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Int J Biol Sci 2024; 20(2):765-783. doi:10.7150/ijbs.91295 This issue Cite

Review

Radiotherapy in Preclinical Models of Brain Metastases: A Review and Recommendations for Future Studies

Wen Shi^1#, Guilong Tanzhu^1#, Liu Chen¹, Jiaoyang Ning¹, Hongji Wang¹, Gang Xiao¹, Haiqin Peng¹, Di Jing¹, Huadong Liang², Jing Nie², Min Yi², Rongrong Zhou^1,3,4

1. Department of Oncology, Xiangya Hospital, Central South University, Changsha, 410008, Hunan Province, China.
2. Department of Technology, Hunan SJA Laboratory Animal Co., Ltd., Changsha, Hunan Province, China.
3. Xiangya Lung Cancer Center, Xiangya Hospital, Central South University, Changsha, 410008, Hunan Province, China.
4. National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, 410008, Hunan Province, China.
# Wen Shi and Guilong Tanzhu contributed equally to this work.

✉ Corresponding author: Rongrong Zhou, Department of Oncology, Xiangya Hospital, Central South University, No. 87 Xiangya Road, Kaifu District, Changsha, 410008, China, E-mail: zhourredu.cn.

Abstract

Brain metastases (BMs) frequently occur in primary tumors such as lung cancer, breast cancer, and melanoma, and are associated with notably short natural survival. In addition to surgical interventions, chemotherapy, targeted therapy, and immunotherapy, radiotherapy (RT) is a crucial treatment for BM and encompasses whole-brain radiotherapy (WBRT) and stereotactic radiosurgery (SRS). Validating the efficacy and safety of treatment regimens through preclinical models is imperative for successful translation to clinical application. This not only advances fundamental research but also forms the theoretical foundation for clinical study. This review, grounded in animal models of brain metastases (AM-BM), explores the theoretical underpinnings and practical applications of radiotherapy in combination with chemotherapy, targeted therapy, immunotherapy, and emerging technologies such as nanomaterials and oxygen-containing microbubbles. Initially, we provided a concise overview of the establishment of AM-BMs. Subsequently, we summarize key RT parameters (RT mode, dose, fraction, dose rate) and their corresponding effects in AM-BMs. Finally, we present a comprehensive analysis of the current research status and future directions for combination therapy based on RT. In summary, there is presently no standardized regimen for AM-BM treatment involving RT. Further research is essential to deepen our understanding of the relationships between various parameters and their respective effects.

Keywords: Brain metastasis, Radiotherapy, Dose fractionation, Radiation, Animal models, Combined modality therapy

Introduction

With prolonged patient survival and advancements in imaging technology, the incidence of brain metastases (BMs) is on the rise ^[1-4]. Common primary tumors associated with BM include lung cancer ^[5-7], breast cancer ^[4,8,9], and malignant melanoma ^{[10, 11]}. Despite multiple interventions, such as surgery, radiotherapy (RT), and chemotherapy, patients with brain metastases face disappointingly short survival, with a 2-year survival rate that is less than 10% ^[12]. In recent years, the application of targeted therapy and immunotherapy has led to improvements in survival ^[4,12,13].

Radiotherapy (RT) is the cornerstone treatment for BM and enhances the local control rate ^[14] and reduces BM recurrence ^[15-18]. The RT options for BM treatment include whole-brain radiotherapy (WBRT) ^[19] and stereotactic radiosurgery (SRS) ^[20-22]. Prophylactic cranial irradiation (PCI), a unique form of RT, is known to delay and reduce the occurrence of BM ^[23-25]. WBRT is typically recommended for patients with multiple brain metastases (usually > 3 lesions ^[19]), while SRS may serve as the standard treatment for oligometastatic lesions (usually ≤ 4 lesions ^{[20-22, 26]}). In addition to its direct impact on the BM, RT alters the tumor microenvironment and the permeability of the blood-brain barrier (BBB), laying the foundation for combination therapies ^[27-29]. Currently, the sequence and timing of combining RT with immunotherapy ^[30-34], targeted therapy, or new treatments such as nanomaterials ^[35-38] are key research areas in the field of BM treatment.

Animal experiments play a pivotal role in preclinical research, offering a theoretical basis for clinical translation. However, distinct treatment regimens yield varied effects, and the parameters from model establishment to treatment delivery are diverse. Although RT parameters (such as RT mode, dose, dose rate, fractionation, etc.) have been explored in subcutaneous models of various tumors, these models are limited by replicating the intrinsic structure of the BBB and the unique immune microenvironment of the BM. Consequently, honest evaluations of RT and drug efficacy for treating BM are challenging. Intracranial patient-derived tumor xenograft (PDX) models, more akin to the phenotype and genotype of BM patients than subcutaneous PDX models ^[39,40], are crucial for assessing local curative effects and their mechanisms ^[13].

Currently, RT parameters in animal models of brain metastases (AM-BMs) lack standardization, and there is a dearth of reviews on this topic. This review, based on AM-BM, systematically summarizes RT regimens for BM for the first time, covering model establishment for RT implementation, and providing a reliable foundation for subsequent research. Additionally, comprehensive treatment is the primary approach for treating BM. We consolidate the schemes and molecular mechanisms of RT combined with other treatments.

