# Overcoming the limits of pediatric brain tumor radiotherapy: The use of preclinical 3D models

Élodie Czuba, Marlene Deschuyter, Natacha Entz-Werlé, Georges Noel, Hélène Burckel

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Élodie Czuba, Marlene Deschuyter, Natacha Entz-Werlé, Georges Noel, Hélène Burckel. Overcoming the limits of pediatric brain tumor radiotherapy: The use of preclinical 3D models. Cancer/Radiothérapie, 2024, 28 (5), pp.424-434. 10.1016/j.canrad.2024.06.003. hal-05380813

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Review article

# Overcoming the limits of pediatric brain tumor radiotherapy: The use of preclinical 3D models

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## Repousser les limites de la radiothérapie des tumeurs cérébrales pédiatriques : utilisation des modèles précliniques tridimensionnels

Élodie Czuba a,b, Marlène Deschuyter c, Natacha Entz-Werlé c,d, Georges Noël a,b,e,
Hélène Burckel a,b,*

## INFO ARTICLE

Historique de l'article :
Reçu le 25 avril 2024
Reçu sous la forme révisée
le 12 juin 2024
Accepté le 13 juin 2024

Keywords :
Pediatric brain tumor
Radiation
Radioresistance
3D models

Mots clés :
Tumeur cérébrales pédiatrique
Irradiation
Radiorésistance
Modèles 3D

## ABSTRACT

Radiotherapy (RT) is an integral part of managing pediatric brain tumors, yet many patients develop tumor radioresistance, leading to recurrence and poor clinical outcomes. In addition, neurocognitive impairment is a common long-term side effect of RT, significantly impairing quality of life. Indeed, increasing evidence suggests that the developing child's brain is particularly vulnerable to the neurotoxic effects of ionizing radiation. Consequently, developing novel preclinical models is crucial for studying radiation's impact on normal brain tissue and predicting patient-specific responses to RT, enabling the development of personalized therapies combined with RT. However, this area remains underexplored, primarily due to the transfer of results gathered from in vitro tumor models from adults to pediatric entities while the location and molecular characteristics of the brain tumor differ. Recent years have seen the emergence of patient-specific 3D in vitro models, which have been established for entities including glioblastoma and medulloblastoma. These models better mimic primary parenteral tumors more closely in their histological, transcriptional, and mutational characteristics, thus approximating their intratumoral heterogeneity more accurately than conventional 2D-models. In this review, we presented the main limits of pediatric brain tumor radiotherapy, including mechanisms of radioresistance, associated tumor relapse, and the side effects of irradiation on the central nervous system. We also conducted an exhaustive review to identify studies utilizing basic or advanced 3D models of pediatric brain tumors combined with irradiation and discussed how these models can overcome the limitations of RT.

© 2024 L'Auteur(s). Published by Elsevier Masson SAS au nom de Société française de radiothérapie oncologique (SFRO). Cet article est publié en Open Access sous licence CC BY (http://creativecommons.org/licenses/by/4.0/).

## RÉSUMÉ

La radiothérapie fait partie intégrante de la prise en charge des tumeurs cérébrales pédiatriques, mais de nombreux patients sont atteints de tumeur radiorésistante qui entraîne une récidive et des résultats cliniques médiocres. De plus, d'importants troubles neurocognitifs dus aux irradiations peuvent survenir, nuisant considérablement à la qualité de vie des patients. En effet, de plus en plus de preuves suggèrent que le cerveau de l'enfant en développement serait particulièrement vulnérable aux effets neurotoxiques des rayonnements ionisants. Par conséquent, le développement de nouveaux modèles précliniques est crucial pour étudier les effets de ces rayonnements sur le tissu cérébral sain, prédire les réponses spécifiques des patients à la radiothérapie et permettre de développer de nouvelles thérapies personnalisées associées à la radiothérapie. À ce jour, ce domaine reste insuffisamment étudié, principalement en raison du transfert des résultats obtenus à partir de modèles tumoraux in vitro de l'adulte à l'enfant, alors

<footnote>a Radiobiology Laboratory, institut de cancérologie Strasbourg Europe (ICANS), 3, rue de la Porte-de-l'Hôpital, 67000 Strasbourg, France</footnote>

<footnote>b Laboratory of Engineering, Informatics and Imaging (ICube), Integrative multimodal imaging in healthcare (Imis), UMR 7357, université de Strasbourg, 4,
rue Kirschleger, 67000 Strasbourg, France</footnote>

<footnote>c CNRS UMR 7021, Laboratory of Bioimaging and Pathologies, Team OnKO-3T, Faculty of Pharmacy, 74, route du Rhin, 67401 Illkirch, France</footnote>

<footnote>d Pediatric Oncohematology Unit, centre hospitalier universitaire de Strasbourg, 1, avenue Molière, 67098 Strasbourg cedex, France</footnote>

<footnote>e Department of Radiation Oncology, institut de cancérologie Strasbourg Europe (ICANS), Unicancer, 17, rue Albert-Calmette, 67200 Strasbourg, France</footnote>

<footnote>* Corresponding author: Radiobiology Laboratory, institut de cancérologie Strasbourg Europe (ICANS), 3, rue de la Porte-de-l'Hôpital, 67000 Strasbourg, France.
Adresse e-mail : h.burckel@icans.eu (H. Burckel).</footnote>
que la localisation et les caractéristiques moléculaires de la tumeur cérébrale diffèrent. En revanche, ces dernières années ont vu l'émergence de modèles in vitro tridimensionnel établis à partir de tumeurs pédiatriques telles que le glioblastome et le médulloblastome. Ces modèles peuvent mimer au plus près les tumeurs primaires dont ils sont issus selon les caractéristiques histologiques, transcriptionnelles et mutationnelles reflétant leur hétérogénéité intratumorale, ce qui n'est pas rapporté dans les modèles classiques bidimensionnels. Dans cette revue, nous avons exposé les principales limites de la radiothérapie des tumeurs cérébrales pédiatriques, notamment les mécanismes de radiorésistance, la rechute tumorale associée ainsi que les effets secondaires des irradiations sur le système nerveux central. Nous avons également mené une revue exhaustive de littérature sur les études utilisant des modèles tridimensionnels de base ou évolués de tumeurs cérébrales pédiatriques associés à l'irradiation et discuté de la façon dont ces modèles peuvent nous aider à surmonter les limites de la radiothérapie.

