April 22, 2026.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

References

  1. ↵
    1. Callahan MK,
    2. Postow MA,
    3. Wolchok JD
    : CTLA-4 and PD-1 pathway blockade: combinations in the clinic. Front Oncol 4: 385, 2015. DOI: 10.3389/fonc.2014.00385
    OpenUrlCrossRefPubMed
  2. ↵
    1. Antonia SJ,
    2. Villegas A,
    3. Daniel D,
    4. Vicente D,
    5. Murakami S,
    6. Hui R,
    7. Yokoi T,
    8. Chiappori A,
    9. Lee KH,
    10. de Wit M,
    11. Cho BC,
    12. Bourhaba M,
    13. Quantin X,
    14. Tokito T,
    15. Mekhail T,
    16. Planchard D,
    17. Kim YC,
    18. Karapetis CS,
    19. Hiret S,
    20. Ostoros G,
    21. Kubota K,
    22. Gray JE,
    23. Paz-Ares L,
    24. de Castro Carpeño J,
    25. Wadsworth C,
    26. Melillo G,
    27. Jiang H,
    28. Huang Y,
    29. Dennis PA,
    30. Özgüroğlu M, PACIFIC Investigators
    : Durvalumab after chemoradiotherapy in stage III non–small-cell lung cancer. N Engl J Med 377(20): 1919-1929, 2017. DOI: 10.1056/NEJMoa1709937
    OpenUrlCrossRefPubMed
  3. ↵
    1. Postow MA,
    2. Callahan MK,
    3. Wolchok JD
    : Immune checkpoint blockade in cancer therapy. J Clin Oncol 33(17): 1974-1982, 2015. DOI: 10.1200/JCO.2014.59.4358
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Syn NL,
    2. Teng MWL,
    3. Mok TSK,
    4. Soo RA
    : De-novo and acquired resistance to immune checkpoint targeting. Lancet Oncol 18(12): e731-e741, 2017. DOI: 10.1016/S1470-2045(17)30607-1
    OpenUrlCrossRefPubMed
  5. ↵
    1. Kamyan D,
    2. Elemam NM,
    3. Alkhayyal N,
    4. Talaat IM,
    5. Bendardaf R
    : Unlocking new frontiers in breast cancer: the role of the tumor microenvironment, cutting-edge therapies, and immunotherapy. Anticancer Res 45(7): 2729-2747, 2025. DOI: 10.21873/anticanres.17643
    OpenUrlAbstract/FREE Full Text
    1. Wu J,
    2. Lagunas AM,
    3. Crowe DL
    : DNA Ligase 4 inhibition sensitizes prostate cancer to immune checkpoint blockade in vivo. Anticancer Res 45(3): 883-896, 2025. DOI: 10.21873/anticanres.17476
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Kure M,
    2. Tazawa H,
    3. Demiya K,
    4. Kondo H,
    5. Mochizuki Y,
    6. Komatsubara T,
    7. Yoshida A,
    8. Uotani K,
    9. Hasei J,
    10. Fujiwara T,
    11. Kunisada T,
    12. Urata Y,
    13. Kagawa S,
    14. Ozaki T,
    15. Fujiwara T
    : P53-armed oncolytic virotherapy promotes the efficacy of PD1 blockade in murine osteosarcoma tumors. Anticancer Res 46(1): 69-84, 2026. DOI: 10.21873/anticanres.17924
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Joiner MC and
    2. van der Kogel AJ
    1. Vens C,
    2. Koritzinsky M,
    3. Wouters BG
    : Irradiation-induced damage and the DNA damage response. In: Basic Clinical Radiobiology. Fifth Edition. Joiner MC and van der Kogel AJ (eds.). Boca Raton, FL, CRC Press/Taylor & Francis Group, pp 9-20, 2018.
