Radio therapy and immunotherapy

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REVIEW ARTICLE OPEN

Radiotherapy combined with immunotherapy: the dawn of


cancer treatment
Zengfu Zhang1, Xu Liu2, Dawei Chen1 ✉ and Jinming Yu1 ✉

Radiotherapy (RT) is delivered for purposes of local control, but can also exert systemic effect on remote and non-irradiated tumor
deposits, which is called abscopal effect. The view of RT as a simple local treatment has dramatically changed in recent years, and it
is now widely accepted that RT can provoke a systemic immune response which gives a strong rationale for the combination of RT
and immunotherapy (iRT). Nevertheless, several points remain to be addressed such as the interaction of RT and immune system,
the identification of the best schedules for combination with immunotherapy (IO), the expansion of abscopal effect and the
mechanism to amplify iRT. To answer these crucial questions, we roundly summarize underlying rationale showing the whole
immune landscape in RT and clinical trials to attempt to identify the best schedules of iRT. In consideration of the rarity of abscopal
effect, we propose that the occurrence of abscopal effect induced by radiation can be promoted to 100% in view of molecular and
genetic level. Furthermore, the “radscopal effect” which refers to using low-dose radiation to reprogram the tumor
microenvironment may amplify the occurrence of abscopal effect and overcome the resistance of iRT. Taken together, RT could be
regarded as a trigger of systemic antitumor immune response, and with the help of IO can be used as a radical and systemic
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treatment and be added into current standard regimen of patients with metastatic cancer.

Signal Transduction and Targeted Therapy (2022)7:258 ; https://doi.org/10.1038/s41392-022-01102-y

INTRODUCTION to combine the two treatment modalities as one is local means


Radiotherapy (RT), a fundamental component of cancer treatment, is and another is systemic. And there are several reviews focusing on
received by about half of all patients with cancer.1–3 RT is a powerful this field and presenting perspective of iRT to guide clinical trials
weapon for both curative purposes as well as for palliation and and make the further processes (Fig. 1).21,33–41 Indeed, iRT has
maintenance of the quality of life.4,5 As we focus on investigations of shown an exciting impact on patients with certain cancers in
new technologies such as FLASH RT, proton RT, and carbon ion RT PACIFIC and other clinical trials.42–48 These convincing clinical data
which aim to improve the therapeutic ratio,6–11 increasing evidence provide the foundation for further exploration of combination
on immunomodulatory effects of RT casts new light on a systemic schedules.
antitumor response. Recent studies have found RT may be similar to Nevertheless, there are still many unresolved issues about iRT.
an “accelerant” by means of inducing in situ vaccination by killing First, although the abscopal effect has been quite well-known
tumor cells and triggering a systemic immune response.12–20 The since its initial description,3,49,50 the underlying mechanisms of
most representative example is the abscopal effect: radiation on one how RT influences immune cells and induces abscopal regression
site may cause regression of tumor at remote and distant non- of tumor remain unknown. Meanwhile, according to the rarity of
irradiated sites.21–23 The potential systemic antitumor capacity the abscopal effect,51 we should take into account how to amplify
provides a sound basis for iRT. the occurrence of this phenomenon so as to expand the beneficial
In recent years, cancer immunotherapy has been considered as population. Second, the optimal patterns of iRT largely remain
one of the most successful approaches in oncologic therapy, unresolved. The agents, sequence, dose, fractionation, and
particularly with regard to immune checkpoint inhibitors (ICIs) in irradiated sites required further exploration.
treating solid tumors. For some certain types of tumors such as This review will attempt to address these questions based on
non-small cell lung cancer (NSCLC), ICIs can significantly improve the latest advances in iRT in the view of rationales and clinical
conditions of survival without interruption and induce long-term practice. Moreover, we will introduce “the radscopal effect” as a
durable remission which means even patients with advanced new concept derived from abscopal effect to show the powerful
tumor can get persistent and stable benefit from immunother- strength of low-dose radiation combined with immunotherapy.
apy.24–31 However, the number of patients who may durably
respond to IO alone just accounts for a minority of cases.32 It is
urgent to broaden the beneficial spectrum of immunotherapy and DEVELOPMENT HISTORY OF RT AND IO
identify appropriate patient selection. iRT can overcome the Since Röntgen discovered X-ray in 1895 and Marie Curie
problems we mentioned above as far as possible. Thus, it is natural discovered radium and polonium, advances of radiation utilization

1
Department of Radiation Oncology, Shandong University Cancer Center, Yantai Road, No. 2999, Jinan, Shandong, China and 2Department of Radiation Oncology, Shandong
Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jiyan Road, No. 440, Jinan, Shandong, China
Correspondence: Dawei Chen ([email protected]) or Jinming Yu ([email protected])
These authors contributed equally: Zengfu Zhang, Xu Liu.

Received: 14 April 2022 Revised: 19 June 2022 Accepted: 30 June 2022

© The Author(s) 2022


Radiotherapy combined with immunotherapy: the dawn of cancer treatment
Zhang et al.
2
The amazing result of PACIFIC
trial to access efficacy and
safety of durvalumab after
First description of the rare chemoradiotherapy in stage III
abscopal effect.3 NSCLC.43
Ongoing investi-
gations of RT
First confirmed statistic combined with
First proof-of-principle trial of
difference of abscopal effect immunotherapy.
Treatment of cancer with radiotherapy combined with
and significantly increased
bacterial products by Coley. immunotherapy (GM-CSF) in
survival benefit by combining
patients with metastatic solid
RT and immunotherapy.376
tumours.70

1893 1895 1953 2012 2015 2016 2017 2019 2020 2021 2022

A doubling of ORR of pembrolizum-


Discovery of X-ray by First case report of ab after SBRT in patients with
Röntgen. the abscopal effect in advanced NSCLC in PEMBRO-RT
a patient.50 trial.377

Exploration of combining The role of low dose RT


immunotherapy and stereotac- with immunotherapy to
tic ablative radiotherapy.71 reverse tumor immune
desertification.348

Fig. 1 Historical timeline of some important developments regarding the iRT

in medicine have never slowed down. Tracing back the FLASH RT, proton RT, and carbon ion RT may benefit more
development history in RT, it can be summarized as two paths patients with reduced toxicities and improved survival outcomes.
(physical development and biological effect) and four major eras Cancer immunotherapy has a history of more than 100 year so
(discovery era, orthovoltage era, megavoltage era, and ion beams far. Although difficult to prove, the initiation of immunotherapy
era).52,53 During the era of discovery, which referred to the period can be traced back to 2400 years ago.61 Nowadays, William B.
from the discovery of X-ray to 1930, the roots of RT were Coley is widely accepted as the father of immunotherapy. The first
established. In addition to the salient discovery by Röntgen in clinical trial using IO (mixtures of Streptococcus pyogenes and
1895, Becquerel reported the phenomenon of radioactivity in Serratia marcescens) to treat tumors was designed by Coley in
1896 and in 1898 the Curies isolated radium, which lay the 1893. However, due to the lack of known mechanisms about the
foundation of RT. Indeed, a patient with breast cancer received the “Coley’s toxins” and immune system, IO has been in a slow
treatment of RT using X-ray in 1896.54 Although X-rays were used development stage and even raises doubts about tumor immunity
in the clinic, biological effects and mechanisms still remain and its treatment. In 1959, the first cancer vaccine study
unclear. Regaud and Coutard found the advantages of fractio- conducted by Ruth and John Graham achieved success and
nated therapy in the research of delivering the total radiation dose aroused interest in this area.62 With the establishment of existence
and their studies promoted the understanding of X-ray’s biological and the key role of T cell in adaptive immunity by Miller in 1967,63
characteristics.55 Another crucial discovery is a practical X-ray tube cancer IO was made in rapid-fire succession. In addition to the
which can promote the delivery of X-rays with higher-energy above-mentioned development, the discovery of dendritic cells
(180–200 kV) by Coolidge.56 The orthovoltage era is from 1930 to and natural killer cells, promoted the understanding of underlying
1950 which is a booming and transitional period of physical and mechanisms of immune system and aided in the process of cancer
machine developments. The cyclotron was developed by Widerøe IO. In the 1980’s, Taniguchi et al. cloned the IL-2 and it showed
in 1927 and invented in 1930 by Lawrence and Livingston.57 promising results in clinical trials.64,65 Furthermore, it was
Another major advance was the synchrotron, conceived by Veksler approved by the US FDA in 1991 for the patients who were
and McMillan during 1944–1945.58 The first synchrotron began suffering from metastatic kidney cancer. In 1987, Brunet et al. first
operation in 1952 at Brookhaven National Laboratory.59 The proved cytotoxic T-lymphocyte antigen 4 (CTLA-4) and its immune
megavoltage era refers to the period from about 1950 to 1985 and checkpoint function was proved by Jim Allison and his colleagues
it seems that this era is still in progress. Cobalt teletherapy and in 1995.66 Until 2010, ipilimumab as the revolutionary checkpoint
megavoltage linear electron accelerators were considered a inhibitor was approved by FDA for the treatment of stage IV
revolution in cancer treatment which produced radiation with melanoma after the definitive clinical study.67 Another checkpoint
high energy to treat deep tumors.60 In the 1960s and 1970s, inhibitor, nivolumab, was approved by FDA in 2014. Atezolizumab,
electron linacs have been widely used. With the development of another checkpoint inhibitor of the programmed death 1 ligand
computer and imaging technologies, computed tomography (PD-L1) protein, was approved by FDA in 2016.
scans and similar imaging technologies were applied to treatment With the deepening research on biological effect of RT, it seems
planning of RT for determining tumor volumes as well as that combining RT and IO become an inevitable strategy to
identifying normal organ anatomy. The appearance of intensity- improve the outcomes of cancer treatment. In 1975, Milas et al.
modulated radiation therapy (IMRT) improves the radiation beams first proposed that the antitumor effect of local irradiation could
adjusted to the three-dimensional shape of the target and reduces be improved by administration of Corynebacterium granulosum
the damage to surrounding normal tissues.2 In addition, image- or Corynebacterium parvum in C3Hf/Bu mice with a syngeneic
guided radiation therapy (IGRT), using frequent pretreatment fibrosarcoma.68 Besides immunosuppressive effect induced by RT,
imaging to increase certainty regarding tumor location, also investigators realized that RT also has a potential to activate
occurred with the technological progress. And due to the advent immune system of human body, which lay a solid foundation for
of these imaging technologies, stereotactic body radiotherapy iRT. In 2005, Demaria and his colleagues first proposed radio-
(SBRT) has been widely used recent years. In the near future, immunotherapy as a novel approach to treat cancer.69 Following