Establishment of AM-BM and the Intervention Time of RT

Various methods are employed for the establishment of AM-BMs including intracerebral ^{[33,39,41-50]}, intracardiac ^{[31,32,51-54]}, carotid artery ^[55] and tail vein injection ^[56,57]. Few studies have explored spontaneous ^[58] or induced AM-BM ^[58,59]. Mice are the most commonly used animals for AM-BM ^{[30,38,41,43,49,56,60-65]}. The growth and therapeutic effects of AM-BM were monitored using in vivo imaging systems (IVIS) or magnetic resonance imaging (MRI), despite the relatively small volume of the BM (Figure 2E). The evaluation indicators for RT efficacy in AM-BM typically include tumor size and lesion size, overall survival, Ki67, Caspase-3, γH2AX expression, and so on (Figure 2G).

Establishing brain-tropic cells (brain metastasis cells) requires in vivo and in vitro screening. The selection process involves modifying cancer cells with reporter genes such as luciferase or GFP, which allows changes to be easily visualized, assessed, and prepared, using IVIS or MRI. Additional rounds of selection are then carried out. The modified cells are then reintroduced into the mice, usually after a period of growth outside the body (Figure 1).

The methods for establishing AM-BMs are multifaceted, each with pros and cons. Intracerebral injection (mostly in the striatum ^{[33,39,41-48]} or cerebral cortex ^[49]) can swiftly cause the formation of a single lesion ^[36], leading to high success rates. This method effectively summarizes BM growth and proliferation ^[66,67]. However, this approach disrupts the BBB, neglects the metastasis and colonization process, and thereby weakens predictive accuracy of treatment efficacy ^[59]. Arterial inoculation (internal carotid artery and intracardiac injection) is complex ^[68] and has low success rate ^[69,70]. Due to hematogenous metastasis, the location or lesions of intracranial tumors are randomized, and the formation of multiple extracranial metastases is unavoidable ^[71]. Intravenous (IV) inoculation (tail vein injection) is uncommon due to the low incidence of BM formation and inevitable lung metastases. Spontaneous models frequently form a single lesion in the BM ^[58], reflecting the actual process from tumor occurrence to metastasis. However, extensive use is hindered by the prolonged experimental period and metastases throughout the body ^[68]. Given these considerations, we highlighted precautions for model construction and detection indicators in the AM-BM (Figure 2).

The chosen modeling method influences the RT mode (Figure 3B). For intracerebral inoculation modeling, SRS ^[42,72,73] or WBRT ^{[30,33,37,38,45,46,49,74,75]} were commonly applied. Arterial inoculation-constructed AM-BM often involves WBRT ^{[31, 32, 51-55]}. Additionally, PCI is used for AM-BMs established through intravenous (IV) inoculation via tail vein injection ^[56,57]. Different model-building parameters and growth characteristics of BMs led to variable RT implementation times (Figure 3B).

The irradiation time of AM-BMs varies based on the inoculation methods (intracerebral earlier than systemic) and animal species (mouse earlier than rats) ^[48]. In syngeneic models, BMs proliferate faster than in xenograft models, suggesting a shorter treatment window ^[68]. For intracerebral injection of lung/breast cancer AM-BM, irradiation time is closely tied to injection cell number, generally, beginning within 2 weeks for 10⁶ cells ^[46,76] and within 2-3 weeks for 10⁴ cells ^[45,75,77]. In melanoma AM-BM, irradiation of 10²-10⁴ cells usually occurs within 1-2 weeks ^{[33,34,42,48,73]}. In particular, PCI is administered within one week of cell inoculation ^[56,57,78]. The relationship between the implanted cell counts and RT intervention time is shown in Table 1.

 Figure 1 

The Process of Establishing Brain Tropic Cells. Establishing brain-tropic cells (brain metastasis cells) requires in vivo and in vitro screening. The selection process involves modifying cancer cells with reporter genes such as luciferase or GFP, which allows changes to be easily visualized, assessed, and prepared, using IVIS or MRI. Additional rounds of selection are then carried out. The modified cells are then reintroduced into the mice, usually after a period of growth outside the body.

 Figure 2 

Precautions in Model Construction and Detection Indicators in the AM-BM. (A-D) Several key parameters significantly influence the tumor formation rate during AM-BM model development. (A) Species including C57BL/6 mice, SCID mice, BALB/c nude mice, and rats are frequently utilized in AM-BM studies. (B) Various cell lines, such as lung cancer (H2030-BrM, PC-9-BrM3), breast cancer (BT474-BrM3, 4T1Br4), and melanoma cell lines (B16-F10, B78), are commonly employed for AM-BM establishment. (C) The quantity of injected cells is a critical determinant of successful model construction and the optimal time window for treatment. (D) Current BM modeling methods encompass intracerebral injection (ICB), intracardiac injection (ICD), internal carotid artery injection (ICA), tail vein injection (IV), and spontaneous or induced models (SP). (E-G) Common parameters assessed in in-vivo studies include: (E) Tumor lesion, tumor number, and tumor volume. (F) Survival. (G) Tumor biomarkers, such as the expression of Ki67, γH2AX, and so on.