  9. ↵
    1. Park HJ,
    2. Griffin RJ,
    3. Hui S,
    4. Levitt SH,
    5. Song CW
    : Radiation-induced vascular damage in tumors: implications of vascular damage in ablative hypofractionated radiotherapy (SBRT and SRS). Radiat Res 177(3): 311-327, 2012. DOI: 10.1667/rr2773.1
    OpenUrlCrossRefPubMed
  10. ↵
    1. Demaria S,
    2. Kawashima N,
    3. Yang AM,
    4. Devitt ML,
    5. Babb JS,
    6. Allison JP,
    7. Formenti SC
    : Immune-mediated inhibition of metastases after treatment with local radiation and CTLA-4 blockade in a mouse model of breast cancer. Clin Cancer Res 11(2 Pt 1): 728-734, 2005.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Dewan MZ,
    2. Galloway AE,
    3. Kawashima N,
    4. Dewyngaert JK,
    5. Babb JS,
    6. Formenti SC,
    7. Demaria S
    : Fractionated but not single-dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody. Clin Cancer Res 15(17): 5379-5388, 2009. DOI: 10.1158/1078-0432.CCR-09-0265
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Dovedi SJ,
    2. Adlard AL,
    3. Lipowska-Bhalla G,
    4. McKenna C,
    5. Jones S,
    6. Cheadle EJ,
    7. Stratford IJ,
    8. Poon E,
    9. Morrow M,
    10. Stewart R,
    11. Jones H,
    12. Wilkinson RW,
    13. Honeychurch J,
    14. Illidge TM
    : Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade. Cancer Res 74(19): 5458-5468, 2014. DOI: 10.1158/0008-5472.CAN-14-1258
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Mohan R,
    2. Grosshans D
    : Proton therapy - Present and future. Adv Drug Deliv Rev 109: 26-44, 2017. DOI: 10.1016/j.addr.2016.11.006
    OpenUrlCrossRefPubMed
  14. ↵
    1. Hu Y,
    2. Paris S,
    3. Sahoo N,
    4. Wang Q,
    5. Wang Q,
    6. Barsoumian HB,
    7. Huang A,
    8. Da Silva J,
    9. Bienassis C,
    10. Leyton CSK,
    11. Voss TA,
    12. Masrorpour F,
    13. Riad T,
    14. Leuschner C,
    15. Puebla-Osorio N,
    16. Gandhi S,
    17. Nguyen QN,
    18. Wang J,
    19. Cortez MA,
    20. Welsh JW
    : Superior antitumor immune response achieved with proton over photon immunoradiotherapy is amplified by the nanoradioenhancer NBTXR3. J Nanobiotechnology 22(1): 597, 2024. DOI: 10.1186/s12951-024-02855-0
    OpenUrlCrossRefPubMed
  15. ↵
    1. Rykkelid AM,
    2. Sinha PM,
    3. Folefac CA,
    4. Horsman MR,
    5. Sørensen BS,
    6. Søland TM,
    7. Schreurs OJF,
    8. Malinen E,
    9. Edin NFJ
    : Combination of proton- or X-irradiation with anti-PDL1 immunotherapy in two murine oral cancers. Sci Rep 14(1): 11569, 2024. DOI: 10.1038/s41598-024-62272-z
    OpenUrlCrossRefPubMed
  16. ↵
    1. Nielsen S,
    2. Sitarz MK,
    3. Sinha PM,
    4. Folefac CA,
    5. Høyer M,
    6. Sørensen BS,
    7. Horsman MR
    : Using immunotherapy to enhance the response of a C3H mammary carcinoma to proton radiation. Acta Oncol 62(11): 1581-1586, 2023. DOI: 10.1080/0284186X.2023.2238550
    OpenUrlCrossRefPubMed
  17. ↵
    1. Iversen AB,
    2. Busk M,
    3. Bertelsen LB,
    4. Laustsen C,
    5. Munk OL,
    6. Nielsen T,
    7. Wittenborn TR,
    8. Bussink J,
    9. Lok J,
    10. Stødkilde-Jørgensen H,
    11. Horsman MR
    : The potential of hyperpolarized 13C magnetic resonance spectroscopy to monitor the effect of combretastatin based vascular disrupting agents. Acta Oncol 56(11): 1626-1633, 2017. DOI: 10.1080/0284186X.2017.1351622
    OpenUrlCrossRefPubMed
  18. ↵
    1. Tozer GM,