Signal Transduction and Targeted Therapy (2022)7:258


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Zhang et al.
3
the concept of iRT, research of combining RT with various kinds of immunomodulatory actions of RT.95,101–105 On the one hand, IR
IO were flourishing. In 2012, first case report of the abscopal effect not only promotes the immunogenic cell death of cancer cells
in a patient with melanoma treated with ipilimumab and RT was which then affects the behavior of immune cells. On account of
reported and iRT has tremendous potential to increase the the stress response induced by IR, some intrinsic biological
occurrence of abscopal effect.50 The first proof-of-principle trial of processes occur in the dying cancer cells including the release of
RT combined with IO (GM-CSF) achieved a successful result to damage-associated molecular patterns (DAMPs).34 The release of
produce abscopal responses in patients who are suffering from DAMPs acts on different immune cells and exert potent
metastatic solid tumors in 2015.70 In 2017, the amazing result from immunomodulatory effects of IR which will be discussed below.
PACIFIC trial showed the significant clinical benefit and changed On the other hand, cellular response driven by DNA damage also
the modality of NSCLC. In addition, Chang et al. proposed the changes the immunogenicity of these irradiated cancer cells.99
combination of IO with stereotactic ablative radiotherapy (SBRT) Then we will discuss the relationship between DNA damage
and showed the significant advantages of this strategy.71 Nowa- response (DDR) and the STING signaling in cancer cells which
days, there are hundreds of ongoing clinical trials to explore results in the formation of inflammatory microenvironment
optimal combination modality of iRT and we expect more remodeling by ionizing radiation and the generation of the
advances and further interest in this field. abscopal effect after RT.
RT is a well-documented trigger of DNA damage and this occurs
via two primary mechanisms: direct breakage of DNA by high-
RATIONALE OF IRT energy photons and the generation of ROS. Under physiological
Except the direct damage to irradiated tumor cells, ionizing conditions, DNA strand breaks can be repaired by the three central
radiation (IR) also induces a series of biological effects which are DDR kinases: DNA-dependent protein kinase (DNA-PK), ataxia
deemed to be systemic, immune-mediated antitumor effects.72–80 telangiectasia-mutated (ATM), and ataxia telangiectasia & Rad3-
In fact, the immunomodulatory effects of IR are exactly theoretical related protein (ATR) which prevent progression of cells with DNA
basis of combination. IR can exert a potent antitumor immune damage into mitosis and avoid exposure of DNA in the cytoplasm.
response by influencing almost all steps in the cancer-immunity These DDR kinases further facilitate two main mechanisms
cycle rather than just several discrete steps with ICIs.81,82 These including non-homologous end-joining (NHEJ) and homologous
effects comprise enhancing tumor antigen release and presenta- recombination (HR) repair. In addition, a myriad of DDR proteins
tion,83,84 promoting priming and activation of immune cells,85,86 repair the genomic lesions when the DNA damage appears.106
increasing density of tumor-infiltrating lymphocytes,87,88 facilitat- However, it is very common for the disorder of DDR processes in
ing recognition of tumor cells by T cells and augmenting cancer which may lead to the persistence of genomic instability.107
antitumor effect,89,90 which have been well summarized and And cell cycle checkpoint disruption occurs commonly in tumor
elucidated in several reviews. Besides, in the process of activating cells leading to formation of micronuclei containing DNA in the
immune system, IR also leads to proinflammatory cytokines cytoplasm.108,109 And it is a well-established phenomenon that IR
released through cyclic GMP–AMP synthase (cGAS)-stimulator of may induce the production of micronuclei in cancer cells and it is
interferon genes (STING) and other inflammatory signals.91–93 also well-known for the production of type I interferon (IFN) caused
Furthermore, the tumor microenvironment is remodeled by by IR.110 Thus, surveillance of micronuclei by cGAS builds a bridge
cytokines and stromal, immunological, and vascular changes between DDR and STING signaling pathway. For the surveillance
induced by IR and mediates an antitumor response.94,95 The function of micronuclei by cGAS, cytoplasmic DNA induced by IR
reprogrammed tumor microenvironment induced by IR plays a promotes type I IFN production through cGAS–STING pathway in
role as a “game changer” to transform “cold” tumors with less cancer cells and triggers subsequent innate immune signaling. One
immune cells infiltration into “hot” tumors with lymphocytic of the most important negative regulators is DNA exonuclease
infiltration and provide a pre-requisite for response to ICIs. This Trex1. Vanpouille-Box and colleagues found the key role of DNA
key role of IR has been discussed and widely accepted.96,97 exonuclease Trex1 in the context of RT which degrades cytoplasmic
However, RT doesn’t work the way we want it to, on the DNA preventing cGAS activation.111
contrary, the immunomodulatory effect induced by RT is a double- In addition to the damaged cytoplasmic DNA, another source of
edged sword that not only enhances systemic antitumor immune immunogenic DNA is exposure of the mitochondrial genome
response but also promotes immunosuppression to some (mtDNA) to the cytoplasm in cancer cells. mtDNA damage and
extent.35,98 Generally, IR is deemed to inhibit the immune function reactive oxygen species (ROS) resulting from RT also provoke a
of the body for its lethality to immune cells in the peripheral series of immune responses and the mtDNA damage may be more
blood. In fact, its damage to the immune system goes far beyond sensitive to the disorder of DDR processes than nuclear DNA
that. IR also influences tumor microenvironment by increasing damage.112,113 However, the underlying mechanism of mtDNA
some inhibitory immune cells that include Treg cells and myeloid- induced by IR remains unclear. In a conclusion, all of these
derived suppressor cells (MDSCs).99,100 Besides, many studies have damaged DNA including cytoplasmic DNA and mtDNA induced by
shown immunosuppressive cytokines are elevated after IR can be recognized by cGAS, which then oligomerizes with DNA
radiotherapy. in the form of a 2:2 complex.114–116 After binding to DNA, cGAS
Subsequently, we will elucidate distinguished notable effects then exerts a catalytic role to promote the synthesis of the second
induced by IR in cancer cells, immune cells, and stromal cells, messenger 2′3′-cyclic GMP–AMP (cGAMP).117,118 Binding of 2′3′-
respectively, and offer a landscape of the whole immune process cGAMP stimulates STING and promotes the translocation to the
after radiation based on immune cells including Treg cells, Golgi which acivates TANK-binding kinase 1 (TBK1).115,119,120 TBK1
neutrophils, macrophages, myeloid-derived suppressor cells, phosphorylates STING and promote the interferon regulatory
dendritic cells (DCs), natural killer cells (NKs) and other subsets factor 3 (IRF3) to translocate to the nucleus which triggers the
of T lymphocytes. expression of IFN-β gene. Type I IFNs induced by cGAS–STING
pathway are immunomodulatory factors to destroy cancer cells as
Effects of IR on cancer cells the essential link of innate and adaptive immune responses
Radiation damages cancer cells via two primary mechanisms: through DCs.121,122 Type I IFNs secreted by cancer cells can
direct breakage of DNA by high-energy photons and the facilitate DCs maturation, increase DCs co-stimulatory molecule
generation of ROS. Besides the direct impact on cancer cells, it expression as well as enhancing DCs lymph-node migratory
has been proved that a series of biological events induced by DNA capacity. Moreover, it also plays a crucial role in priming CD8+
damage which occur in cancer cells play an important role in the T cells and aiding in the following antitumor responses.

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Of note, besides IRF-3, both canonical and non-canonical NF-κB Immunomodulatory effect on various immune cells
pathways play a crucial role in cGAS-STING induced type I IFN Treg cells are a type of CD4+ lymphocyte with the expression of the
expression in this context of RT. Generally, NF-κB is activated transcription factor forkhead box P3 (FOXP3); the existence of Treg
through canonical and non-canonical pathways. The canonical cells has important implications for the maintenance of immune
pathway mediates the activation of NF-κB1 p50, RELA and c-REL homeostasis and self‐tolerance.137–141 In cancer, Treg cells exert
whereas the non-canonical NF-κB pathway selectively activates “unproductive” immunosuppression leading to unwanted immuno-
p100-sequestered NF-κB members, predominantly NF-κB2 p52 suppressive effects or even promoting disease progression.140 There
and RELB. In addition to the generation of ROS, the signaling are numerous studies indicating that Treg cell numbers in the tumor
pathways of IR-induced DNA damages also activate the NF-κB microenvironment increase significantly when tumor cells receive
pathway. Radiation-induced NF-κB is mostly mediated via IKK- various types of radiotherapy.142–144 And elevated levels of Treg cells
dependent canonical pathway.123 IFN-β largely depends on the are related to a suppressive tumor microenvironment, the resistance
facilitation effect of the enhanceosome and canonical NF-κB can of immunotherapy, and poor prognosis.145–149 Although IR leading to
work in conjunction with IRF-3 as well as other enhancer Treg cells infiltration is a generally acknowledged fact, the underlying
components to maximize the expression of IFN-β gene.124,125 mechanism is still not revealed. Muroyama et al.143 observed that the
Abe et al. observed a 50% decrease in IFN-β production in primary immunosuppressive function of Treg in the tumor microenvironment
mouse embryonic fibroblast cells when canonical NF-κB expres- is potentiated by RT. Furthermore, mechanisms of Treg modulation
sion was partially silenced via RNA interference (RNAi).119 Similarly, by radiation are elucidated by Oweida and his colleagues in an
canonical NF-κB and IRF3 are essential to induce type I IFN in DCs orthotopic mouse model of head and neck cancer (HNC).150 They
stimulated by irradiated tumor cell after IR.126 This is also demonstrated that signal transducer and activator of transcription 3
consistent with another research which proved impaired canoni- (STAT3), as a key molecule to regulate FOXP3,151,152 promotes the
cal NF-κB pathway may recede IR-induced antitumor immunity.126 conversion induced by radiation from CD4+ T cells to Treg cells and
In contrast, it is reported that the release of IFN induced by IR may STAT3 inhibition in combination with radiation changes tumor
be inhibited by the non-canonical pathway in DC cells activated microenvironment dramatically in the aspect of decreasing Treg cells,
by STING pathway. Investigators demonstrated that cancer cells MDSCs and M2 macrophages along with enhancing effector T cells
exposed to IR may promote the activation of the non-canonical and M1 macrophages. This result also corroborates previous studies
NF-κB pathway in DCs through the process we discussed above. implicating that STAT3 signaling may play a crucial role in enhanced
Taken together, the canonical and non-canonical NF-κB pathways Treg function and conversion.153 The underlying mechanism is IL-
may play the opposite role in regulating the type I IFN expression 10R-mediated STAT3 signaling pathway. IL-10 binds to its receptor
induced by the cGAS-STING signaling pathway in the and activates STAT3 signaling which then promotes the proliferation
context of RT. of Foxp3(+) iTregs with increased level of CTLA-4 and potentiated
The delivery of cGAS between cancer cells and non-cancer cells immunosuppressive function with the help of TGF-β. Similarly, this
has been demonstrated. Like other IR-generated DAMPs, DNA process may occur when using blockade PD-1 in vitro. It is reported
derived from cancer cells can be recognized by DCs to exert that PD-1 blockade also increases the release of IL-10 by T cells,
immune response which is similar to Type I IFNs.127 For instance, it leading to higher Tregs proliferation.154 However, Woods found that
is reported that tumor-derived exosomes containing DNA can be Treg suppression is reduced in vitro by PD-1/PDL1 blockade and
transferred to DCs from tumor cells exposed to IR which leads to enhanced by STAT3 inhibition.155 And in accordance with their
an upregulation of co-stimulatory molecules and STING- research, previous studies proved that STAT3 expression in Tregs has
dependent activation of Type I IFN.128 In addition to DNA, it is a close relation to decreased suppressive function.156 These contrary
reported that cytoplasmic tumor-derived cGAMP as the secondary data show that more investigations are needed to explore the role of
messenger can diffuse to adjacent cells via gap junctions.129 The STAT3 expression for patients with tumor in response to different
intercellular signaling may aid in the systemic immune treatments but STAT3 signaling and IL-10 production may play a
response of RT. critical role in the proliferation and function of Treg. Of note, the type
I IFN production and signaling pathway and NF-κB pathway involve
Effects of IR on stromal cells in the production of IL-10.157 As we discussed above, cGAS-STING
Cancer-associated-fibroblasts (CAFs) are a heterogeneous popula- induced by IR activates type I IFN and NF-κB signaling pathway
tion that make up the majority of stromal cells in many tumors. which then lead to IL-10 production. Specifically, IL-10 binds to its
CAFs are relatively resistant to IR which stay alive in tissue culture receptor and activates the JAK–STAT3 signaling which then exerts
when they are expose to ablative doses of radiation (18 Gy). Of the biological effect.158,159 Moreover, increased TGF-β production
note, following radiation, CAFs have persistent DNA damage and induced by RT may favor Treg accumulation in the tumors.160 ROS
become senescent.130 DDR may activate downstream signaling induced by RT may promote the conformational change of the
and result in changes in molecules secreted by CAFs. Moreover, IR latency-associated peptide/TGFβ complex which leads to the
can recruit CAFs and myofibroblasts in the tumor microenviron- secretion of TGF-β when tumor cells receive RT.161 Many studies
ment undergo to phenotypic transformation to CAFs.131 In have confirmed the critical role of TGF-β for iTreg development and
particular, the expression of some angiogenic molecules is IR greatly increases the level of TGF-β in the tumor microenviron-
downregulation while the expression of fibroblast growth factor ment.162–166 In Treg cells, TGF-β activates Smad2 and Smad3 through
(FGF), and macrophage migratory inhibitory factor (MIF) is TGF-β receptors and promotes the formation of a heterotrimer with
upregulation when CAFs are exposed to radiation.132 In addition, Smad4. The heterotrimer translocates into the nucleus and binds to
CAFs can promote the conversion to radioresistance cancer stem the conserved non-coding sequence 1 (CNS1) which is located in the
cells through the release of transforming growth factor-β (TGF- Foxp3 gene locus as an intronic enhancer.167 Importantly, there are
β).133 CAFs are known as immunosuppressors in tumor micro- two consecutive Smad-binding sites CNS1 it has been demonstrated
environment and generally, CAFs promote tumor progression.134 that CNS1 plays an important role in iTreg/pTreg generation.168 In
However, it is reported that irradiated CAFs may change addition, miR10a/10b expression has been demonstrated to be
protumorigenic features which may lead to reduction of tumor associated with TGF-β expression and it is reported that TGF-β
engraftment and angiogenesis in vivo models.135. Of note, both induces miR10 expression in Treg cells.169 And miR-10a can increase
irradiated CAFs and intact CAFs show the ability to mediate the expression level of FOXP3 and facilitate the differentiation of Treg
immunosuppression by reducing human T cells.136 More investi- from naive CD4+ T cell.170 IR not only increases the amount of Treg
gations and exploration are needed to characterize the specific cells, but also enhances the radioresistant capacity of Treg. It was
immunosuppressive role of irradiated CAFs in this context. shown that Tregs surviving after RT had an upregulated Akt

