Low-Dose Cyclophosphamide Enhances the Tumoricidal Effects of 5-Day Spacing Stereotactic Ablative Radiotherapy by Boosting Antitumor Immunity
Article information
Abstract
Purpose
This study aimed to investigate the potential role of low-dose cyclophosphamide (Cy) as a radiosensitizer by evaluating its impact on the immune response and the abscopal effect of stereotactic ablative radiotherapy through preclinical models.
Materials and Methods
CT26 tumors (immunologically hot) and 4T1 tumors (immunologically cold), grown in immunocompetent BALB/c and immunodeficient BALB/c–nude mice, were irradiated with 20 Gy in two fractions with 5-day spacing followed by intraperitoneal injections of 9 mg/kg Cy every 3 days. Immunological changes in CT26 tumors caused by the treatments were assessed using flow cytometry. Changes in the expression of hypoxia-inducible factor-1α (HIF-1α) in tumors were also assessed. Splenocytes and bone marrow–derived dendritic cells (DCs) were exposed to various concentrations of Cy to assess T cell proliferation and DC differentiation.
Results
The combination of Cy with radiotherapy (RT+Cy) significantly suppressed tumor growth compared to RT alone in immunocompetent mice, while that effect was not observed in immunodeficient mice. Additionally, RT+Cy effectively induced abscopal effects in hot and cold tumors, with increased CD8+ T cells in blood and tumors. Significantly higher expression levels of granzyme B, interferon γ, and tumor necrosis factor α were observed in RT+Cy group compared to the RT alone group. In vitro data indicated that low-dose Cy promotes DC differentiation. Low-dose Cy suppressed the radiation-induced upregulation of HIF-1α in the tumors.
Conclusion
Low-dose Cy enhances tumoricidal effects of 5-day spacing high-dose RT by increasing antitumor immune responses.
Introduction
Stereotactic ablative radiotherapy (SABR) has been successfully utilized to control various types of cancers, including lung, liver, prostate, and oligometastatic cancer [1-4]. Notably, SABR has shown greater efficacy than prediction based on the conventional radiobiological principle such as the 5R’s [5]. Increasing evidence suggests that SABR not only kills tumor cells by inducing DNA damage but also causes vascular damage, leading to secondary tumor cell death [5]. Furthermore, SABR has been reported to enhance the antitumor immune response by inducing immunogenic cell death, which releases tumor-specific antigens and various immune cytokines, thereby boosting cytotoxic T-cell activation [5-7]. To maximize the immunogenic response induced by SABR, several strategies are under investigation. Preclinical models have reported that SABR with intervals of 5-10 days between treatments improves antitumor immunity [8-11]. One preclinical trial suggested that ‘pulsed’ SABR, with an optimal interval of 7-10 days between sessions, enhances the efficacy of checkpoint blockade [10]. Our previous preclinical study demonstrated that a 5-day interval between radiotherapy (RT) sessions, termed ‘spacing SABR’, was far more effective in enhancing the host immune response, including abscopal effects, compared to daily SABR [11], especially in immunologically hot tumors. In immunologically cold tumors, however, the impact of 5-day spacing SABR is limited, indicating a need for additional enhancement strategies.
Cyclophosphamide (Cy) was previously used as an effective anticancer drug for various cancers, including breast cancer and lymphoma, at high doses. On the other hand, although low-dose Cy is not strictly defined, it is generally used at approximately 30-100 mg/kg/wk or less in preclinical studies for tumors and 2 mg/kg/day or less in clinical studies for rheumatic diseases [12,13]. It has been reported to act as an immune-modulating agent by selectively depleting immunosuppressive regulatory T cells (Tregs) and enhancing effector T cell functions [14-16]. Additionally, low-dose Cy increases dendritic cell (DC) function to prime and enhance cytotoxic T-cell potency [17,18]. In this regard, increasing evidence suggests that low-dose Cy alone or in combination with other antitumor agents downregulates hypoxia-inducible factor-1α (HIF-1α) in tumors, a master inducer of immune suppression [19-23].
