Proton radiation therapy

Proton therapy uses a beam of protons of primarily medium LET which are positively charged particles (essentially hydrogen nuclei) with a very well-defined range of tissue penetration, to deliver radiation to a targeted tumour. Higher-LET values do exist across the track of the proton beam particularly towards the distal edge of the spread-out Bragg peak (SOBP), compared with conventional X-ray radiation (Ref. Reference Vanderwaeren21). This radiation modality allows for a better distribution of the delivered dose of radiation compared to photon (X-Ray) radiotherapy and has led to an expansion of the treatment options in radiation oncology. The SOBP is a combination of several Bragg peaks in such a way, that an entire tumour can be targeted with minimal dose deposition to adjacent normal tissues. The idea to use protons in cancer treatment was conceived by the physicist Robert R. Wilson in 1946 (Ref. Reference Wilson22) and the first patients were treated in 1954 (Ref. Reference Lawrence23). We have now amassed a substantial amount of clinical evidence on the efficacy of proton radiation treatment (Ref. Reference Yuan, Zhan and Qian24). In a recent comparative analysis of protons versus photons in combination with chemotherapeutic regimens, proton chemoradiotherapy presented significantly reduced acute adverse events that were causing unplanned hospitalisations, with similar disease-free and overall survival (Ref. Reference Baumann25).

A new exciting development in the field of radiotherapy is the so-called ‘FLASH’ radiotherapy which has quickly evolved into a highly promising radiation modality. Proton and other particles (such as electrons or carbon ions) are delivered at ultra-high (‘FLASH’) dose rates (currently at >40 Gy/sec of higher) whereas in the clinic, the ‘standard’ dose rates are at approximately 5 Gy/min (Ref. Reference Hughes and Parsons26). FLASH radiotherapy has demonstrated increased sparing of the normal tissue while maintaining an equipotent antitumour efficacy compared to conventional radiotherapy in multiple preclinical studies (Refs Reference Favaudon27–Reference Diffenderfer34). The medium LET that characterises proton radiation, along with the SOBP can enhance the potency of ‘FLASH’ effect during proton radiotherapy (Ref. Reference Hughes and Parsons26). A major advancement in the field of FLASH radiotherapy is the onset of the world's first human clinical trial of FLASH RT by the Cincinnati Children's/UC Health Proton Therapy Center. The study named as FAST-01 (FeAsibility Study of FLASH Radiotherapy for the Treatment of Symptomatic Bone Metastases) (NCT04592887), will be delivering a single radiotherapy session to the recruited cancer patients in a fraction of a second. For a recent analytical review on FLASH radiotherapy and potential biological mechanisms underlying its biological effects the reader is directed to (Ref. Reference Hughes and Parsons26).

Carbon ion therapy

Carbon ion radiotherapy (CIRT) is another form of particle beam therapy which provides a series of physical and biological advantages (Refs Reference Malouff35, Reference Brown and Suit36). The larger mass of carbon allows for even more reduced beam scattering, characterised by a steeper dose distribution area with minimal penumbra and sharper Bragg peak (Refs Reference Rackwitz and Debus37–Reference Durante and Loeffler39) compared to protons, theoretically allowing for better tolerance of radiosensitive organs (Refs Reference Kamada40–Reference Imada42). Moreover, carbons demonstrate higher LET than protons, something which leads to a two to three fold-higher RBE compared to protons and photons (Ref. Reference Raju and Carpenter43) due to the more dense and complex DNA damage (Ref. Reference Mohamad38). Other differences of CIRT compared to other radiotherapies include the lack of oxygen effect, sub-lethal damage repair and the reduced impact of the cell cycle on radiosensitivity (Ref. Reference Ebner and Kamada44). Today, there are 13 CIRT centres in five countries treating tumours, such as prostate, head and neck, lung, liver, bone, soft tissue sarcomas, locally recurrent rectal cancer and pancreatic cancer (Refs Reference Kamada40, Reference Shinoto45). Unfortunately, there is still scarce evidence (e.g. comparative clinical trials) comparing head-head the efficacy and toxicity of CIRT to other particle or photon RT.

