IMPACT OF PROTON THERAPY ON THE TIME

In this section, we review the impact of proton therapy on the TIME from three perspectives: (i) infiltrating immune cells, (ii) cytokines and chemokines, and (iii) immune checkpoints. Figure 1 shows the TIME profile, and Table 2 presents the impact of proton therapy.

FIGURE 1: Landscape of the TIME.

Tumor Types | Radiotherapy Strategy | Changes in the TIME | References
COAD | 7 days after 16.4 Gy | CD8+ T cells, CD8+ T cells, TAM1s increased | 81
 |  | Tregs decreased | 
COAD | 7 days after 8 Gy | CD8+ T cells, CD8+PD1+T, NK cells decreased | 82
 |  | CD4+PD1+T, MDSCs increased | 
NSCLC | 8 days after 60 Gy/s | CD8+ T cells, TAM1s increased | 83
 |  | Tregs, TAM2s decreased | 
HNSCC | 12 hours after 5 Gy | IFN‐γ increased | 84
 |  | Exosomes decreased | 
HCC | 2 days after 3 Gy | CD8+ T cells increased | 85
 | 2 days after 6 Gy | MDSCs, TAM2s increased | 
 | 3 days after 12 Gy | IL‐6 increased | 
Glioma | 8 days after 257 ± 2 Gy/s | CD4+ T cells, CD8+ T cells, Tregs, CD8+ TRMs, NK cells, B cells increased | 74
Medulloblastoma | 5 days after 100 Gy/s | CD8+ T cells, TAM1s increased | 86
 |  | TAM2s decreased | 
GBM | 7 days after 30 Gy | CD8+ TRMs, B cells, CD8+ TAMs, cDC1 increased | 87

Impact on infiltrating immune cells

Photon therapy is a double‐edged sword for anti‐tumor immunity within the TME. 88 , 89 Recent studies have focused on the influence of proton therapy on immune cells within the TME. For instance, a study involving BALB/c mice with CT26 colon tumors demonstrated that proton irradiation led to increased immune cell infiltration, particularly at 7 and 14 days post‐treatment. RNA sequencing revealed the upregulation of genes associated with immune response and interferon signaling, suggesting that proton therapy not only induces direct tumor cell death, but also modulates the immune landscape, promoting a more favorable environment for immune activity. 81 Similarly, Iturri et al. found that proton minibeam radiotherapy (pMBRT) enhanced antigen presentation and immune memory formation in a rat model of glioblastoma. Moreover, pMBRT significantly increased the density of tissue‐resident memory CD8+T cells (CD8+TRMs), B cells, conventional dendritic cells type 1 (cDC1), and CD8+ tumor‐associated macrophages (CD8+ TAMs). 87 A subsequent study indicated that the preservation of specific immune cell subpopulations may result from proton therapy reducing the radiation exposure to the lymph nodes. 53 However, in the HCC model, although proton therapy decreased the tumor size, it also recruited immune suppressive cells such as CD163+ M2 and myeloid‐derived suppressor cells (MDSCs) into the TME. 85 Likewise, proton radiation triggered a stronger immunosuppressive response in colon tumors, manifested as an increase in MDSCs and CD4+PD1+T cells and a decrease in NK and CD8+T cells. 82 FLASH therapy, which has an excellent capacity to improve radiotherapy efficiency and reduce toxicity to normal tissues, 90 , 91 has been applied to proton therapy in the laboratory. 92 In a lung cancer model, pFLASH (60 Gy/s) enhanced the infiltration of cytotoxic CD8+T cells and M1‐type iNOS+ macrophages within the TME compared with conventional proton therapy (CPT, 1 Gy/s). Additionally, pFLASH significantly decreased the proportions of FOXP3+ regulatory T cells (FOXP3+ Tregs) and M2‐type CD163+ macrophages. Together, these changes led to a more effective reduction in the lung tumor burden and inhibited tumor cell proliferation. 83 Furthermore, Ni et al. discovered that pFLASH had a significant impact on TAM. Specifically, pFLASH eliminated lipid oxidase expression and reduced low‐density lipid oxidation. This decreased PPARγ activity and arginase 1 expression, thereby inhibiting immunosuppressive macrophage polarization. 86 In a rat model of orthotopic glioma, the advantages of pFLASH (257 Gy/s ± 2) over CPT (4 Gy/s ± 0.02) were not in tumor control but in alleviating neuroinflammation‐induced memory damage and increasing the infiltration of tumor‐infiltrating lymphocytes (TILs, including CD4+T cells, CD8+T cells, Tregs, CD8+TRMs, NK cells, and B cells) in the TME 8 days after radiation. 74 The effects of pFLASH on specific immune cells were complex. For example, compared to CPT, pFLASH treatment reduced Tregs in the TME. 83 However, this reduction was not statistically significant in the orthotopic glioma rat model (P>0.05). 74 The varied effects of proton therapy on tumor‐infiltrating immune cells may be due to two factors: (i) immune cell heterogeneity within the TME across tumors 93 and (ii) heterogeneity of the radiation dose. 94 Moreover, overemphasizing the effects of proton therapy on single‐type cells may be inappropriate, because the interactions of immune cells in the TME are complex and critical. 95 Hypoxia causes immune suppression in the TME. 96 However, oxygen supplementation did not reverse the immune suppression caused by proton therapy in glioma‐bearing rats. 31 , 97