 Figure 3 

Animal Species, Irradiation Methods Selection, and Effects of RT on AM-BM. (A) Animal species currently employed in RT studies of AM-BM encompass mice, chicken embryos, monkeys, rats, dogs, and rabbits. (B) Different irradiation methods are utilized based on the modeling approach: SRS or WBRT is commonly applied for local inoculation modeling, WBRT for intracardiac and internal carotid artery injections, and PCI for tail vein injection. (C-D) The prognostic factors influencing survival were as follows: (C) RT-induced side effects on brain tissue, such as radiation edema, necrosis, neurotoxicity, and hippocampal damage. (D) Factors such as radiation resistance genes (TopBP1 and Claspin), secretion of S100A9, and the overexpression of RAGE limit the survival benefits of RT. (E) In vivo studies reveal differential responses of the blood-brain barrier/blood-tumor barrier in various mouse strains to RT. Notably, doses of 3 Gy/1F, 12 Gy/3F, 15 Gy/1F, 15.5 Gy/1F, and or 20 Gy/2F did not significantly alter the permeability of the blood-brain barrier/blood-tumor barrier in BALB/c nude mice. However, doses of 15.5 Gy/1F and 30 Gy/5F can induce changes in the blood-brain barrier/blood-tumor barrier permeability in C57BL/6 mice. The time window during which RT induces BBB/BTB opening in AM-BMs has not been determined.

Dose and Fractionation of RT in AM-BM

RT is a conventional therapy for BM ^[17]. However, the diversity of RT regimens in AM-BM across multiple studies underscores the necessity for standardization. Currently, WBRT is widely applied in AM-BM ^{[30,33,34,37,38,45,46,49,54,74-77,79]}, followed by SRS ^[42,72,73]. Various parameters influence RT efficacy, including the RT method, dose, fractionation, dose rate, and intervention time. A comprehensive summary of these parameters was obtained from available radiotherapy studies in AM-BM (Table 2-6). Additionally, a comprehensive and scientific template for reporting experiments involving AM-BM and RT is shown in Table 7.

The linear-quadratic (L-Q) model, typically used to calculate the biologically effective dose (BED) of different fractionation schemes employs the formula BED = D [1 + d/(α/β)] ^[80]. The alpha/beta ratio(α/β), total dose (D), and fractional dose (d) are integral components ^[81]. The criteria for RT schemes in AM-BM include (1) clinical regimens ^[62,74,76], (2) BED equivalent schemes ^{[33,34,38,45,54,75,76,79]}, (3) previous experience based on different research objects ^{[35-37,50,52,56]}, and (4) protection of normal tissues ^[49,55,82]. The first two criteria are generally applied.

WBRT

WBRT is widely employed in AM-BMs. The 30 Gy/10F or 20 Gy/5F regimens ^[83] are recommended by the National Comprehensive Cancer Network (NCCN) guidelines for BM patients ^[84], while preclinical studies typically administer WBRT at a total dose ranging from 15 Gy to 20 Gy in a single or fractionated high-dose irradiation format ^{[33,34,38,45,54,75,76,79]}.

As indicated by previous reports, lower doses (< 15 Gy) of WBRT have been explored due to their ability to inhibit tumors and prolong survival ^{[36,37,52,56]}. Notably, a study implementing WBRT (12 Gy/3F) significantly restricted tumor volume but failed to reduce the number of BM lesions ^[52]. In AM-BM of breast cancer, Choi et al. demonstrated that 10 Gy/1F exhibited a stronger inhibitory effect than 5 Gy/1F, with no significant difference observed with 20 Gy/1F ^[65]. Compared with those treated with 5 Gy/1F, 15 Gy/1F, or 20 Gy/1F, animals treated with BM via the 10 Gy/1F regimen had the longest survival ^[50]. In combination therapy, the use of WBRT (7 Gy/1F) with nanoparticles for AM-BM of melanoma demonstrated a reduced RT dose and prolonged survival ^[36].

Using the L-Q model, the BED of RT regimens (15-16 Gy/1F, 20 Gy/2F) in AM-BM was found to be comparable to the clinical regimens of 30 Gy/10F, assuming the α/β value of 10 ^{[54,76,78,79]}. Zarghami et al. and Murrell et al. employed 16 Gy/1F and 20 Gy/2F, respectively, and showed significant reductions in BM lesions and tumor volume ^{[54, 79]}. Due to the limited access to synchrotron radiation sources, some studies have applied single-dose fraction RT ^[85], which saves time but may increase the risk of edema and necrosis.

To mitigate side effects, certain studies have adopted regimens with lower BED for normal tissue ^[49,55,82]. Martínez-Aranda et al. demonstrated that the 16.5 Gy/3F protocol ^[55], which involves a BED lower than 30 Gy/10F, significantly alleviated brain toxicity and reduced the frequency of intraperitoneal anesthesia, partially circumventing accidental death ^[55]. Similarly, Prociss et al. verified that the 10 Gy/5F regimen effectively avoided neurotoxicity in AM-BM ^[49]. To overcome radiation resistance in melanoma and prevent radiation necrosis simultaneously, Wall et al. raised the single dose to evaluate the effect of RT (12 Gy/3F) ^[82].