Signal Transduction and Targeted Therapy (2022)7:258


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Radiation TGF-β
IL-10

TβRII TβRI

miR-10a P

JAK
P
JAK T3
3 S TA
T smad7
P
S TA P P

smad3
F-қB

smad2
N

P P
STAT3 STAT3
smad4

Nucleus

P P

smad3
smad2
P P smad4
STAT3 STAT3
CNS1
Foxp3

Expansion ↑
Differentiation ↑ Expansion ↑
T cell suppression ↑ Differentiation ↑
CTLA-4 expression ↑ Treg cell Development ↑

Fig. 2 Immunomodulatory effect of radiation on Treg cells. Radiation promotes the conversion from CD4+ T cells to Treg cells and enhances
Treg function through IL-10R-mediated STAT3 signaling pathway. Radiation also increases the secretion of IL-10 which can bind to IL-10
receptors. This binding activates JAKs and activated JAKs, such as JAK1 and JAK2, phosphorylate STAT3 at Tyr705, resulting in translocation of
activated STAT3 dimers to the nucleus. STAT3, as a cotranscription factor with FOXP3, promotes expansion, differentiation, and T cell
suppression and increases CTLA-4 expression of Treg cells. In addition, miR-10a induced by radiation can enhance the expression level of
FOXP3 and promote the differentiation of Treg from naive CD4+ T cell. Radiation increases the level of TGF-β in the TME greatly and TGF-β
recognizes and binds TGFβRII, which then phosphorylates TGFβRI. TGF-β activates Smad2 and Smad3 and promotes the formation of a
heterotrimer with Smad4. Smads are recruited to the CNS1 region which has been identified at the Foxp3 gene locus. The CNS1 promotes
generation, expansion, differentiation, and development of Treg cells. Parts of this figure were drawn with aid of Servier Medical Art (http://
www.servier.com), licensed under a Creative Commons Attribution 3.0 Unported License. Parts of this figure were drawn with aid of Servier
Medical Art (http://www.servier.com), licensed under a Creative Commons Attribution 3.0 Unported License

expression, which rendered them more resistant to RT-induced According to functional differences, tumor-associated neutro-
apoptosis.171,172 In addition to the increasing count of Treg cells, IR phils (TANs) can be divided into two subtypes: antitumorigenic or
can modulate the phenotype and function of human Treg cells. protumorigenic neutrophils, termed N1 and N2, respec-
Beauford and colleagues found that IR can attenuate the function of tively.173–177 TANs can be a component of tumor-promoting
Treg to suppress the proliferation of CD8+ T cells through inflammation by promoting angiogenesis, immunosuppression,
downregulating Foxp3 expression and modulating the expression remodeling of the extracellular matrix, and metastasis. The
of Treg signature molecules, for example, increasing the expression interaction between neutrophil polarization and tumor micro-
of lymphocyte activation gene 3 (LAG-3) and decreasing the environment has been well summarized but there are few studies
expression of CD25 and CTLA-4.142 Kumagai et al.147 found Treg focusing on the relationship between TANs polarization and
cells has indicative significance for the prediction of PD-1 therapeutic IR.178–184 Generally, TGF-β, an immunosuppressive cytokine over-
effect. They confirmed that the frequency of PD-1+CD8+ T cells has a expressed by tumor cells, polarizes neutrophils to a protumori-
close relation to that of PD-1+ Treg cells in the tumor microenviron- genic phenotype (N2) and inhibits N1 phenotypic polarization. On
ment and this ratio may be a better predictor for PD-1 therapeutic the contrary, IFN-β polarizes neutrophils to an antitumorigenic
effect. The signaling pathways of immunomodulatory effect on Treg phenotype (N1) while inhibits N2 polarization.185 The function of
cells are summarized in Fig. 2. N1 involves in promoting tumor cell cytotoxicity/apoptosis,

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Cytotoxicity Genetic
instablity

ROS
IFN-β Radiation
ROS
Type 1 TGF-β
interferon
receptor TGFβR1

neutrophil elastase neutrophil elastase

Cancer cell
CTL-mediated lysis proliferation
Neutrophil
N1 TAN N2 TAN
Type-1 IFN
Treg cell

M1 φ M2 φ
CD8+ T cell
CD8+ T cell
Immunostimulation Immunosuppression
NK cell

Fig. 3 Immunomodulatory effect of radiation on TANs. Radiation shows complexity regarding the immunomodulatory effect on TANs. On the
one hand, radiation may induce TANs to exhibit the antitumor characteristic (N1) by IFN-β. N1 phenotype induces tumor cells cytotoxicity/
apoptosis through ROS and activates CD8+ T cells and M1 macrophage. On the other hand, radiation may induce TANs to exhibit the pro-
tumor characteristic through TGF-β. N2 phenotype promotes genetic instability by ROS, cancer proliferation, and immunosuppression effect
by inhibiting CD8+ T cells and NK cells and enhancing Treg cells. Parts of this figure were drawn with aid of Servier Medical Art (http://
www.servier.com), licensed under a Creative Commons Attribution 3.0 Unported License

strengthening the antibody-dependent cellular cytotoxicity polarization.179 Once more, the contradictory roles of neutrophils
(ADCC), and activating T cells.186–189 N2 phenotype shows the in RT responses may reflect differences in tumor phenotypes
protumorigenic characteristics including promoting the tumor (Fig. 3).
growth, stemness, angiogenesis, invasion, and suppressing Similar to TANs, tumor-associated macrophages (TAMs) can be
immunity.190,191 However, to date our knowledge on the role of classified into two categories, a tumor-cell-killing phenotype of
N1 and N2 in RT responses remains limited and contradictory. macrophage called M1, and the other being a tumor-promoting
Similar to DCs, IR activates neutrophils through toll-like receptors phenotype called M2,201–208 although in reality there is a
(TLR)-dependent mechanisms.192 Neutrophils recognize DAMPs spectrum-like level of activation that cannot be simply classified
which are TLRs agonists after RT and the ligation of TLRs enhances as M1 and M2.209 Brown et al.210 reviewed a series of evidence
the immune responses directed against tumor-associated anti- that had explored the significance of the increasing TAMs in the
gens (TAAs). On the one hand, RT may induce TANs to exhibit the tumor microenvironment after IR in preclinical studies and clinical
pro-tumor characteristic, since neutrophils may promote resis- trials of newly diagnosed glioblastoma patients. Akkari et al.211
tance to RT.193–195 Wisdom and his colleagues found that elevated reported the dynamic changes in glioma macrophage populations
neutrophil levels have a close relation to poor outcome of patients after RT and altered expression of several genes and proteins in
with cervical cancer after chemoradiation. Similarly, others have recurrent human glioblastoma. The mechanisms for radiation-
found that genetic depletion of neutrophils improves RT response induced alteration of TAMs are unclear. Generally, IR elicits a high
in a genetically engineered mouse model of sarcoma. Notably, recruitment of TAMs through chemokine (C-C Motif) ligand 2
evidence has verified that anti-Ly6G antibody-mediated neutro- (CCL2) and colony-stimulating factor 1 (CSF1) pathways, and IR
phil depletion may lead to an improvement of the RT can also cause oxygen deprivation and upregulate hypoxia-
efficacy.196,197 These data suggest that neutrophils play a crucial inducible factor (HIF) which recruits TAMs to infiltrate tumor sites,
role in the tumor microenvironment following RT which may especially hypoxia sites through stromal cell-derived factor 1 (SDF-
promotes the conversion of N1 to N2. Thus, targeting neutrophils 1)/C-X-C chemokine receptor type 4 (CXCR4)-dependent signaling
and their immunosuppressive effector molecules, TGFβ and pathways (Fig. 4).212–214 These recruited macrophages adopted an
nicotinamide phosphoribosyltransferase (NAMPT) might be crucial M2-like pro-tumoral phenotype with enhanced pro-survival and
to reversing immunosuppression.198 Besides the pro-tumor pro-angiogenic activities, often leading to tumor recurrence and
characteristics, TANs also exhibit antitumor characteristics by treatment failure.215 In addition to the slight changes of IR to the
inhibiting the growth of tumor cells, and interacting with other viability of macrophages, it is reported that IR can also modify the
immune cells which is induced by RT. Takeshima et al.199 observed macrophage phenotype. Genard et al.216 systematically summar-
that RT may lead to sterile inflammation by infiltrating CD11b+Gr- ized the dose-dependent effects of IR to polarize macrophages.
1high+ neutrophils into the tumors rapidly and transiently and Generally, numerous studies have implicated that TAMs may
sterile inflammation eventually activated tumor-specific cytotoxic polarize to M2 phenotype in the context of low-dose IR while
T cells which may result in tumor regression. Liu et al.200 proved high-dose IR may promote the polarization to M1 in vitro.217,218
that RT can promote the recruitment of neutrophils and that the Thus, IR promotes not only M1 activation of TAMs but also
radiosensitivity can be improved by neutrophils which inhibit the facilitates M2 activation. Although Meng et al.219 have investi-
process of epithelial-mesenchymal transition via the ROS- gated that a large single dose (20 Gy) or at 2 Gy in 10 fractions
mediated PI3K/Akt/Snail signaling pathway. As we discussed (10 × 2 Gy) promotes the conversion of immunosuppressive TAMs,
above, TGF-β polarizes neutrophils to a protumorigenic pheno- but some studies revealed a contrary effect of short-course or low-
type (N2) and inhibits N1 phenotype. However, RT has dose RT on TAMs. A study shows low-dose radiation programs
also been demonstrated as an inhibitor of the TGF-β pathway, macrophage differentiation to an inducible nitric oxide synthase
thereby stimulating the antitumor-N1 neutrophil phenotype (iNOS)+/M1 phenotype and these iNOS+/M1 macrophages can

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M0 φ
CD4+ T cell
IκBα IκBα Radiation
NF-қB
p65 p50 p50
p50

