Targeted alpha therapy: a comprehensive analysis of the biological effects from “local-regional-systemic” dimensions

Abstract

Targeted alpha therapy (TAT) has emerged as a promising radiopharmaceutical modality in precision oncology. Compared to beta-emitters, alpha-emitters exhibit superior properties, including higher linear energy transfer, shorter penetration range, enhanced resistance to hypoxic conditions, and convenient radiation protection. Notably, alpha-emitters also demonstrate therapeutic efficacy against a subset of tumors exhibiting resistance to beta-radiotherapy. In 2013, the first α-particle therapeutic agent, ²²³RaCl₂ (Xofigo^®), was approved by the FDA for treating bone metastases in advanced castration-resistant prostate cancer, marking a milestone in clinical translation of alpha-emitters. However, the biological mechanisms underlying alpha-particle-mediated therapeutic effects remain incompletely elucidated, which has hindered the optimization of precision treatment strategies. This review systematically analyzes TAT’s tripartite antitumor mechanisms—targeted effects, bystander effects and abscopal effects—thereby constructing a “local-regional-systemic” multidimensional antitumor network. This framework not only clarifies the radiobiological principles of α-emitters but also provides innovative perspectives for advancing TAT applications in tumor precision therapy.

Graphical abstract

Introduction

Recently, targeted alpha therapy has advanced greatly in tumor therapy. Table 1 provides a systematic summary of the commonly used medical alpha-emitters. Compared with beta-emitting radionuclides, alpha-emitting radionuclides have shown potential advantages. The alpha particles emitted by alpha-nuclides are doubly charged helium-4 nuclei ( He²⁺) characterized by substantial mass and high energy levels (4–9 MeV). These particles exhibit remarkably higher linear energy transfer (LET) than beta particles, with LET values ranging from 50 to 230 keV/µm, in contrast to the 0.2 keV/µm LET typical of beta radiation. This high LET characteristic enables a single alpha particle trajectory to induce irreparable DNA double-strand breaks (DSB). In comparison, beta particle irradiation predominantly causes single-strand breaks (SSB), which are generally reparable, resulting in cytotoxic potency approximately 500-fold lower than that of alpha particles [1]. Moreover, since stronger interactions with matter lead to a shorter path, the substantial mass and charge of alpha particles cause them to interact extensively with the surrounding medium, limiting their range in tissue to about 45–100 μm—roughly the diameter of 5 to 10 cells—thereby effectively reducing damage to neighboring healthy cells compared to the longer range of beta particles [2]. It is worth noting that the short range of alpha-emitters may result in inferior cross-fire effects compared to beta-emitters, leading to suboptimal efficacy in large masses or antigen heterogeneous tumors [3]. Furthermore, the cytotoxicity induced by alpha-emitters is independent of oxygen free radicals generated by radiolysis of water and unaffected by cell cycle phases. The underlying mechanism lies in the dominant direct DSB induced by alpha particles. Significantly, the hypoxia tolerance of alpha-emitters confers significant advantages in treating radiation-resistant refractory tumors due to hypoxic tumor microenvironments [4–7]. TAT can also effectively overcome resistance issues associated with beta-particle therapies, driving a paradigm shift in radionuclide treatment approaches [8] (Fig. 1). With the aforementioned radiobiological advantages, alpha-emitters are gradually transitioning from preclinical trials to clinical applications. Currently, Several clinical trials based on alpha-emitters have been approved (Table 2), marking an advancement in the clinical translation of this therapeutic strategy.

Table 1.

Common characteristics, delivery strategies, and clinical applications of alpha-emitters

Alpha emitter	Half-life	Range (um)	Emissions Per Decay	Max Energy (MeV)	Delivery strategies	Current clinical applications
223Ra	11.4 d	46 ~ 70	4 α, 2β-	5.716	Natural bone-targeting properties	mCRPC
225Ac	9.92 d	50 ~ 90	4 α, 2β-	5.830	Monoclonal antibody, peptides, bispecific antibody, small molecules,	mCRPC, SCLC, LCNEC GEP-Nets, advanced solid tumours, glioma
211At	7.21 h	55 ~ 80	1 α, 1 EC	5.869	Monoclonal antibody	Acute leukemia, myelodysplastic syndrome
212Pb	10.64 h	600	1 α, 2β-	6.09	Small molecules, peptides	mCRPC, solid tumors, neuroendocrine tumors, melanoma, atypical lung carcinoids
212Bi	60.6 min	40 ~ 100	1 α, 1β-	6.051	/	/
213Bi	45.6 min	40 ~ 100	1 α, 2β-	5.875	Monoclonal antibody	Leukemia, myelodysplastic syndrome
149Tb	4.1 h	25 ~ 30	1 α, 1 β+	3.967	/	/
227Th	18.7 d	50 ~ 70	5 α, 2β-	6.038	Antibody	mCRPC

Fig. 1.

Comparison of the characteristics of alpha particles and beta particles. (Created with Biorender.com)

Table 2.