Alterations in the tumor microenvironment following radiation exposure also led to varied effects on tumor eradication. Different RT fractions with equivalent doses led to diverse responses of immune cells ^[31,32]. Schulz et al. reported that compared with that of 10 Gy/1F, 10 Gy/5F increased monocyte-derived macrophage (TAM-MDM) infiltration. However, the former regimens reduced TAM-MDMs and, more significantly, altered the expression of genes related to pro-inflammatory host defense responses in peripheral myeloid cells ^[32]. Distinct RT fractions with the same BED generated diverse inhibitory effects. For instance, 30 Gy/10F significantly reduced intracranial metastases, while 15 Gy/1F was more beneficial for median survival ^[76].

RT was found to delay the progression of BM and prolong survival in various studies ^{[33,36,38,45,55,76,77,86]}. Hofland et al. demonstrated that in AM-BM of Ehrlich's ascites cancer, RT significantly prolonged survival, with the 10 Gy/1F regimen showing more apparent effects ^[50]. In AM-BM of breast cancer, RT may achieve long-term survival ^[76]. However, individual studies reported conflicting conclusions ^{[44,49,75,87]}. For instance, Chae et al. showed that WBRT (10 Gy/1F) reduced tumor volume but had no significant effect on survival ^[53]. Similarly, regimens such as 30 Gy/10F, 16.5 Gy/3F, and 9 Gy/3F did not improve tumor progression and survival due to radiation resistance ^[88].

Reasons for Conflicting Results in WBRT Research in AM-BM:

(1) Impact of Model Establishment on Survival. Some studies suggest that the mode of model establishment may influence survival. Arterial inoculation, which can lead to widespread tumor metastasis, raises the concern that animal death may not be solely attributed to BM ^[76].

(2) Side Effects Caused by RT. The side effects of RT such as radiation edema, necrosis, nerve damage, and hippocampal damage can vary and impact study outcomes. WBRT (10-15 Gy) has been observed to inhibit nerve growth by inducing DNA double-strand breaks (DSBs) and apoptosis ^[53,76,89]. Additionally, single high-dose RT may lead to hippocampal toxicity ^[76,90] (Figure 3C).

(3) Inconsistent Radiosensitivity among Various Cancer and Cell Subtypes. The calculation of the biologically effective dose (BED) by the linear-quadratic (L-Q) model may be inaccurate, especially after standardizing the empirical rule α/β=10 across various tumors ^[91]. Moreover, the L-Q model is not suitable for relatively high doses (>13 Gy) ^[92].

(4) Radiation-Resistant Genes. The presence of radiation-resistant genes, such as TopBP1, Claspin, and Caveolin 1, in AM-BM of NSCLC, may influence the effectiveness of RT (Figure 3D). These genes alleviate inhibition, leading to improved survival following RT ^[75,93]. Furthermore, the expression and secretion of S100A9 from BM cells, which binds to the RT-induced RAGE receptor, activate NF-κB mediated RT resistance ^[88] (Figure 4K).

In conclusion, these factors highlight the complexity of interpreting and comparing results across different studies. A summary of the relevant parameters for WBRT in AM-BM is provided in Table 2 and Table 3.

 Figure 4 

Changes in Molecular Pathway Induced by RT with or without Additional Treatments in Animal Models of Brain Metastases from Lung Cancer, Melanoma, and Breast Cancer. (A-C) Lung Cancer BM. (A) AZD-3759 enhances NSCLC radiosensitivity by inhibiting EGFR and JAK1; (B) AZD7762 promotes NSCLC radiosensitivity by suppressing CHK1; (C) RT inhibits phosphorylation of JAK2 and STAT3, inducing apoptosis. (D-F) Melanoma BM. (D) Thymoquinone (TQ) increases radiation-induced apoptosis by inhibiting JAK2 phosphorylation; (E) Riluzole enhances radiosensitivity of melanoma BM cells by inhabiting GRM1; (F) 5-ALA increases melanoma BM radiosensitivity by increasing the porphyrin content. (G-K) Breast Cancer BM. (G) L-arginine mediates radiosensitivity through NO-dependent inhibition of GAPDH and PARP activation; (H) Metformin enhances tumor suppression when used as an adjuvant in RT; (I) LRRC31 importation via nanomaterials inhibits DNA repair and radiosensitivity in breast cancer BM; (J) Vorinostat, a histone deacetylase inhibitor, increases radiation sensitivity by inhibiting HDAC; (K) BM secreting S100A9, which binds to the RT-induced RAGE receptor, activates NF-κB-mediated RT resistance.