    2. Kanthou C,
    3. Baguley BC
    : Disrupting tumour blood vessels. Nat Rev Cancer 5(6): 423-435, 2005. DOI: 10.1038/nrc1628
    OpenUrlCrossRefPubMed
  19. ↵
    1. Siemann DW,
    2. Chaplin DJ,
    3. Walicke PA
    : A review and update of the current status of the vasculature-disabling agent combretastatin-A4 phosphate (CA4P). Expert Opin Investig Drugs 18(2): 189-197, 2009. DOI: 10.1517/13543780802691068
    OpenUrlCrossRefPubMed
  20. ↵
    1. Folkes LK,
    2. Christlieb M,
    3. Madej E,
    4. Stratford MRL,
    5. Wardman P
    : Oxidative metabolism of combretastatin A-1 produces quinone intermediates with the potential to bind to nucleophiles and to enhance oxidative stress via free radicals. Chem Res Toxicol 20(12): 1885-1894, 2007. DOI: 10.1021/tx7002195
    OpenUrlCrossRefPubMed
  21. ↵
    1. Horsman MR,
    2. Wittenborn TR,
    3. Nielsen PS,
    4. Elming PB
    : Tumors resistant to checkpoint inhibitors can become sensitive after treatment with vascular disrupting agents. Int J Mol Sci 21(13): 4778, 2020. DOI: 10.3390/ijms21134778
    OpenUrlCrossRefPubMed
  22. ↵
    1. Horsman M,
    2. Overgaard J
    : Hyperthermia: a potent enhancer of radiotherapy. Clin Oncol (R Coll Radiol) 19(6): 418-426, 2007. DOI: 10.1016/j.clon.2007.03.015
    OpenUrlCrossRefPubMed
  23. ↵
    1. Elming PB,
    2. Sørensen BS,
    3. Oei AL,
    4. Franken NAP,
    5. Crezee J,
    6. Overgaard J,
    7. Horsman MR
    : Hyperthermia: the optimal treatment to overcome radiation resistant hypoxia. Cancers (Basel) 11(1): 60, 2019. DOI: 10.3390/cancers11010060
    OpenUrlCrossRefPubMed
  24. ↵
    1. Huang L,
    2. Li Y,
    3. Du Y,
    4. Zhang Y,
    5. Wang X,
    6. Ding Y,
    7. Yang X,
    8. Meng F,
    9. Tu J,
    10. Luo L,
    11. Sun C
    : Mild photothermal therapy potentiates anti-PD-L1 treatment for immunologically cold tumors via an all-in-one and all-in-control strategy. Nat Commun 10(1): 4871, 2019. DOI: 10.1038/s41467-019-12771-9
    OpenUrlCrossRefPubMed
  25. ↵
    1. Oei AL,
    2. Korangath P,
    3. Mulka K,
    4. Helenius M,
    5. Coulter JB,
    6. Stewart J,
    7. Velarde E,
    8. Crezee J,
    9. Simons B,
    10. Stalpers LJA,
    11. Kok HP,
    12. Gabrielson K,
    13. Franken NAP,
    14. Ivkov R
    : Enhancing the abscopal effect of radiation and immune checkpoint inhibitor therapies with magnetic nanoparticle hyperthermia in a model of metastatic breast cancer. Int J Hyperthermia 36(sup1): 47-63, 2019. DOI: 10.1080/02656736.2019.1685686
    OpenUrlCrossRefPubMed
  26. ↵
    1. Ibuki Y,
    2. Takahashi Y,
    3. Tamari K,
    4. Minami K,
    5. Seo Y,
    6. Isohashi F,
    7. Koizumi M,
    8. Ogawa K
    : Local hyperthermia combined with CTLA-4 blockade induces both local and abscopal effects in a murine breast cancer model. Int J Hyperthermia 38(1): 363-371, 2021. DOI: 10.1080/02656736.2021.1875059
    OpenUrlCrossRefPubMed
  27. ↵
    1. Overgaard J
    : Simultaneous and sequential hyperthermia and radiation treatment of an experimental tumor and its surrounding normal tissue in vivo. Int J Radiat Oncol Biol Phys 6(11): 1507-1517, 1980. DOI: 10.1016/0360-3016(80)90008-5
    OpenUrlCrossRefPubMed
  28. ↵
    1. Sørensen BS,
    2. Bassler N,
    3. Nielsen S,
    4. Horsman MR,
    5. Grzanka L,
    6. Spejlborg H,
    7. Swakoń J,
    8. Olko P,
    9. Overgaard J