TNF-α IFN-γ CCL2 CSF-1

ROS
TNFR
IFNγR CSF-1R
CCR2

M2 φ
M1 φ
CXCR4
CXCL12
SIRPα LILRB1 Radiation
IL-10
MHC
CD47

IL-12
TGF-β

IL-1β CCL22
Tumor cell

Treg cell

Dendritic cell

Fig. 4 Immunomodulatory effect of radiation on TAMs. Under the effect of p50–p50 NFκB homodimer induced by radiation, M2 macrophages
acquired their phenotype. Meanwhile, increased ROS caused by radiation also promotes the polarization to M2 macrophage. The activation of
p50–p50 dimer promotes the conversion towards M2 phenotype, leading to the secretion of IL-10 and TGF-β which inhibit DCs. And CCL22 secreted
by M2 macrophage also recruits Treg cells to exert immunosuppressive function. Radiation elicits a high recruitment of TAMs through CCL2/CCR2
and CSF1/CSF1R pathways. And it can also recruit TAMs to infiltrate tumor sites, especially hypoxia sites through SDF-1/CXCR4-dependent signaling
pathways. Moreover, M1 macrophage ban be activated by CD4+ cells through TNF and IFN-γ and then kill tumor cells via phagocytosis which plays
a crucial role in abscopal effect. Parts of this figure were drawn with aid of Servier Medical Art (http://www.servier.com), licensed under a Creative
Commons Attribution 3.0 Unported License

orchestrate effective T cell recruitment and kill tumor cells through reducing M1-surface markers expression in the context of RT. And
iNOS.220 Moreover, total body low-dose radiation also promotes tumor-vasculature development via endothelial-to-mesenchymal
the polarization of TAMs towards an iNOS+/M1 phenotype.221 transition after RT may play a crucial role in macrophage
Another study shows short-course neoadjuvant RT reprograms polarization.228
macrophages towards an M1 phenotype in rectal cancer MDSCs are the “queen bee” of the tumor microenvironment
patients.222 These conflicting results may reflect the complexity that protect the tumors and contribute to tumor progres-
and plasticity of TAMs and more investigations into how RT affects sion.229–235 In normal conditions, bone marrow-derived myeloid
macrophage polarization are needed. The mechanisms about the cells can differentiate into cells of the innate immune system
effects of radiation dose on the polarization of TAMs remain including macrophages, dendritic cells, and granulocytes. But in a
unclear but there are several possible interpretations including pathological condition, myeloid cell precursors may be prolifer-
ROS, NF-κB signaling, and MAPK phosphorylation (Fig. 5). One of ated and differentiated into MDSCs that migrate to the tumor
the most critical mechanisms is the NF-κB balance. It is reported microenvironment and develop into TAMs which are induced by
that p50–p50 NF-κB homodimer may promote the polarization inflammatory cytokines, for instance, soluble tumor necrosis factor
towards M2 macrophages while p50–p65 NF-κB heterodimer (sTNF). MDSCs have a close relation with TAMs and TANs in the
favors the polarization towards M1 macrophages.223 Crittenden tumor microenvironment. Polymorphonuclear (PMN)-MDSCs seem
et al. demonstrated that M2 phenotype emerged in the context of to develop into N2 TANs while monocytic (M)-MDSCs seem to
high-dose irradiation (60 Gy) which activated p50–p50 dimer and proliferate and develop into TAMs under chemotaxis of several
promoted the secretion of IL-10.224 Of note, ROS is known as a factors, such as CCL2, CXCLs, and S100A8/A9, etc.236 And signals
secondary messenger which leads to secretion of a series of underlying the development, differentiation, and recruitment of
proinflammatory cytokines. However, it has been proved for the MDSCs are complex and involve granulocyte-macrophage colony-
M2 activation.225 Tabraue et al.226 demonstrated the role of liver X stimulating factor (GM-CSF), granulocyte (G)-CSF, macrophage
receptor (LXR) nuclear receptors on radiation-induced polarization (M)-CSF and vascular endothelial growth factor (VEGF); cytokines
of macrophages in LXR double knock-out mice models. Moreover, such as IL-4, IL-6, IL-10, IL-1β, interferon IFN-γ, TGF-β, and
the regulatory function of CAFs on macrophages after RT has been prostaglandin E2 (PGE2); chemokines such as CCL2, CXCL5, and
proved.227 The interaction between CAFs and M1-macrophages CXCL12. MDSCs can exert the immunosuppressive effect through
showed that CAFs can inhibit the function of M1-macrophages by releasing TGFβ, and IL-10, and inducing Treg cells.35,237–243 In

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Radiation

CXCL12

Gβ Gγ Gβ Gγ

PI3K
JAK
RAS

Akt

P P
RAF STAT3 STAT3

MEK ERK

NF-κB
Chemotaxis
Migration
Proliferation
Cytokines expression

M2 φ

Fig. 5 Signaling pathways of radiation on TAMs. Radiation increases the level of CXCL12 which then binds to CXCR4, a kind of G-protein-
coupled receptor (GPCR). Similarly, CCL2 and its receptor, CCR2 are also activated by radiation. Next, the receptor undergoes a second
conformational change that activates the intracellular trimeric G protein by the dissociation of Gα subunit from the Gβ/Gγ dimer. Then, the
phosphatidylinositide 3-kinases (PI3Ks) can be activated by both Gβ/γ and Gα subunits. PI3Ks regulate gene transcription, migration, and
adhesion of TAMs by the phosphorylation of AKT and of several focal adhesion components. In addition, Gα subunit also activates the Ras and
Rac/Rho pathways, leading to the phosphorylation of ERK. Activated ERK can phosphorylate and regulate other cellular proteins, as well as
translocate into the nucleus and phosphorylate and regulate transcription factors, leading to migration, proliferation, and cytokines
expression of TAMs. Gβ/Gγ dimer also activates JAK/STAT signaling pathway to promote changes in cell morphology leading to chemotactic
responses. Of note, CSF-1/CSF-1R activates all the three signaling pathways we discussed above. Parts of this figure were drawn with aid of
Servier Medical Art (http://www.servier.com), licensed under a Creative Commons Attribution 3.0 Unported License

almost all patients with tumors, MDSCs seem to be the major may enhance the transcription of CSF1 gene which lead to the
factor to protect the tumors and contribute to tumor progression. significant release of CSF1 in prostate post-RT. And Liang et al.
Zeng et al. found that caspase-1 from human MDSCs may lead to found a mechanism by which extrinsic resistance develops after
T cell-independent tumor proliferation through inactivating T cells local ablative radiation that relies on the immunosuppressive
and NK cells.244 Moreover, MDSCs also have the function to action of STING of MDSCs.249 The STING/type I interferon pathway
facilitate the expansion and activation of Treg cells. A study enhances suppressive inflammation in tumors by recruiting
showed that monocytic MDSCs which is isolated from transplant myeloid cells in part via the CCR2 pathway (Fig. 6). Intriguingly,
patients can suppress the proliferation of CD4+ T cells and it is reported that activation of STING pathway can reprogram
promote the expansion of Treg cells.245,246 It is reported that a MDSCs into immunostimulatory cells.250 However, majority of
significant increase of MDSCs in some organs or tissues of the studies showed that STING signaling pathway in the tumor
human body such as spleen, lung, lymph nodes, and peripheral microenvironment can inhibit the recruitment, differentiation, and
blood was observed when primary tumor sites received RT.247 function of MDSCs.100 Cheng et al. observed that the activation of
However, other groups reported that there was a dramatical STING signaling pathway by cGAMP may enhance the secretion of
decrease of MDSCs 7–14 days after the murine colon tumors IFN-γ but decrease the number of MDSCs in vivo.251 Therefore,
received a single high dose of RT.196,248 These contrary data might these studies suggest the suppression function of IR on MDSCs. In
be related to the various tumor models and difference of radiation summary, cGAS/STING signaling is vital in the context of RT and its
doses, fractionation, irradiated sites as well as the timings of the complex effect on MDSCs need to be exploited. In pancreatic
analyses. CSF1/CSF1R signaling pathway plays a key role in the cancer, the potential mechanism of intensive immunosuppression
infiltration of MDSCs into tumors in the context of RT. The of MDSCs after RT is by increasing lactate secretion induced by
blockade of this signaling pathway can improve tumor recurrence Warburg effect.252 And the essential factor to mediate lactate-
post local RT. Xu and colleagues proved the importance of CSF1/ regulated activation of MDSCs is HIF-1α which activates HIF-1α/
CSF1R signaling in the recruitment of MDSCs which can limit the STAT3 pathway. Another study showed that silencing information
efficacy of RT.247 The further studies revealed that the accumula- regulator 2 related enzyme 1 (SIRT1) can deregulate function and
tion and translocation of the DNA damage-induced kinase ABL1 differentiation of MDSCs through orchestrating HIF-1α-dependent

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Radiation
DNA damage
CSF-1
CCL2

CSF-1R CCR2

Gα Gβ Gγ

Gβ Gγ

cGAS

GTP PI3K
JAK
ATP STING

cGAMP
P P Akt
STAT3 STAT3

TBK1 IKK

IRF3

IFNβ P
IFN-β1 NF-κB

Chemotaxis
Recruitment
Migration
myeloid cells
Proliferation

MDSC

Fig. 6 Immunomodulatory effect of radiation on MDSCs. DNA damage resulted from radiation may activate two signaling pathways in
MDSCs: cGAS-STING and JAK/STAT signaling pathways. Similar to signaling pathways in TAMs, JAK/STAT signaling and PI3K/AKT signaling
pathway are activated in MDSCs caused by CSF-1/CSF-1R and CCL2/CCR2. Upon DNA damage, all of these damaged DNA including
cytoplasmic DNA and mtDNA induced by IR can be recognized by cGAS, which then oligomerizes with DNA in the form of a 2:2
complex.114–116 After binding to DNA, cGAS then exerts a catalytic role to promote the synthesis of the second messenger 2′3′-cyclic
GMP–AMP (cGAMP). Binding of 2′3′-cGAMP stimulates STING and promotes the translocation to the Golgi which acivates TANK-binding kinase
1 (TBK1). TBK1 phosphorylates STING and promote the interferon regulatory factor 3 (IRF3) to translocate to the nucleus which triggers the
expression of IFN-β gene. Parts of this figure were drawn with aid of Servier Medical Art (http://www.servier.com), licensed under a Creative
Commons Attribution 3.0 Unported License

glycolysis.253 Of note, IR generally induces the recruitment and addition, IR can strengthen tumor cross-presentation of DCs which
infiltration of MDSCs to yield immunosuppressive effect on the may promote the activation and proliferation of T cells.267,268 The
immune system (as discussed above). release of DAMPs and TAAs promotes DCs to migrate towards
Dendritic cells are the most potent antigen-presenting cells for lymph nodes and further results in the emergence of systemic
their specialized dendritic morphology, as the essential compo- antitumor immune responses. On the other hand, inflammatory
nent to link innate and adaptive immunity.254–260 The most cytokines modified by RT may augment the function of DCs and
common contributors to activate DCs are pathogen-associated potentially exert an immunostimulatory response to radiation.34
molecular pattern molecules (“danger signals”) through the Moreover, new mechanisms have been proved by researchers
respective receptors. But in the case of RT, DCs are mainly (Fig. 7). Yu and colleagues found that in vitro when DCs are
activated by DAMPs including HMGB1 and calreticulin. Gupta and exposed to 0.2 Gy radiation, there is an increase of migration
colleagues observed that after tumor RT, there is an upregulation mediated by CCR7 and IL-12 production induced by the ATM/NF-
of two co-stimulatory molecules, CD70 and CD86 on DCs.85 On the κB pathway.269 For high dose of radiation, Zhou found that it may
one hand, the initial factors are increased by IR and the promote DCs homing and T cells priming through facilitating the
mechanisms that IR can upregulate the expression of class I ROS-induced cytoskeletal reorganization.86 In addition to the
MHC have been well established.84,261–263 And immunogenic cell positive immune effect of radiation to DCs, DCs may also play a
death caused by radiation may release large amounts of antigens crucial role in radiation-induced immunosuppression. Liu et al.
and DAMPs including HMGB1, ATP, calreticulin, heat shock observed a decrease of CD8+ DC both in patients and mice after IR
proteins, etc.264 These molecules can further activate DCs through which causes shift from Th1 to Th2 immunity and this process
binding to the receptors which comprise TLRs, retinoic acid- might be mediated by Fms-like tyrosine kinase 3 ligand (FLT3
inducible gene 1 (RIG1)-like receptors, and nucleotide-binding and ligand), a CD8+DC-inducing cytokine.270
oligomerization domain (NOD)-like receptors. For instance, calre- Generally speaking, B cells and T cells are sensitive to IR, so IR
ticulin increased by RT acts as a pro-phagocytosis eat-me signal in damage to these lymphocytes which may lead to adverse events
opposition to CD47.265 Release of HMGB1 from tumor cells, via of RT. Within the lymphocyte population, B cells are the most
TLR4 activation, promotes antigen presentation by DCs.266 In sensitive to IR. Compared with B cells, T cell subsets show different