Overall, low-dose Cy enhances antitumor immunity, making it potentially suitable for a wider range of tumors, including immunologically cold tumors. We hypothesized that Cy could act as a radio-immune sensitizer to amplify the tumor immune response induced by spacing SABR, potentially enhancing the antitumor effects of RT. Our specific aim was to elucidate the efficacy of spacing SABR combined with low-dose Cy on both primary and secondary tumors, as well as to shed lights on the immunological profile changes caused by Cy in mouse tumor models.
Materials and Methods
1. Mice
Female BALB/c and BALB/c-nude mice (6-8 weeks old) were obtained from Orient Bio Co. BALB/c mice are immunocompetent, whereas BALB/c-nude mice are immunocompromised. The mice were housed at 22°C±3°C and 50%±20% humidity for at least 1 week before experiments. They were provided with food (Purina) and purified water ad libitum. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC). Euthanasia was performed in cases of tumor necrosis or cannibalism.
2. Cell lines
The CT26 colorectal carcinoma (immunologically hot tumor model) and 4T1 mammary cancer (immunologically cold tumor model) cell lines were obtained from the American Type Culture Collection (ATCC). CT26 cells were cultured in Dulbecco’s modified Eagle’s medium, and 4T1 cells in RPMI 1640 medium, both supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.
3. Tumor growth and Lung micrometastasis
CT26 and 4T1 cells (2×105 cells) were injected subcutaneously into the right hind leg of mice. When tumors reached 50-150 mm3 (7-10 days post-inoculation), mice were assigned to treatment groups: low-dose Cy alone, RT alone, and RT with Cy (RT+Cy). Control groups received no treatment or sham treatment. To assess the abscopal effect, primary tumors were induced by injecting tumor cells into the right hind leg, followed by an additional injection (1×105 cells) into the flank 4 days later. When primary tumors reached 50-150 mm3, the mice were assigned to treatment groups. Tumor sizes were measured every 2-3 days using calipers, and volumes were calculated with the formula: volume=0.523×(longest axis)×(shortest axis)2. Lungs from mice bearing CT26 and 4T1 tumors were extracted on days 26, 15, and 22 for CT26 (BALB/c), CT26 (BALB/c-nude), and 4T1 (BALB/c), respectively, following initial tumor irradiation. Lungs were fixed, dehydrated, embedded in paraffin, sliced, stained with hematoxylin and eosin, and metastatic lesions were quantified using Motic DSAssistant Lite 1.0 software.
4. Irradiation and Cy administration
Mice were anesthetized with Alfaxalone and Rompun (10:1 ratio), and the tumors were irradiated with 2×10 Gy with a 5-day spacing using a 60Co unit at 1.0 Gy/min. Cy was administered intraperitoneally at 9 mg/kg every 3 days until sacrifice. To determine the optimal timing of Cy administration in combination with irradiation, low-dose Cy was compared when administered 3 days before, on the same day, or 2 days after the first irradiation.
5. Flow cytometry and blood sampling
Primary and secondary CT26 tumors and spleens from mice were collected on days 8, 15, 22, and 45 after the first irradiation. On day 45, samples from the RT or RT+Cy groups were the only ones available, as the control and Cy alone groups were euthanized on day 22. Tumors were digested with tumor dissociation enzyme (Miltenyi Biotec) for 1 hour at 37°C and filtered through a 70-µm strainer. Spleens were homogenized, red blood cells (RBC) lysed with RBC lysis buffer (Sigma-Aldrich), and filtered. Blood samples collected on days 8, 15, and 22 post-irradiation were lysed with RBC lysis buffer. Cells were incubated with anti-CD16/32 antibodies, washed, and stained with labeled antibodies. Flow cytometry data were analyzed using CytoFLEX (Beckman Coulter) and FlowJo software (BD). Precision counting beads (#424902, BioLegend) were used for quantification.
