Utilizing Carbon Ions to Treat Medulloblastomas that Exhibit Chromothripsis

Novel radiation therapies with accelerated charged particles such as protons and carbon ions have shown encouraging results in oncology. We present recent applications as well as benefits and risks associated with their use. We discuss the use of carbon ion radiotherapy to treat a specific type of aggressive pediatric brain tumors, namely medulloblastomas with chromothripsis. Potential reasons for the resistance to conventional treatment, such as the presence of cancer stem cells with unique properties, are highlighted. Finally, advantages of particle radiation alone and in combination with other therapies to overcome resistance are featured. Provided that future preclinical studies confirm the evidence of high effectiveness, favorable toxicity profiles, and no increased risk of secondary malignancy, carbon ion therapy may offer a promising tool in pediatric (neuro)oncology and beyond.


Introduction
Radiotherapy is one of the most frequently applied cancer treatment modalities, which is based on the medical use of ionizing radiation. One fundamental principle of radiotherapy is to maximize the dose deposition within the target volume, leading to tumor shrinkage, while minimizing the radiation exposure of the surrounding healthy tissue. Emerging radiation strategies with accelerated charged particles such as protons or heavy ions (e.g., carbon ions) have shown promising results in different tumor entities [1,2].
Favorable physical and radiobiological characteristics of charged particles could provide potential clinical benefits over conventional radiotherapy utilizing photons [3]. Charged particles have a distinct depth-dose profile characterized by a low entrance dose and maximum dose deposited within the so-called Bragg peak. The subsequent steep dose fall-off allows sparing of normal tissue beyond the target volume [2,4]. In comparison, photons deposit their dose continuously while crossing the tissue, resulting in a high entrance and considerable exit dose. This limits the prescribed dose of photons to the maximum tolerable dose for healthy tissue, which may not suffice to eliminate aggressive tumors, potentially leading to treatment failure.
Unlike photons, which deposit most of their dose upon entering the tissue, particle radiotherapy provides the opportunity to treat deep-seated tumors and perform precise irradiation of tumors close to organs at risk (e.g., brain stem, optic nerve, or intestine) due to the properties of the Bragg peak. The characteristic depth-dose profile of particles decreases the risk of treatment-related late toxicities often associated with photon radiotherapy [2,3].
An advantage of heavier charged particles such as carbon ions as compared to other radiotherapies is the high relative biological effectiveness (RBE) [2,5]. The RBE is defined as the ratio of the photon dose to the charged particle dose with This article is part of the Topical Collection on Radiation Biology the same biological effect. The high RBE of carbon ions can be explained by its high linear energy transfer (LET), which results in an increased ionization density compared to irradiation with photons or protons. The dense ionization of DNA molecules leads to complex DNA damage, due to the formation of multiple clustered DNA double-strand breaks (DSBs) [6,7]. Clustered DSBs are repaired less efficiently in cells as compared to less complex damage and thus lead to higher effectiveness in cell killing [6,8].
Furthermore, particle radiation could be used to overcome radioresistance to conventional radiotherapy. In particular, the sensitivity of cells to high-LET irradiation with carbon ions is less dependent on the presence of oxygen compared to photon irradiation, which potentially provides an advantage to carbon ions to eliminate hypoxic tumors [8,9]. As the radiation response to carbon ions is largely independent of the cell cycle phase of the irradiated cells, slowly proliferating and quiescent cells could be targeted as well [7,10]. In addition to these effects, carbon ion radiation has shown the potential to prevent metastatic spread in several tumor types, such as sarcoma or glioma [11,12]. In contrast, irradiation with photons could potentially lead to increased tumor cell migration and more metastases [13].
Despite a number of advantages that advocate the use of particles, radiation with photons remains the standard approach in radiooncology for several reasons, including the broad availability of this treatment modality and safety considerations inherent to alternative new technologies. Due to the limited access to particle beam treatment facilities, especially those offering carbon ion radiation, most of the up-to-date research and clinical trials utilizing particles have been based on protons rather than on heavy ions. As of 2019, a total of 222,425 cancer patients were treated with protons and 34,138 patients with carbon ions worldwide [14].
While indications for proton radiotherapy are increasing [15], carbon ions are utilized more reluctantly, especially in pediatric (neuro)oncology. This is mainly due to safety concerns linked with novel treatment strategies and still uncertain radiobiologic effects on normal tissues. Nevertheless, considering the advantageous physical and radiobiologic characteristics of carbon ions, it will be of critical importance to achieve a better understanding of the mechanisms of action and of putative side effects for the future use of carbon ions for cancer treatment.