SRS

SRS, which is commonly administered to single lesions ^{[35,42,72,73]} in AM-BM, has received limited research attention. The dosage of SRS tends to decrease when combined with other treatments ^[35,42]. Numerous studies have demonstrated that SRS contributes to prolonged survival ^[42,72,73]. Notably, Nakahara et al. recently reported a significant increase in survival when SRS (32 Gy/1F) was combined with immunotherapy ^[72]. Additionally, they observed the inhibition of JAK2 and STAT3 phosphorylation after SRS (15 Gy/1F). This inhibition, in turn, triggered cell death by regulating apoptosis-related proteins, such as increased Caspase-3 and BAX and decreased BCL-2 and Survivin ^[42] (Figure 4C). A summary of relevant parameters from SRS studies in AM-BM is presented in Table 4.

PCI

In contrast to therapeutic irradiation, the primary objective of PCI is to reduce the incidence of BM ^[25]. This perspective is supported by various preclinical studies and computer models ^[56,57,78]. In preclinical studies targeting breast cancer, 4 Gy/1F at 3.2 Gy/min and 20 Gy/2F significantly lowered the occurrence of BM ^[56,78]. However, eliminating dormant cells in BM has shown to be challenging with PCI, which accounts for subsequent tumor occurrence ^[78].

A critical consideration involves determining the optimal timing for PCI intervention in AM-BM. Studies demonstrate that performing PCI within 1-5 days after tumor cell injection ^[56,57,78] consistently reduces the incidence of BM. In contrast, executing PCI either before or 3-6 weeks after systemic inoculation poses challenges and may not achieve optimal outcomes ^[56]. Premature PCI interventions may even promote tumor progression and metastatic formation by inducing alterations in the brain microenvironment. For instance, administering RT (10 Gy/1F) seven days before injection results in damage to normal brain tissue, which becomes more susceptible to the growth of BM ^[51]. The relevant parameters of the PCI research are detailed in Table 5. Moreover, for those that do not specify the specific radiotherapy method, the parameters are detailed in Table 6.

RT and the BBB/BTB

Both clinical and preclinical studies have consistently demonstrated that RT induces an increase in the permeability of the BBB or blood-tumor barrier (BTB), resulting in elevated intracranial drug concentrations. These findings form the theoretical basis for combination therapy ^{[27-29,94-98]}. In addition, RT (30 Gy/5F) combined with focused ultrasound (FUS) disrupts BBB integrity ^[99]. Interestingly, a subset of in-vivo studies has reported contradictory conclusions. Notably, high-dose irradiation whether delivered as a single dose or in a fractionated manner, has shown limited impact on BBB/BTB permeability in certain scenarios ^[52,54]. In AM-BM of lung/breast cancer (nude mouse models), after 3 Gy/1F ^[74], 12 Gy/3F ^[52], 15 Gy/1F ^[74], 15.5 Gy/1F ^[100], or 20 Gy/2F ^[54] irradiation did not significantly alter BBB permeability and, in some cases, even led to a short-term decrease (within 24 hours) ^[74]. The immune function of the BM model may contribute to this observed phenomenon ^[100]. For instance, twelve hours after 15.5 Gy/1F irradiation, changes in the integrity of the BBB and the activity of efflux transporters were noted in immunocompetent mice, while no such differences were observed in immunocompromised mice. This finding implies a potential association between the immune response and BBB damage after RT ^[100]. It is essential to consider the time interval during which RT induces BBB opening, and variations in sensitivity among different detection methods should be taken into account (Figure 3E).

Combination therapy in AM-BM

RT Combined with Chemotherapy

Chemotherapy has been shown to augment the radiosensitivity of BM ^{[50,55,101,102]}. Temozolomide (TMZ) ^[55,102], etoposide ^[50], and dexrazoxane ^[50], when combined with RT, effectively inhibit the progression of AM-BM. TMZ, recognized for its ability to penetrate the blood-brain barrier, is recommended as a chemotherapeutic drug for intracranial tumors ^[103]. Furthermore, TMZ enhances the radiosensitivity of brain metastatic tumor cells by inhibiting DNA damage repair after RT and amplifying mitotic catastrophe ^[102]. RT combined with TMZ has been shown to prolong survival in AM-BM of breast cancer ^[55]. Meanwhile, in the AM-BMs of NSCLC, non-ablative radiation (2 Gy) enhances the delivery of anti-MGMT morpholino oligonucleotides (AMONs) improving TMZ efficacy by inhibiting MGMT ^[43]. Etoposide plus dexrazoxane, combined with WBRT (10 Gy/1F) increased the median survival by 60% with no additional toxicity ^[50]. Similarly, an antibody-drug conjugate such as BR96-DOX in combination with RT significantly prolonged survival in AM-BMs of SCLC ^[77]. Furthermore, compared with concurrent chemoradiotherapy, antibody-drug conjugates administered before RT improved survival ^[77].

RT Combined with Targeted Therapy

The combination of RT and targeted therapy typically has synergistic effects ^[104]. WBRT enhances the therapeutic effect of single domain antibody fragment (Anti-HER2 VHH 5F7) on human epidermal growth factor receptor type 2 (HER2) positive BM by increasing vascular permeability ^[49]. Overall, targeted c-Met and RT inhibit tumors and prolong the overall survival of tumor-bearing mice ^[44].