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Radiation

CCL19/CCL21
IL-10

CCR7

DAMPs CD28
B7
Tumor cell TCR
TLR4 CD4+ T cell
MHC

Dendritic cell

TAAs

Immunmmunogenic CD155 IL-12


cell death
CD112
TIGIT NK cell-induced
IFN-γ CD112R DC recruitment

NK cell

CCL5/CXCL1

Fig. 7 Immunomodulatory effect of radiation on DCs. Radiation on DCs significantly increases the expression of chemokines CCL19 and
CCL21 which then bind to CCR7 and mediate migration of DCs. In addition, immunogenic cell death caused by radiation may release large
amounts of antigens and DAMPs including HMGB1, ATP, calreticulin, heat shock proteins, and other cellular factors which bind specific
pattern-recognition receptors on the dendritic cell, including Toll-like receptors, RIG1-like receptors, and NOD-like receptors. And radiation
also results in the release of TAAs which promotes DCs activation, migration, and proliferation of T cells. Parts of this figure were drawn with
aid of Servier Medical Art (http://www.servier.com), licensed under a Creative Commons Attribution 3.0 Unported License

sensitivity to IR. Thus, the sensitivity to IR for T cells has been patients who receive SBRT combined with IO, which better
explored in a large body of studies but there are a large of facilitates the abscopal effect and increases the occurrence rate in
conflicting results.271 Arina et al.272 found that RT can reprogram a patients with metastatic disease in iRT.280 Moreover, RT induces
huge proportion of T cells which show increased motility and the production and release of cytokines. The most important
increased secretion of IFN-γ. In addition, T cells within the tumor mechanism is to activate the cGAS-STING and NF-κB signaling
may be more resistant to IR compared with naive and circulating leading to an increase of cytokines.128,281,282 Some investigations
T cells. Generally, CD4+ T cells are considered more radioresistant show that IR promotes the secretion of CXCL16 in tumor cells
than CD8+ T cells, with Treg cells even more resistant to which can be combined with Th1 cells and CXCR6 on activated
RT.271,273,274 Except the direct damage to T cells of IR, it also CD8+ T cells to increase the infiltration of local immune cells.283,284
activates T cell through increasing major histocompatibility class I On the other hand, IR may promote the migration of T cells
(MHC-I), DAMPs, and TAAs and enhances T-cell infiltration. towards irradiated tumor sites resulting in a positive immunolo-
Moreover, these molecules as well as increased proinflammatory gical outcome. In this process, the intercellular adhesion of
cytokines augment the prime of T cells to exert an antitumor molecule-1 (ICAM-1) may promote leukocytes migration to the
response with the migration of DCs towards adjacent lymph endothelial cells, and reprogram an inflammatory tumor micro-
nodes95. It has been proven that IL-1β, TGF-β, fibroblast growth environment. This is enhanced by IR and this leads to more T-cell
factor (FGF), and TNF, as well as NACHT, LRR, and PYD domains- infiltration into tumor tissues.285–287 It is reported that the density
containing protein 3 (NALP3)-inflammasome activation and of CD3+ and CD8+ lymphocytes in tumor site is associated with
signaling are key components to mediate the response to disease-free and overall survival in patients treated with chemor-
IR.275–278 Recently, Lin and colleagues found that radiation- adiotherapy for rectal cancer.87 More recently, Dovedi et al.288
induced small extracellular vesicles may play a role as efficient found that low-dose RT combined with PD-1 blockade can
carriers containing TAAs and DAMPs in promoting tumor antigen promote the migration of T-cells to primary treated sites and
release and triggering antitumor immunity.83 Moreover, a study augment tumor regression. Thus, RT enhances the secretion of
has proved that radiation can promote tumor cells to release more cytokines which may promote T-cell infiltration and augmenting
MHC-I molecules in murine tumor models, which facilitates cross- T-cell priming.
presentation and T-cell priming.279 In fact, the high release of TAAs NK cells are cytotoxic lymphocytes in the innate immune
and MHC-I has been already reported in human carcinoma cell system to kill cancerous cells. The two most well-characterized
lines when researchers use sublethal irradiation on human tumor subsets of NK cells are the CD56brightCD16− and CD56dimCD16+
cells.89 This result just accords with the role of low-dose RT in populations.289–292 In the subsets of NK cells, the CD56dim NK cell

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TGF-β
Radiation
Granzyme B
Perforin
TGF-βR

CD112 CD112R
NKG2D NKG2DL
NK cell Tumor cell
Dendritic cell

CD155 TIGIT
KIR PD-1 FASL FAS

PD-L1
IL-12

MHC
TNF-α

Tumor cell
IFN-γ

Fig. 8 Immunomodulatory effect of radiation on NK cells. On the one hand, radiation inhibits NK cells through increasing TGF-β. Moreover,
the exposure of MHC-I molecules on the surface of tumor cells caused by radiation also inactivates NK cells through KIR. In addition, radiation
induces the upregulation of activating receptors NKG2D and NKG2D ligands (NKG2DLs) to enhance the function of NK cells via granzyme B
and perforin, FAS and FAS ligand and ADCC. Parts of this figure were drawn with aid of Servier Medical Art (http://www.servier.com), licensed
under a Creative Commons Attribution 3.0 Unported License

population is the major contributor to kill infected and malignant signaling pathways especially cGAS-STING signaling, secretion of
cells. The main mechanisms are granzyme B and perforin, FAS and chemokines and cytokines (Table 1), formation of hypoxia
FAS ligand and ADCC. For the direct effect on tumor cells and condition, and activation of the classical and alternative comple-
cellular cross-talk, NK cell-based immunotherapies have been ment system play a crucial role in reprogramming tumor
explored well.293–300 The activation of NK cells depends on the microenvironment induced by IR.308–312 The process of repro-
balance between activation and suppression signals.301 The gramming the tumor microenvironment is suffused with complex
activating receptors of NK cells such as NKG2D can promote the and profound changes, which is not only limited to a change in
secretion of cytokines and enhance the cytotoxicity and an quantity and form, but also full of various interactions and
increasing expression of NKG2D ligands was observed by modulations. We have discussed this process in the above article.
investigators in several human cancer cell lines after IR.301,302 Besides, many reviews have given an unambiguous and detailed
The increasing expression of NKG2D ligands (NKG2DLs) induced interpret of reprogrammed tumor microenvironment and have
by radiation may further activate the NK pathway and NKG2D- shown potential benefit of reprogrammed tumor microenviron-
based CAR T cells combined with RT can exert synergistic efficacy ment induced by RT to combine with IO.123,146,313–318
in glioblastoma (Fig. 8).303 Meanwhile, inhibitory signaling path-
ways including PD-1, NKG2A and killer immunoglobulin-like iRT enhcances abscopal effect
receptors (KIRs) play a key role in function of NK cells so ICIs Traditionally, abscopal effect refers to an interesting phenomenon
can attenuate suppression of NK cell through binding to these that local radiation may exert a systemic antitumor immune
inhibitory signaling pathways.304 Furthermore, other molecules response and lead to the regression of non-irradiated distant
such as MHC-I induced by RT may also inhibit the function of NK tumors. Unfortunately, it is so rare for the occurrence rate of
cells. In addition to increasing NKG2D ligands, IR enhances NK cell abscopal effect that it has no broad application value seemingly.
homing and cytotoxicity in canine models of sarcoma.305 Whereas, with the advent of immunotherapy, it seems that using
Researchers observed significantly increased NK cells homing to radiotherapy combined with immunotherapy to enhance absco-
tumors in vivo and increased activation of circulating NK cells after pal effect is available. A large number of studies about abscopal
RT which yield tumor regression and abscopal responses. This has effect induced by iRT has come out.21,48,319–326 Nowadays, the
also been shown in a human triple-negative breast cancer criterion for determining whether abscopal effect has occurred is
xenograft model.306 whether the tumor regression at distant non-irradiated sites can
be observed. In view of the combination of RT with IO, the
IR reprograms the tumor microenvironment immune state of the whole human body and have already
IR not only reprograms the tumor microenvironment from “cold” changed before tumor regression at distant non-irradiated sites
to “hot”, but also exerts immunoinhibitory effects in the tumor (macroscopic abscopal effect). This force is not powerful enough
microenvironment.307 In the above, the authors have discussed to decrease tumor volume although molecules and gene
and summarized the effects of IR on cancer cells, stromal cells, and expression of tumor cells or normal tissue cells nearby really do
immune cells which lay a basis for reprogramming the tumor change. Thus, we propose a broad concept of abscopal effect from
microenvironment. Along with this process, activation of intrinsic three aspects: macro level, molecular and genetic levels. From this

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Table 1. Immunomodulatory factors and their functions in TME Table 1. continued
induced by radiation
Factors Receptors Function
Factors Receptors Function
CSF1 CSF1-R TAM mobilization and
Chemokines proliferation
CXCL2 CXCR2 TAM infiltration and M1 → M2 TAM polarization
differentiation MDSCs recruitment
CXCL12 CXCR4, CXCR7 TAM infiltration and G-CSF G-CSF receptor Neutrophiles mobilization
differentiation
CCL2 CCR2 TAM infiltration and
differentiation
point of view, the occurrence of abscopal effect is 100%, since
Monocyte recruitment
most non-macro-level abscopal effects occur microscopically and
MDSCs recruitment in a subclinical manner (Fig. 9).
CCL3, CCL5 CCR1, CCR4, CCR5/ TAM infiltration and Heretofore, we have elaborated on immunomodulatory effect
CCR5 differentiation of IR acting on the primary irradiated sites. It is widely accepted
CCL7 CCR1, CCR2, DCs migration that in situ vaccination induced by IR is the key mechanism
CCR3, CCR5 transforming local effects into abscopal responses.13,99 Generally,
CCL21 CCR7 DCs migration the immunogenic death of tumor cells caused by IR releases
CCL22 CCR4 Treg recruitment plenty of neoantigens and DAMPs leading to an increasing of
antigen presentation through DCs.323 With the undergoing
CCL28 CCR3, CCR10 Treg recruitment
maturation of DCs, activated cytotoxic T lymphocytes (CTLs) and
Cytokines NKs then recognize and attack both primary and abscopal tumor
TGF-β TGFβRI, TGFβRII M1 → M2 TAM polarization cells.319,327 Moreover, damaged nuclear DNA in the cytosol can be
N1 → N2 TAN polarization sensed by cGAS-STING pathway leading to IFN production along
Naive CD4+ T cell into Treg this pathway which has a crucial role in T-cell cross-priming to
initiate an antitumor response.328 Similar to the above mentioned,
NK suppression
the activated CTLs and NKs move to tumor sites via blood
TNFα TNFR T cell proliferation circulation and control tumor growth at both irradiated and no-
T cell action irradiated sites.320 The abscopal effect has a close relation with the
M1 macrophage polarization discussed rationale of systemic immune response induced by IR
IL-1 IL-1R MDSCs induction above which includes the infiltrating immune cells and repro-
grammed tumor microenvironment. Local IR triggers systemic
Macrophage recruitment
antitumor immune response through these rationales, so next we
IL-2 IL-2R T cell proliferation and effector will focus on the alteration of non-irradiated sites in molecule and
function gene level.
IL-4 IL-4Rα Macrophage recruitment The molecular abscopal effect mainly refers to the alteration of
IL-10 IL-10R Proinflammatory cytokines cytokines and chemokines induced by IR at distant non-irradiated
inhibition sites. There are two conditions: high-dose RT or low-dose RT. High-
Antigen presentation inhibition dose RT causes immunogenic cell death and leads to a systemic
Treg action antitumor response. We have already certified the changes of
cytokines and chemokines including TNFα, IL-1, IL-6, MCP-1, IL-8 at
Macrophage inhibition
irradiated sites34 and that IR forms inflammatory tumor micro-
IL-12 IL-12R DCs migration environment remodeling by tumur cells.95 However, little is known
T cell priming about the expression levels of these immunomodulatory factors in
Upregulates MHC I tissues distant from the irradiated zone. Siva et al. proved that
Promotes CD8+ T cell-mediated unirradiated out-of-field tissues exhibited delayed “abscopal” DNA
cytotoxicity damage response.329 This may induce a series of response to the
IL-18 IL-18R NK enhancement
DNA damage and change distant tumor microenvironment at
abscopal sites. Similarly, Ventura et al.330 observed oxidatively
IL-33 IL-33R Activation of immune cells induced clustered DNA lesions and apoptotic cell death were
IL-37 IL-18R NK suppression elevated in a wide variety of unirradiated tissues and these events
IFN-β Type I interferon N2 → N1 TAN polarization were accompanied by changes in plasma concentrations of
receptor cytokines including macrophage-derived cytokine, eotaxin, IL10,
IFN-γ Type II interferon Proinflammatory cytokine VEGF, TGFβ1, and TGFβ2. These alterations not only exhibit RT
receptor release toxicities as a cytotoxic agent but also shows potential to mediate
Growth factors abscopal effect by reshaping the tumor environment in many
VEGFA VEGFR-1 Treg proliferation
ways.331,332 One of the possible mechanisms of is the immuno-
modulatory effect by tumor-derived exosomes. Tumor-derived
MDSC accumulation exosomes contain genetic materials and also carry immunomo-
Myeloid cell differentiation dulatory molecules. These tumor-derived exosomes may carry
T cell inhibition these signals to distant sites and exert an interaction with other
CTLA-4, PD-1 expression by immune cells, leading to an abscopal effect.333
CD8+ T cell Moreover, expression levels of cytokines and chemokines in
PGE2 Prostanoid E (EP) Proinflammatory cytokine plasma make a difference after treatment with definitive intensity-
receptor release modulated RT.334 Abscopal gene expression in the out-of-field skin
after synchrotron RT was monitored in which gene expression