Carbon Ion Radiotherapy: a Potential New Strategy to Treat High-Risk Medulloblastomas?
Medulloblastoma is the most frequent malignant brain tumor in children. It can be stratified into four major molecular subgroups: Wingless, Sonic hedgehog, Group 3, and Group 4 [16]. Each subgroup presents unique molecular and clinically relevant features, and the subgroup determination is an integral part of the diagnosis and treatment decision [17]. The front-line treatment for medulloblastoma involves maximum safe surgical resection, followed by risk-adapted postoperative radiotherapy and conventional chemotherapy [17]. Clinical outcomes vary largely across the molecular subgroups and are also associated with specific gene mutations or cytogenetic features [18].
Sonic hedgehog medulloblastoma patients who carry germline TP53 mutations belong to a very-high-risk group with dismal prognosis, with a 5-year overall survival rate of 27% [19]. Germline mutations in TP53 lead to a cancer predisposition syndrome termed Li-Fraumeni syndrome (LFS) [20,21]; hence, medulloblastomas are also regarded as an LFS-associated disease. Current standard treatments show limited efficacy and pose a risk of treatment-induced secondary tumors due to the germline TP53 mutations. One characteristic feature of these aggressive medulloblastomas is extensive genome instability, seen as massive genomic rearrangements caused by a phenomenon termed chromothripsis [22].
Chromothripsis is commonly described as a form of genome instability which occurs as a single catastrophic event and leads to the shattering of one or a few chromosomes through the formation of multiple locally clustered DNA DSBs [23]. The subsequent error-prone repair of shattered chromosomes can lead to numerous aberrations, including the loss of chromosome fragments, gene fusions, chromosomal rearrangements, the loss of gene function, or formation of double minute chromosomes. In Sonic hedgehog medulloblastomas, chromothripsis was proposed to occur as a consequence of TP53 mutations in the germline [22]. Beyond pediatric medulloblastoma, chromothripsis was linked with dismal prognosis for the patients in a number of other cancer entities including multiple myeloma [24], neuroblastoma [25], and acute myeloid leukemia [26].
By utilizing orthotopic patient-derived xenograft (PDX) mouse models, a recent study in our group investigated the use of carbon ions as a novel strategy for the treatment of matched primary and recurrent pediatric Sonic hedgehog medulloblastomas with a germline TP53 mutation [27]. The extensive genome instability and pronounced homologous recombination repair deficiency of these tumors provided a potential vulnerability that we decided to target using carbon ions in combination with PARP inhibitors. In the treatmentnaive primary tumor model, carbon ions prolonged survival and resulted in a complete response, allowing for a longterm follow-up (300-day post-irradiation). The analysis of the putative long-term side effects in cured mice showed no detectable impact of carbon ions on neurogenesis in the mouse brain and no detectable radiation-induced secondary tumors in the surrounding healthy brain tissue. In order to mimic the TP53 germline mutation in medulloblastoma patients with Li-Fraumeni syndrome, we performed the same targeted carbon ion irradiation in a genetic Trp53 + / − mouse model. Magnetic resonance imaging and histopathological analysis performed 200 days after irradiation did not detect structural brain impairments or radiation-induced lesions.
These encouraging results and, in particular, the complete tumor response and the favorable toxicity profile warrant further investigation of carbon ion radiotherapy in primary medulloblastomas with chromothripsis. Acquiring further data on the potential neurological and developmental consequences as well as the absence of late radiation-induced tumors will be important for advocating the use of carbon ions in children.
Furthermore, it is important to point out that not all tumor types demonstrate the same radiosensitivity and complete response to carbon ions. For instance, in patient-derived mouse models of primary glioma [28] and soft tissue sarcoma [29], carbon ions did not lead to complete response. In addition, even though the recurrent medulloblastoma model in our study showed radiosensitivity associated with a period of tumor growth delay, the effect of carbon ions was not curative as in the matched primary tumor model [27]. While potential reasons for the treatment resistance of the recurrent medulloblastoma model are discussed later on, it will be essential to achieve a better understanding of the reasons for the sensitivity and remarkable response in the context of primary medulloblastoma. It will be critical to investigate the effects of the treatment in a broad range of medulloblastoma models with and without chromothripsis, to evaluate the vulnerability and the response to carbon ions. Ultimately, this could potentially allow to indicate carbon ion radiotherapy for the treatment of further tumor types.