Current clinical trials of targeted alpha therapy are underway

Clinical trial	Alpha-emitter	Target	Drug	Approved indications	Primary outcome measures
NCT03746431	225Ac	IGF-1R	225Ac-FPI-1434	Locally advanced or metastatic solid tumours	AE, DLT, ORR
NCT05902247	225Ac	PSMA	225Ac-PSMA I&T	mCRPC	AE, SAE, Safety, Tolerability
NCT04946370	225Ac	PSMA	225Ac-J591	mCRPC	DLT, Optimal dose, CR
NCT04597411	225Ac	PSMA	225Ac-PSMA-617	CRPC	RP2D
NCT05496686	225Ac	/	225Ac-MTI-201	Metastatic uveal melanoma	MTD, DLT, AE, SAE
NCT06732505	225Ac	SSTR	225Ac-DOTATATE	Inoperable, locally advanced or metastatic, progressive, well-differentiated, SSTR + GEP-Nets	Safety, Tolerability, AE, DLT
NCT06147037	225Ac	EGFR-cMET	225Ac-FPI-2068	Metastatic and/or recurrent solid tumors	Safety, Tolerability, AE, MTD, RP2D
NCT06975332	225Ac	NK-1R	225Ac-DOTA-SP	Recurrent glioblastoma (WHO G3-G4)	Clinical progression
NCT06492122	225Ac	PSMA	225Ac-FL-020	mCRPC	DLT, RP2D, AE, SAE
NCT06736418	225Ac	DLL3	225Ac-ABD147	SCLC and LCNEC of the lung following platinum-based chemotherapy	Safety, AE, Tolerability, DLT, RP2D, ORR, DCR, DOR, PFS, OS
NCT06939036	225Ac	SSTR2	225Ac-SS0110	ES-SCLC and recurrent locally advanced or metastatic MCC	Safety, Tolerability, RP2D, SAE, PFS, DCR, BOR, ORR, DoR
NCT05636618	212Pb	SSTR2	212Pb-ADVC001	Advanced SSTR2 positive neuroendocrine tumors	DLT, AE, AUC
NCT05655312	212Pb	MC1R	[212Pb]VMT01	Unresectable and metastatic melanoma	DLT, ORR, AE, SAE
NCT06427798	212Pb	SSTR	[212Pb]VMT-alpha-NET	Gastrointestinal neuroendocrine tumors	MTD, ORR
NCT05283330	212Pb	GRPR	212Pb-DOTAM-GRPR1	Recurrent or metastatic GRPR-expressing Tumors	RP2D
NCT03670966	211At	CD45	211At-BC8-B10	Relapsed or refractory high-risk acute leukemia or myelodysplastic syndrome	Toxicity

mCRPC metastatic castration-resistant prostate cancer

While the inherent radiobiological advantages of alpha-emitters establish a foundation for precision therapy, the multifaceted mechanisms of TAT remain incompletely elucidated. Current research primarily focuses on alpha particle-induced nuclear DNA damage as the dominant on-target effect [9]. However, emerging evidence has increasingly revealed TAT-mediated subcellular structures targeting, although it’s still not comprehensive [10]. Significantly, beyond these direct cytotoxic actions, TAT exerts cytotoxic effects on non-irradiated tumor cells through off-target mechanisms. These effects are predominantly orchestrated by the radiation bystander effect and systemic abscopal immune activation, both critically contributing to therapeutic efficacy. This review systematically synthesizes recent advances in TAT’s biological mechanisms, integrating organelle-specific targeting with systemic off-target regulatory networks to decode its multi-layered tumor eradication principles, thereby providing a theoretical framework for optimizing TAT clinical translation strategies.

Targeted effects

DNA centered effects

For decades, nuclear DNA has been regarded as the primary target of irradiation [11]. DNA damage induced by radionuclides occurs through both direct and indirect mechanisms. Among them, the radiolysis of water produces reactive oxygen species (ROS) including hydroxyl radicals (•OH), superoxide anions (O₂•⁻), and hydrogen peroxide (H₂O₂) [12], a process commonly referred to as the “indirect effect” of radiation. In addition, charged particles can directly ionize biomolecules (DNA, proteins, and lipids). This direct effect is particularly significant in high LET particles (such as α-particles), as alpha particles induce localized high-density ionization in biological tissues. Unlike beta particles that primarily induce repairable SSB in tumor cells through radical-mediated mechanisms, alpha particles directly cause complex, irreparable clustered DSB. This highly efficient mode of DNA damage makes alpha particles uniquely advantageous in radiotherapy [4, 13].

Generally, DNA damage triggers the DNA damage response pathway, which safeguards cell viability and preserves genomic integrity by activating DNA repair mechanisms [14].The repair mechanisms for alpha particle-induced DSBs relies on two distinct molecular pathways: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) [15]. NHEJ mediates rapid DNA restoration through direct ligation of broken DNA termini, offering immediate damage resolution at the cost of reduced fidelity, often resulting in accumulation of insertion/deletion mutations. In contrast, HR operates as a high-precision repair system strictly confined to S and G2 phases of the cell cycle, requiring a homologous template [16]. Notably, high-LET alpha particles generate complex DNA lesions that are particularly challenging for cellular repair systems due to their dense ionization patterns.

The high LET properties of alpha particles, arising from their elevated ionization density and localized energy deposition, induce more complex biological damage compared to low-LET radiation (β particles and γ rays) [17]. Such radiation not only generates DNA DSBs but also causes SSBs, base oxidation, and clustered lesions. During damage recognition, the MRE11-RAD50-NBS1 (MRN complex) localizes to DNA damage sites [18], facilitating the recruitment and activation of serine/threonine protein kinases (ATM/ATR). This triggers phosphorylation cascades in key signaling molecules such as Chk2, γ-H2 AX, and p53 [19, 20]. In response to DNA damage, cells can activate repair mechanisms such as HR through the recruitment of repair proteins like 53BP1 and BRCA1 [21, 22]. Simultaneously, cell cycle arrest mediated by proteins such as p21 and Chk1 provides critical time for DNA repair, thereby preventing abnormal replication of damaged DNA [23]. When damage exceeds reparable thresholds, the p53 signaling network shifts to activate a pro-apoptotic program by upregulating effector molecules like Bax, Puma, Noxa, and Fas, driving cells toward irreversible apoptosis [24, 25]. Marques et al. have elucidated that ²²³RaCl₂ induces irreparable DNA double-strand breaks, which activates cell cycle checkpoint through the ATM/CHK2 signaling pathway, consequently triggering G2/M phase arrest and initiating apoptotic cascades [26]. This regulatory mechanism, which coordinates DNA repair and programmed cell death in accordance with the biological relevance of DNA damage [27], establishes a homeostatic equilibrium between genome integrity maintenance and the elimination of genomically compromised cells (Fig. 2). While existing review articles have systematically elucidated the mechanisms underlying ionizing radiation-induced DSBs and their dynamic coordination with cell cycle progression and DNA repair processes, the precise molecular mechanisms underlying TAT regulation remain incompletely elucidated [28].