Similarly, targeted agents enhance the efficacy of radiotherapy. Targeting EGFR ^[62], CHK1 ^[45], HDAC ^[47], CXCR4 ^[71], ATR ^[47], and GRM1 ^[82] significantly improved RT efficacy. For instance, AZD3759 (zorifertinib) amplifies the antitumor effect of RT by interfering with EGFR and JAK1 ^[62] (Figure 4A). AZD7762 enhances radiosensitivity in vitro and in vivo by inhibiting CHK1 ^[45] (Figure 4B). Vorinostat improves the median survival of AM-BMs by blocking histone deacetylases (HDACs), leading to DSBs repair inhibition and mitotic catastrophe ^[47] (Figure 4J). Riluzole (a glutamate signaling blockade) sensitized cells to RT (Figure 4E). Mechanistically, inhibition of glutamate signaling led to G2/M phase arrest in melanoma cells ^[82].

Additionally, targeted drugs modulate the tumor microenvironment ^[71,82]. Endostar enhances RT efficacy by blocking RT-induced CXCR4. Subsequently, TAM infiltration and macrophage M2 polarization are inhibited, and the percentage of CD4+T/CD3+T cells increases ^[71].

RT Combined with Immunotherapy

Immunotherapeutic strategies, including immune checkpoint inhibition (ICI), adoptive cells, tumor vaccines, oncolytic viruses, and cytokine therapy, are integral components of AM-BM treatment. Currently, the integration of immunotherapy with RT in AM-BM primarily involves ICI and in-situ vaccination (ISV), both of which enhance RT efficacy. Notably, combining a tumor vaccine with RT (15 Gy) significantly reduces tumor volume and delays BM progression ^[33].

RT contributes to immunotherapy efficacy by regulating the tumor microenvironment ^[30-32]. WBRT recruits myeloid cells and enhances their proinflammatory response ^[32]. Meanwhile, WBRT significantly elevated TNF-α and CXCL1 in the serum of immunocompetent and immunocompromised mice ^[100]. The levels of proinflammatory cytokines (TNF-α, IL-2, and IL-12p70) increase after WBRT in immunocompetent mice but not in nude mice ^[100]. Furthermore, increasing the radiation dose (from 15 Gy to 18.5 Gy) improved immunotherapy efficacy in AM-BM, resulting in longer survival and tumor dormancy periods ^[34]. Relatively low-dose WBRT (4 Gy) or targeted radionuclide therapy increased the number of T cells (CD4+ and CD8+) and the monocyte/macrocytic phagocytic (F4/80+) population, enhancing the immunotherapy response in melanoma BM ^[30].

The sequencing of immunotherapy and RT needs to be further explored. Transcriptome analysis revealed that RT following ICI treatment is involved in cell death and inflammation signaling in melanoma BM. Preclinical studies have demonstrated that RT followed by anti-PD-L1 therapy is preferable ^[105], which has also been confirmed in clinical trials ^{[106, 107]}.

Combination of RT and Novel Technologies

Ongoing exploration by radiation biologists has led to the application of novel technologies to AM-BM. Compared with conventional RT, FLASH radiotherapy (FLASH-RT) and heavy ion radiotherapy exhibit superior curative effects with relatively fewer adverse events ^[108], holding promise for BM treatment ^[109].

The optimization of drug carriers has also advanced the field of BMs treatment. RT combined with ultrasound-mediated rupture of oxygen-carrying microbubbles (MBs) delays tumor progression and improves survival in AM-BM of breast cancer ^[86]. In addition, nanoparticles enhance the therapeutic effect of RT on BM by modulating radiosensitivity ^[35-37] or increasing the dose absorption of RT ^[38] (Figure 4I).

The development of novel medicines is equally compelling for researchers. 5-aminolevulinic acid (5-ALA), a novel photodynamic drug, enhances the radiosensitivity of melanoma BM by upregulating protoporphyrin IX (PpIX) ^[101] (Figure 4F). Thymoquinone (TQ) improves the efficacy of gamma knife therapy on melanoma BMs, by enhancing apoptosis through regulation of the JAK2/STAT3 pathway ^[42]. Moreover, TQ induces the secretion of inflammatory growth factors ^[42] (Figure 4D). Metformin increases the concentration of lactate in MDA-MB‐231-Br cells by suppressing the MCT4 protein, thereby enhancing the anti-tumor effect of RT ^[65] (Figure 4H). In the AM-BM of breast cancer, L-arginine amplifies RT efficacy by modulating nitric oxide metabolism ^[110] (Figure 4G). Additionally, magnetic field therapy (athermal radiofrequency electromagnetic fields) combined with RT significantly inhabits radiation-resistant cells and prolongs the survival of AM-BMs ^[111].

Future and Prospects

The two-year survival rate for patients with BM is typically less than 10% ^[12]. RT, including WBRT and SRS, is one of the essential treatments for BM. The topic of brain metastases has attracted much attention in the 2023 oncology conferences (such as ASCO, ASTRO, and WCLC). Dose exploration remains a key topic, particularly in the field of radiotherapy for brain metastases. This review, based on the AM-BM of various tumors, presents a comprehensive summary of preclinical research on BM and RT for the first time. We focused on RT parameters, including modality, total dose, fractionation, dose rate, and their corresponding effects. Additionally, we highlight recent advancements in the study of BM with RT, emphasizing combination with chemotherapy, targeted therapy, immunotherapy, and novel technologies.