Nuclear radiotherapy

The biological effects of external beam Radiotherapy are sufficiently described by the ionisation effects occurring while accelerated ions traverse and interact with the biological matter based on nuclear physics laws. Many decades ago, a combinatorial Radiotherapy modality was proposed, one where in addition to primary ionisations, accelerated particles would also cause, nuclear reactions to atoms of a suitable substrate, thus creating heavier ions (commonly α-particles), which are characterised by higher LET values. In this review, we use the term Nuclear Radiotherapy to distinguish this modality from conventional external beam RT. The adjective ‘Nuclear’ within the term implies the need of nuclear physics to describe the reactions taking place, the possible nuclear energy states of implicated particles as well as their kinetic energy and finally their LET values. High LET ions are expected to cause more severe damage to the targeted cancer cells. In this review, we will focus on proton-boron (fusion/) capture therapy (PBCT) and boron-neutron capture therapy (BNCT) (see below and Supplementary data). Although initially there was a great deal of promise in the use of Nuclear Radiotherapy approaches, these have not fulfilled their promise for reasons we will outline below. A major shortcoming of Nuclear Radiotherapy has been attributed to the limited possibility of a nuclear interaction in practice. Even if an accelerated charged particle has the exact amount of energy to excite the targeted molecule/atom and its trajectory indeed points towards the target, the possibility (cross section) for the nuclear reaction to be triggered is at least 10⁴ times smaller than the corresponding one for the ionisation process, even when high-energy ions or electrons are used as primary particles (Refs Reference Kaganovich46, Reference Tawara and Kato47). In the case of fast neutrons, an elastic and inelastic scattering are in general the dominant mechanisms for energy moderation, since human tissues act as an excellent absorber for neutron capture (n,γ) reactions in the thermal (0.025 eV) and epithermal energy region (0.025–4 eV). Thus the predominant mechanisms for radiation damage and energy deposition are usually not the intended ones via the specific, useful (n, α) nuclear reactions. Additionally, the delivery of adequate amounts of boronated compounds to the tumour while avoiding any significant uptake in normal tissues constitutes one of the most challenging issues for a successful treatment with the BNCT or PBCT techniques (Ref. Reference Malouff48). Thus, there are still many issues open for scientific investigation before the practical clinical stage is reached. Despite the clinical failure of these approaches, they have laid the foundations for a more efficacious approach based on similar principles such as PBCT (see below and in Supplementary data).

Proton–boron capture therapy

PBCT is an innovative method towards increased proton biological effectiveness. PBCT is based on pure nuclear physics principles and uses a nuclear fusion reaction between low-energy protons and ¹¹B atoms, that is

(1)$${\rm p} + ^{11}{\rm B}\;\to \;^{12}{\rm C} ^\ast \;\to 3\alpha $$

(2)$${\rm p} + ^{11}{\rm B}\to ^{12}{\rm C} ^\ast \to \alpha _0 + ^8Be, \;^8Be\to 2\alpha $$

(3)$${\rm p} + ^8{\rm B}\to ^{12}{\rm C} ^\ast \to \alpha _1 + ^8Be ^\ast , \;^8Be ^\ast \to 2\alpha $$

The reaction produces high LET α-particles of enhanced damaging potential ideally solely on the desired target tumour area that is SOBP, without damaging neighbouring normal tissues (Refs Reference Bláha49, Reference Maliszewska-Olejniczak50).