© 2024 L'Auteur(s). Published by Elsevier Masson SAS au nom de Société française de radiothérapie oncologique (SFRO). This article is published in Open Access under a Creative Commons Attribution 4.0 International (CC BY 4.0).

## 1. Abbreviations

<table><tr><td>ABC</td><td>ATP-binding cassette</td></tr><tr><td>ATRT</td><td>atypical teratoid rhabdoid tumor</td></tr><tr><td>BO</td><td>brain organoids</td></tr><tr><td>CNS</td><td>central nervous system</td></tr><tr><td>CRT</td><td>conventional radiotherapy</td></tr><tr><td>CSC</td><td>cancer stem cells</td></tr><tr><td>DIPG</td><td>diffuse intrinsic pontine glioma</td></tr><tr><td>DNET</td><td>dysembryoplastic neuroepithelial tumors</td></tr><tr><td>ESC</td><td>embryonic stem cells</td></tr><tr><td>GBM</td><td>glioblastoma</td></tr><tr><td>GLICO</td><td>cerebral organoid glioma</td></tr><tr><td>GSC</td><td>glioma stem cells</td></tr><tr><td>HGG</td><td>high-grade glioma</td></tr><tr><td>HIF</td><td>hypoxia-inducing factors</td></tr><tr><td>hiPSC</td><td>human-induced pluripotent stem cells</td></tr><tr><td>IMRT</td><td>intensity-modulated radiation therapy</td></tr><tr><td>IQ</td><td>intelligence quotient</td></tr><tr><td>IR</td><td>irradiation</td></tr><tr><td>LGG</td><td>low-grade glioma</td></tr><tr><td>MB</td><td>medulloblastoma</td></tr><tr><td>MRI</td><td>magnetic resonance imaging</td></tr><tr><td>NEOCOR</td><td>neoplastic corticoid</td></tr><tr><td>NS</td><td>neurosphere</td></tr><tr><td>PBRT</td><td>proton beam radiotherapy</td></tr><tr><td>PDO</td><td>patient-derived organoids</td></tr><tr><td>PNET</td><td>primitive neuroectodermal tumor</td></tr><tr><td>ROS</td><td>reactive oxygen species</td></tr><tr><td>RT</td><td>radiotherapy</td></tr><tr><td>TME</td><td>tumor microenvironment</td></tr><tr><td>VMAT</td><td>volumetric modulated arc therapy</td></tr><tr><td>WHO</td><td>World Health Organization</td></tr></table>

## 2. Introduction

Brain tumors represent the most common solid tumor in children aged 15 years or younger, accounting for 25% of all tumors, and 10% in adolescents and young adults (15-25 years old) [1-3]. They stand as the first leading cause of death in children in high-income countries, with a median overall survival ranging between 12 and 24 months for the most aggressive and resistant types [4]. According to the World Health Organization (WHO) classification of tumors of the central nervous system, pediatric brain tumor can be divided into several categories. The most prevalent class consists of embryonal tumors, which encompass especially medulloblastoma representing 20% of all pediatric brain tumors [5]. The second common class is diffuse astrocytic and oligodendro-

glia tumors, encompassing a range of entities, such as astrocytoma (low- and high-grade glioma [LGG or HGG]), glioblastoma (HGG), glioma diffuse intrinsic pontine glioma (DIPG, HGG), oligodendroglioma (LGG) or dysembryoplastic neuroepithelial tumors (DNET, LGG). Additionally, ependymal tumors, most commonly ependymomas, as well as neural and mixed neuronal-glial tumors such as gangliogliomas are observed. Other notable types include meningiomas, mesenchymal tumors such as primitive neuroectodermal tumor (PNET), tumor of the sellar region with craniopharyngiomas, germ cell tumors or choroid plexus tumors [6]. Initially, these tumors are surgically excised whenever feasible, although complete resection is often hindered by their localization in critical brain regions [7]. Subsequently, patients may be recommended additional therapeutic modalities such as radiotherapy (RT) in case of incomplete resection for low-grade glioma or as an additional therapy after complete tumor removal for most tumor types, depending on patient age [8]. Chemotherapy or targeted therapy is integral components of treatment plans, varying based on the specific chemotherapeutic agents and tumor type [7]. Within this multimodal approach, irradiations remain a major therapeutic tool to treat most of pediatric brain tumors [9,10]. However, the efficacy of this therapeutic approach is significantly limited by tumor cell radioresistance, leading to tumor relapse in patients surviving their primary disease [10-13]. For instance, the phenomenon of radioresistance is observed in approximately 40% of medulloblastoma (MB), 90% of grade II gliomas, and 20-40% of ependymomas, yet it is not clearly elucidated at oncogenic and microenvironmental levels. These tumor relapses present a significant challenge characterized by cells acquiring a more aggressive phenotype. Additionally, the current radiation approach, considering quality and dosage, frequently results in neurocognitive disorders, greatly impacting the patients' quality of life [14]. Addressing the challenge to predict patient-specific responses to RT, understanding the impact of radiation on the central nervous system (CNS), optimizing RT schedules, and developing new personalized therapeutics combined with RT, the use of appropriate preclinical models is required. Despite in vitro 2D-models being widely used combined to irradiation (IR), these models have many limitations: absence of cell-cell interaction and oxygen/nutrient gradient, lack of extracellular environment or morphology changes can be cited [15,16]. All these 2D-model limitations impede understanding the role played by intratumor heterogeneity dynamics in the processes of relapse.

Thus, using 3D in vitro models is the promising approach to develop realistic and functional preclinical models derived from patient brain cells. Indeed, these models should accurately replicate physiological characteristics, considering cellular and molecular heterogeneities, and showing differences in accessibility to nutrients as well as oxygen and pH variations. Various methodologies have come into use that have allowed the validation of models,
such as neural spheroids, the most widely used model, which can be cultured with or without a scaffold. Spheroid models can be derived with stem-like cell properties, such as neurospheres, to approximate the structure of the brain tumor and its microenvironment. More recently, more complex models such as brain organoids have been developed. These are self-organizing 3D structures that closely mimic an organ or tissue at a morphological, cellular, and functional level, and are commonly derived from human-induced pluripotent stem cells (hiPSC), cancer stem cells (CSCs) or patient-derived tumors to model healthy or tumoral tissues.