Fig. 2.

A concept map of TAT-induced targeted effects. (Created with Biorender.com). The direct ionization by alpha particles induces clustered DNA DSBs, which recruit and activate the ATM/ATR kinase cascade, initiating DNA damage response signaling network. The extent of damage dynamically regulates downstream pathways to determine cellular fate: mild damage activates cell cycle arrest to facilitate DNA repair, while severe damage triggers irreversible cell death via the intrinsic apoptotic pathway. Furthermore, α-particle irradiation specifically activates ASMase, catalyzing sphingomyelin hydrolysis to generate ceramide, which aggregates into lipid rafts, thereby activating the MAPK signaling cascade (e.g., p38/JNK/ERK phosphorylation) to amplify pro-apoptotic signals. The ionization of α-particles induces mitochondrial membrane potential collapse, triggering mitochondrial dysfunction and subsequent calcium overload. Moreover, α-particles provokes endoplasmic reticulum stress and initiates lysosomal-mediated autophagy, forming a coordinated chain of cellular damage responses

It is noteworthy that studies on alpha-particle-based radiopharmaceuticals provide critical insights into elucidating the aforementioned mechanisms. A ²¹³Bi-labeled PSMA inhibitor (²¹³Bi-PSMA I&T) demonstrated rapid tumor-targeting kinetics, with its alpha-particle emissions inducing persistent accumulation of DNA DSBs in tumor tissues [29]. Similarly, the ²¹¹At-labeled heterodimeric peptide (iRGD-C6-lys(²¹¹At-ATE)-C6-DA7R) has been demonstrated to significantly inhibit the viability of glioma cells. This compound effectively induces cellular apoptosis and triggers cell cycle arrest at the G2/M phase [30]. Recent research has demonstrated that ²²³RaCl₂ induces DNA damage and activates repair pathways in various prostate cancer cells. The finding revealed that it activates DSB repair via the NHEJ mechanism, while simultaneously inducing high levels of apoptosis. The potent cytotoxic effect of ²²³Ra may be attributed to alpha-particle-induced clustered DNA damage sites [31].

Non-DNA centered effects

Cell membrane

DNA is not the only target of radiation-induced damage. The cell membrane, a dynamic bilayer structure, is composed of lipids (30–80%), proteins (20–60%), and carbohydrates (0–10%). The lipids in the cell membrane contain polyunsaturated fatty acids (PUFA), which are susceptible to oxidation by ROS induced by radiation. This oxidation process generates lipid-derived metabolites, such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE). These breakdown products serve as biomarkers of lipid peroxidation, indicating that irradiation has disrupted the biological functions of the cell membrane [32].

Alpha particle-induced ROS can activate acid sphingomyelinase (ASMase) [33, 34], which hydrolyzes sphingomyelin, a type of membrane phospholipid, to produce ceramide. Ceramide accumulation leads to the formation of large signaling platforms known as lipid rafts. The formation of lipid rafts can activate a variety of signaling pathways [35, 36] and ion channels (such as potassium [37] and calcium [38] channels), ultimately resulting in nuclear DNA damage (Fig. 2).

Several studies have revealed the mediating role of the cell membrane in alpha particle-induced cell death. Seideman et al. indicated that alpha particles released by ²²⁵Ac-DOTA-anti-CD3 IgG antibodies can induce dose-dependent apoptosis through activation of the sphingomyelin pathway [39]. Ladjohounlou and his colleagues also demonstrated that the cell membrane serves as a radiosensitive target for radiation-induced damage through their studies using antibodies labeled with alpha-emitting radionuclides (²¹³Bi, ²¹²Pb/²¹²Bi) [40]. Their research further revealed that alpha radioimmunotherapy can induce cell death by activating p38 and Jun N-terminal kinase 1/2 (JNK1/2) signaling pathways through lipid raft-mediated mechanisms. Disrupting the integrity of lipid rafts leads to the absence of p38 and JNK1/2/3 phosphorylation. When pharmacological inhibitors of p38 (SB203580) and JNK1/2/3 (SP600125) were employed in conjunction with alpha particle-irradiated cells, an enhancement in cell viability was observed. This finding suggests that the p38 and JNK mitogen-activated protein kinase (MAPK) signaling pathways are pivotal in the targeted cytotoxicity of alpha-particle irradiation [41]. Additionally, signaling pathways such as NF-κB and PI3 K/AKT have been shown to be activated [36].

Mitochondria

In the cytoplasm, mitochondria, which play a crucial role in cellular metabolism and energy homeostasis, occupy 25% of the cell volume, indicating a high probability of being hit by particles [42]. Mitochondrial DNA, being unprotected by histones, is often observed to have mutations or deletions under radiation exposure. Schilling-Toth et al. elucidated this phenomenon as radiation-induced mitochondrial genomic instability [43, 44]. Notably, intact mitochondrial function is essential for the cells to induce nuclear signaling responses upon cytoplasmic-specific targeting interventions [22].