Proton Boron reaction involves the production of ¹²C in one of its excited (nuclear) states (¹²C*). For the resonance occurring at 0.675 MeV (projectile energy) (Ref. Reference Ajzenberg-Selove51) there are three main options of de-excitation. Two of them are described by the 2-step reaction ((1) and (2)) and the third by the 1-step reaction (1) (Ref. Reference Stave52). According to Stave et al. the predominant one is the 2-step reaction, which involves the production of a Be nucleus (Ref. Reference Stave52). In order to investigate the α-particles biological effects, exact knowledge of their LET is required. To calculate the LET values we need to know the energy values that they are emitted with and this can be considered as a rather complicated problem. In the following, we describe why this issue is a bit complicated. Figure S1 (Supplementary data) illustrates the phenomenon. The p + ¹¹B → 3α reaction releases an amount of energy Q = 8.59 MeV for example [http://nrv.jinr.ru/nrv/webnrv/qcalc/], which has to be shared (along with a fraction of approx. 11/12 of the incoming proton's kinetic energy) among the three ejectiles, that is the α-particles with varying energies as shown in Table S1 (maximum 231 keV/μm).

BNCT is a radiotherapeutic modality which targets cancer cells via a two-step procedure. During the first stage, the patient is injected with a tumour-localising drug which carries the stable isotope boron-10 (¹⁰B) which has a high tendency to capture low energy ‘thermal’ neutrons (n _th). The most widely used boron delivery agents are the sodium borocaptate (BSH) and the boronophenylalanine (BPA), while the latter can be associated to fructose to yield BPA-F with enhanced solubility (Ref. Reference Barth, Mi and Yang53). In the second stage, the patient is subjected to irradiation with neutrons and the subsequent nuclear reactions occurring when ¹⁰B is hit with neutrons, lead to the emission of high-energy α-particles (⁴He) and recoiling lithium-7 (⁷Li) nuclei that kill the cancer cells that have accumulated sufficient quantities of ¹⁰B (Refs Reference Barth, Mi and Yang53–Reference Barth55). The basic interaction equations are described below:

(4)$$^{10} {\rm B} + n_{th}\to ^{11}{\rm B}^\ast \to \alpha _0 + ^7Li$$

(5)$$^{10} {\rm B} + n_{th}\to ^{11}{\rm B}^\ast \to \alpha _1 + ^7Li^\ast $$

Using catkin 2.03 and jinr Q-value_calc [http://nrv.jinr.ru/nrv/webnrv/qcalc/] one can easily determine the levels of the kinetic energy of implicated particles, considering for neutrons T_n = 0.025 eV:

$${\rm T}_{\alpha _0\;\;}is \,1.776\,{\rm MeV}\;{\rm while}\;{\rm T}_{Li} = 1.014\,{\rm MeV}\;{\rm T}_{\alpha _1} = 1.472\,{\rm MeV\ and}\;{\rm T}_{Li\ast } = 0.840\,{\rm MeV}$$

Figure S2 (Supplementary data) illustrates the energy levels implicated in boron-neutron fusion. The major benefit of this approach is the protection it affords to critical normal tissues from radiation-induced damage, even at the margins of the tumour (Ref. Reference Barth, Zhang and Liu54). This supports the use of BNCT as an alternative therapeutic option for the treatment of multiple, locally invasive malignant tumours such as high-grade gliomas (Ref. Reference Miyatake56), recurrent cancers of the head and neck region (Ref. Reference Wang57) and primary tumours or cerebral metastases of melanoma (Ref. Reference Hiratsuka58). Current clinical evidence mostly revolves around the use of non-radioactive ¹⁰B. Gadolinium-157 (Gd), another non-radioactive isotope has also been used but the present data have been strictly derived from in vivo studies without further examination of its efficacy in the clinic (Refs Reference De Stasio59–Reference Shih and Brugger61).