This review highlights some identified limitations of RT, a major aspect of pediatric brain tumor treatment, including radioresistance mechanisms, associated tumor relapse, as well as the side effects of irradiation on the central nervous system. We have conducted an exhaustive review of studies using 3D models of pediatric brain tumors with radiation and discuss the potential advantages of using these models to understand and improve current RT treatments.

### 3. Limits of pediatric brain tumor radiotherapy

Focal or craniospinal (CS) RT is utilized in the upfront treatment of many pediatric brain tumors, as adjuvant therapy for resected tumors or definitive treatment for unresectable malignancies. Despite the high cure rate in most of pediatric brain tumors, RT side effects significantly impact the patient's quality of life and tumor relapses cannot be controlled by conventional RT treatments, often resulting in fatalities.

#### 3.1. RT and its side effects

Children diagnosed with brain tumors are now exhibiting prolonged survival rates, allowing for the occurrence of late effects from irradiation. Three to twelve weeks post-IR, somnolence syndrome may occur, characterized by symptoms like fatigue, nausea, vomiting, fever, dysphagia, and ataxia [10,17-19]. Months or even years later, 50-90% of patient may experience cognitive dysfunction such as hearing loss, reduced intellectual quotient (IQ) scores, impaired neurocognition, memory deficits, or attention problems [10,11,14,20]. A clinical study on patients diagnosed with MB monitored long-term side effects of treatment. Thus, patients were treated with maximum surgical resection of the primary tumor, followed by craniospinal irradiation (CSI) of 36 Gy and an 18 Gy boost to the posterior fossa for a total dose of 54 Gy (1.8 Gy fraction per day, five days per week) with adjuvant chemotherapy. Aktekin et al. showed that 55% of patients had neurological sequelae, 90% patients had psychiatric issues such as mental retardation, 70% patients had two or more neuropsychiatric problems simultaneously and 60% had hearing loss [21].

In addition, a heightened risk of tumor relapse was frequently observed after RT in around 30% of patients with pediatric brain tumor [22]. Ritzmann et al. studied the relapse rate in ependymoma patients treated with focal RT of 54 Gy in daily fractions of 1.8 Gy over six weeks after complete tumor resection and chemotherapy for children over three years. They found a 62% relapse rate, with younger patients exhibiting a higher risk of recurrence [23]. Indeed, the probability of developing radiotoxicity is mainly influenced by factors including age at exposure, sex, the volume of irradiated brain tissue, type of irradiation and the delivered dose [24]. Patients receiving radiation to larger volumes and higher doses tend to exhibit more severe and frequent cognitive deficits over time. For patient with MB, the current dose-fractionation protocol includes 23.4 Gy in 13 fractions of craniospinal irradiation (CSI). A study comparing this protocol to a lower dose (18 Gy) showed a decrease in five-year event-free survival by reducing the CSI dose in

young children with average-risk MB, but is associated with better neurocognitive outcomes, such as a late decline in IQ [25].

To balance treatment efficacy and toxicity, conventional external beam RT (CRT) using X-ray radiation has evolved significantly, with advancements such as intensity-modulated radiation therapy (IMRT) or volumetric modulated arc therapy (VMAT). Additionally, the use of proton radiotherapy is taking on an increasingly significant role in pediatric brain tumor treatment due to its potential advantages stemming from its distinct dose distribution capabilities. This technique holds the potential to deliver higher doses to the targeted area while simultaneously protecting surrounding healthy tissue from damage, thereby preserving the central nervous system (CNS). To support this, Mash et al. explored neuropsychological performance in patients mainly with low-grade glioma, embryonal tumors or ependymoma and treated with focal or CSI using proton beam therapy (PBRT) (mean 53.5 Gy) or CRT (mean 53.4 Gy). Thus, superior cognitive outcomes have been reported following proton PBRT compared to XRT [20]. Similarly, PBRT and CRT were compared in MB patients, and those receiving PBRT achieved significantly higher scores on most analyzed neurocognitive outcomes (including IQ, verbal comprehension and perceptual reasoning indices, visual motor integration, and verbal memory) than those receiving CRT [4]. In a different approach, patients with pediatric brain tumor located in the supratentorial (17.6%), midline (28.8%), or posterior fossa (53.6%) compartments received radiation and revealed that the PBRT cohort was associated with higher IQ and processing speed index (speed of mental processing, using visual stimuli and graphomotor skills) compared to the CRT cohort [26]. Furthermore, pediatric patients undergoing cranial radiation are at risk of developing arterial vasculopathies, years to decades after CRT or PBRT treatment, increasing the likelihood of late stroke [24,27,28].

Therefore, all these identified RT side effects have considerable repercussions on the functional outcomes and quality of life for childhood cancer patient. They may lead to poor social skills, difficulty adjusting to new environments, social withdrawal, and mild deficits in academic functioning [29].

The inerrant mechanisms behind neurocognitive decline due to RT are complex and vary according to acute or late effects. Immediately after IR, the primary effects include direct damage to progenitor cells (neural and glial) as well as to vascular, inflammatory, and stromal cells [30]. The induced neuroinflammation and microvascular damage cause apoptosis of endothelial cells of blood brain barrier and oligodendrocytes, leading to demyelination and white-matter necrosis [20,31]. In long-term, vascular abnormalities also contribute to hypoxia and oxidative stress in the myelin membrane, which is critical in pediatric tumors, given that full myelination of the cerebral cortex occurs in early adulthood [32]. An upregulation of tumor necrosis factor-alpha, IL-1β, and NF-kB contributes to an inflammatory microenvironment, which was also highlighted as contributing to radiation-induced side effects observed in multiple organs, including the brain [33]. A preclinical study on rodent model displayed a significant decrease of oligodendrocyte density after irradiation of the spinal cord at a total dose of 22 Gy, associated with demyelination observed 19 weeks post-IR [34]. Mash et al. studied white-matter integrity in various patients with pediatric brain tumors treated with CRT or PBRT and showed that white-matter integrity was diminished [20]. In another clinical study that enrolled 75 pediatric brain tumor patients who received PBRT, five patients were identified with large cerebral vessel vasculopathy such as stenosis or occlusion. Among them, four patients presented with acute stroke and showed magnetic resonance imaging evidence of acute infarcts [35].

Despite numerous studies, the complete understanding of the quality and dose radiation effect on the developing human CNS remains elusive, necessitating the development of adequate human in vitro models.
## 3.2. Tumor radioresistance mechanisms

Last years, considerable research efforts have been directed towards understanding the mechanism of radioresistance in brain tumor including pediatric ones and multitude of mechanisms have been proposed to explain this phenomenon. Thus, hypoxia, tumor heterogeneity and the presence of cancer stem cells, are among the key factors contributing to the emergence of radioresistance in these tumors.