    : Relative biological effectiveness (RBE) and distal edge effects of proton radiation on early damage in vivo. Acta Oncol 56(11): 1387-1391, 2017. DOI: 10.1080/0284186X.2017.1351621
    OpenUrlCrossRefPubMed
  29. ↵
    1. Horsman MR,
    2. Chaplin DJ,
    3. Overgaard J
    : Combination of nicotinamide and hyperthermia to eliminate radioresistant chronically and acutely hypoxic tumor cells. Cancer Res 50(23): 7430-7436, 1990.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Lindegaard JC,
    2. Overgaard J
    : Factors of importance for the development of the step-down heating effect in a C3H mammary carcinoma in vivo. Int J Hyperthermia 3(1): 79-91, 1987. DOI: 10.3109/02656738709140375
    OpenUrlCrossRefPubMed
  31. ↵
    1. Horsman MR,
    2. Christensen KL,
    3. Overgaard AJ
    : Winner of the 1988 Lund Science Award: Hydralazine-induced enhancement of hyperthermic damage in a C3H mammary carcinoma in vivo. Int J Hyperthermia 5(2): 123-136, 1989. DOI: 10.3109/02656738909140442
    OpenUrlCrossRefPubMed
  32. ↵
    1. Salmon HW,
    2. Siemann DW
    : Effect of the second-generation vascular disrupting agent OXi4503 on tumor vascularity. Clin Cancer Res 12(13): 4090-4094, 2006. DOI: 10.1158/1078-0432.CCR-06-0163
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Verbrugge I,
    2. Hagekyriakou J,
    3. Sharp LL,
    4. Galli M,
    5. West A,
    6. McLaughlin NM,
    7. Duret H,
    8. Yagita H,
    9. Johnstone RW,
    10. Smyth MJ,
    11. Haynes NM
    : Radiotherapy increases the permissiveness of established mammary tumors to rejection by immunomodulatory antibodies. Cancer Res 72(13): 3163-3174, 2012. DOI: 10.1158/0008-5472.CAN-12-0210
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Lukacova S,
    2. Khalil AA,
    3. Overgaard J,
    4. Alsner J,
    5. Horsman MR
    : Relationship between radiobiological hypoxia in a C3H mouse mammary carcinoma and osteopontin levels in mouse serum. Int J Radiat Biol 81(12): 937-944, 2005. DOI: 10.1080/09553000600567616
    OpenUrlCrossRefPubMed
  35. ↵
    1. Guha P,
    2. Heatherton KR,
    3. O’Connell KP,
    4. Alexander IS,
    5. Katz SC
    : Assessing the future of solid tumor immunotherapy. Biomedicines 10(3): 655, 2022. DOI: 10.3390/biomedicines10030655
    OpenUrlCrossRefPubMed
  36. ↵
    1. Topalian SL,
    2. Drake CG,
    3. Pardoll DM
    : Immune checkpoint blockade: a common denominator approach to cancer therapy. Cancer Cell 27(4): 450-461, 2015. DOI: 10.1016/j.ccell.2015.03.001
    OpenUrlCrossRefPubMed
  37. ↵
    1. Overgaard J
    : Influence of sequence and interval on the biological response to combined hyperthermia and radiation. Natl Cancer Inst Monogr 61: 325-332, 1982.
    OpenUrlPubMed
  38. ↵
    1. Lee Y,
    2. Auh SL,
    3. Wang Y,
    4. Burnette B,
    5. Wang Y,
    6. Meng Y,
    7. Beckett M,
    8. Sharma R,
    9. Chin R,
    10. Tu T,
    11. Weichselbaum RR,
    12. Fu YX
    : Therapeutic effects of ablative radiation on local tumor require CD8+ T cells: changing strategies for cancer treatment. Blood 114(3): 589-595, 2009. DOI: 10.1182/blood-2009-02-206870
    OpenUrlAbstract/FREE Full Text
    1. Dovedi SJ,
    2. Melis MHM,
    3. Wilkinson RW,
    4. Adlard AL,
    5. Stratford IJ,
    6. Honeychurch J,
    7. Illidge TM
    : Systemic delivery of a TLR7 agonist in combination with radiation primes durable antitumor immune responses in mouse models of lymphoma. Blood 121(2): 251-259, 2013. DOI: 10.1182/blood-2012-05-432393