6. T cell proliferation and DC differentiation assay
For T cell proliferation, lymphocytes from splenocytes obtained from naïve BALB/c mice were separated using nylon wool fiber columns [24], and subsequently isolated using the EaseSep Mouse CD8+ T cell isolation kit (#19853, EasySep) and EaseSep MouseCD4+CD25+ Regulatory T cell isolation kit II (#18783, EasySep). The isolated CD8+ T cells were stained with 5 µM CellTrace CFSE (#C34554, Thermo Fisher Scientific), stimulated with Dynabeads Mouse T Activator CD3/CD28 (#11452D, Gibco) at a 1:1 cell-to-bead ratio in a U-bottom 96-well plate (1.0×105 cells/200 µL/well), and treated with serial dilutions of Cy, starting from 6.4 mM. The 9 mg/kg Cy administered every 3 days in the preclinical study corresponds to approximately 3.2 mM, while the daily dose of 3 mg/kg corresponds to approximately 1.0 mM. For isolated Tregs proliferation, CD3/CD28 activator stimulated T cells were additionally cultured with 300 U/mL interleukin (IL)-2 (#130-120-330, Miltenyi Biotec) and 5 ng/mL transforming growth factor β1 (TGF-β1; #7666-MB, R&D system). After 3 days, T cells were analyzed for CFSE dilution by flow cytometry. For DC differentiation, bone marrow cells were harvested from BALB/c femurs, resuspended at 2.5×106 cells/mL in medium with 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; #130-095-739, Miltenyi Biotec) and serial dilutions of Cy. The media, GM-CSF, and Cy were changed every 3 days. On day 6, mature bone marrow‒derived DCs were analyzed by flow cytometry. The gating strategy is shown in S1 and S2 Figs.
7. HIF-1α expression in tumor tissues
CT26 tumors were excised on days 8 and 15, fixed in formalin, embedded in paraffin, and sectioned at 3 µm thickness. Sections were deparaffinized, underwent antigen retrieval in 0.01 M citrate buffer (pH 6.0) for 10 minutes, and were incubated overnight at 4°C with HIF-1α antibodies (1:100, sc-13515, Santa Cruz Biotechnology). Sections were rinsed with phosphate buffered saline (PBS), incubated with peroxidase reagent and anti‒mouse IgG (ImmPRESS, Vector Laboratories) for 20 minutes, washed with PBS, and stained with DAB (Vector Laboratories). Sections were counterstained with Mayer’s hematoxylin and observed under a light microscope. Images were captured at 400× magnification and analyzed using Image-J software ver. 1.38 (National Institutes of Health).
8. Statistics
All the results are presented as mean±standard error of the mean, except the DC differentiation analysis, which is presented with standard derivation. Tumor growth delay was compared using two-way ANOVA. For multiple groups, one-way ANOVA was employed, whereas Student’s t test was utilized for comparing continuous data between two groups. Statistical significance was defined as a p-value less than 0.05. Data analysis was performed using the GraphPad Prism software ver. 9 (GraphPad). Detailed descriptions of the number of animals and all statistical tests are shown in the figure legends.
Results
1. Two days after the first irradiation was optimal time for Cy administration to achieve the best tumor control
Cy was administered (9 mg/kg every 3 days) either 3 days before, on the same day, or 2 days after the first irradiation. Administering Cy 2 days after the first RT most effectively suppressed primary tumor growth (Fig. 1A). Based on the results, subsequent experiments used the 2-day post-RT regimen.
Cyclophosphamide with radiotherapy (RT+Cy; 2 days post-RT) significantly reduced tumor growth delay and prevented lung micrometastasis compared to RT alone in BALB/c immunocompetent mice, an effect not observed in BALB/c‒nude mice. These results demonstrated that the tumoricidal effect of adding Cy to radiation resulted from an immune reaction. (A) CT26 tumors were treated with low-dose Cy in three schemes: 3 days before, simultaneously, or 2 days after first day of RT (20 Gy in 2 fractions, 5-day interval). Among them, the most effective treatment schedule was administering Cy 2 days after the first irradiation. Data from two independent experiments were pooled and presented. (B) Tumor growth curve of CT26 in BALB/c mice (one of three independent experiments is shown) and BALB/c-nude mice (one experiment). (C) To investigate lung metastasis, CT26 tumors were treated with the same scheme in BALB/c-immunocompetent or BALB/c-nude mice. The microscopic images represent H&E-stained sections of lungs (10× magnification, scale bar=0.3 cm). Bar plots show the total number of metastatic lesions (> 15 or 50 µm) in whole lung (one experiment). Data in A and B are presented as mean±standard error of the mean, with two-way analysis of variance with Sidak’s multiple comparisons at the endpoint. Data in C are presented as mean±standard error of the mean, with a one-way ANOVA test. *p < 0.05, **p < 0.01, ***p < 0.001.