### 3.2.1. Tumor heterogeneity

Tumor heterogeneity is a hallmark of the majority of cancer and is partially responsible to therapy failures, including RT. Tumor heterogeneity may be divided into intertumoral or intratumoral heterogeneity [36]. Intertumoral heterogeneity arises from different patients, whose altered genotype and phenotype are induced by diverse etiological and environmental factors, leading to various subgroups in most pediatric brain tumors with distinct clinical characteristics, genetic aberrations, and prognoses. For MB tumors, four principal subgroups exist: wingless (WNT), sonic hedgehog (SHH), group 3 and group 4, each with distinct molecular and clinical signatures [37–39]. WNT MBs have an excellent outcome with a non-mutated WNT gene and a mutation of CTNNB1 gene, while group 3 is considered the most aggressive subgroup due to its high metastatic potential and poor survival. It is characterized by *MYC* amplifications, *GABRA5* overexpression, and *SMARCA4* mutations. In the SHH group, mutations in the sonic hedgehog signaling pathway, including those affecting ATP synthase-binding cassette (ABC) transporter genes, have been observed. These genes are involved in transporting various substrates across cellular membranes using ATP. Therefore, the use of ABC inhibitors has been shown to enhance radiosensitivity, indicating a correlation between these mutations and the radiation resistance observed in this subgroup of MB [40].

Contrary to adults, pediatric high-grade glioma (pHGG) is not classified within standard groups. However, a molecular classification of pHGG has identified unique genetic and epigenetic features. Six distinct epigenetic and biological subgroups have been defined through DNA methylation studies, resulting in six main methylation clusters: WT, G34, LGG like/PXA like, IDH and K27 [41–43]. Each group presents a distinct age distribution. For example, the K27 cluster appears predominantly in the adolescent population, which presents a very low survival of 6 months, while LGG/PXA like group shows a mean survival superior to 100 months and is more frequently detected in children (mean age of 4 years). Each subgroup presents specific gene mutations associated with their radiotherapy response, explaining their specific survival level.

For instance, TP53, a tumor suppressor gene is mutated in all groups except LGG/PXA like group. It has been observed that patients carrying a TP53 mutation had a significantly worse clinical and radiological response to RT, explaining the higher clinical and radiation response for patient in LGG/PXA like group compared to the others [44,45]. Similarly, the H3K27M gene, a major oncogenic driver, presents mutation in the K27 pHGG subgroup, conferring radioresistance due to the dysregulation of DNA repair machineries [46]. Malignant molecular subgroups of ependymoma are uniformly classified using DNA methylation profiling according to the anatomical compartment of the CNS, spine, posterior fossa, supratentorial, each with three subgroups [47,48]. For supratentorial (ST) ependymomas, prototypic fusion genes involving RELA and YAP1 are observed in two subgroups and are associated with favorable clinical outcomes for YAP1 subgroup. Posterior fossa subgroups are characterized by a young median age at diagnosis, an overall balanced genome, and poor clinical outcomes. EZHIP overexpression, somatic mutations and chromosomal instability are detected affecting the cerebellum and brainstem [49]. EZHIP overexpression leads to the loss of H3K27me3-specific histone demethylases, which can

be correlated with radioresistance [50]. For spinal ependymoma, a subgroup presenting MYCN amplification has been associated with a poor prognosis.

Intratumoral heterogeneity is defined by distinct genetic alterations present in individual tumors and can be explained by the presence of different cell populations or clones having distinct genetic or molecular profiles within a tumor. Clonal evolution is the process by which a benign neoplasia forms, where successive mutations accumulated in a cell give rise to clonal outgrowths that proliferate in response to tumor microenvironment selective pressures, such as hypoxia, acidosis, competition for space and resources, immune evasion, and treatments leading to malignant transformation [51]. Furthermore, intratumoral heterogeneity is further complicated by the presence of a variety of different cell types, including cancer stem cells, a pool of cells capable of differentiation and increased proliferation. A clinical study is currently underway to define radiogenomic signatures that are predictive of patient outcome with ependymoma tumors (NCT05151718).

### 3.2.2. Hypoxia

Tumor hypoxia is a hallmark of pediatric and adult brain tumors and mainly in high-grade glioma such as GBM. As for adult, hypoxia in pediatric tumor occurs due to the abnormal neovascularization observed within these tumors [52]. As tumors develop, their demand for oxygen increases while the supply remains unchanged. Tumors exploit the host organ's vascular system to create their own vascular network through angiogenesis, but these new blood vessels are unstable and cause microvascular thrombose and vessel occlusions creating hypoxic areas called hypoxic niches [53]. These niches are resistant to conventional anticancer treatments such as chemotherapy and RT. Indeed, hypoxia limit reactive oxygen species formation necessary for cell killing by IR, hyperactivate DNA damage response machinery and regulate cell cycle. To achieve this, hypoxia is characterized by hypoxia-inducible factors (HIFs) which are heterodimeric transcription factors composed of a regulating α subunit, which is sensitive to oxygen with three different homologous α subunits namely HIF-1, HIF-2 and HIF-3. As oxygen level decreases, HIF-α subunit is no more degraded and accumulates in the nucleus, inducing various genes expression mediating cell response to hypoxia. HIFs was characterized in pediatric brain tumor where it was found that hypoxia protects cells against anticancer drug or tumor necrosis and that HIF-1α plays a key role in conferring apoptosis resistance under hypoxia [54]. Additionally, in MB cell lines, upregulation of HIFs under hypoxic conditions was expressed, and a decrease in cellular proliferation was observed in response to knockdown of the *HIF-1α* gene [55]. Always in MB cells, chronic hypoxia (five days in 1% oxygen conditions) induced radio- and chemoresistance mediated with the expression of a number of genes involved in the detection and repair of double strand breaks (DSBs) such as Nibrin (NBN) and MRE11, which are significantly down-regulated [56].

Moreover, stem cells are present in large quantities in hypoxic niches of glial brain tumor and this lack of oxygen plays a role by increasing cell proliferation, self-renewal, and promoting stemness maintenance in glioma stem cells (GSCs) with intrinsic radioresistance mediated by *HIF-1α* and *HIF-2α* [57].