Although numerous studies have demonstrated that radiation induces mitochondrial DNA damage, the specific mechanisms by which mitochondrial function contributes to radiation-induced cytoplasmic toxicity remain unclear. This is primarily due to the challenge of selectively targeting the cytoplasm without affecting the nucleus. Radiation exposure leads to a sustained increase in mitochondrial oxidative stress, this change is associated with mitochondrial membrane potential, mitochondrial respiration, and the ATPase encoded by the mitochondria [43]. Bo Zhang et al. reported that targeted cytoplasmic irradiation with alpha particles can specifically induce upregulation of DRP1 expression, thereby driving mitochondrial fission, accompanied by impaired mitochondrial respiratory chain function (such as a significant reduction in the activities of cytochrome c oxidase and succinate dehydrogenase within 4 h after irradiation) [45]. It is worth noting that mitochondrial fission gradually recovers to a nearly normal morphology within 24 h, while the respiratory chain function partially recovers. This dynamic change may reflect the adaptive stress response of mitochondria through the fission-fusion balance. Treatment with the DRP1 inhibitor mdivi-1 can block radiation-induced mitochondrial fission but fails to reverse the impairment of respiratory chain function. This suggests that DRP1-dependent fission may indirectly exacerbate functional defects by limiting the clearance of damaged mitochondria. This finding reveals the complex association between mitochondrial dynamics and function, indicating that DRP1-mediated fission is a compensatory stress adaptation mechanism after alpha-particle irradiation. However, excessive mitochondrial fission can lead to reduced respiratory chain function and irreversible mitochondrial damage. Evidence suggests that aberrant mitochondrial fission contributes to impaired respiratory chain activity in neurodegenerative diseases such as Alzheimer’s and Parkinson’s [46, 47]. Furthermore, study had demonstrated that under alpha-particle irradiation, the transcriptional expression of mitochondrial biomarkers such as LONP1 and TFAM, as well as mtDNA-encoded genes MT-CYB and MT-RNR1, was significantly upregulated during the differentiation of human induced pluripotent stem cell-derived cardiomyocytes, which may represent an additional adaptive response mechanism to radiation stress [48]. Yang et al. developed a novel ²²³Ra-loaded Mg/Al-layered double hydroxide (LDH) nanomaterials (²²³Ra-LDH) through a coprecipitation strategy. This radiopharmaceutical delivery system induces mitochondrial dysfunction via ROS generation and stimulates endogenous Ca²⁺ release. The resultant Ca²⁺ overload ultimately triggers apoptosis, demonstrating precise ²²³Ra delivery within the tumor microenvironment [49].

Other subcellular structures

Current research primarily focuses on these established radiobiological targets, while investigations into the functional mechanisms of subcellular structures such as the endoplasmic reticulum (ER) and lysosomal system in alpha-particle therapy remain limited. Although studies have observed damage responses in these organelles following alpha-irradiation, their underlying molecular regulatory mechanisms have yet to be systematically elucidated. Diniz Filho et al. demonstrated that ²²³RaCl₂ irradiation of MDA-MB-231 cells triggered canonical autophagy pathway activation, characterized by autophagosome-lysosome formation, which represents one of the principal mechanisms underlying radiation-induced cell death [50]. Furthermore, transmission electron microscopy (TEM) revealed ultrastructural alterations, including dilation of both ER and mitochondria following alpha-particle exposure [51]. A recent study demonstrated that ²²³Ra induces ER stress in tumor cells, which markedly upregulates calreticulin (CRT) expression and facilitates its ectopic surface translocation, thereby potentiating susceptibility to cytotoxic T lymphocyte (CTL)-mediated cytolysis [52]. Functioning as both a canonical ER chaperone and pivotal damage-associated molecular patterns (DAMP) [53], CRT plays a pivotal role in radiation-induced immunogenic modulation. This finding elucidated the pivotal role of ER stress in ²²³Ra-mediated immune modulation and established a theoretical framework for developing ER-focused TAT strategies. Besides, research demonstrated that monoclonal antibodies (mAbs) labeled with alpha-emitters (such as ²¹²Bi, ²¹³Bi, and ²¹²Pb) can migrate from the cell surface to lysosomes, accompanied by the gradual accumulation of alpha-emitting radionuclides within lysosomes, thereby ensuring optimal cellular radiation exposure. This discovery provides valuable insights for developing lysosome-targeted strategies based on alpha-emitters [54, 55].

Bystander effects

In addition to the direct cytotoxicity of TAT, alpha-emitters also cause biological effects through non-targeted effects (NTEs), which commonly include bystander effects and abscopal effects induced by immune response activation. Numerous studies have demonstrated that non-irradiated cells can exhibit a series of biological responses when their neighboring cells are exposed to radiation, the phenomenon known as bystander effect. In the 2006 report by the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR), the term “bystander effect” is defined as “the ability of irradiated cells to convey manifestations of damage to neighboring cells not directly irradiated” [56]. Notably, research has confirmed a close correlation between this bystander effect and the hyper-radiosensitivity of cells to low-dose radiation exposure [57].

In 1992, Nagasawa and Little performed a pioneering study that first revealed bystander effect induced by alpha-radiation. They exposed Chinese hamster ovary cells to low doses of alpha-particle irradiation (0.31 mGy), where only a small fraction of cell nuclei (1% or less) were traversed by alpha particles, yet 30% of the cells demonstrated an increased frequency of sister chromatid exchanges. Under normal conditions, approximately 2.0 Gy of X-ray dose is typically required to achieve a similar biological effect [58]. This study highlighted the potential value of radiation-induced bystander effect (RIBE) in reducing radiation dosage.

Experimental evidence

In recent years, research on alpha-emitting radionuclides in the field of RIBE has been continuously advancing. The study revealed that when lymphocytes were irradiated with ²¹³Bi-labeled monoclonal antibodies, the percentage of cell mortality was significantly higher than the percentage of nuclei actually hit by alpha particles, indicating the presence of a pronounced bystander effect [59]. Leung et al. provided evidence through the establishment of an in vivo model that ²²³Ra-induced cytotoxic bystander effects are involved in suppressing the growth of disseminated tumor cell xenografts. Their findings demonstrate the therapeutic potential of alpha-emitters in disseminated tumor cells [60]. Subsequently, the team achieved stratification of the direct effects of alpha particles and radiation-induced bystander effects through in vivo measurements of the biological effects of alpha-particles emitted by ²²³RaCl₂, further highlighting the critical regulatory role of bystander effects in enhancing the overall therapeutic response [61]. Moreover, Rajon et al. developed a model to investigate the role of the bystander effect induced by ²²³Ra in delaying the growth of breast cancer xenografts in mice [62]. These studies contribute to understanding the mechanisms by which ²²³Ra slows the progression of metastatic lesions. Other researchers have used ²¹²Pb/²¹²Bi-labeled monoclonal antibodies to reveal the roles of ROS and cell membranes in alpha-particle-emitter-induced bystander effects [40]. Additionally, Yang et al. focused on the biological role of mitochondrial transfer in RIBE, revealing that damaged mitochondria mediate alpha-particle-induced bystander effects through intercellular transmission, though the precise molecular pathways remain to be elucidated [63].