Recurrent Medulloblastoma
Tumor recurrence due to treatment resistance is unfortunately still a common fate for patients across different cancer entities. In medulloblastoma, tumor recurrence leads to universally poor outcomes (5-year overall survival rate < 10% in the context of recurrent medulloblastoma) [30,31]. There is currently no effective treatment for recurrent medulloblastoma. Patients are subjected to repeated surgical resection, irradiation, and high-dose chemotherapies [32,33]. One of the reasons for recurrence could be the ineffectiveness of current treatment strategies to eliminate medulloblastoma stem cells, which may survive the treatment and cause tumor regrowth [34,35].
Morrissy et al. [36] found that recurrent tumors substantially differ from matched primary tumors in terms of genetic composition. In particular, whole-genome sequencing analyses showed genetic divergence and a distinct clonal composition in relapsed post-treatment tumors as compared to matched primary tumors. In line with this, another study showed that metastases from medulloblastoma were relatively similar to each other from a genomic perspective but differed substantially from the matched primary tumor, suggesting that clonal selection leads to the escape of a treatment-refractory clone [37]. These findings question the selection of therapeutic targets based solely on the analysis of the primary tumor, without taking the "metastatic compartment" into consideration [37] and advocate the necessity to re-biopsy recurrent medulloblastoma for treatment management, as actionable targets identified in the initially diagnosed tumor may not be present in the major clone in the relapsed tumor [36].
Using fractionated carbon ion radiation (5 × 3 Gy), we were able to significantly prolong survival of a heavily pretreated relapse PDX model of medulloblastoma with chromothripsis [27]. After a period without detectable tumor (> 50 days of growth delay), all animals experienced a relapse. The detection of putative cancer stem cells (CSCs) positive for SOX2 as well as the prior therapy given to the patient to treat the tumor (before the establishment of the PDX models) provides possible explanations for the observed resistance to carbon ions in our PDX mouse model of relapsed medulloblastoma.

Properties of Cancer Stem Cells
Cancer stem cells (CSCs) comprise a small subpopulation of cells within the tumor that have the ability for self-renewal, tumor initiation, and differentiation into multiple cell types [38]. These cells can promote resistance to treatment due to their high adaptive abilities [39] and possess the features required for metastasis [40,41]. CSCs also play an important role in maintaining tumor growth and can reconstitute the tumor mass. CSCs usually proliferate slowly and can potentially stay in the G0 phase of the cell cycle for an extended period [42,43]. In a number of tumor types, these tumorinitiating cells display a certain phenotypic plasticity that can be influenced by stimuli from the tumor microenvironment [44].
Elucidating the molecular mechanisms associated with treatment resistance is crucial for developing more effective treatment options that can successfully eliminate the cells that lead to tumor recurrence. One of the first steps towards novel therapeutic strategies which could eliminate CSCs while protecting normal tissue [45] is the identification of reliable markers and molecular signatures for CSCs.