2. The effective tumoricidal effect of Cy observed exclusively in immunocompetent mice suggests that this response is mediated by an immune reaction
In BALB/c mice bearing CT26 tumors, addition of Cy effectively suppressed primary tumor growth compared to RT alone (Fig. 1B, left). This effect was not observed in BALB/c-nude mice (Fig. 1B, right). Regarding lung metastasis, Cy exhibited a preventive effect on micrometastasis in immunocompetent mice, compared to the control group. In contrast, in nude mice, Cy had no significant impact on either primary tumor growth or lung metastasis in these mice. Therefore, it is suggested that the effect of Cy relies on the presence of an immune response (Fig. 1C).
3. Cy exhibited the abscopal effects in 4T1 (cold tumor) as well as CT26 (hot tumor)
Primary tumors were locally irradiated at 5-day intervals, and 2 days after the first irradiation, Cy (9 mg/kg) was administered every 3 days until the mice were sacrificed (Fig. 2A). The RT+Cy treatment effectively suppressed the growth of secondary tumors as well as primary tumors. No differences in the size of secondary tumors were observed between the control, Cy alone, or RT alone groups (Fig. 2B, right). To elucidate the long-term abscopal effect of Cy, the primary and secondary tumor responses were observed up to 45 days after the first irradiation. The average growth rate of the secondary tumors in the RT+Cy group was much slower than that in the RT group (Fig. 2C). Using the 4T1 cell line, RT+Cy also significantly suppressed primary and secondary tumor growth than RT alone or Cy alone (Fig. 2D).
The 5-day spacing cyclophosphamide with radiotherapy (RT+Cy) significantly improved both primary and secondary tumor control (abscopal effect) in both CT26 (hot tumor) and 4T1 (cold tumor) models. (A) The primary tumors (irradiated) were implanted in the right thigh, and the secondary tumors (non-irradiated) were implanted 4 days after primary tumor injection in the flank to assess the abscopal effects of RT and Cy treatments. (B) The CT26 tumor growth curves of primary and secondary tumors in BALB/c mice. (C) Long-term observation of primary and secondary tumors in CT26 models (n=5 per group, one experiment). (D) The 4T1 tumor growth curve in BALB/c mice. Data in B from three independent experiments and D from two independent experiments were pooled and presented as mean±standard error of the mean, with 2-way ANOVA with Sidak’s multiple comparisons at the endpoint. Data in C are presented as mean±standard error of the mean, with a multiple 2-tailed t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.
4. The concentration of Cy impacted DC differentiation and proliferation of T cell differently
The effects of various Cy doses (0-6.4 mM) on DC differentiation were evaluated in vitro. The 1.0 mM concentration used in vitro is equivalent to the daily animal dose in this study. DC differentiation was significantly enhanced by 0.8 to 1.6 mM Cy compared to 0, 3.2, or 6.4 mM (Fig. 3A). Moreover, the proportions of DCs that express activation markers, CD80 and MHCII, were highest at 0.8 mM (Fig. 3A). Changes in the proliferation of isolated CD8+ T cells and Tregs from splenocytes were measured in response to varying doses of Cy. CD8+ T cells showed no difference in proliferation at concentrations ranging from 0.8 to 3.2 mM, with a reduction observed at 6.2 mM. In contrast, Treg proliferation was significantly reduced starting at a Cy concentration of 3.2 mM (Fig. 3B). These in vitro results indicated that low-dose Cy (0.8-3.2 mM) enhanced DC differentiation without significantly affecting the proliferation of CD8+ T cells.