Animal models for BM encompass diverse species, including mice ^{[30,38,41,43,49,56,60-65]}, rats ^{[48,72,77,112, 113]}, monkeys ^[114], dogs ^[115], rabbits ^[116], and chick embryos ^[117-119], with mice being predominantly the predominant ones used (Figure 3A). The organ tropism of tumor cells in chick embryos was recently found to be consistent with that in mice ^[118]. The location of the BM in most studies is limited to the cerebral cortex ^{[49, 50]} or the striatum ^{[33, 39, 41-48]}. Establishing the AM-BMs in specific anatomical locations, such as the leptomeninges ^{[120, 121]} and cerebellum ^[122], requires further exploration. The "seed" and "soil" interactions during tumor metastasis endow the primary tumors and metastases with different characteristics, the selection and establishment of brain-tropic cells necessitate attention ^[68]. Radiation-resistant models have also received limited research attention. In recent years, the application of humanized mice, microfluidic chips mimicking ^{[123, 124]}, PDX models ^[39,40], and organoids ^[125] has emerged, enhancing the translatability of research in the field of BM. The application of emerging models, diagnostic methods, and treatment techniques to study brain metastasis may catalyze its clinical transformation and change treatment paradigms, which deserves further attention. Single-cell sequence and spatial transcriptomics offer promising avenues for obtaining more authentic information about BMs. Meanwhile, the difference in the organ affinity of primary tumor metastases to brain tissue needs to be further explored. Owing to the rarity of brain metastatic cells, certain studies have resorted to employing cell lines derived from primary tumors to investigate the relevant mechanisms involved. Although validated in animal models, the exploration of brain metastasis mechanisms outside the brain microenvironment has somewhat compromised the persuasiveness of the conclusions. This issue is currently a focus in the field of brain metastasis research. Continuous optimization of animal model construction and the development of emerging models may offer a potential solution.

For clinical transformation, preclinical studies have mostly used mice, which have certain differences in genetics, radiation sensitivity, and other aspects compared to humans. Like the parameters of chemotherapy and immunotherapy, preclinical radiation dosimetry parameters are difficult to convert and apply to humans. In terms of dosage, preclinical models can only provide positive (effective tumor suppression) or negative (failure to tumor suppression) results, which is an unavoidable problem in clinical transformation. Utilizing and optimizing models with personalized patient information, such as PDX models and organoids, is more convincing and easier to use for achieving clinical conversion.

The choice of the RT method is influenced by the establishment of animal models. BM formed by intracerebral inoculation is commonly treated with WBRT or SRS, whereas intracardiac and carotid artery injections (systemic injection) may generate multiple intracranial metastases, often treated with WBRT in AM-BM. In addition, intravenous injection is relatively more frequently used in PCI research. However, whether the carotid artery or intracardiac injection is suitable for PCI research still needs convincing evidence.

The timing of RT intervention is also worthy of attention. The AM-BMs of breast/lung cancer (10⁶ tumor cells) or melanoma (10⁴ tumor cells) constructed by intracerebral injection are generally administrated RT within two weeks. Meanwhile, for systemic inoculation, the irradiation time for intracardiac injection modeling is generally later than that for intracerebral transplantation. Notably, the timing of RT intervention and the definition of the irradiation field between AM-BM and clinical application pose challenges but advances in animal imaging technology and RT may provide solutions.

Current RT regimens for AM-BM include four criteria: (1) clinical regimens ^[62,74,76], (2) BED equivalent regimens ^{[33,34,38,45,54,75,76,79]}, (3) experience-based regimens ^{[35-37,52,56]}, and (4) protection of normal tissues ^[49,55,82]. Dose and fractionation emerge as critical factors. Presently, BED equivalent schemes (based on the L-Q model) are predominantly applied. Most studies employ either single high-dose irradiation or high-dose fractional irradiation (15-16 Gy/1F) ^{[33,34,38,45,54,75,76,79]}. However, some researchers argue that the L-Q model is unsuitable for high-dose irradiation ^[126], in which the dose-survival curve of tumor cells is significantly shifted, accounting for decreased predictive accuracy ^[81,92]. Meanwhile, for BED calculations, α/β is not entirely consistent across different tumors and cell subtypes ^[91]. While RT alone or combined with other treatments effectively inhibits BM, inevitable adverse events, including radiation necrosis, cerebral edema, and neuronal damage, can occur ^[127]. Due to the substantial heterogeneity among various studies, drawing definitive conclusions regarding optimal RT regimens or combination therapies for maximum benefit is challenging.

Owing to the limited volume of BM in animals, only a few preclinical studies have reported IMRT in AM-BM ^[86,128]. Conformal magnetic resonance imaging (MRI) in rats contributes to precise RT to some extent ^[112]. Currently, the Small Animal Radiation Research Platform (SARRP) (Xstrahl, Camberley, UK) ^[129,130], X-RAD SmART (Precision X-ray, North Branford, Connecticut, USA) ^[131], and the SAIGRT system ^[132] have been validated to achieve small-volume precise radiotherapy in AM-BM. Moreover, Delaney et al. conducted IMRT for mice BMs using SARRP combined with cone-beam computed tomography guidance ^[86]. Interestingly, they demonstrated that the clinical linear accelerator Novalis TX (Brainlab AG, Feldkirchen, Germany) could also achieve IMRT in AM-BM ^[128].