Cancer Stem Cell Markers
Several cell surface markers enriched in CSCs have been identified in different tumor entities, with CD133 [46,47] and CD44 [48] being some of the most prominent ones across cancer types. Other markers such as CD24, CD166, EpCAM [48], CD13 [49], and CD144 [50] have been reported in different tumor types (e.g., pancreatic, breast, and colorectal cancer). A subset of these markers is associated with prognosis and treatment outcome [51]. To identify CSCs, a combination of markers is recommended, as single markers lack specificity, and a number of these markers are expressed in normal cells as well [52].

Cancer Stem Cells in Brain Tumors
The beforementioned CSC marker CD133 was also identified in brain tumors [53]. In glioblastoma, a radioresistant fraction of CD133 + cells showed increased abilities to repair DNA damage by upregulating the activity of checkpoint kinases [54]. Calabrese et al. [55] reported that brain tumor stem cells positive for Nestin and CD133 occupy a perivascular niche where endothelial cells communicate with self-renewing CSCs and maintain the stem-like state of CSCs by secreting specific factors. Aldehyde dehydrogenase (ALDH) was identified as another CSC marker in primary brain tumors by Choi et al. [56].

Cancer Stem Cells in Medulloblastoma
In pediatric brain tumors, including medulloblastoma, Hemmati et al. [57] identified a population of multipotent, self-renewing cells similar to neural stem cells, but unlike these, with a high capacity to proliferate. These putative medulloblastoma stem cells were found to express CD133, SOX2, and musashi-1, among other markers. Panosyan et al. [58] found a correlation between neurosphere formation of tumor cells, reflecting the proportion of tumorigenic cells, and clinical outcome, which was especially strong in embryonal tumors including medulloblastoma. The aggressiveness of embryonal tumors was proposed to be partly attributed to an increased proportion of self-renewing tumorigenic CSCs: the high expression of CSC markers in medulloblastoma was correlated with worse prognosis in a number of studies [34,58].
In medulloblastoma, a number of markers beyond the widely used CD133 enrich for tumorigenic cell subpopulations [45]. Several studies [59,60] identified a population of CD15 + cells as tumor-initiating cells, but also actively proliferating cells. In Sonic hedgehog medulloblastoma, Nestin + and SOX2 + cells were identified as putative cells of origin [34]. Vanner et al. [34] showed that a quiescent SOX2 + cell subset drives tumor propagation and shows resistance to both chemotherapy and vismodegib, an inhibitor of the Sonic hedgehog pathway. Zhang et al. [35] demonstrated that OLIG2 + progenitor cells drive tumor initiation in Sonic hedgehog medulloblastoma, become quiescent in full-blown medulloblastoma, are enriched in therapyresistant tumors, and represent the cell population that reemerges at relapse. Oncogenic pathways such as HIPPO-YAP and AURORA-A/MYCN are active in these putative tumor stem cells, and co-targeting of these pathways led to arrest of tumor growth and improved survival in vivo. Singh et al. [61] found that PI-3 K inhibition targeted putative CD15 + CSCs in Sonic hedgehog medulloblastoma. Furthermore, PTEN and PI-3 K play a crucial role in tumorigenicity, survival, and response to therapy of these CSCs. CD15 + cells were found to be resistant to several drugs currently used to treat medulloblastoma, such as cisplatin, temozolomide, and Sonic hedgehog inhibitors. In line with these findings, Hambardzumyan et al. [62] found that in several in vivo models of medulloblastoma, the PI3K pathway plays a role in CSCs in the perivascular niche. Importantly, Nestin + stem cells survived γ-radiation due to the activation of the PI3K/Akt pathway and can give rise to recurrence following radiation.

What Makes Putative CSCs Resistant to Irradiation?
Cancer stem cells have a number of features that potentially explain their survival to treatment. In the following section, we mention some of the main mechanisms leading to resistance to conventional radiotherapy with photons in CSCs.