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Zhang et al.
13

RT

Macro abscopal effect Tumor cells shrink

Molecular abscopal effect Changes of cytokines

Genetic abscopal effect Genetic changes

Fig. 9 Macro, molecular, and genetic abscopal effect. Abscopal effect in the traditional sense refers to the tumor regression at distant non-
irradiated sites which can be observed in clinic. However, when the primary tumor is irradiated, cytokines and immune cells at distant non-
irradiated sites also changes due to the systemic immune response caused by IR. In addition, there are alterations of gene expression at
distant non-irradiated sites. Parts of this figure were drawn with aid of Servier Medical Art (http://www.servier.com), licensed under a Creative
Commons Attribution 3.0 Unported License

levels of TNF and TGFβ1 increased indicating a systemic about potential mechanisms of low-dose RT. In summary, both
inflammatory response and there was a decrease in Ccl2, Mdm2, high-dose and low-dose radiation can mediate molecular altera-
and Trp53 gene expression.335 Similarly, Aravindan et al. found a tion at abscopal tumor site and exert systemic antitumor response.
robust increase in p65 and cMyc expression in distant heart after However, mechanisms about how molecular changes of abscopal
the radiation of lower abdomen.336 These results indicate that the sites remains largely unclear and what happened at abscopal sites
status of abscopal sites has possibly started to change before the in the condition of high-dose radiation is unknown. We expect
observed tumor regression. Hence, the alteration of non-irradiated there is much direct and powerful evidence to explore the
sites in molecule and gene level may reflect the sensitivity of abscopal effect at the molecular level and we hold the view that
patients to RT and become predictors of abscopal effect and this will increase occurrence rate of abscopal effect and provide
response to the treatment. For instance, recent studies indicated new approach to overcome the resistance of immunotherapy.
that tumor-derived exosomes may become a biomarker in
gastrointestinal cancer.337 The contents of exosomes also had a Immunomodulatory effect of low-dose radiation and radscopal
strong association with survival.338 However, the clinical meanings effect
of molecular and genetic changes of abscopal sites induced by RT With a view to immune effect of conventional dose RT, low-dose
remains unclear for the lack of clinical data. We expect more RT may play a distinct role in antitumor immune response
investigations to explore the molecular and genetic changes of combined with IO. Many studies have affirmed the immunomo-
abscopal sites to benefit more patients. dulatory effect of low-dose RT,220,339–341 sometimes termed the
Fortunately, the authors observed the effect of low-dose RT on “radscopal effect”. Radscopal effect refers to the systemic
non-irradiated tumor stroma and discussed possible mechanisms antitumor effects which are strengthened by low-dose RT on the
and values.280 Combined with SBRT and immunotherapy, low- basis of stereotactic RT. This radiation strategy with high-dose
dose RT (doses below the threshold thought to physically damage stereotactic RT and low-dose RT was proposed by James Welsh
DNA or kill cancer cells directly) stimulated abscopal tumor stroma and this strategy was named as “RadScopal” technique.342 Unlike
throughout the body and facilitated abscopal tumor regression. tumoricidal-dose RT, low-dose RT can reprogram the tumor
This provides circumstantial evidence for the existence of microenvironment and reactivate the immune microenvironment,
molecular abscopal effect and thereout it triggers new thoughts thus reversing the resistance of patients to IO. Herrera et al. also
that based on pre-existing abscopal effect in molecule level, low- reviewed that innate and adaptive immunity can be mobilized
dose radiation and immunotherapy offer a force more powerful when all lesions are treated with low-dose RT combined with
method to activate abscopal antitumor immune response causing IO.343 Generally, conventional RT may produce potent immuno-
macroscopic tumor regression. Moreover, Yin et al.321 discussed suppressive factors but low-dose radiation may be a strong

Signal Transduction and Targeted Therapy (2022)7:258


Radiotherapy combined with immunotherapy: the dawn of cancer treatment
Zhang et al.
14
weapon to address these limitations. We have discussed the city. However, the radscopal effect of low-dose RT gathers soldiers
immunomodulatory effects of IR in the above part. Of note, low- together and opens the city gates to bring the soldiers into
dose RT shows a similar but distinguishing effect on human the city.
immune system. The underlying mechanisms of distinguishing Radscopal effect has been proven in preclinical and clinical
effect of low-dose RT may be initiated from DNA damage. Low- studies. In mice with lung adenocarcinoma tumors, Barsoumian
dose RT may lead to various lesions of DNA. DNA damage after and his colleagues observed low-dose RT improved the outcomes
such low doses is not sufficiently severe to induce cells death but of ICIs and identified potential mechanisms by which low-dose
can initiate danger signaling.344 As we discussed in the part of radiation promotes M1 macrophage polarization, enhances NK
“Effects of IR on cancer cells”, defects of DNA repair may activate cells infiltration and reduces TGF-β levels.342 In a phase 2
cGAS-STING pathway which induces inflammatory response. In prospective trial, it is reported that the combination of high and
addition, DAMPs released from these damaged cells may further low-dose RT limits tumor growth at primary and secondary
activate immune responses. sites.351 Furthermore, in a phase II trial of SBRT and ipilimumab,
However, there are few studies to investigate the effect of low- researchers found that lesions receiving low-dose radiation were
dose RT on immune cells. A large body of evidence has implicated more likely to respond to the combined therapy.352 In addition, a
that low-dose RT may result in the damage of CD4+ T cells and recent clinical trial reported that LDRT plus high-dose RT safely
promote the conversion to a Th2 phenotype.345 Just like we improved lesion-specific response in patients with immune-
discussed above, low-dose RT leads to generation of an iNOS+/M1 resistant solid tumors by promoting infiltration of effector immune
phenotype and these iNOS+/M1 macrophages can orchestrate cells into the tumor microenvironment which is consistent with
effective T cell recruitment and kill tumor cells through iNOS.220 Yu previous data.353 In a phase I clinical trial administering low-dose
and colleagues found that in vitro when DCs are exposed to 0.2 Gy RT, low-dose cyclophosphamide and immune checkpoint block-
radiation, there is an increase of migration mediated by CCR7 and ade to patients with immune scarce tumors, researchers observed
IL-12 production induced by the ATM/NF-κB pathway.269 Persa that the combinatorial treatment triggered T-cell infiltration,
et al.346 showed that low and high-dose irradiation-induced predominantly of CD4+ cells with Th1 signatures.348 Similarly,
qualitatively different functional changes in murine splenic DCs Yin et al.321 proved that hypo-fractionated RT of the primary tumor
in vivo. They found that low-dose RT stimulated antigen uptake plus low-dose RT of the abscopal tumor as well as administering
and lowered antigen presentation while high doses did not PD1 blockades enhances the abscopal response and the triple
influence. A recent study showed that targeted radionuclide therapy group achieved optimal abscopal tumor growth control.
therapy can render immunologically cold syngeneic B78 mela- Thus, we believe that low-dose RT will further improve regimen
noma tumors sensitive to ICIs.347 Researchers observed a of iRT and benefit more people from iRT as a trigger of systemic
significant increase in tumor-infiltrating myeloid (CD11b+), and antitumor immune response.
NK cells and an increase in the ratio of effector CD8+ to
suppressor Treg cells after low-dose RT. Moreover, Herrera et al.348
found that low-dose RT reprogrammed the tumor microenviron- BIOMARKERS OF TREATMENT RESPONSE
ment of tumors with scarce immune infiltration and elicited Although iRT has shown advantages in many kinds of tumors, just
predominantly CD4+ cells with features of exhausted effector part of patients can get benefit from the combination. Thus, it is
cytotoxic cells, with a subset expressing NKG2D and exhibiting crucial and urgent to investigate efficient and precise biomarkers
proliferative capacity, as well as a unique subset of activated which predict and evaluate response to treatment. Besides,
dendritic cells expressing the NKG2D ligand Rae1. Interestingly, validated biomarkers may promote the selection of patients and
low-dose splenic radiation may reduce CTLA-4 expression on the prediction of outcomes. So far, some biomarkers for IO alone have
Treg cell surface to inhibit liver tumor development and been widely tested including tumor mutation burden, DNA repair
researchers observed a significant decrease of the percentage of deficiencies, PD-L1 expression, and gut microbiome.354–356 For
CD4+CD25+Treg/CD4+ cells in the blood and the expressions of instance, several studies reported that the higher tumor mutation
Foxp3, IL-10, TGF-β, and CTLA-4 in spleen and liver tumors in this burden is associated with better outcomes of ICIs.357,358 However,
diethylnitrosamine-induced rat liver tumor model.349 Low-dose RT due to the later response of IO and several limitations, there is a
also sensitizes tumor cells to immune rejection by locally activated lack of evidence to support them as predictors of iRT. It is
CAR T cells. In a model of pancreatic adenocarcinoma hetero- necessary to find novel biomarkers or further explore the efficacy
geneously expressing sialyl Lewis-A (sLeA), DeSelm and colleagues of former biomarkers.
found that not only sLeA+ but also sLeA− tumor cells exposed to In view of the rational of iRT, DAMPs reflecting the key events of
low-dose RT become susceptible to CAR therapy, reducing RT-induced immunogenic cell death might be potential biomar-
antigen-negative tumor relapse.350 kers to predict the efficacy of iRT. A large body of evidence has
These results show potential prospect of low-dose radiation to shown that RT leads to immunogenic cell death by upregulating
promote antitumor response which could be called radscopal calreticulin, as one of DAMPs.359,360 In addition, calreticulin
effect. In consideration of low occurrence of abscopal effect, there induced by RT may play a key role in uptake tumor cells and
are several possible mechanisms: the insufficient immune augmenting immune cells.48 Thus, the level of calreticulin after RT
components to exert effective antitumor response and the may indicate the sensitivity of tumor cells to T cells and it has the
inhibitory immune barriers caused by tumors at abscopal site. potential to act as a biomarker of iRT.
Low-dose RT can overcome these limitations exactly. On the one Absolute lymphocyte count (ALC) may serve as another
hand, low-dose RT can increase secretion of chemokines involved biomarker to predict abscopal effect. The circulating lymphocyte
in the attraction of T cells (e.g., CXCL9, CXCL10, CXCL11, CCL4, and population plays as the most crucial component in the anticancer
CCL5).321 Thus, the CD8+ T cells primed by primary RT could be immune response, governing immune response to RT as well as
attracted to the remote secondary tumor and low-dose RT response to ICIs. Early clinical data suggest that higher lymphocyte
amplifies the potential of abscopal effect. Besides, low-dose RT counts are associated with a higher response rate and more
can reprogram the tumor microenvironment by increasing T cells, durable treatment response in patients treated with ICIs.361 In an
NK cells, polarizing M1 macrophage, upregulating immunostimu- analysis of 165 patients from three prospective trials evaluating
latory factors such as NKG2D and its ligand and downregulating the combination of RT and IO, investigators found that pre-RT ALC
inhibitory factor.342,348 Figuratively speaking, abscopal effect of RT was significantly associated with progression-free survival (PFS) in
increases the number of soldiers (immune cells and effective both the traditional RT and SBRT groups.362 In a study with 11
molecules) but sometimes their force is insufficient to enter the patients administering ipilimumab after RT, Grimaldi et al. found