There are prerequisites for the ideal boron-containing candidate such as low systemic toxicity, much higher tumour uptake compared to the normal tissue and rapid clearance from normal tissues. Currently, the most successful boron delivery agent, BPA selectively accumulates in tumours through cancer-related amino acid transporters including LAT1 (Ref. Reference Nomoto62). However, this approach also carries disadvantages such as reduced solubility and a short retention time (Ref. Reference Fukuda63). The demand for better tumour targeting properties and a noticeable difference in the rate of uptake of boron-containing agents by different patients, have ushered in the production of several generations of boron-containing agents including boron-containing porphyrins, amino acids, polyamines, nucleosides, peptides, monoclonal antibodies, liposomes, nanoparticles of various types, boron cluster compounds and co-polymers (Refs Reference Barth, Mi and Yang53, Reference Barth55, Reference Farr64–Reference Coderre67). The current list of malignancies for which BNCT has shown at least some clinical benefit is quite long (Ref. Reference Malouff48) and includes glioblastoma multiforme (Refs Reference Miyatake56, Reference Chanana68–Reference Kawabata70), meningioma (Ref. Reference Miyatake71), head and neck cancers (Refs Reference Wang57, Reference Kankaanranta72–Reference Wang76), lung cancer (Ref. Reference Suzuki77), sarcomas (Ref. Reference Futamura78), cutaneous malignancies (Refs Reference Menéndez79–Reference Yong81), extramammary Paget's disease (Ref. Reference Hiratsuka58), paediatric cancers (Refs Reference Nakagawa82–Reference Kageji84) and metastatic disease (Refs Reference Wittig85, Reference Zonta86). These studies underscore the promise of PBCT as a therapeutic strategy, especially for radiotherapy-resistant tumours. Moreover, if coupled with proton FLASH radiotherapy modalities, BPCT could further enhance the proton therapy therapeutic index. (Ref. Reference Bláha49).

Proton Based therapies

Proton therapy is a quickly expanding radiotherapy modality where accelerated proton beams are utilised to accurately transport their maximum of energy and dose to the tumour but is usually ineffective against highly radioresistant tumours. In the case of protons, typical energies used in the clinic regularly range from 60–250 MeV including the case of the latest advances in the field that is, the FLASH method where ultra-high dose rates 40–200 Gy/s can be achieved through the delivery of very short proton ms-pulses (Refs Reference Darafsheh91, Reference Kourkafas92).

Theoretical considerations

In an effort to have a better understanding of the primary molecular effects of high-LET radiations, we have mined accumulated data on the induction rates of double and single-strand breaks (DSBs and SSBs respectively) for a variety of biological systems and both experimental and simulation data (Tables 1–3). Theoretical predictions of biological damage from DNA to cellular survival are always very helpful and have aided significantly the planning of experiments and interpretations of results. By the word of theoretical predictions, we refer to the well-accepted and acknowledged stochastic modelling using Monte Carlo (MC) simulations and particle track structure codes [for recent reviews please see (Refs Reference Nikjoo93, Reference Chatzipapas94)] as well as others non-stochastic using mathematical or machine learning-based calculations (Refs Reference Kalospyros95–Reference Papakonstantinou97). It must be mentioned though that all simulations of any type are optimised based on experimental data. For example, using the Geant4DNA MC code and an improved DNA geometry model as well as clustering algorithms, DNA damage yields were calculated and compared to literature (Ref. Reference Zhao98). The model was tested against published experimental and computational results for DSB rates and RBE values. A general agreement was observed over the whole simulated proton energy range. The DSB yield was 8.0 ± 0.3 DSB Gy⁻¹Gbp⁻¹ for ⁶⁰Co, and 9.2 ± 0.2 DSB Gy⁻¹Gbp⁻¹for 250 kVp X-rays, and between 11.1 ± 0.9 and 8.1 ± 0.5 DSB Gy⁻¹Gbp⁻¹for 2–100 MeV protons. The specific results suggest that DSBs consist of direct and indirectly-induced (through ROS) SSBs that account approximately for half of the total DSBs (Ref. Reference Zhao98).