These findings not only reveal the existence of bystander effects induced by alpha-emitter irradiation, but also highlight the indispensable role of intercellular communication in mediating this phenomenon, as cells can be killed without being directly traversed by particles. With the continuous advancement of research in recent years, both direct cell-to-cell communication via gap junctions and the release of signaling molecules have been identified as two primary mechanisms involved in bystander effects (Fig. 3).

Fig. 3.

RIBE is mediated by multiple mechanisms of intercellular communication. (Created with Biorender.com). The GJIC formed by connexins, which directly transmit oxidative stress signals to bystander cells. Irradiated cells can also signal through the secretion of soluble mediators, or by transporting extracellular vesicles that carry miRNAs and damaged organelles like mitochondria and lysosomes, leading to bystander cell death. These mechanisms collectively result in genomic instability, reduced clonogenic capacity, and non-targeted cell death in bystander cells

Gap junction intercellular communication

Early studies primarily focused on the role of gap junction intercellular communication (GJIC) mediated by direct cell-cell contact in radiation-induced bystander effects [64]. Azzam et al. first identified the critical function of GJIC in bystander signal transmission between cells in 1998 [65], and their follow-up study in 2001 further demonstrated that connexin43 (CX43), as a core regulatory molecule of GJIC, plays an essential mediating role in intercellular communication. Of particular significance, pharmacological inhibition of GJIC using γ-hexachlorocyclohexane (lindane) resulted in a marked reduction of alpha-particle-induced bystander effects, directly confirming the necessity of GJIC in this process [66]. Subsequent research by the Autsavapromporn team expanded the understanding in this field, revealing that GJIC not only transmit radiation-induced damage signals but also amplify oxidative stress-related signaling pathways, thereby serving as central regulators in the cascade reactions of radiation bystander effects [67].

However, RIBE is mediated not only by gap junction channels but also through extracellular mediator-mediated communication, including soluble mediator secretion and vesicle-based molecular transport.

Extracellular mediator-mediated communication

Soluble mediators

Substantial evidence from multiple studies has demonstrated that various soluble signaling factors play crucial regulatory roles in RIBE. Such as reactive species [68, 69] (e.g., ROS, NO), cytokines [70–72] (e.g., IL-12, IL-15, IL-18, TNF-α, IFN-γ, TGF-β1), ionic signaling mediators [73] (e.g., Ca²⁺).

Among various signaling molecules, ROS and reactive nitrogen species (RNS) play crucial regulatory roles in the bystander effect [36, 74]. Previous study has demonstrated that ROS/RNS are generated in the mitochondria of target cells through a Ca²⁺-dependent mechanism [75]. In RIBE, ROS serve as the initial triggering factor that initiates the cascade reaction, while nitric oxide radical (NO•) and its active metabolites function as key effector molecules, regulating the activation and transduction of downstream signaling pathways [76]. Research data suggest that under alpha-particle radiation conditions, ROS may participate in the activation process of both the p53-dependent pathway and multiple members of MAPK superfamily in bystander cells through the regulation of redox-sensitive signaling pathways, and this mechanism may be associated with micronucleus formation in bystander cells [77]. ROS act as pivotal signaling molecules that induce cyclooxygenase-2 (COX-2) expression [64]. Research has demonstrated that the COX-2 signaling cascade plays a central regulatory role in RIBE. Zhou et al. substantiated that pharmacological inhibition of COX-2 activity using NS-398, a specific COX-2 inhibitor, significantly attenuated bystander responses. Furthermore, given the pivotal role of COX-2 in activating MAPK pathway, this investigation further elucidates the critical involvement of MAPK signaling cascades in bystander effect mechanisms. Notably, COX-2-associated pathways are indispensable for mediating cellular inflammatory responses, suggesting potential molecular crosstalk between inflammatory mechanisms and bystander effects [78]. NO, serving as a core component of RNS, is synthesized and released through enzymatic catalysis by distinct isoforms of nitric oxide synthase (NOS) [64, 79]. Shao et al. demonstrated that NO is involved in mediating the regulatory effects of radiation-induced bystander effects on cell proliferation and micronucleus formation in human salivary gland cells [80].

Significantly, research findings indicate that dimethyl sulfoxide (DMSO), a ROS scavenger, failed to significantly suppress the bystander effect following irradiation, whereas Filipin, a cell membrane signaling inhibitor, exhibited pronounced inhibitory effects. This discovery suggests that beyond signaling molecule mechanisms, membrane-dependent signal transduction pathways may play a more critical role in regulating bystander effects [71].

In vitro experiments demonstrated that disruption of lipid raft structures using Methyl-β-cyclodextrin, filipin, or ROS scavengers significantly suppressed non-targeted cytotoxicity, confirming the critical role of the cell membrane in radiation-induced NTEs [81]. The co-culture experiments utilizing U937 macrophages and bystander cell HL-7702 hepatocytes demonstrated that alpha-radiation triggers a bidirectional cAMP communication mediated through cell membrane signaling pathways. However, this interaction was not observed when the membrane signaling pathway was blocked using filipin, which inhibits cAMP transmission [82]. Further studies have revealed that alpha-particle irradiation activates membrane-dependent signal transduction pathways, such as the MAPK signaling cascade, which plays a central regulatory role in bystander effects. Key components of this pathway include JNK, ERK1/2, and p38 MAPK [38, 77, 83]. Relevant data revealed that lipid raft-dependent mechanisms contribute approximately 50% to the NTEs induced by alpha-particle irradiation, highlighting the importance of membrane architecture in mediating these biological responses [40, 81].