Signal Transduction and Targeted Therapy (2022)7:258


Radiotherapy combined with immunotherapy: the dawn of cancer treatment
Zhang et al.
15
that the occurrence of abscopal effect is associated with the patients. A preclinical study reported that RT in 7.5 Gy/fraction
median ALC before RT.363 This result also corroborates another may achieve a better outcome along with maintaining low Treg
study using data from 3 institutional phase 1/2 trials to examine numbers in mice bearing B16-ovalbumin murine melanoma, but
the predictive capacity of recorded parameters in patients not the 5 Gy.379 Another similar study showed the use of 15 Gy
undergoing combined RT and IO. They found that post-RT single-dose irradiation resulted in a greater number of host
absolute lymphocyte count, when analyzed as a continuous immune cells infiltrating tumors, compared with the 3 Gy × 5
variable, correlated with abscopal responses.364 For post-RT ALC, fractionated schedule261. However, high-dose RT (≥15 Gy) may
the abscopal response rate was 34.2% in the cohort with ALC increase the proportion of splenic Tregs suppressing the
higher than the median value, compared with 3.9% in patients antitumor immune response.380 In addition, other preclinical
with ALC lower than the median. These data indicate the studies favored that conventional fractionation might have better
predictive role of ALC on systemic abscopal effects induced by iRT. efficacy to combine with IO.111 In clinical practice, it seems that
hypofractionated RT may show advances in some certain cancers
and the combination of IO + SBRT may be more potential in the
CLINICAL PRACTICE modality of iRT.71 In the subgroup analysis of PACIFIC study, the
Based on the significant immunomodulatory effect of IR, we may investigator has proved that patients may get a dramatical survival
broadly apply the use of iRT. In fact, iRT has been a great success benefit no matter what radiation doses are used.381 In a
to treat patients with cancers including NSCLC, melanoma, and randomized phase I/II trial for lung and liver lesions of NSCLC,382
some solid tumors.365–371 Compared with monotherapy, iRT shows there were better out-of-field ORRs and longer median PFS times
significant perspectives to the local control of lesion sites and of pembrolizumab + SBRT (50 Gy in 4 fractions) compared with
abscopal effect. Here we systematically summarize the clinical pembrolizumab + traditional RT (45 Gy in 15 fractions) which may
trials of iRT in recent years and explore the potential value in indicate that hypofractionated radiotherapy can better coordinate
clinical application. Since the initial proof-of-principle trial about the effect of immunotherapy. In the concurrent pembrolizumab +
iRT, Golden and his colleagues first proved that RT combined with RT groups, the out-of-field ORRs were 38% in the pembrolizu-
granulocyte-macrophage colony-stimulating factor (GM-CSF) does mab + SBRT group and 10% in the pembrolizumab + traditional
produce a high objective abscopal effect in patients who are RT group. This result also corroborates other clinical trials.383
suffering from metastatic solid tumors and this trial provides the Nevertheless, SBRT with PD-1 inhibitor and low-dose cyclopho-
proof-of-principle for the next clinical trials using iRT to treat sphamide showed no significant clinical benefit compared with
metastatic tumors.70 Based on the evidence of KEYNOTE-001 for conventional RT in patients with metastatic colorectal cancer.384
efficacy and safety of pembrolizumab, Shaverdian et al.372,373 The intriguing results of clinical trials may be associated with
further investigated that patients with advanced NSCLC treated different tumor types and heterogeneity. In conclusion, these data
with RT previously get longer PFS and OS with pembrolizumab show SBRT and, hypofractionated RT may exert a more effective
than patients who did not receive previous RT, with an acceptable antitumor immune response but the optimal dose for patients still
safety profile. And this study was the largest to report the effects needs more clinical data to identify. And there are a large body of
of previous RT on the activity and toxicity of ICIs at the time. In clinical trials in this field (Table 2). The data from these ongoing
addition to PD-1/PD-L1 inhibitors, the combination of RT with clinical trials may contribute to the selection of RT modalities.
CTLA-4 inhibitors has also been shown to have an abscopal effect The next question is lesion selection. Poleszczuk described a
and therapeutic effect. In addition, Formenti et al.374 found that mathematical model that incorporates physiologic information
for patients with NSCLC who have not responded to IO before, about T-cell trafficking to estimate the distribution of focal
some patients respond to IO, and some respond with significantly therapy-activated T cells between metastatic lesions.385 Their
longer survival time when they receive treatment of RT combined study showed that not all metastatic sites participate in systemic
with CTLA-4 blockade. The most powerful evidence supporting iRT immune surveillance equally and therefore the success in
was the phase III PACIFIC trial, which observed that the median triggering the abscopal effect depends on the selection of
PFS of patients with locally advanced, unresectable NSCLC is metastatic site to receive the treatment. Likewise, an open-label,
16.8 months with the PD-L1 antibody durvalumab versus phase I trial determining SBRT and ipilimumab in patients with
5.6 months of patients with placebo.42 Furthermore, they found metastatic solid tumor refractory to standard therapies and ≥1
that durvalumab significantly prolonged overall survival, as lesion in the liver or lung amenable to SBRT with ≥1 additional
compared with placebo and the following results including non-contiguous lesion for monitoring found that liver (vs. lung)
patient-reported outcomes, 3-year survival rates and 4-year irradiation produced greater T-cell activation, reflected as
survival rates confirmed that a clinical benefit can be attained increases in the proportions of peripheral T cells expressing ICOS,
through IO combined with chemoradiotherapy in PACIFIC study GITR, and 4-1BB which is associated with better clinical benefit.386
indeed.43–45,375 Based on Pembro-RT trial, a pooled analysis of two Therefore, we need to consider how to select an appropriate
randomized trials from the Netherlands and MD Anderson lesion to activate immune response, or another way to think about
including Pembro-RT showed that the effect of RT combined it, we can use multi-site RT to achieve systemic disease
was statistically valid in advanced NSCLC.376,377 However, the control.387–389 The multi-site RT is based on our cognition to the
choice of ICIs and RT including agents, sequence, dose, fractiona- most tumors and rationales of iRT. Achievement of abscopal effect
tion, and irradiated sites to exert the best synergies needs to be depends on shared TAAs from an irradiated tumor recognized by
explored and optimized.378 Hereafter, we attempt to summarize other lesions, but it is precise because of the heterogeneity of
the existing clinical trials to look at the possible optimal tumors that not the entire cellular population can exert an
combination of iRT and the broad prospect of iRT to treat locally effective immune response at these other lesions.390,391 Even for
advanced and advanced tumors. TAAs that could be recognized by entire cellular population, the
immunosuppressive tumor microenvironment may be the barrier
Radiotherapy dose/fractionation for CD8+ T cells to access the lesions.392 If we irradiate multiple
Concerning the rationale and some crucial clinical trials, the lesions, or even all tumor lesions, it is possible to overcome these
preponderance of iRT has been established. However, there are barriers to immune activation. As noted above, we can use low-
still many questions worth to be discussed such as the dose, lesion dose radiotherapy to overcome immunosuppressive tumor
selection, and scope of RT. As we discussed above, the immune microenvironment for access of CD8+ T cells and multi-site
response induced by RT is “dose dependent” so the optimal dose radiation might active more shared TAAs with the heterogeneity
should maximize tumor immunity and could be tolerant by of different tumor lesions. Multiple retrospective studies in

Signal Transduction and Targeted Therapy (2022)7:258


16
Table 2. Representative trials using combination of radiotherapy and immunotherapy

ClinicalTrials.gov Trial Phase Condition or disease Sequence RT IO Results Sponsors Estimated/actual


identifier study
completion date

NCT02474186 Phase 1 Various Concurrent 35 Gy in 10 fractions GM-CSF Abscopal responses in NYU July 2015
Phase 2 27.6% of patients Langone Health
NCT02125461 Phase 3 NSCLC RT, IO 54 to 66 Gy Durvalumab Durable PFS and AstraZeneca December
sustained OS benefit 30, 2022
with durvalumab after
chemoradiotherapy
NCT02608385 Phase 1 Solid tumors SBRT, IO SBRT dosing varied by Pembrolizumab Well tolerated with University of July 2022
site and ranged from acceptable toxicity Chicago
30 to 50 Gy in three to
five fractions
NCT02221739 Phase 1 NSCLC Concurrent 6 Gy x5, later changed Ipilimumab Objective responses NYU October 27, 2015
Phase 2 to 9.5 Gy x3 were observed in 18%, Langone Health
and 31% had disease
control
NCT02434081 Phase 2 NSCLC Concurrent 66 Gy in 33 fractions Nivolumab The addition of European Thoracic March 31, 2020
nivolumab to Oncology Platform
concurrent CRT is safe
and tolerable
NCT02492568 Phase 2 NSCLC RT, IO SBRT 3 doses of 8 Gy Pembrolizumab Well tolerated and a The Netherlands June 2018
Zhang et al.
Radiotherapy combined with immunotherapy: the dawn of cancer treatment

doubling of ORR Cancer Institute


NCT02444741 Phase 1 NSCLC Concurrent Various Pembrolizumab Safe and more M.D. Anderson September
Phase 2 beneficial for patients Cancer Center 17, 2022
with low PD-L1
expression
NCT02343952 Phase 2 Carcinoma, NSCLC RT, IO 59.4 to 66.6 Gy Pembrolizumab PFS and OS Nasser Hanna, M.D. September 2022
improvement with
consolidation
pembrolizumab
NCT03631784 Phase 2 NSCLC Concurrent 60 Gy in 30 daily Pembrolizumab Promising antitumor Merck Sharp & May 15, 2023
fractions activity and Dohme Corp.
manageable safety
Ongoing or completed clinical trials using combination of radiotherapy and immunotherapy for NSCLC
ClinicalTrials.gov Trial Phase Condition or disease Sequence RT IO Status Sponsors Estimated/Actual
identifier study
completion date
PD-1 inhibitor
NCT03035890 Not Metastatic NSCLC Concurrent Hypo-fractionated Immuno-Therapeutic Active, not recruiting West Virginia June 30, 2023
Applicable Radiation Agent (Nivolumab/ University
pembrolizumab/
atezolizumab)

Signal Transduction and Targeted Therapy (2022)7:258


NCT05111197 Phase 3 Locally advanced or IO, RT SBRT Anti-PD-1 or anti-PD- Not yet recruiting Institut December 2024
metastatic NSCLC L1 immunotherapy Cancerologie de
l’Ouest
NCT03523702 Phase 2 Locally Advanced NSCLC Concurrent Selective personalized Pembrolizumab Recruiting Albert Einstein September 2022
radiotherapy College of Medicine
Table 2. continued
ClinicalTrials.gov Trial Phase Condition or disease Sequence RT IO Results Sponsors Estimated/actual
identifier study
completion date

NCT03383302 Phase 1 NSCLC Stage II and Stage I RT, IO SBRT Nivolumab Recruiting Royal Marsden NHS January 2022
Phase 2 Foundation Trust
NCT03825510 Not Metastatic NSCLC RT, IO SBRT Nivolumab/ Recruiting Crozer-Keystone August 28, 2021
Applicable pembrolizumab Health System
NCT04577638 Phase 2 NSCLC Stage III Concurrent Intensity Modulated Nivolumab Recruiting Center Eugene February 1, 2024
Radiotherapy Marquis
NCT03168464 Phase 1 NSCLC Metastatic Concurrent 6 Gy x 5 fractions Ipilimumab/ Recruiting Weill Medical December
Phase 2 nivolumab College of Cornell 30, 2022
University
NCT03867175 Phase 3 Stage IV NSCLC Concurrent SBRT Pembrolizumab Recruiting Wake Forest December
University Health 31, 2027
Sciences

Signal Transduction and Targeted Therapy (2022)7:258


NCT03110978 Phase 2 Stage I-IIA or Concurrent SBRT Nivolumab Recruiting M.D. Anderson June 30, 2022
Recurrent NSCLC Cancer Center
NCT03812549 Phase 1 Stage IV NSCLC RT, IO SBRT/LDRT Sintilimab Recruiting Sichuan University December
31, 2022
NCT03313804 Phase 2 NSCLC, Squamous Cell IO, RT SBRT Nivolumab/ Recruiting John L. Villano, June 30, 2028
Carcinoma of the Head pembrolizumab/ MD, PhD
and Neck atezolizumab
NCT04929041 Phase 2 Stage IV NSCLC Concurrent SBRT Ipilimumab/ Recruiting National Cancer December
Phase 3 nivolumab/ Institute (NCI) 31, 2027
pembrolizumab
NCT03774732 Phase 3 NSCLC Metastatic Concurrent SBRT Pembrolizumab Recruiting UNICANCER September
21, 2024
Zhang et al.