Lower Susceptibility to DNA Damage and Increased Repair Capacity
In several tumor types (e.g., breast and brain), significantly less radiation-induced damage was observed in CSCs as compared to tumor cells negative for CSC markers. This was shown by a decreased number, and faster resolving of γH2AX foci [54,63], used as a marker to detect DNA DSBs [64]. In glioma, it has been shown that in CSCs, DNA damage checkpoints were activated via phosphorylation of Chk1/2 proteins as a response to radiation. Improved treatment sensitivity was observed when adding a specific inhibitor of Chk1 /Chk2 checkpoint kinases which regulate DNA repair and cell cycle progression [54]. Furthermore, it is also known that radiation-induced reactive oxygen species (ROS) cause an indirect damage to the DNA molecules [65]. Proteins responsible for the removal of ROS, like glutathione [66], can be highly expressed in CSCs. This phenomenon was observed in breast cancer, where lower levels of ROS were measured in CSCs as compared to non-CSCs [63]. Therefore, a higher tolerance of ROS in CSCs may contribute to radioresistance [67].

Quiescence and Cell Cycle Dependence of the Radiation Response
Highly proliferative tumor cells are more sensitive to the anti-tumor effects of radiation. Since a number of CSC populations are reported to remain mostly in the G0 phase and cycle slowly, this quiescence gives CSCs enough time to repair IR-induced DNA damage prior to replication, if they re-enter the cell cycle [42,68]. This potentially provides an advantage for the survival of CSCs following irradiation.

Pathways with Pro-survival Signaling Activity in CSCs
In glioma, the Notch pathway has been shown to play a role in the radioresistance of CSCs. Notch inhibition with γ-secretase led to reduced Akt activity and Mcl-1 levels, while knockdown of Notch1/Notch2 sensitized glioma CSCs to radiation and impaired tumor reformation in xenograft models [69]. In mouse mammary CSCs, the WNT/betacatenin pathways have been shown to influence radioresistance [70]. In medulloblastoma [62], the PI3K pathway plays a role in CSCs in the perivascular niche, which can contribute to recurrence following radiation. In the tumor bulk, the treatment with γ-radiation leads to apoptotic cell death, whereas the Nestin-expressing stem cells survived the radiation due to activation of the PI3K/Akt pathway. Upon inhibition of Akt signaling by a small molecule inhibitor called perifosine, CSCs in the perivascular niche could be sensitized to radiation and displayed increased apoptotic cell death. In another solid tumor type, namely head and neck squamous cell carcinoma, Hedgehog signaling is upregulated in the tumor, where it contributes to stromal-mediated resistance. The use of Hedgehog inhibitors was able to sensitize the tumor to radiotherapy [71].

The Role of CSCs Niches and Microenvironmental Factors
The microenvironment, including blood vessels, fibroblasts, extra-cellular matrix, and immune cells, can protect the CSCs [72]. As mentioned above, in a number of tumor types, such as medulloblastoma and glioma, CSCs reside in a perivascular niche [55,62]. Calabrese et al. found that disruption of this niche destroys the self-renewing cell population in brain tumors; thus, antiangiogenic drugs could, at least in part, inhibit brain tumor growth [55].

Potential Advantages of Carbon Ion Radiotherapy to Target CSCs
The use of high-LET radiation such as radiation with carbon ions could be beneficial in the context of targeting CSCs, as some of the mechanisms of resistance to photons may not apply to high-LET irradiation, due to its distinct physical and biological properties. As mentioned above, DNA damage induced by carbon ions is more difficult to repair. By causing a higher proportion of DNA damage by direct effects, it is also less dependent on ROS, which is an advantage to target CSCs, as these cells show increased ROS scavenging properties [67]. Furthermore, as the effects of carbon ions are less dependent on the cell cycle as compared to conventional radiotherapy, this represents an additional benefit to eliminate quiescent CSCs [10,73]. Oonishi et al. found that pancreatic CSCs showed increased G2/M cell cycle arrest, caused by the induction of complex DSBs and reduced DNA damage repair after carbon ion irradiation [74]. High-LET radiation was also able to suppress survival signaling, i.e., AKT signaling, which is often active in CSCs, with AKT inhibition leading to increased apoptosis [75]. In addition, glioma CSCs resistant to irradiation with photons were found to be sensitive to carbon ion radiation. Importantly, carbon ions led to a reduction in the number of CD133 + cells [76]. In line with this, Takahashi et al. showed stronger effects of carbon ions on glioblastoma sphere-type cells as compared to photons [77]. As a further advantage of particle therapy, Chiblak et al. showed that carbon ions could change the glioma niche towards being more immunopermissive and antiangiogenic [28].