Changes in cytokines and chemokines

Proton therapy has been linked to significant alterations in cytokine and chemokine levels within the TME. Proton therapy increases pro‐inflammatory cytokines such as interleukin‐6 (IL‐6), interleukin‐1β (IL1β), and tumor necrosis factor‐α (TNFα), which regulate immune responses and tumor progression. For example, a study on HCC indicated that after 3 days of proton therapy, the tumor volume decreased, which was accompanied by an increase in IL‐6, suggesting that a pro‐inflammatory environment may contribute to tumor control. 85 , 98 A recent study using a rat GBM model also revealed an increase in pro‐inflammatory IL1β, chemokine ligand 1, and TNFα 7 days after proton radiation. 87 Furthermore, in patients with HNSCC, proton therapy has been found to decrease the production of exosomes that suppress immune function, thereby potentially enhancing overall immune surveillance against tumors. 84 These changes in cytokine profiles highlight the key role of proton therapy in TIME remodeling, shaping a complex inflammatory and immune response against tumors.

Regulation of immune checkpoint expression

Immune checkpoints such as programmed cell death 1 (PD1), programmed cell death–ligand 1 (PD‐L1), cytotoxic T‐lymphocyte–associated protein 4 (CTLA4), and lymphocyte activation gene‐3 (LAG3) are essential for tumor immune evasion. 99 , 100 Recent studies have suggested that proton therapy affects anti‐tumor immunity by altering immune checkpoint expression. For instance, Chen et al. observed that photon therapy increased PD‐L1 expression in HCC cells both in vitro and in vivo. 85 Although an increase in PD‐L1 was positively correlated with the RBE in HCC, 85 this finding has been challenged by other studies. Shukla et al. found that after 8 days of pFLASH (60 Gy/s), the proportion of PD‐1+CD3+ T cells decreased, accompanied by a reduction in PD‐L1 expression in lung cancer cells. 83 Therefore, arbitrarily increasing the proton irradiation dose is not recommended because of its effects on immune checkpoint expression.

The TIME, including infiltrating immune cells, cytokines, chemokines, and immune checkpoints, has been illustrated in Figure 1. For immune cells, we mainly focused on T cells, B cells, NK cells, TAMs, and neutrophils. For cytokines and chemokines, we illustrated the alterations in IL‐6 and exosomes. We have also introduced the expression of PD‐L1, which changes dynamically with the dose of proton irradiation. Abbreviations: Tregs, regulatory T cells; CD8+ TRMs, CD8+ tissue‐resident memory cytotoxic T cells; TAMs, tumor‐associated macrophages; NK Cells, natural killer cells; PD‐1, programmed cell death 1; PD‐L1, programmed cell death ligand 1; TIME, tumor immune microenvironment.