### 3.2.3. Cancer stem cells

CSC are an heterogenous subpopulation of cells within the tumor, comprising less than 3% of the cell population in some tumors and typically found in the most oxygen-deprived regions of the tumor [57]. CSC are characterized by biomarkers such as CD133, Nestin, SOX2 or ALDHα depending on the subpopulation. They possess self-renewal properties, can proliferate continuously, form neurospheres and differentiate into various cell types, contributing to tumor growth. In pediatric brain tumors, particularly in
GBM, ependymoma and MB tumors, intertumoral heterogeneity has confirmed the presence of cancer stem cells. Studies have demonstrated that Nestin overexpressing progenitor-like cells in transgenic mouse models form glioma and MB tumors [58]. Additionally, CSCs have been identified in group 3 MB and shown to be capable of forming neurospheres in culture, with upregulation of CD133, Nestin, and Musashi markers [59]. Importantly, it has been well established that CSCs are significantly more radioresistant as compared to non-CSCs leading to tumor recurrence [60]. Indeed, RT is more effective in rapidly dividing cells than in slow-dividing, quiescent, or stem-like cells. This radioresistance could be due to several factors, including the expression of drug efflux transporters, the regulation of cell death pathways, and the activation of DNA repair mechanisms via the presence of ChK1 and ChK2 enzymes in brain tumors. ChK1 and ChK2 proteins serve as DNA damage checkpoints and are overexpressed in response to IR in the fraction of glioma cells expressing CD133, found in most pediatric brain tumors such as MB, glioma or ependymoma [61,62]. Hambardzumyan et al. discovered Nestin+ MB CSC in the perivascular niche of different post-irradiated (2 Gy) SHH-MB mouse models. To better understand the resistance mechanism via CSC, they tested an inhibitor of Akt signaling, perifosine, and demonstrated cell death induction after combined treatment with IR. Thus, they concluded that PI3K/Akt pathway is implicated in MB CSCs' radioresistance through p53-dependent cell cycle arrest [63].

Another potential resistance mechanism described in brain tumor CSCs is increased aldehyde dehydrogenase (ALDH) activity. ALDH proteins play a role in the maintenance of CSCs and have been demonstrated to be a marker of radioresistance in gliomas [62]. Thus, a study on ATRT in vitro models showed that ATRT cells displayed high expression of ALDH and that combination treatment of an ALDH inhibitor had a possible additive effect when combined with RT [64].

Overall, the existence of CSCs in pediatric brain tumors highly influences radioresistance, other signaling elements that directly contribute to maintain cancer stem cells in tumors are also implied.

### 3.3. Signaling pathways

#### 3.3.1. NOTCH

Notch plays complex roles during normal vertebrate neural development. It promotes neural stem cell survival and self-renewal while inhibiting the differentiation of neural progenitor cells into neurons [65]. However, Notch signaling also promotes the terminal differentiation of astrocytes from glial progenitor cells while inhibiting the differentiation of oligodendrocytes [66]. Over the past decade, accumulating evidence has also shown a critical role for Notch signaling in the maintenance of glioma stem cells through different mechanisms such as the activation of the PI3K/AKT pathway [67]. Following exposure to IR, inhibiting Notch resulted in decreased cell growth, proliferation, and clonogenicity, while promoting apoptosis, indicating a strong correlation between Notch signaling and radioresistance in glioma cells [68]. Taylor et al. determined in a pediatric tumor model of DIPG, a high expression of Notch receptors, ligands, and downstream effectors. In addition, treatment of the DIPG cell lines with a γ-secretase inhibitor or shRNA suppressed the level of Notch effectors, inhibiting cell growth by 75% and enhancing the efficacy of IR at a dose of 2 Gy [69].

#### 3.3.2. Wnt/β-catenin

Another pathway implicated in the maintenance of stem cells is the canonical Wnt/β-catenin pathway, which is an important regulator of embryonic development as well as adult stem cells and has been found to be altered in cancers [70]. In terms of radioresistance,

the Wnt/β-catenin pathway is involved in GBM radioresistance by maintaining the stem cell phenotype and its expression is correlated with the degree of malignancy, thus may serve as a prognostic biomarker [71]. Recently, Zhang et al. found that TOP2A was significantly upregulated in MB and related to poor prognosis in patients and may be directly correlated to Wnt/β-catenin activity, which was inhibited after knockdown of TOP2A alone or in combination with IR treatment at 8 Gy [72].

#### 3.3.3. STAT3

The signal transducer and activator of transcription 3 (STAT3) protein is a member of a family of transcription factors that transmit intracellular signals from the cell membrane to the nucleus [73]. STAT3 activation is mediated by phosphorylation of a tyrosine residue (Tyr705) located in the SH2 structural domain and a serine phosphorylation site at residue 727 (Ser727) within the C-terminal structural domain. It has been demonstrated that aberrant STAT3 activation is associated with radioresistance and a high risk of recurrence after IR, in gliomas [72,74]. Chang et al. demonstrated in isolated CD133+ MB cell lines, an increase in tumorigenicity due to a higher capacity to form spheroid-like bodies and an upregulation of STAT3 expression and STAT3-related pathways. In addition, MB-derived CD133+ cells treated with a potent STAT3 inhibitor significantly reduce the survival rate and number of cells after IR (2 to 10 Gy) [75].

## 4. 3D model of pediatric brain tumor combined with irradiation

Conventional 2D in vitro culture for tumor models involves growing cells in standard petri dishes, flasks, or multi-well plates, arranged in a single layer and adapted to oxygen levels around 20%. These models tend to favor cell populations that grow quickly and are compatible with culture conditions, leading to a loss of cellular diversity. Consequently, the resulting cell populations often lack the inter- and intra-heterogeneous nature, or the cellular diversity of the tumor microenvironment (TME) seen in the original tumors. Indeed, the interplay of tumor cells with the various components of TME including various non-tumoral cell populations and the extracellular matrix (ECM), affects the response to treatment, including RT and tumor recurrence, as seen previously. As a result, it has become essential to develop models which include these key characteristics and allow tumor-TME interactions with essential element gradients as find in in vivo tumor. In recent years, various models have emerged, but sphere and organoid-based models remain the primary 3D structures used in studies in combination with irradiation in pediatric brain tumor studies, as depicted in Table 1.