Various doses of cyclophosphamide (Cy) were administered to evaluate the direct effects on dendritic cells (DCs) and T cells through in vitro assay. The concentration of 1.0 mM in vitro is equivalent to the daily dose used in vivo. Low-dose Cy at 0.8 or 1.6 mM markedly enhanced DC differentiation, while at a higher concentration (6.4 mM), Treg proliferation was inhibited. (A) Bone marrow‒derived cells were cultured for 6 days in the absence or presence of gradient concentration of Cy (mM) and soluble granulocyte-macrophage colony-stimulating factor for DC differentiation. Representative contour plots show the enhanced capacity of DCs from bone marrows at low-dose Cy. The percentage of DCs is indicated within the contour plots, while the absolute number and proportion of DCs, CD80+ DCs, and MHCII+ DCs are displayed in the bar graph to the right (data from 2 independent experiments are shown). (B) The proliferative capacity of CD8+ T cells and Tregs was assessed after 3 days in the absence or presence of gradient concentrations of Cy (mM) and cytokines for immune cell activation. Among the doses tested (0, 0.8, 1.6, 3.2, and 6.4 mM), 6.4 mM Cy significantly suppressed Treg proliferation. Other doses, excluding 6.4 mM Cy, showed no effect on T cell proliferation (n=3). Data in A are presented as mean±standard deviation, with a one-way ANOVA test. Data in B are presented as mean±standard error of the mean, with two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
5. Cy suppressed radiation-induced HIF-1α, and increased DCs and T cells
Fig. 4A shows that the expression of HIF-1α in tumors markedly increased on days 8, 15, and 45 after the first irradiation. However, the HIF-1α expression in tumors treated with RT+Cy was significantly lower than in tumors treated with RT alone. These results indicated that Cy suppressed the radiation-induced HIF-1α expression in the tumors.
Cy reduced radiotherapy (RT)-induced hypoxia-inducible factor-1α (HIF-1α) on days 8, 15, and 45 after first RT. In the cyclophosphamide with RT (RT+Cy) group, an increasing number of dendritic cells (DCs) and T cells were observed in the primary tumors on day 45 compared to the RT group. Similar outcomes of T cells were observed in the blood on day 22. Notably, low-dose of Cy significantly increased the number of DCs in blood on day 22 after the first irradiation compared to other groups. (A) Immunohistochemical staining for HIF-1α in CT26 tumors on days 8, 15, and 45 after initial irradiation. At least 10 images per section were captured, and the percentage of positive areas was quantified. Scale bars=100 μm. Data represent the means of 3-4 tumors±minimum and maximum values. The number of mice used in each experiment is as follows: one experiment is shown (day 8 and 15: control 4, Cy 4, RT 3, RT+Cy 3; day 45: RT 5, RT+Cy 4). (B) Flow cytometry analysis for CT26 tumor tissue on days 8, 15, 22, and 45 after the initial irradiation. Bar plots represent the sequential changes in the number of immune cells within tumors. RT+Cy increased tumor-infiltrating DCs on days 8 and 15 compared to RT alone. The number of mice used in each experiment is as follows: one experiment is shown (days 8 and 15: control 4, Cy 4, RT 3, RT+Cy 3; day 22: n=6 per group; day 45: n=5 per group). Tregs, regulatory T cells. (C) Flow cytometry analysis of blood on days 8, 15, and 22 after initial irradiation. Serial changes in the number of immune cells between the treatment groups. The asterisks on day 15 indicate values comparing the RT group with the RT+Cy group. One experiment is shown (n=6 per group). The data in A from days 8 to 22 were analyzed using one-way ANOVA, while the data in A from day 45 were analyzed using a t test. The data in B and C are presented as mean±standard error of the mean, with a one-way ANOVA test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Flow cytometry analysis of immune cells in CT26 tumor tissues on days 8, 15, 22, and 45 following the first irradiation is shown in Fig. 4B. No significant differences in tumor-infiltrating immune cells, including DCs, CD8+ T cells, CD4+ T cells, and Tregs, were observed among the groups on the days 8, 15, and 22. However, on day 45, a notable increase in the number of DCs and T cells, including CD8+ T cells, CD4 T cells, or Tregs, was observed in the RT+Cy group compared to the RT group.
The immunological profiles in the blood of mice were assessed on days 8, 15, and 22 after the first irradiation (Fig. 4C). On day 22, the number of DCs in the blood of Cy group was higher than that in other groups. Additionally, the number of T cells in the blood of the RT+Cy group was significantly higher than in the RT alone group.