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Schaue D,
    2. Ratikan JA,
    3. Iwamoto KS,
    4. McBride WH
    : Maximizing tumor immunity with fractionated radiation. Int J Radiat Oncol Biol Phys 83(4): 1306-1310, 2012. DOI: 10.1016/j.ijrobp.2011.09.049
    OpenUrlCrossRefPubMed
  41. ↵
    1. Huang Y,
    2. Dong Y,
    3. Zhao J,
    4. Zhang L,
    5. Kong L,
    6. Lu JJ
    : Comparison of the effects of photon, proton and carbon-ion radiation on the ecto-calreticulin exposure in various tumor cell lines. Ann Transl Med 7(20): 542, 2019. DOI: 10.21037/atm.2019.09.128
    OpenUrlCrossRefPubMed
  42. ↵
    1. Mirjolet C,
    2. Nicol A,
    3. Limagne E,
    4. Mura C,
    5. Richard C,
    6. Morgand V,
    7. Rousseau M,
    8. Boidot R,
    9. Ghiringhelli F,
    10. Noel G,
    11. Burckel H
    : Impact of proton therapy on antitumor immune response. Sci Rep 11(1): 13444, 2021. DOI: 10.1038/s41598-021-92942-1
    OpenUrlCrossRefPubMed
  43. ↵
    1. Baatout S
    1. Baeyens A,
    2. Abrantes AM,
    3. Ahire V,
    4. Ainsbury EA,
    5. Baatout S,
    6. Baselet B,
    7. Botelho MF,
    8. Boterberg T,
    9. Chevalier F,
    10. Da Pieve F,
    11. Delbart W,
    12. Edin NFJ,
    13. Fernandez-Palomo C,
    14. Geenen L,
    15. Georgakilas AG,
    16. Heynickx N,
    17. Meade AD,
    18. Michaelidesova AJ,
    19. Mistry D,
    20. Montoro A,
    21. Mothersill C,
    22. Pires AS,
    23. Reindl J,
    24. Schettino G,
    25. Socol Y,
    26. Selvaraj VK,
    27. Sminia P,
    28. Vermeulen K,
    29. Vogin G,
    30. Waked A,
    31. Wozny AS
    : Basic concepts of radiation biology. In: Radiobiology Textbook. Baatout S (ed.). Cham, Springer International Publishing, pp 25-81, 2023.
  44. ↵
    1. Paganetti H
    : Mechanisms and review of clinical evidence of variations in relative biological effectiveness in proton therapy. Int J Radiat Oncol Biol Phys 112(1): 222-236, 2022. DOI: 10.1016/j.ijrobp.2021.08.015
    OpenUrlCrossRefPubMed
  45. ↵
    1. Oei AL,
    2. Kok HP,
    3. Oei SB,
    4. Horsman MR,
    5. Stalpers LJA,
    6. Franken NAP,
    7. Crezee J
    : Molecular and biological rationale of hyperthermia as radio- and chemosensitizer. Adv Drug Deliv Rev 163-164: 84-97, 2020. DOI: 10.1016/j.addr.2020.01.003
    OpenUrlCrossRefPubMed
  46. ↵
    1. Ando K,
    2. Suzuki Y,
    3. Kaminuma T,
    4. Yoshimoto Y,
    5. Oike T,
    6. Okonogi N,
    7. Sato H,
    8. Tamaki T,
    9. Noda SE,
    10. Mimura K,
    11. Nakano T
    : Tumor-specific CD8-positive T cell-mediated antitumor immunity is implicated in the antitumor effect of local hyperthermia. Int J Hyperthermia 35(1): 226-231, 2018. DOI: 10.1080/02656736.2018.1492027
    OpenUrlCrossRefPubMed
  47. ↵
    1. Fabian KP,
    2. Padget MR,
    3. Fujii R,
    4. Schlom J,
    5. Hodge JW
    : Differential combination immunotherapy requirements for inflamed (warm) tumors versus T cell excluded (cool) tumors: engage, expand, enable, and evolve. J Immunother Cancer 9(2): e001691, 2021. DOI: 10.1136/jitc-2020-001691
    OpenUrlAbstract/FREE Full Text
  48. ↵
    1. Petitprez F,
    2. Meylan M,
    3. de Reyniès A,
    4. Sautès-Fridman C,
    5. Fridman WH
    : The tumor microenvironment in the response to immune checkpoint blockade therapies. Front Immunol 11: 784, 2020. DOI: 10.3389/fimmu.2020.00784
    OpenUrlCrossRefPubMed
  49. ↵
    1. Carvalho HA,