Vesicles

Additionally, radiation-induced bystander signals can be transmitted between cells via extracellular vesicles (EVs), such as exosomes (50–150 nm), the smallest extracellular vesicles [84]. Compared to other soluble factors, exosomes possess a protective lipid bilayer that enables stable systemic propagation without degradation [85, 86]. They can carry bioactive molecules, including nucleic acids, proteins, and lipids, which are delivered to recipient cells through various mechanisms. This dual functionality allows exosomes to mediate both material transfer and signaling in intercellular communication [87]. Studies have shown that radiation enhances the release of exosomes and promotes the migration of recipient cells [88–90]. Molecular profiling further reveals that exosomes are enriched with critical signaling pathway molecules associated with cell migration [88]. Furthermore, as a novel mediator of RIBE, exosomes participate in the systemic immunomodulatory effects of radiation. Exosomes released from irradiated cells can deliver immune-active molecules (e.g., MHC-I molecules, DAMPs, and double-stranded DNA) to unirradiated cells, triggering innate immune responses and reshaping local or systemic immune microenvironments [91].

Study indicated that when high-LET radionuclides are used for intracellular irradiation, RIBE is constrained by a threshold radiation dose. Alpha particles and Auger electrons can induce bystander effects at low radiation doses, but the effects diminish at higher doses [92]. Under conditions where direct radiation effects are limited, low average absorbed doses can trigger high bystander effects [93, 94]. This mechanism expands the killing range of tumor cells in heterogeneous regions (such as areas with uneven radiopharmaceutical biodistribution or clusters of drug-resistant clones), providing an innovative “controlled-by-less” strategy to reduce the recurrence risk of residual lesions.

Abscopal effects

In addition to the bystander effect, NTEs can also produce biological effects through abscopal effect. Although the abscopal effect is not the primary target of irradiation, it may significantly enhance the overall therapeutic outcome. In 1905, Heineke et al. first proposed that radiation might transmit signals from the irradiated area to unirradiated regions through the bloodstream. This suggested that an underlying biological mechanism could allow radiation therapy to exert effects beyond the localized treatment area, potentially influencing distant tissues or organs. The term “abscopal effect (AE)” in its true sense was first coined by British scientist Mole in 1953. He observed that when a local tumor was treated with radiation therapy, distant tumors that had not been irradiated also showed signs of shrinkage or regression. He defined the AE as “the gradual regression of distant tumors that were not irradiated as a result of local radiation” [95].

Accumulating evidence now suggests that the abscopal effect is mediated by the immune system [96]. Studies confirmed that systemic immune responses play a crucial role in radiation-induced anti-tumor effects [97, 98]. Ferreira’s team revealed the unique immunomodulatory advantages of alpha-emitters through comparative studies in immunocompetent prostate cancer models. The alpha-emitting radiopharmaceutical ²²⁵Ac-NM600 demonstrated significantly stronger antitumor efficacy compared to the beta-emitting radiopharmaceutical ¹⁷⁷Lu-NM600. This enhanced therapeutic effect is attributed to the alpha-emitter’s specific reduction of immunosuppressive regulatory T cell (Treg) infiltration, whereas no such remodeling of the immune microenvironment was observed in the ¹⁷⁷Lu-NM600 treatment group [99]. Leung et al. intravenously administered ²²³RaCl₂ at doses of 0, 50, or 600 kBq/kg to Swiss Webster mice and collected spleens on days 5, 12, and 19 to evaluate changes in immune cell populations. The results demonstrated that ²²³RaCl₂ affected the functional activity of NK cells and led to a reduction in splenic CD8⁺ T cells in Swiss Webster mice, depending on both administered activity and time post-administration [100]. Current clinical trials have demonstrated the significant therapeutic efficacy of ²²³Ra in mCRPC patients, and this success has laid the foundation for other radionuclide therapies in mCRPC treatment. In-depth investigation of the dynamic changes in immune microenvironment before and after treatment, as well as the radionuclide-specific immunomodulatory mechanisms, will provide critical insights for optimizing personalized treatment strategies [101].

Radiation-induced immunogenic cell death (ICD) constitutes a pivotal mechanism underlying abscopal effects [102]. Ionizing radiation triggers the release of DAMPs from tumor cells, including key mediators such as high mobility group box 1 (HMGB1), adenosine triphosphate (ATP), and CRT. These DAMP molecules activate pattern recognition receptors, thereby promoting the maturation and differentiation of antigen-presenting cells (APCs). Subsequently, the matured APCs migrate to tumor-draining lymph nodes, where they present processed tumor-associated antigens to T lymphocytes. This antigen presentation cascade ultimately activates tumor-specific CTL-mediated immune responses [103] (Fig. 4).

Fig. 4.