6. Addition of Cy after RT increased CD8+ T cell activation associated with DCs
The potency of CD8+ T cells in the primary tumors, secondary tumors, and spleens was assessed using flow cytometry on day 22 (Fig. 5). The percentage of interferon γ (IFN-γ)+ CD8+ T cells was significantly higher in the RT+Cy group compared to in the RT group in the primary tumors, secondary tumors, and spleen. And the percentage of GzmB+ CD8+ T cells was marginally increased in the RT+Cy group compared to RT group in primary and secondary tumors. Interestingly, the percentage of tumor necrosis factor α (TNF-α)+ CD8+ T cells of RT+Cy group mice was significantly increased in the spleen, not in the primary tumor or secondary tumor. In the tumors, almost CD8+ T cells in all groups expressed TNF-α.
On day 22 after initial irradiation, the number of CD8+ T cells and the percentage of functional CD8+ T cells were assessed in the primary tumor (A), secondary tumor (B), and spleen (C). The combination of 5-day spacing radiotherapy (RT) and low-dose cyclophosphamide (Cy) significantly increased T cell activation compared to RT alone or Cy alone, even though the number of CD8+ T cell showed no difference between each group. IFN-γ, interferon γ; TNF-α, tumor necrosis factor α. One experiment is shown (n=6 per group). All data are presented as mean±standard error of the mean, with an unpaired 2-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
On day 45, the number of DCs, CD8+ T cells, and IFN-γ+ CD8+ T cells in primary tumors was significantly greater in the RT+Cy group compared to the RT alone group (Fig. 6A), even though there was no observed difference in CD8+ T cell functionality. Similarly, in secondary tumors, the number of DCs was significantly increased in the RT+Cy group, while the numbers of CD8+ T cells and IFN-γ+ CD8+ T cells showed a marginal increase (Fig. 6B).
Flow cytometry analysis was conducted on primary tumors (A), secondary tumors (B), and spleen tissues (C) on day 45 after first irradiation to evaluate the long-term immune effects of cyclophosphamide (Cy). The Cy with radiotherapy (RT+Cy) group showed a significant increase in the number of DCs and activated CD8+ T cells compared to the RT alone group. One experiment is shown (n=5 per group). The tumor growth delay curve is shown in Fig. 2C. All data are presented as mean±standard error of the mean, with an unpaired 2-tailed t test. *p < 0.05, **p < 0.01.
In the spleen, the number of CD8+ T cells and CD44-positive effector CD8+ T cells on day 45 was higher in the RT+Cy group than in the RT alone group (Fig. 6C). These results demonstrate that Cy enhances and sustains the antitumor immune response induced by RT.
Discussion
Considerable evidence suggests that local radiation induces immunological changes in the tumor microenvironment, depending on the radiation dose and fractionation schedule [10,25,26]. We previously reported that extending the interval between two fractions of 20 or 40 Gy from 1 day to 5 days increased the abscopal effect and antitumor immune response in a hot tumor model. Specifically, the 1-day interval RT induced both immunosuppressive myeloid-derived suppressor cell (MDSC) and cytotoxic CD8+ T cell infiltration in the tumors, whereas the 5-day spacing RT reduced MDSC infiltration and increased tumor-specific CD8+ T cell activation [11]. However, it was ineffective in decreasing lung metastasis or inducing abscopal effects in immunologically cold tumors. In the present study, we investigated whether the antitumor immunity induced by spacing RT could be further enhanced by combining it with low-dose Cy, which has been demonstrated to elevate antitumor immunity [13,27,28] by increasing cytotoxic T cells and depleting Treg [15,16]. The feasibility of enhancing antitumor immunity by combining Cy with other treatment modalities, including RT, has been investigated [13,27,28]. Interestingly, low-dose Cy enhance the effect of irradiation, leading to tumor growth delay and induction of immune cells [13]. In light of these observations, we hypothesized that low-dose Cy could be used as a radio‒immune sensitizer for the 5-day spacing RT regimen. As we hypothesized, in the present study, the RT+Cy was more effective than RT alone in suppressing primary tumor growth and inducing abscopal effects, even for the immunologically cold tumors.