Table 1. Protons and electromagnetic irradiation

Table 2. Carbons and electromagnetic radiations

Table 3. α-particles and electromagnetic radiations

Table 1 contains data from in vitro and in silico works using protons and electromagnetic irradiation. Variations among different simulations can be attributed to different initial conditions fed into the algorithm, and especially the concentration of antioxidants, oxygen levels and nuclear DNA density. As a general conclusion, it can be deducted that an increase in LET leads to an increase of DSBs and to a reduction of SSB levels as well as of other non-DSB oxidative damage (Refs Reference Zhao98–Reference Pater101). This of course suggests a higher level of complexity for DSBs at least.

Carbon Ion radiotherapy

CIRT is the treatment of choice for proton-resistant tumours like sarcomas (Ref. Reference Malouff35). Table 2 contains data from in vitro and in silico studies of carbons. Keta et al. (Ref. Reference Keta102) irradiated three malignant cell lines with protons or carbons and they evaluated DNA damage by quantifying γH2AX foci. No significant differences were observed in the γH2AX foci formation between the two radiation modalities and they attributed this result to the limited resolution that ICC offers when performed using conventional fluorescence microscopy. Yokota et al. (Ref. Reference Yokota105) using pulsed-field gel electrophoresis (PFGE) quantified DSBs yields in protoplasts from the tobacco BY-2 cell line. They identified a difference in the induced DSB between γ-rays and carbon ions, but they could not satisfactorily detect a difference in the DSB yields among the several LET values of carbons. Moreover, their approach failed to exhibit expected results that is higher DSBs for the highest LET used. They interpreted this result by the phenomena occurring to the carbons penumbra region and to the possible dosimetry inaccuracies. Back in 1995, Heilmann et al. (Ref. Reference Heilmann, Taucher-Scholz and Kraft106) irradiated CHO-K1 cells with carbons of different energies and assessed DNA damage using constant-field gel electrophoresis. Their results, although not revealing an increased number of DSBs, underline the possible inability of electrophoretic approaches to detect very small DNA fragments as a result of highly complex DSBs, a phenomenon also discussed in (Ref. Reference Hada and Georgakilas8). Shiina et al. (Ref. Reference Shiina107) compared carbons of 60 keV/μm LET and X-rays using agarose gel electrophoresis (AGE), in several Tris scavenger concentrations. Comparing results for minimum (1.0 × 10⁶/s) and maximum (Tris 1.0 × 10¹⁰/s) scavenging capacities of Tris. Corresponding values of Tris scavenging capacity that mimics cellular environment, that is 3.0 × 10⁸/s are also presented in Table 2. An increase in scavenging capacity causes a decrease of both DSB and SSB levels, for every radiation modality.

α-particles Produced during nuclear radiotherapy

α-particles are produced during nuclear radiotherapy as well as during brachytherapy. They have short ranges in the body, depending on their emission energy, usually less than 100 μm. Their energy and consequently their LET change dramatically with distance, when traversing tissues or water, which is considered roughly radiologically equivalent to tissue. Table 3 includes studies using plasmid DNA, protoplasts, cells or simulations: Pater et al. (Ref. Reference Pater101) using MC simulations observe similar dependence of DSB and SSB with LET for α-particles and for protons: LET increase leads to DSB increase against SSB levels which decrease. Yokota et al. (Ref. Reference Yokota105) counted DSB yields after exposure to α-particles or γ-rays, by using protoplasts from the tobacco BY-2 cell line and by performing PFGE: LET increase resulted to increase of DSB levels. In another study, by using plasmid pUC18 and AGE, it was found that LET increase leads to an increase of DSB yields but it was not detected to affect the number of SSBs per Gy and per Gbp (Ref. Reference Urushibara110). Prise et al. (Ref. Reference Prise, Pullar and Michael111) compared α-particles and γ-rays irradiation using pNSG-CAT plasmid and AGE at two concentrations of the ROS scavenger Tris. At both concentrations, similar results with (Ref. Reference Pater101) for both scavenging conditions were acquired. A study using V79-379A hamster cells and PFGE shows increased DSB levels for α-irradiation compared to X-rays (Ref. Reference Claesson112). To summarise, all the studies show that the LET increase leads to DSB increase against SSB, at least for moderate LET values.