### 4.1. Brain 3D models with spheres

Sphere-based models are simple, made of cancer cells and can be categorized into three groups: spheroids derived exclusively from established or primary tumor cell lines, organotypic cultures based on tissue culture, and neurospheres cultured under stem cell conditions. These diverse models offer several advantages for studies involving radiation, which will be elaborated upon in this section (Fig. 1).

#### 4.1.1. Spheroids

Pediatric brain tumor spheroids, like other spheroids, are clusters of cells that self-assemble and are cultivated in specialized tissue culture plates, such as hanging drop microplates or low attachment plates [76]. Initially, spheroids can grow without the need of a matrix. These 3D tumor models are found to recapitulate 3D cell-cell interactions, including tight junctions, as well as
<table><caption>**Table 1**
Exhaustive studies of the literature using pediatric brain 3D-models with photon radiation.</caption><thead><tr><th>Brain tumor</th><th>3D model</th><th>Cell origin</th><th>Matrix</th><th>Dose (and dose rate)</th><th>Aim of study</th><th>Ref</th></tr></thead><tbody><tr><td rowspan="2">Glioblastoma</td><td rowspan="2">Tumoroids</td><td rowspan="2">Tissue dissociated from surgical specimens</td><td>Matrigel®</td><td>3 Gy</td><td>Radioresistance of CSC</td><td>[86]</td></tr><tr><td>None</td><td>10 Gy</td><td>Tumoroids response to conventional therapy</td><td>[85]</td></tr><tr><td rowspan="6">Medulloblastoma</td><td rowspan="2">Neurospheres</td><td rowspan="2">Tissue dissociated from surgical specimens</td><td>Matrigel®</td><td>3 Gy</td><td>Tumoroids response to conventional therapy</td><td>[87]</td></tr><tr><td>None</td><td>2 Gy to 8 Gy (1.1 Gy/min)</td><td>Radioresistance of CSC</td><td>[81]</td></tr><tr><td rowspan="4">Tumoroids</td><td>Matrigel® (for tumoroid model)</td><td>2 Gy</td><td>Targeted treatment efficacy in combination with IR</td><td>[90]</td></tr><tr><td>Matrigel® (for tumoroid model)</td><td>2 Gy</td><td>Targeted treatment efficacy in combination with IR</td><td>[90]</td></tr><tr><td>Matrigel®</td><td>2 Gy (2 or 4 Gy/min)</td><td>Targeted treatment efficacy in combination with IR</td><td>[89]</td></tr><tr><td>None</td><td>5 or 10 Gy</td><td>Tumoroids response to conventional therapy</td><td>[88]</td></tr><tr><td rowspan="2">Organotypic multicellular tumor spheroids</td><td rowspan="2">Mouse cerebellums and cell lines</td><td>None</td><td>8 Gy</td><td>Targeted treatment efficacy in combination with IR</td><td>[78]</td></tr><tr><td>None</td><td>1.8 to 10 Gy</td><td>Impact of IR on healthy brain</td><td>[77]</td></tr></tbody></table>

DNET: dysembryoplastic neuroepithelial tumor; NS: neurosphere; CSC: cancer-stem cells; IR: irradiation.

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Type, origin and characteristics of brain spheres and brain organoid-based models. Brain spheres encompass spheroid, organotypic multicellular, and neurosphere models. Spheroids are often derived from established cell lines or primary cells and exhibit a hypoxic core. They can be cultured with a matrix to study cell-matrix interactions and can be grafted onto brain slices to produce organotypic multicellular tumor spheroids, creating a coculture model of healthy brain tissue with tumor cells. Neurospheres are often derived from established cell lines or primary brain cells and are enriched stem-like cell cultures. Brain organoids encompass tumoroid and mini-brain models: tumoroids present intratumoral heterogeneity and share numerous genomic similarities with primary tumors, whereas "Mini-brains" are derived from iPSCs or ESCs-derived brain cells and enable the generation of cocultured models such as GLICO or NeoCOR. NS: neurosphere; GLICO: cerebral organoid glioma; neoCOR: neoplastic corticoid. Created with BioRender.com.</figcaption></figure>
transport properties, mimicking real tumors [77]. The cell origin of spheroid is immortalized or patient-derived cells under conditions which do not select for stem cell renewal. Depending on their size, the spheroid structure is organized into several layers: an external layer accessible to nutrients and oxygen containing proliferative cells, an intermediate layer composed of senescent cells, and a core mainly necrotic, creating a gradient of oxygen and nutrients similar to those found in in vivo tumors [78].

Alternatively, spheroids can also be cultured in a matrix made from natural extracts or synthetic scaffolds, which act as porous support structures allowing cells to produce their own ECM. This approach is advantageous for brain tissue culture as it leads to a gene expression profile closer to the original tissue, and enables the examination of cell-matrix interactions [76]. With a matrix, the oxygen gradient is increased, resulting in a severe hypoxic core and a more realistic treatment sensitivity compared to traditional 2D cell cultures. Indeed, hypoxia is a cause of treatment resistance including RT, as previously mentioned, making this model very attractive for studies using IR. Despite the characteristic oxygen gradient, spheroids are frequently derived from cell lines, and few models are available for pediatric brain tumors, explaining the lack of studies with spheroid monoculture in this field.

### 4.1.2. Organotypic multicellular tumor spheroids

Organotypic multicellular tumor spheroids is a coculture model of spheroid with organotypic brain slice and presents as a promising model to simultaneously study efficacy of radiation on a 3D tumor model and its potential side effects on the peripheral brain. Thus, two studies used this model to evaluate promising therapeutic strategy for the treatment of pediatric MB. Recently, Rossi et al. used an organotypic multicellular tumor spheroids to study the effect of β-blockers treatment, known to inhibit angiogenesis and tumor cell proliferation in various type of cancer in combination with IR. For this purpose, they used a panel of MB cell lines (HD-MB03, UW228-2, DAOY, ONS-76, D283 Med and D341 Med cells) to generate spheroids. They were then grafted on mouse cerebellar slice and daily treated with β-blockers and/or IR (1.8 to 10 Gy) for five days. Tumor growth and invasion within the cerebellum slices were then analyzed over time by imaging. Thus, data displayed that the combinatorial treatment resulting in a reduction of tumor growth, no histological lesions, and no DNA damages in the non-tumor cerebellar tissue, including in the MB periphery. These results suggest that the combination is significantly effective for reducing MB tumor mass without inducing additional damage to the cerebellum [79]. Similarly, Penco-Campillo et al. worked on the same coculture model using two MB radioresistant cell lines (Daoy and ONS-76) to generate spheroids transplanted onto mouse cerebellar slices. Thus, the team proved the blood brain barrier crossing of CXCR1/2 inhibitor over 14 days and the inhibitor efficacy on spheroid growth invasion within the cerebellum tissue and suspect that it might act as a radiosensitizer of tumor cells [80].