Cy has been reported to deplete Tregs, thereby enhancing antitumor immunity [15,16]. However, in our in vitro study, low-dose Cy did not significantly impact Treg proliferation, while high-dose of Cy reduced Treg proliferation. Previous studies have shown that low-dose Cy enhances antitumor immunity by promoting the migration of DCs from bone marrow and maturation of DCs mediated by type I interferons [17,18]. Nakahara et al. [18] demonstrated that Cy suppressed resident DCs while having no impact on migratory or plasmacytoid DCs. This imbalance in DCs led to increased antigen presentation to naïve T cells by DCs, thereby elevating the antitumor immune response. It has been reported that irradiation of tumors promotes the activation of intratumoral DCs in radio‒immunogenic tumors, but not in poorly radio-immunogenic tumors [29].
In the present study, low-dose Cy promoted DC differentiation, and irradiation of tumors increased the number of DCs in tumors on day 22 after the first irradiation. Furthermore, the number of DCs in both primary and secondary tumors in the RT+Cy groups was markedly greater than in the RT alone group on day 45 after the first irradiation. The activated DCs in tumors engulf tumor-associated antigens released from dying tumor cells, migrate to tumor-associated lymph nodes, and prime cytotoxic CD8+ T cells [22]. It is therefore reasonable to suggest that the greater effectiveness of RT+Cy in suppressing the growth of irradiated primary tumors and the abscopal effect in suppressing the growth of secondary tumors was due, at least in part, to the elevation of antitumor immunity caused by low-dose Cy.
A pertinent question is how Cy elevates the antitumor immunity by increasing DC activity, which in turn leads to enhanced CD8+ T cell activation. As shown in Fig. 4A, HIF-1α expression in tumors treated with RT+Cy was significantly lower than in those treated with RT alone at days 8, 15, or 45 after the first irradiation. This suggests that radiation-induced HIF-1α expression was downregulated by low-dose Cy. HIF-1α is known to suppress DC recruitment and activation, T cell priming, and the immunological lysis of tumor cells by CD8+ T cells [22,23]. Additionally, HIF-1α enhances Treg activation, increases the production of immunosuppressive TGF-β and IL-10, induces immune checkpoint expression and their ligands, and suppresses the expression of GzmB, TNF-α, and IFN-γ in CD8+ T cells [22,23]. In the present study, the expressions of GzmB, TNF-α, and IFN-γ in the RT+Cy group tended to be generally higher than those in the RT-alone groups, related to increased activation of CD8+ T cells by Cy. While our in vitro data indicate that low-dose Cy played a direct role in the differentiation of DCs, but it did not have a direct effect on CD8+ T cells. Our findings suggest that low-dose Cy may promote DC differentiation, which is accompanied by a down-regulation of HIF-1α, and that this DC differentiation subsequently leads to CD8+ T cell activation. However, directly assessing the temporal sequence of these events would be challenging, making it difficult to establish a clear causal relationship between DC differentiation and HIF-1α reduction. Additionally, a further limitation is that the experiments were performed exclusively in the BALB/c mouse model, which exhibits a Th2-dominant immune response. Validating these findings in other models, such as C57BL/6 mice, which are Th1-dominant, would be important for providing a more comprehensive understanding of the observed effects.
In conclusion, our findings demonstrate that 5-day spacing high-dose RT combined with Cy enhances the tumoricidal effect through increasing antitumor immunity.
Electronic Supplementary Material
Supplementary materials are available at Cancer Research and Treatment website (https://www.e-crt.org).
Notes
Ethical Statement
Mice were housed in particular pathogen-free facilities. All investigations involving mice have been carried out in line with the applicable ethical standards for animal testing and research as well as the IACUC-approved protocols (Kirams2022-0125). Consent to participate was waived.
Author Contributions
Conceived and designed the analysis: Kim H, Chun SJ, Yang K, Kim MS.
Collected the data: Kim H, Sun S, Cho H, Kim TJ, Lee YJ.
Contributed data or analysis tools: Kim H, Chun SJ, Chie EK, Yang K.
Performed the analysis: Kim H, Chun SJ.
Wrote the paper: Kim H, Chun SJ, Kim MS.
Revised the paper: Sun S, Cho H, Kim TJ, Lee YJ, Chie EK, Yang K.
Conflict of Interest
Conflict of interest relevant to this article was not reported.
Funding
This research was supported by the Korea Institute of Radiological & Medical Sciences (KIRAMS, Seoul, Korea) (No.50572‒2023, 50572-2024, 50552-2024).