The Role of apoptosis, autophagy, necrosis and senescence in high-LET radiotherapy

Cells exposed to IR can exhibit a multitude of responses. Depending on the radiation type, LET (low- or high-), dose, dose rate and radiosensitivity of the exposed cell type, IR may induce cell death through apoptosis, autophagy or necrosis, mitotic catastrophe or even cell cycle arrest and accelerated senescence (Ref. Reference Golden138). For most cancer cell types, mitotic catastrophe is the most common end result of IR, whereas apoptosis (programmed cell death) is associated primarily with hematopoietic cancers (Refs Reference Eriksson and Stigbrand139, Reference Radford140) which have a lower apoptotic threshold. The primary aim of RT is to clonogenically kill cancer cells, causing the minimum disruption to healthy ones; (Refs Reference Di Pietro141, Reference Jinno-Oue142). Apoptotic pathways after IR have been defined and these are the membrane stress pathway, the intrinsic pathway and the extrinsic pathway (Ref. Reference Rahmanian, Hosseinimehr and Khalaj143). Different studies for example on the head and neck cancer underline the differences between cell death mechanisms for low or high-LET radiation therapies (for a recent review please see (Ref. Reference Fabbrizi and Parsons144)).

The intrinsic pathway of apoptosis is mediated by DNA SSBs and DSBs. When DNA lesions are repairable, the emerging growth arrest is transient, eventually resulting in DNA repair, cell cycle continuation, and a return to cell replication and growth. In contrast, when DNA lesions are severe or irreparable (e.g. severe DNA damage or complex damage), prolonged DDR signalling is triggered, resulting in apoptosis or senescence (permanent growth arrest) (Ref. Reference Campisi and d'Adda di Fagagna145). The extrinsic pathway on the other hand, involves signalling through death receptors (DRs), which belong to the tumour necrosis factor (TNF) receptor superfamily. The accumulation of p53 is critical to IR-induced apoptosis or senescence (Ref. Reference Alvarado-Ortiz146). Several target genes of the p53 transcription factor encode proteins, such as p21 and p27 that induce cell-cycle arrest, PAI1 and CDKN1b which are involved in the induction of senescence, or PUMA, BAX and NOXA which are mediators of apoptosis (Ref. Reference Kastenhuber and Lowe147). Most solid tumour cells have either inactivating p53 mutations or defective apoptotic machinery, which makes them radioresistant and might affect the response to RT (Refs Reference Rödel148, Reference Cuddihy and Bristow149). Necrosis, another type of cell death, is an outcome usually appearing in response to very large doses of radiation (>55 Gy), although as a rare side-effect (Ref. Reference MacDonald, Gunderson and Tepper150). There are cases though, that even doses as low as 2–4 Gy can cause significant necrosis. Most replicating cells die by the mitotic catastrophe which mistakenly some researchers equate to necrosis [the reader can consult this seminal review (Ref. Reference Brown and Wouters151)].

It may occur in lower doses as well, but it is often viewed as an unregulated event. Autophagy is a mechanism responsible to maintain metabolic homoeostasis through the segregation of damaged or unwanted cytoplasmic constituents into autophagosomes, destined for lysosomal degradation. Autophagy is directly correlated to the DDR, since there is evidence that is activated by DNA damage and is required for several functional outcomes of DDR signalling, including repair of DNA lesions, senescence, cell death and cytokine secretion (Ref. Reference Eliopoulos, Havaki and Gorgoulis152). In response to IR, it is yet unclear whether autophagy promotes cell survival or cell death (Refs Reference Wu153–Reference Palumbo and Comincini155) although it could be either of the two depending on cell context and dose. Senescence is the process by which cells irreversibly stop proliferating and enter a state of permanent growth arrest (Ref. Reference Gorgoulis156). IR has been shown to induce senescence especially via the induction of DNA damage. Understanding the mechanisms that result in one of the above cellular responses is extremely important especially for an effective RT. Patients that have received stereotactic body radiation therapy (SBRT), frequently experience recurrence [e.g. non-small cell lung cancer (NSCLC) patients, stage I], reducing the overall effectiveness of radiotherapy effective (Ref. Reference Spratt157). It has been postulated that tumour recurrence is can derive from senescent transformed cells in the tumour area which reverse or escape the senescent phenotype (Ref. Reference Liao158).