Innovative irradiation methods such as HA-WBRT, FLASH-RT ^[108], and heavy iron therapy ^[109], may yield improved therapeutic effects in BM. Recent studies have explored tumor-treating fields ^[133] and athermal radiofrequency electromagnetic field ^[111]. Targeted therapy, immunotherapy, and novel technologies like nanoparticles and oxygen-containing microbubbles have been extensively studied in primary tumors, but their exploration in metastatic tumors is limited (Figure 5). Tumor cells evolve during metastatic periods, and the characteristics of metastatic lesions are not consistent with those of primary tumors. Moreover, the BBB establishes a particular intracranial immune environment. Consequently, the application of novel treatment methods and technologies for treating BM warrants further investigation. The mechanisms by which RT modulates the BBB and regulates the microenvironment of the BM demand in-depth exploration.

In conclusion, the choice of RT regimens in BM depends on the model establishment. It is imperative to focus on refining RT or comprehensive treatment protocols for AM-BM and strive for the standardization of preclinical research on RT to facilitate its clinical application. However, further studies are needed to elucidate how to optimize the efficacy of RT in BM.

 Figure 5 

Combination Treatment with RT in Current AM-BM Research. Current research on AM-BMs explores diverse combination treatments with RT, including immunotherapy, novel drug applications, targeted therapy, surgery, chemotherapy, nanomaterial applications, ultrasound (mediating oxygen-containing microbubble rupture), magnetic field therapy, and electric field therapy. These comprehensive approaches signify the multifaceted strategies being investigated to optimize the efficacy of RT in AM-BM.

Abbreviations

BMs: brain metastases; AM-BM: animal model of brain metastases; WBRT: whole brain radiation therapy; SRS: stereotactic radiosurgery; PCI: prophylactic cranial irradiation; RT: Radiotherapy; IMRT: Intensity-modulated radiation therapy; ICB: intracerebral injection; ICD: intracardiac injection; ICA: intracarotid artery injection; IV: Intravenous inoculations; TVI: tail vein injection; PDX: Patient-derived tumor xenograft; MRI: magnetic resonance imaging; BBB: the blood-brain barrier; L-Q: The linear-quadratic; BED: the Biologically effective dose; D: total Dose; d: single dose; TAM-MDM: monocyte-derived macrophages; TAM-MDM: monocyte-derived macrophages; BTB: blood-tumor barrier; FUS: focused ultrasound; TMZ: temozolomide; NSCLC: non-small cell lung cancer; AMONs: anti-MGMT morpholino oligonucleotides; EGFR: Epidermal growth factor receptor; HER2: growth factor receptor type 2; HDACs: Histone deacetylases; ES: Endostar; ICI: immune checkpoint inhibitors; ISV: in situ vaccination; IVIS: In vivo Imaging System; NF-κB: nuclear factor kappa-light-chain-enhancer of Activated B cells signaling; FLASH-RT: FLASH radiotherapy; RAGE: receptor for advanced glycation end products; MBs: microbubbles; 5-ALA: 5-aminolevulinic acid; PpIX: protoporphyrin IX; TQ: Thymoquinone; GRM1: metabotropic glutamate receptor 1; SARRP: the Small Animal Radiation Research Platform.; LLC: Lewis lung cancer; IT: immunotherapy; TT: targeted therapy; CTR: Chemotherapy; GK: Gamma Knife; NA: Not available.

Acknowledgements

We thank the team members for their valuable and constructive comments. Also, we thank all those involved in writing and revising this manuscript. Also, we thank members of Hunan SJA laboratory animal Co., Ltd. for their help. The figures are created with BioRender.com.

Funding

This work was supported by the Natural Science Foundation of Hunan (2022JJ30992 to R.R.Z) and the National Multidisciplinary Cooperative Diagnosis and Treatment Capacity Building Project for Major Diseases of China (Lung Cancer, z027002).

Availability of data and materials

Data sharing does not apply to this article as no datasets were generated or analyzed during the current study.

Author contributions

WS collected the original data, performed the literature review, wrote the manuscript, and revised the tables. GT conceived the structure of the manuscript and critically revised the manuscript. LC and WHJ designed figures. JN, XG, and HP revised the tables. DJ, HL, JN, and MY made critical revisions, modified the grammar, and polished the paper. WS and GT contributed equally to the manuscript. All authors reviewed and approved the final manuscript to be published.

Competing Interests

The authors have declared that no competing interest exists.

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Author contact

Corresponding author: Rongrong Zhou, Department of Oncology, Xiangya Hospital, Central South University, No. 87 Xiangya Road, Kaifu District, Changsha, 410008, China, E-mail: zhourredu.cn.

Received 2023-10-19
Accepted 2023-12-14
Published 2024-1-1

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