Despite numerous advantages, spheroids poorly represent the cellular heterogeneity in tumor such as stem cells which are known to be radioresistant and the main cause of second malignancies in pediatric brain tumor. Therefore, spheroids 3D-model incorporating CSC appears to be pertinent.

### 4.1.3. Neurosphere

To selectively favor the proliferation of stem-like cells, cell lines (either immortalized or brain tumor-derived) used to form spheroids are switched to a serum-free medium with growth factors and cultured in specialized tissue culture plates to obtain free-floating aggregates called neurosphere (NS) [81]. The resulting neurospheres formed under these culture conditions are harvested, dissociated, and plated again to obtain an enriched stem-like cell culture that can be maintained for longer times. Thus, NS

can be used to explore responses to anticancer treatment and have multiple applications in pediatric neuro-oncology, such as early neuronal differentiation to evaluate the side effects on brain development or as tumor initiating-like cells capable of initiating tumors, in the study of second malignancies among others [82]. An advantage is that GSC could differentiate into non-tumorigenic cancer-associated cells, such as vascular cells, that contribute to the formation of the tumor niches. Furthermore, NS can contribute to a deeper understanding of the role of brain tumor CSCs in mechanisms of radioresistance. Lu et al. investigated MB NS to test a potential radiosensitizer, resveratrol. Firstly, cells were dissociated from samples of MB patients and cultured in suspension in serum-free medium with growth factors (stem cell conditions) to obtain NS. The results obtained by flow cytometry exhibited that NS could be positively stained for the CD133 marker of brain tumor stem cell and showed an upregulation of expression levels of stem-cell-related genes (Oct-4, Nanog, Nestin, and Musashi-1) and antiapoptotic (Bcl-2 and Bcl-xL). After resveratrol treatment with various dose of IR (2 to 10 Gy), some significant decrease in survival fraction, migration and colony formation were observed at 2 Gy for the treatment combination compared to each treatment alone [83].

The effect of radiation quality and dose on the developing human CNS was explored with stem cell-derived models. Mayer et al. utilized hESC-derived NS model to examine the potential impact of IR exposure on the capability of hESC to differentiate into neural stem cells and subsequently into neurons. For this purpose, hESC was exposed with 1 Gy X-rays and NS formation was initiated in ultra-low attachment plates and cultured in stem cell conditions. Neural differentiation of hESC into neural stem cells was initiated 4 days after exposure to IR. Then, the expression of neural stem cells and neuron-specific marker proteins was analyzed in NS neuronal differentiation, confirming that the capacity of hESC was not impaired. However, the proliferation capacity of neural stem cells seems altered, as indicated by NS size measurements and functional capacity using microelectrode array (MEA) chips [84]. Klatt et al. employed a similar approach to investigate the effects of low-dose X-ray IR (1 Gy) in a NS model derived from hiPSC and identified a significant impact of radiation on cells surviving, suggesting a negative impact on the early developing human brain with potential later detrimental effects [85].

Despite their self-renewal and differentiation capacities, NS have a major drawback related to the absence of interactions among GSC and other cells, such as non-tumor cells (stromal cells, inflammatory cells), which are implicated in radioresistant mechanisms, as well as vascular cells belonging to the TME [86]. Moreover, both spheroids and NS require significant optimization of culture conditions to achieve uniformity in size, enhance reproducibility, and establish optimal gradients while preventing necrosis. These reasons explain the lack of studies using these models.

### 4.2. Brain organoids-based models

The use of 3D organoids may help overcome limitations of traditional sphere culture, which are organized in a random 3D structure. Indeed, organoids self-arrange their cell types into micro-moieties that more closely approximate organ tissue structure and function. In addition, the lack of cellular diversity in spheroids is overcome with organoids models. These models can be divided into two major groups: (I) organoids starting only from tumor tissue, called “tumoroids” or patient-derived organoids (PDOs) and (II) organoids composed of a non-tumor compartment from hiPSCs or hESC.

#### 4.2.1. Tumoroids/Patient-derived organoids

There are two main reported methods to generate brain tumors: Jacob et al. describe tumoroid formation from a small piece of
GBM tumor dissected and dissociated to generate initial tumor cells (patient derived, patient-derived metastases, or genetically engineered) and cultured for two weeks without matrix and growth factors [87]. Alternatively, Hubert et al. generated GBM tumoroids by dissociation of tumor piece and culture in matrix for two months [88]. Compared to spheroids, tumoroid model maintains cellular heterogeneity with a mixture of CSCs and non-CSCs, quiescent and proliferative cells, and even immune cells such as macrophages, microglia and T cells. Moreover, the histological characteristics of the primary tumor are maintained with non-cellular areas filled with fluid or extracellular matrix, which can be observed. For studies using IR, brain tumoroids present multiple advantages such as a nutrient and hypoxic gradient. Hubert et al. characterized GBM tumoroids and the presence of in vitro "hypoxic niches" containing primarily non-stem senescent cells and diffuse quiescent CSCs due to microenvironmental gradients and "perivascular niches" characterized by the presence of SOX2+ stem cells. They explored whether different regions of GBM organoids exhibit varying radiation sensitivities by examining caspase 3 cleavage. Thus, they demonstrated a significant increase in GBM cells around the organoid rim, but not within the organoid core after irradiation at 3 Gy, where apoptotic cells were also almost exclusively SOX2 negative (non-stem). These findings functionally validate the ability of SOX2β cells within the organoid to display a common CSC phenotype radioresistance [88]. Additionally, tumoroids present an in vivo like-cell diversity creating a radiosensitivity/radioresistance like most in vivo tumor and not found in spheroids [89]. Recently, the team of Lago et al. reported the successful generation and characterization of tumoroids from MB pediatric brain tumor biopsies to evaluate response to treatment protocols, including RT. The chosen patient was 15 years old, and the tumor was characterized by MYC amplification, making it strongly aggressive. Fresh surgically resected tumor samples were chopped into small pieces (0.5-2 mm diameter), and tumor pieces were transferred to a low-cell-adhesion culture plate containing PDOS in stem cell conditions. They demonstrated a significant difference in the fluorescence intensity of living cells, especially in treated PDOS with two doses of RT (5 and 10 Gy), and a decrease in Ki67+ proliferative cells in treated PDOS one month after the last dose of RT [90]. Similarly, Sundar et al. have generated pediatric GBM tumoroids in a matrix and demonstrated their resistance to standard-of-care therapy, including RT. To date, tumoroids have been irradiated with 3 Gy X-rays and treated with temozolomide. It was demonstrated that organoid specimens were able to maintain their structure and overall viability compared to matched spheres. However, it was observed that the organoids continued to exhibit proliferation in the rim, as evidenced by phosphorylated Histone H3 staining [89]. This inerrant radioresistance of organoids was also therapeutically exploited. For instance, Frisira et al. used MB tumoroids to validate the therapeutic potential of a proteasome inhibitor (NPI-0052) combined with IR. They created tumor organoids from primary cells derived from the most aggressive MB subgroups and grown in Matrigel®. The tumoroids were treated with 2 Gy γ-radiation and different concentrations of NPI-0052. The team demonstrated that all organoids exhibited the morphological characteristics of MBs by H&E staining, inducing, enhanced p53 stabilization, cleaved caspase-3, and increased γ-H2AX expression levels compared to treatment alone. These data suggested that NPI0052 treatment enhances radiation effect in the most aggressive MB subgroups via DNA damage and p53-induced apoptosis [91]. In a similar manner, Li et al. generated three tumoroids from three subgroup MB patient specimens, R403 (Group 4-MB, subtype VI), R901 (Group 4-MB, subtype VIII) and R902 (SHH-MB), and treated them with CT-179, an OLIG2-positive inhibitor, combined with IR (2 Gy). They demonstrated that the combination treatment affected the structure of tumoroids differently and the response to treatment was assessed with cleaved caspase-3 staining. Additio-