α-particle–emitting radiopharmaceuticals induce systemic anti-tumor abscopal effects. (Created with Biorender.com). Alpha particle irradiation triggers tumor cells to release DAMPs, including HMGB1, ATP, and CRT. These DAMPs bind to pattern recognition receptors on the surface of APCs, driving the maturation and differentiation of APCs. Mature APCs migrate to tumor-draining lymph nodes, presenting processed tumor-associated antigens to T lymphocytes and activating CTLs. Activated CTLs eliminate tumor cells by infiltrating primary tumor sites and distant metastases through the peripheral circulation, while inducing long-term immune memory formation

Evidence suggested that cellular exposure to alpha irradiation (²²⁷Th) induced the production of DAMPs, triggering immunogenic cell death. This indicates the activation of the immune system under alpha irradiation [104]. Previously, the alpha-emitter ²¹³Bi was also reported to produce similar results. Alpha-particle therapy for multiple myeloma using ²¹³Bi can induce irradiated cells to secrete soluble factors that activate dendritic cells (DCs) and trigger the occurrence of ICD in tumor cells [105]. Gorin et al. also observed that treatment of MC-38 cells with ²¹³Bi induced the release of DAMPs and activated DCs, leading to a systemic and lasting anti-tumor response [106]. Furthermore, irradiation therapy using ²¹¹At on small cell lung cancer induced the release of MHC-I molecules and CRT from tumor cells. This process activates endogenous anti-tumor immune responses by modulating the functionality of immune cells within the tumor microenvironment [107]. Similarly, ²²³Ra has been shown to exhibit similar regulatory mechanisms [52]. Moreover, Yang et al. demonstrated that alpha-emitter radiopharmaceuticals (²²³Ra-LDH) can activate the STING signaling pathway in vivo, eliciting a cascade effect that effectively induces ICD while concomitantly enhancing DCs maturation and T-lymphocyte activation [49]. The above studies demonstrate that TAT not only enhance T-cell cytotoxic capacity by promoting antigen presentation, but also activate antitumor immune responses through inducing tumor cell ICD. It is noteworthy that tumor immunomodulatory effects triggered by different types of alpha-emitter therapies may vary, therefore further in-depth exploration of their mechanisms of action remains necessary. Intriguingly, TAT can further establish long-term immunological memory by promoting the formation of effector memory T cells, thereby effectively preventing tumor recurrence [108].

Multiple clinical studies evaluating the immunomodulatory effects of alpha-emitters are currently underway. In 2019, a case of multiple cutaneous squamous cell carcinoma was reported, where complete remission of the treated lesion was observed 76 days after implantation of ²²⁴Ra-loaded seeds. Two distant lesions showed significant shrinkage. One year after treatment, the treated lesion remained in complete remission, while the untreated distant lesions spontaneously regressed. This may be related to an immune-mediated response [109]. Kim et al. enrolled 15 male patients with metastatic castration-resistant prostate cancer (mCRPC) who received a regimen of ²²³Ra at 50 kBq/kg. Peripheral blood mononuclear cells were collected before treatment and 3–4 weeks after treatment to analyze the phenotype and functional characteristics of CD8⁺ T cells. The results showed that after a single ²²³Ra treatment, the proportion of effector memory CD8⁺ T cells expressing PD-1 decreased from 20.6 to 14.6% [110]. However, the study had a small sample size, and the significance of the immunological changes and the long-term effects of treatment still require further investigation in future research. Currently, a phase I/II study combining ²²³Ra with the anti-PD-L1 antibody (atezolizumab) has been completed [111]. Numerous clinical trials exploring the combined use of radionuclide ²²³Ra and immunomodulatory drugs in the treatment of mCRPC are also actively advancing [112–115]. Evidently, combination regimens employing different alpha-emitting radionuclides with immunotherapy have exhibited heterogeneous clinical outcomes, while the synergistic effects between TAT and immune checkpoint inhibitors (ICIs) (Table 3) point to a potentially promising treatment approach [116–120]. Existing clinical studies have reported that the combined administration is typically performed on the same day. If concurrent administration is not tolerated, the radiopharmaceuticals treatment is administered first, followed by immunomodulatory drugs. This sequencing strategy may be attributed to the potential of prior radioligand therapy to prime the tumor microenvironment. However, there is still insufficient clinical evidence or established guidelines to determine whether this specific administration sequence yields superior clinical benefits. Further clinical trials are needed to explore the optimal treatment sequencing strategy [111].

Table 3.

Recent studies on the combination of TAT and ICIs

Alpha radionuclide	Combination	Tumor type	Study type	Efficiency
Bismuth-213 [121]	Anti-PD-1 mAb (CD279)	Melanoma	Preclinical study	Slow down tumor growth rate by 1.5 times (compared to monotherapy)
Astatine-211 [122]	Anti-PD-1 mAb	Glioblastoma	Preclinical study	Significantly prolong PFS and 100% complete response
Astatine-211 [108]	Anti-PD-L1 mAb	Breast cancer, Colorectal cancer	Preclinical study	Prolong overall survival, Form long-term immune-memory effects
Actinium-225 [123]	Anti-PD-1 mAb	mCRPC	Preclinical study	Prolong TTP and survival
Actinium-225 [124]	Anti-PD-L1 mAb	Melanoma	Preclinical study	Delay tumor growth
Lead-212 [117]	Anti-PD-1 mAb, Anti-CLTA-4 mAb (Dual ICIs)	Melanoma	Preclinical study	43% complete tumor response
Thorium-227 [119]	Anti-PD-L1 mAb	Colorectal cancer, Ovarian cancer, Mesothelioma	Preclinical study	58.3% (7/12) complete tumor response (Significantly better than monotherapy)
Radium-223 [111]	Atezolizumab (Anti–PD-L1 mAb)	mCRPC	Clinical trial	No clear evidence of clinical benefit
Radium-223 [125]	Pembrolizumab (Anti-PD-1 mAb)	mCRPC	Clinical trial	No efficacy improvement
Actinium-225 NCT04946370	Pembrolizumab (Anti-PD-1 mAb)	mCRPC	Clinical trial	Incomplete
Actinium-225 NCT06939036	Atezolizumab, Durvalumab, Avelumab, Pembrolizumab, Retifanlimab (Anti-PD-1/PD-L1 mAb)	ES-SCLC, MCC	Clinical trial	Incomplete
Lead-212 NCT05655312	Nivolumab (Anti-PD-1 mAb)	Melanoma	Clinical trial	Incomplete

PFS, progression-free survival, TTP time to progression, mCRPC metastatic castration-resistant prostate cancer

Challenge and future perspectives

Given the short range of alpha particles, TAT is generally considered to have a high safety profile, with a low incidence of severe acute or subacute adverse events (AEs). However, hematologic toxicity remains the most characteristic AE of TAT, primarily manifested as cytopenia due to bone marrow suppression [126]. Clinical data show that in prostate cancer patients with bone metastases treated with ²²³RaCl₂, the incidence of neutropenia and thrombocytopenia is significantly increased, indicating hematopoietic suppression [127]. Parlani et al. concluded from animal experiments that the myelotoxicity induced by ²²³Ra therapy is acute and reversible, without causing permanent damage to the hematopoietic system [128]. However, this conclusion requires validation through extensive clinical trials. This hematologic toxicity remains a key bottleneck limiting the clinical translation of TAT.