Due to the importance of RT induced-cell death, many studies have been performed with proton, carbon and even FLASH radiation in an attempt to improve the efficacy of RT. For example, Zhang et al. showed that irradiation of chemoresistant cancer stem cells (CSCs) with 4Gy of protons significantly lowered cell viability, invasiveness and a number of tumour spheres compared to gamma rays. This effect was accompanied by increased apoptosis and higher ROS level (Ref. Reference Zhang159). In general, there is an agreement in the literature that proton radiation causes increased levels of apoptosis than photon radiation (Refs Reference Di Pietro141, Reference Zhang159, Reference Ristic-Fira160). Miszczyk et al. investigated the rate of cell killing by apoptosis or necrosis in human peripheral blood lymphocytes and found that irradiation with 60 MeV SOBP caused higher levels of cell death compared to 250 kV photons through necrosis (Ref. Reference Miszczyk161). Wang et al. assessed cell death in four HNSCC cell lines after a single dose of 4 Gy 6 MV photons or 200 MeV SOBP protons and didn't find any differences between the levels of necrosis and apoptosis. They did however, observe increased senescence and mitotic catastrophe following proton irradiation (Ref. Reference Wang162). These differences can be attributed to the different cell types used for the experiments. Normal human diploid lung fibroblasts exposed to FLASH dose rate protons, showed that proton dose rate did not influence cell killing, but by increasing the dose or the dose rate they reported different rates of senescence. After they exposed cells to 20 Gy 4.5 MeV protons, they found that compared to 0.05 Gy/s, the FLASH dose rates of 100 Gy/s or 1000 Gy/s led to a reduced number of senescent cells (Ref. Reference Buonanno, Grilj and Brenner163). Han et al. used FLASH radiation (~10⁹ Gy/s, with doses in the range of ~10–40 Gy) on normal mouse embryonic fibroblast cells and analysed early apoptosis, late apoptosis and necrosis of cells under both normoxic and hypoxic conditions (Ref. Reference Han164). Their results showed that FLASH-IR compared to conventional proton therapy induced significant early apoptosis, late apoptosis and necrosis, and the levels of apoptosis increased with time, in either hypoxic or normoxic conditions. Also, in hypoxic conditions, apoptosis and necrosis were significantly lower. As also shown conclusively in Table 4, in most cases, protons induce mainly apoptosis and not necrosis. This could impact radiotherapy because cells undergoing necrosis lose membrane integrity and leak their intracellular components some of which serve as danger signals that stimulate inflammation and strong innate immune responses (immunogenic cell death) (Ref. Reference Rock and Kono165) impacting tumour radiosensitisation and radiation toxicity (Ref. Reference Golden138).

Table 4. Reported cell death responses after high-LET radiation

Atsushi et al. irradiated human glioma cells (NP-2), with carbon ions and found that at 6 Gy the majority of the cells die by apoptosis and autophagy. The residual cells could not form a colony and showed a morphological phenotype consistent with cellular senescence (Ref. Reference Jinno-Oue142). The detection of senescent cells was also confirmed with SA-β-gal staining. The same group also found that irradiation with carbon ions induced cellular senescence in the same cell line lacking functional p53. Finally, one study has provided significant data in response to carbon ion radiation and autophagy, implying that targeting autophagy might enhance the effectiveness of heavy-ion radiotherapy (Ref. Reference Jin173). They also found that by inhibiting autophagy apoptotic cell death was accelerated, resulting in increased radiosensitivity. The biological cell death effects discussed in this section are summarised below in Table 4.