    2. Villar RC
    : Radiotherapy and immune response: the systemic effects of a local treatment. Clinics (Sao Paulo) 73(suppl 1): e557s, 2018. DOI: 10.6061/clinics/2018/e557s
    OpenUrlCrossRef
  50. ↵
    1. Formenti SC,
    2. Demaria S
    : Combining radiotherapy and cancer immunotherapy: a paradigm shift. J Natl Cancer Inst 105(4): 256-265, 2013. DOI: 10.1093/jnci/djs629
    OpenUrlCrossRefPubMed
  51. ↵
    1. Weichselbaum RR,
    2. Liang H,
    3. Deng L,
    4. Fu YX
    : Radiotherapy and immunotherapy: a beneficial liaison? Nat Rev Clin Oncol 14(6): 365-379, 2017. DOI: 10.1038/nrclinonc.2016.211
    OpenUrlCrossRefPubMed
  52. ↵
    1. Sharabi AB,
    2. Nirschl CJ,
    3. Kochel CM,
    4. Nirschl TR,
    5. Francica BJ,
    6. Velarde E,
    7. Deweese TL,
    8. Drake CG
    : Stereotactic radiation therapy augments antigen-specific PD-1-mediated antitumor immune responses via cross-presentation of tumor antigen. Cancer Immunol Res 3(4): 345-355, 2015. DOI: 10.1158/2326-6066.CIR-14-0196
    OpenUrlAbstract/FREE Full Text
  53. ↵
    1. Arnold KM,
    2. Flynn NJ,
    3. Raben A,
    4. Romak L,
    5. Yu Y,
    6. Dicker AP,
    7. Mourtada F,
    8. Sims-Mourtada J
    : The impact of radiation on the tumor microenvironment: effect of dose and fractionation schedules. Cancer Growth Metastasis 11: 1179064418761639, 2018. DOI: 10.1177/1179064418761639
    OpenUrlCrossRefPubMed
    1. Akanda ZZ,
    2. Neeson PJ,
    3. John T,
    4. Barnett S,
    5. Hanna GG,
    6. Miller A,
    7. Jennens R,
    8. Siva S
    : A narrative review of combined stereotactic ablative radiotherapy and immunotherapy in metastatic non-small cell lung cancer. Transl Lung Cancer Res 10(6): 2766-2778, 2021. DOI: 10.21037/tlcr-20-1117
    OpenUrlCrossRefPubMed
    1. Demaria S,
    2. Ng B,
    3. Devitt ML,
    4. Babb JS,
    5. Kawashima N,
    6. Liebes L,
    7. Formenti SC
    : Ionizing radiation inhibition of distant untreated tumors (abscopal effect) is immune mediated. Int J Radiat Oncol Biol Phys 58(3): 862-870, 2004. DOI: 10.1016/J.IJROBP.2003.09.012
    OpenUrlCrossRefPubMed
  56. ↵
    1. Belka C,
    2. Ottinger H,
    3. Kreuzfelder E,
    4. Weinmann M,
    5. Lindemann M,
    6. Lepple-Wienhues A,
    7. Budach W,
    8. Grosse-Wilde H,
    9. Bamberg M
    : Impact of localized radiotherapy on blood immune cells counts and function in humans. Radiotherapy and Oncology 50(2): 199-204, 1999. DOI: 10.1016/S0167-8140(98)00130-3
    OpenUrlCrossRefPubMed
  57. ↵
    1. Lugade AA,
    2. Moran JP,
    3. Gerber SA,
    4. Rose RC,
    5. Frelinger JG,
    6. Lord EM
    : Local radiation therapy of B16 melanoma tumors increases the generation of tumor antigen-specific effector cells that traffic to the tumor. The Journal of Immunology 174(12): 7516-7523, 2005. DOI: 10.4049/JIMMUNOL.174.12.7516
    OpenUrlCrossRefPubMed
  58. ↵
    1. Zhao X,
    2. Shao C
    : Radiotherapy-mediated immunomodulation and anti-tumor abscopal effect combining immune checkpoint blockade. Cancers (Basel) 12(10): 2762, 2020. DOI: 10.3390/cancers12102762
    OpenUrlCrossRefPubMed
  59. ↵
    1. Thangamathesvaran L,
    2. Shah R,
    3. Verma R,
    4. Mahmoud O
    : Immune checkpoint inhibitors and radiotherapy-concept and review of current literature. Ann Transl Med 6(8): 155, 2018. DOI: 10.21037/atm.2018.03.09
    OpenUrlCrossRefPubMed
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