Although the incidence of hematologic toxicity with ²²⁵Ac-labeled therapies (e.g., ²²⁵Ac-PSMA-617) is relatively low [129], its longer half-life may increase the risk of cumulative toxicity. Some studies have observed nephrotoxicity in the clinical application of ²²⁵Ac [124], which may be related to the redistribution of daughter nuclides (e.g., ²¹³Bi) due to recoil effects, leading to off-target radiation damage. In one clinical trial of ²²⁵Ac-PSMA-617 radioligand therapy for mCRPC, delayed nephrotoxicity was observed [130]. However, data on long-term adverse events remain limited, and future studies should expand clinical trial cohorts and extend follow-up periods to clarify its safety boundaries.

In addition to the aforementioned AEs, the multiple challenges in accurate dosimetry assessment of alpha-emitting radionuclides further hinder their clinical translation.

Alpha-emitters predominantly rely on γ rays emitted during decay for imaging. However, most therapeutic alpha-emitters are administered at low activities, with γ photon yields being particularly low (γ ray abundance < 1%) and the emitted photon energy spectrum is complex. These characteristics result in poor signal-to-noise ratios in conventional SPECT/CT imaging, thereby limiting the reliability of quantitative imaging. Early MIRD monographs made adjustments to dosimetry for alpha-emitters, but calculations for alpha-particle radiopharmaceutical therapy in human tissues were based on assumptions that prove inaccurate. These assumptions fail to account for the heterogeneous microscopic distribution of alpha-particle radiopharmaceutical therapy agents, making dose calculations challenging [131]. Certain alpha-emitters with multiple decay progeny (e.g., ²²³Ra, ²²⁵Ac, ²²⁷Th) feature daughter radionuclides with long half-lives. The high-energy nuclear recoil released during alpha decay may cause redistribution of these daughter nuclides within the body [8]. For instance, ²¹³Bi from ²²⁵Ac decay tends to relocate to kidneys [132]. Accurate dosimetry of alpha-emitter radiopharmaceuticals requires simultaneous imaging of both the target parent nuclides and their redistributed daughters. Understanding the distribution of long-lived progeny is equally crucial for toxicity assessment, as they may induce off-target organ toxicity [133]. Dosimetric measurement of alpha-emitters remains an urgent challenge that needs to be addressed. Due to the short path length, high LET, and heterogeneous distribution of alpha-emitters within target volumes, small-scale dosimetry and microdosimetry assessments has become an essential approach for accurately evaluating the therapeutic potential of TAT and risks to normal tissues. Current research in targeted radionuclide therapy utilizes both Monte Carlo simulations and analytical methods for small-scale dosimetry and microdosimetry assessments. These computational approaches enable evaluation of radiopharmaceutical biodistribution, energy deposition, and radiobiological modeling. While achieving accurate dose measurements remains challenging, reliable dosimetric quantification is critical for ensuring both therapeutic efficacy and patient safety [134].

Conclusion

TAT offers a highly promising strategy for precision oncology by integrating the multidimensional biological effects of targeted effect, bystander effect, and abscopal effect. The targeted effect achieves efficient eradication of localized lesions through α-particles, while the bystander effect overcomes therapeutic blind spots caused by tumor heterogeneity via intercellular signaling transfer, significantly expanding therapeutic coverage. The abscopal effect further enhances systemic therapeutic potential by activating systemic anti-tumor immune responses, inducing regression of distant metastases. The synergy of these three effects establishes a three-dimensional anti-tumor network at the “local-regional-systemic” levels, pioneering novel pathways for precision cancer therapy. However, the clinical translation of TAT still faces critical bottlenecks: despite the precise tumor cell eradication enabled by alpha-emitters through their high LET and short penetration range, the delivery efficiency and stability of radiopharmaceutical carriers remain critical limitations for the broad application of targeted effects. While the bystander effects extend therapeutic impact beyond directly irradiated cells, it introduces potential risks: the nonlinear dose-response characteristics, distant tissue toxicity, and optimization of personalized dosing require in-depth investigation, necessitating high-precision in vivo models to define safety thresholds. Furthermore, although the abscopal effects induced by alpha-emitters have garnered significant interest, their combination with ICIs has yet to demonstrate substantial clinical benefits, highlighting the need for systematic exploration of synergistic strategies with immunotherapy. In conclusion, overcoming these key challenges is essential to translate the biological advantages of alpha-emitters into clinically accessible precision anticancer tools.

Author contributions

All authors contributed to the study conception and design. Zhiling Song, Jiajia Zhang and Shanshan Qin conceived the ideas and wrote the initial draft. Xiaohui Luan, Han Zhang reviewed and edited the manuscript. Mengdie Yang, Yao Jin and Gang Yang created the figures and tables. Fei Yu coordinated the project and supervised the work. All authors read and approved the final manuscript.

Funding

This work was conducted with the supports by National Natural Science Foundation of China (Grant No. 82272030 and No. 82472028) and China University Industry-University-Research Institute Innovation Fund (No.2023HT066).

Data availability

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

Declarations

Ethics

This is a systematic literature review; therefore, no ethical approval is required.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.