Particle Radiotherapy and Targeting Pathways Active in CSCs
As radiotherapy with particles, and especially carbon ions, seems to be promising in the context of targeting resistant cells such as CSCs, adding targeted inhibitors to pathways active in CSCs could potentially further improve the response to radiation therapy. Bertrand et al. [78] tested two targeted pharmacological strategies to overcome the resistance of head and neck squamous cell carcinoma CSCs to photons as well as to carbon ion radiation. Firstly, the strategy using a checkpoint kinase (Chk1) inhibitor (UCN-01) was able to overcome the G2/M arrest and induce radiosensitization of CSCs. Secondly, the use of all-trans retinoic acid (ATRA) inhibited the activity of ALDH, which induced differentiation and radiosensitization of CSCs highly expressing ALDH. When combining both inhibitors together with irradiation, the surviving fraction of cells decreased drastically after irradiation with both photons and carbon ions, indicating promising radiosensitizing effects.
Another emerging treatment option is the combination of carbon ions with the effect of radiosensitizing miRNAs [79]. MiRNAs are small non-coding regulatory RNAs that bind to the 3′UTR of mRNAs, affecting translation as well as transcription. As the deregulation of miRNAs plays an important role in a variety of cancer entities, they have also been proposed as novel targets for anti-tumor therapies [80,81]. The underlying hypothesis is to either downregulate upregulated oncogenic miRNAs with antisense miRNA oligonucleotides or to complement downregulated tumor suppressing miRNAs with miRNA mimics [82]. Sai et al. used a miR-200c mimic in combination with carbon ions, which led to the elimination of pancreatic CSCs in vitro and in vivo. In particular, the treatment suppressed colony and spheroid formation abilities, increased the expression of apoptosis-related genes, and suppressed xenograft tumor growth in vivo [83]. Another study [84] showed that overexpression of a downregulated miRNA (miR-374) was able to sensitize human pancreatic cancer cell lines towards carbon ion irradiation. Using a similar approach, Vares et al. [85] recently observed that a combination treatment utilizing carbon ions, a miRNA (miRNA-34), and a mTOR inhibitor was able to control high-grade chondrosarcoma CSCs.
VEGF-mediated signaling in tumor cells contributes to the function of CSCs and tumor initiation [86]. Targeting angiogenesis via VEGF inhibition with the monoclonal antibody bevacizumab was able to increase the radiosensitivity of melanoma cells to proton irradiation [87]. Exploiting synthetic lethality, multiple studies have shown that PARP inhibitors, known as potent radiosensitizers, can also sensitize cancer cells to carbon ion irradiation [88,89]. We found that PARPi sensitized Sonic hedgehog medulloblastoma with chromothripsis to proton irradiation in a primary tumor model [27] and to carbon ions in an aggressive relapse model (unpublished). As mentioned above, the Hedgehog signaling pathway can play a prominent role in CSCs. Inhibition of Hedgehog signaling sensitized medulloblastoma cells to proton and carbon ion irradiation [90].