nally, SHH-MB organoids showed a higher response, confirmed by significantly reduced proliferation (Ki67), without inducing a compensatory increase in the SOX2+ population (stem cell). These data proved that inhibition of OLIG2-positive cells in combination with IR significantly slows tumor cell progression [92].

Additionally, tumoroids exhibit similarities in the transcriptional and genomic levels with primary tumors, providing an advantage to study radioresistance mechanisms, where numerous pathways are implicated, as mentioned above [93]. Moreover, despite the lack of studies, tumoroids derived from brain tissue are described to contain oligodendrocytes, astrocytes, neurons cells, where oligodendrocyte cells are implicated in the neurotoxicity mechanism of RT. This makes tumoroids a potential model to study the side effects of RT on non-malignant cells.

### 4.2.2. Organoids/"Mini-brain"

Brain organoid (BOs), also known as "mini-brain", are defined as "healthy" organ-like 3D models generated from hESC, hiPSCs or adult stem cells [94]. They have the capability of unlimited proliferation and the potential to differentiate using sequential changes in media and additives. Brain organoids comprise multiple cell types with organization, promoting the maintenance of a stem-like cell state, organ-like function, and gene expression indicative of the organ being modeled, thus mimicking the heterogeneity found in the TME [88]. Unlike tumoroids, organoids, by definition, comprise only 'normal' cells. However, for studying tumor invasion and cell-cell interactions between tumors and normal cells, brain organoids can be cocultured with tumor cells from spheroids or genetically modified to form invasive protrusions and microtube networks in brain tumor cells. Thus, brain models composed of a tumor and a non-tumor compartment, such as cerebral organoid glioma (GLICO) or neoCOR using CRISPR-Cas9-based genetic engineering, have emerged, and are exploited to study the role of genetic mutation in tumorigenesis and tumor development including invasiveness. However, these models are under-utilized in the pediatric brain tumor field. Thus, Bian et al. reported a neoCOR PNET or GBM model from hESC by introducing various oncogene mutations with plasmid electroporation for evaluation of targeted drug such as EGFR inhibitors or chemotherapeutic drug such as afatinib or erlotinib [95]. Similarly, Ballabio et al. utilized human cerebellar organoids based on hiPSC electroporated with plasmids encoding for multiple genes to validate and understand the role of these genes found altered in MB patients [96]. At the time of writing this review, no study using pediatric BO combined with RT has been exploited, although this model seems appropriate. Indeed, BO models preserve multiple TME cell types, including oligodendrocytes and endothelial cells, cells which undergo apoptosis after IR. Thus, we can easily envision studying the side effects of IR on the healthy brain with this model. Moreover, under specific conditions of culture with oligodendrocytes, a myeloid model can be generated to study the myelin disruption after IR [97]. Additionally, coculture models of BO with brain tumor cells seem appropriate to assess both the efficacy of IR on brain tumors and its impact on peripheral tissues.

Even though some BOs could maintain some vascular endothelial cells, no fully formed blood vessels were observed, and this lack of a functional vascular system is a major limitation of these models.

## 5. Conclusions

In summary, due to the overall rarity of pediatric brain tumors and the limited availability of samples, only a few 3D models of these diseases have been established, characterized and treated with conventional therapies, including IR. "Simple" models such as spheroids/NS remain pertinent models for studies involving IR
due to their ability to mimic hypoxia gradients or contain cancer stem cells, providing a valuable starting point for understanding radioresistance mechanisms and exploring potential therapeutic treatments. However, more complex models such as organoids closely mimic in vivo tumors with cellular diversity from the tumor and its microenvironment, hypoxic niches created by vascularization, and the potential for coculture between malignant and non-malignant cells. Thus, organoid models appear to be valuable tools for investigating both the effectiveness and potential toxicities of RT. No fractionated RT protocol was found across the different studies utilizing both 3D-models, and no non-conventional RT was examined using these diverse models. These two aspects require deeper investigation in the future to replicate the radiation treatment protocols of pediatric brain tumor patients more accurately.

Despite the absence of a perfect in vitro model that exactly mimics tumor cells and their TME, it is imperative to enhance the utilization of 3D models, especially organoids, in the future to improve the effectiveness of RT efficacy and decrease long-term devastating side effects, thereby improving the quality of life for all pediatric brain tumors patients.

## Funding

This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

## Disclosure of interest

The authors did not disclose their relationships/activities/interests.

## Author contributions

É.C.: literature search, manuscript writing – initial draft, revision of the manuscript; H.B., G.N.: manuscript writing – initial draft, revision of the manuscript; M.D., N.E.W.: revision of the manuscript.

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