Combining Particle Radiotherapy with Immunotherapy
Another potentially promising treatment concept is to combine particle irradiation with immunotherapy. Several lines of evidence suggest that radiation can lead to an increased immunogenic response to the tumor [91].
Abscopal effects, such as the disappearance of metastatic lesions outside the directly irradiated field, have been reported in the context of radiotherapy [92], which was later understood as the induction of anti-tumor immunity [93]. Autophagy was shown to play an important role in the immunogenic response and particle irradiation was reported to increase autophagy [94,95]. Carbon ions were also shown to sensitize tumor cells to cytotoxic T-cell-mediated cytolysis [96]. Furthermore, hypofractionated carbon ion irradiation can lead to an increased immune response when higher doses per fraction are applied [97,98]. Lastly, the expression of immunogenic modulation such as cell surface neoantigen presentation was shown to be upregulated following radiation with either photons or protons. Proton radiation additionally led to the translocation of calreticulin on the cell surface, which enhanced interactions between CD8 + T cells and tumor cells, leading to T-cell-mediated killing of the tumor cells. Interestingly, CSCs which were resistant to direct cytolytic effects of proton irradiation also upregulated calreticulin in a similar way as non-CSCs, rendering them more sensitive to cytotoxic T-cells [99].
Approaches used in cancer immunotherapy include therapeutic cancer vaccines and targeted antibody therapies [100]. Dendritic cell-based vaccination and the use of immune checkpoint inhibitors were in particularly tested in combination with carbon ion irradiation. In combination with the injection of dendritic cells, carbon ion irradiation led to the suppression of distant metastasis. Two studies [101,102] using a combination of particle radiotherapy and immunotherapy, in form of bone marrow-derived dendritic cells, were able to show that the combination therapy was successful in inducing anti-tumor immunity in murine models of squamous cell carcinoma.
Helm et al. [103] compared the effects of x-ray and highenergy carbon ion therapy in combination with two immune checkpoint inhibitors in a murine osteosarcoma model. The authors showed that a combination of carbon ions with immune checkpoint inhibitors was the most effective to reduce abscopal tumor growth, whereas the single treatments were not sufficient. The abscopal tumors of animals treated with combination treatments showed a higher infiltration of CD8 + cells, suggesting increased immunogenic response.
In a recent study, Takahashi et al. [104] showed that combining carbon irradiation with a dual immune checkpoint blockade improved radiosensitivity in a murine osteosarcoma model. Carbon ion irradiation was able to increase the effects of anti-PD-L1 and anti-CTLA-4 antibodies. Importantly, the combination treatment inhibited distant metastasis and showed a survival benefit.

Conclusion
Carbon ion radiotherapy was identified as a promising treatment strategy in a patient-derived xenograft model of aggressive pediatric medulloblastoma with chromothripsis. This approach may provide significant benefits in pediatric (neuro)oncology, after thoroughly investigating putative side effects. Sparing surrounding normal tissue would avoid neurological and developmental side effects, which are a major concern with current treatment options for medulloblastoma. In the context of cancer predisposition due to germline variants, it is critical to avoid radiation-induced secondary tumors. Carbon ion radiation seems to carry a reduced risk of developing secondary malignancies as compared to photons in adult patients [105], as shown by a direct comparison trial in prostate cancer. In pediatric patients, two studies utilized carbon ions in chordoma and low-grade chondrosarcoma of the skull base [24] as well as various brain tumors [25]. Importantly, these studies reported favorable toxicity profiles, as no severe toxicities occurred, and the treatment was well tolerated. For inoperable osteosarcoma, carbon ion radiation showed encouraging results, and evaluating carbon ion radiotherapy could also be justified in pediatric patients for this tumor type [106]. After performing further preclinical studies and detailed investigations of the molecular effects and potential risks, carbon ion radiotherapy as well as other particle therapies may offer promising strategies in the fight against cancer.
Funding Open Access funding enabled and organized by Projekt DEAL. AE received support from the DFG, Wilhelm Sander Foundation, and Fritz Thyssen Foundation.

Conflict of Interest
The authors declare no conflict of interest.

Human and Animal Rights and Informed Consent
All reported studies/ experiments with human or animal subjects performed by the authors have been previously published and complied with all applicable ethical standards (including the Helsinki declaration and its amendments, institutional/national research committee standards, and international/ national/institutional guidelines).
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