NCT05229614 Phase 2 NSCLC, Head and Neck IO, RT Carbon ion therapy Pembrolizumab Not yet recruiting CNAO National August 2026
Squamous Cell Carcinoma, Center of
Melanoma, Urothelial Oncological
Carcinoma Hadrontherapy
NCT03705806 Stage IV NSCLC IO, RT 30 Gy in 10 fractions PD-1 inhibitor Recruiting University Health September
Network, Toronto 15, 2022
NCT03224871 Early Metastatic NSCLC Concurrent Hypo-fractionated Intralesional IL-2, Completed University of January 10, 2020
Phase 1 Radiotherapy nivolumab, California, Davis
pembrolizumab
NCT05265650 Phase 1 Metastatic NSCLC Concurrent SBRT Nivolumab Not yet recruiting Clinica Universidad June 2024
Phase 2 de Navarra,
Universidad de
Navarra
NCT04513301 Phase 2 Recurrent or IV NSCLC after Concurrent 50-60 Gy/25-30 f Sintilimab Recruiting Shanghai Cancer December 1, 2022
failure of platinum-based Hospital, China
chemotherapy
NCT05222087 Phase 1 Metastatic NSCLC RT, IO SBRT Pembrolizumab Not yet recruiting Peter MacCallum April 2024
Cancer Centre,
Australia
NCT02444741 Phase 1 Stage IV NSCLC Concurrent SBRT Pembrolizumab Active, not recruiting M.D. Anderson September
Phase 2 Cancer Center 17, 2022
Radiotherapy combined with immunotherapy: the dawn of cancer treatment

17
18
Table 2. continued
ClinicalTrials.gov Trial Phase Condition or disease Sequence RT IO Results Sponsors Estimated/actual
identifier study
completion date

NCT03217071 Phase 2 Stage I-IIIA NSCLC IO, RT SBRT Pembrolizumab Active, not recruiting Sue Yom February 28, 2022
NCT02492568 Phase 2 Advanced NSCLC RT, IO SBRT 3 doses of 8 Gy Pembrolizumab Completed The Netherlands June 2018
Cancer Institute
NCT04892849 Not HNSCC, NSCLC, Esophageal IO, RT RT PD-1/PD-L1 inhibitor Recruiting University of December
Applicable Cancer, Urothelial Erlangen-Nürnberg 31, 2027
Carcinoma, Renal Cell Medical School
Carcinoma, Squamous Cell
Carcinoma of the Skin, Small
Cell Bronchial Carcinomas
NCT03223155 Phase 1 Stage IV NSCLC Concurrent or SBRT Ipilimumab/ Recruiting University of December 2024
sequential nivolumab Chicago
NCT04013542 Phase 1 Stage II–III NSCLC Concurrent RT Ipilimumab and Recruiting M.D. Anderson February 1, 2022
nivolumab Cancer Center
NCT04902040 Phase 1 Advanced Bladder RT, IO RT Atezolizumab, Recruiting M.D. Anderson June 1, 2025
Phase 2 Carcinoma, Advanced avelumab, Cancer Center
NSCLC, Advanced Malignant durvalumab,
Solid Neoplasm, Advanced nivolumab and
Melanoma, Advanced Merkel pembrolizumab
Cell Carcinoma, Advanced
Zhang et al.
Radiotherapy combined with immunotherapy: the dawn of cancer treatment

Renal Cell Carcinoma


NCT04271384 Phase 2 Stage 1 NSCLC Concurrent SBRT Nivolumab Recruiting Hospital Israelita June 29, 2023
Albert Einstein
NCT04291092 Phase 2 NSCLC Stage IV WBRT Camrelizumab Recruiting Zhejiang Cancer June 30, 2023
Brain Metastases Hospital
NCT04167657 Phase 2 Advanced NSCLC RT, IO RT Sintilimab Recruiting Peking Union April 15, 2023
Medical College
Hospital
NCT02818920 Phase 2 Stage IB, II or IIIA NSCLC IO, RT RT Pembrolizumab Active, not recruiting Neal Ready March 2026
NCT03589339 Phase 1 Advanced cancers IO, RT SBRT Nivolumab/ Recruiting Nanobiotix March 30, 2023
(Metastatic NSCLC) pembrolizumab
NCT04977453 Phase 1 NSCLC, Head and Neck SBRT Pembrolizumab Recruiting GI Innovation, Inc. December 2025
Phase 2 Squamous Cell Carcinoma,
Renal Cell Carcinoma,
Urinary Bladder Cancer,
Melanoma, Sarcoma
PD-L1 inhibitor
NCT03965468 Phase 2 NSCLC Concurrent SBRT Durvalumab Recruiting European Thoracic December 2021
Stage IV Oncology Platform
Oligometastasis
NCT03035890 Not Metastatic NSCLC Concurrent Hypo-fractionated Immuno-Therapeutic Active, not recruiting West Virginia June 30, 2023

Signal Transduction and Targeted Therapy (2022)7:258


Applicable Radiation Agent (Nivolumab/ University
pembrolizumab/
atezolizumab)
NCT05000710 Phase 2 Metastatic or Locally Concurrent 11 fractions of 3 Gy Durvalumab/ Recruiting Sheba December 2026
Advanced NSCLC tremelimumab Medical Center
Table 2. continued
ClinicalTrials.gov Trial Phase Condition or disease Sequence RT IO Results Sponsors Estimated/actual
identifier study
completion date

NCT05111197 Phase 3 Locally advanced or IO, RT SBRT Anti-PD-1 or anti-PD- Not yet recruiting Institut December 2024
metastatic NSCLC L1 immunotherapy Cancerologie de
l’Ouest
NCT04765709 Phase 2 Large volume stage III NSCLC Concurrent RT Durvalumab Not yet recruiting Mario Negri June 2026
Institute for
Pharmacological
Research
NCT04549428 Phase 2 NSCLC Stage IV Concurrent a single fraction of Atezolizumab Recruiting Oncology Institute July 31, 2022
8 Gy of Southern
Switzerland
NCT04245514 Phase 2 NSCLC Concurrent 20 × 2 Gy (weekdaily, Durvalumab Recruiting Swiss Group for March 2025
4 weeks) Clinical Cancer
5 × 5 Gy (weekdaily, Research

Signal Transduction and Targeted Therapy (2022)7:258


1 week)
3 × 8 Gy (on alternate
days, 1 week)
NCT05267392 Phase 1 Early stage or locally IO, RT Standard of care RT/ Durvalumab Recruiting Instituto Portugues January 2024
Phase 2 advanced, RCT de Oncologia,
unresectable NSCLC Francisco
Gentil, Porto
NCT05128630 Phase 2 NSCLC, Stage III Concurrent reduced-dose hypo- Durvalumab Recruiting IRCCS Policlinico November
fractionated S. Matteo 28, 2025
thoracic RT
NCT04989283 Phase 2 Stage IIB Lung Cancer AJCC Concurrent External Beam Atezolizumab Recruiting National Cancer May 10, 2031
v8, Stage IIIA Lung Cancer Radiation Therapy Institute (NCI)
Zhang et al.

AJCC v8, Superior Sulcus


Lung Carcinoma
NCT03313804 Phase 2 NSCLC, Squamous Cell IO, RT SBRT Nivolumab/ Recruiting John L. Villano, June 30, 2028
Carcinoma of the Head pembrolizumab/ MD, PhD
and Neck atezolizumab
NCT03275597 Phase 1 NSCLC Stage IV IO, RT SBRT Durvalumab + Active, not recruiting University of July 2025
tremelimumab Wisconsin, Madison
NCT04372927 Phase 2 Locally Advanced NSCLC Concurrent Adaptive mediastinal Durvalumab Recruiting University of November
radiation Washington 30, 2026
NCT03916419 Phase 2 Stage IIB, IIIA, and Select IIIB Concurrent MR-Linear Accelerator- Durvalumab Recruiting Washington December
and IIIC NSCLC Radiation University School of 31, 2024
Medicine
NCT04230408 Phase 2 Stage III NSCLC IO, RT, IO 54 to 66 Gy Durvalumab Recruiting Latin American May 2024
Cooperative
Oncology Group
NCT04992780 Phase 2 NSCLC RT, IO Hypo-Fractionation Durvalumab Not yet recruiting University of Kansas November 2023
62.5 Gy in 25 fractions Medical Center
of 2.5 Gy/fraction;
Standard-Fractionation
60 Gy in 30 fractions of
2 Gy/fraction
Radiotherapy combined with immunotherapy: the dawn of cancer treatment

19
20
Table 2. continued
ClinicalTrials.gov Trial Phase Condition or disease Sequence RT IO Results Sponsors Estimated/actual
identifier study
completion date

NCT03446547 Phase 2 Stage I NSCLC RT, IO SBRT Durvalumab Recruiting Vastra July 2023
Gotaland Region
NCT05034055 Phase 2 Metastatic NSCLC RT, IO SBRT Atezolizumab/ Not yet recruiting Yonsei University December 2023
tiragolumab
NCT05157542 Phase 1 Stage III NSCLC Concurrent Low dose radiation Durvalumab Recruiting Juan LI, MD June 10, 2023
therapy
NCT03391869 Phase 3 Stage IV NSCLC IO, RT Local consolidation Nivolumab and Recruiting M.D. Anderson December
therapy ipilimumab Cancer Center 31, 2022
NCT03818776 Early Unresectable NSCLC Concurrent Proton beam Durvalumab Recruiting Case November 1, 2023
Phase 1 therapy RT Comprehensive
Cancer Center
NCT03801902 Phase 1 Locally advanced NSCLC IO, RT Hypofractionated Durvalumab Active, not recruiting National Cancer January 5, 2023
Radiation Therapy/ Institute (NCI)
Fractionated
Stereotactic Radiation
Therapy
NCT02463994 Early Metastatic NSCLC RT, IO Hypo-fractionated PD-L1 antibody Completed University of November 7, 2018
Phase 1 Image-guided Michigan Rogel
Radiotherapy Cancer Center
Zhang et al.
Radiotherapy combined with immunotherapy: the dawn of cancer treatment

NCT02888743 Phase 2 Metastatic Colorectal IO, RT high dose radiation Tremelimumab and Active, not recruiting National Cancer December
or NSCLC therapy/low dose durvalumab Institute (NCI) 31, 2022
radiation therapy
NCT04944173 Phase 2 Stage I NSCLC Concurrent SBRT Durvalumab Not yet recruiting University of British December 2024
Columbia
NCT04310020 Phase 2 Stage II or III NSCLC IO, RT Hypo-fractionated Atezolizumab Recruiting National Cancer March 15, 2022
Radiation Therapy Institute (NCI)
NCT04081688 Phase 1 Refractory NSCLC Stage IV SBRT Atezolizumab/ Recruiting Rutgers, The State June 30, 2023
varlilumab University of
New Jersey
NCT04889066 Phase 2 Brain metastases NSCLC Concurrent Personalized ultra- Durvalumab Not yet recruiting University of Texas January 2025
fractionated Southwestern
stereotactic adaptive Medical Center
radiotherapy or
Fractionated
Stereotactic
Radiotherapy
NCT04892849 Not HNSCC, NSCLC, Esophageal IO, RT RT PD-1/PD-L1 inhibitor Recruiting University of December
Applicable Cancer, Urothelial Erlangen-Nürnberg 31, 2027
Carcinoma, Renal Cell Medical School
Carcinoma, Squamous Cell

Signal Transduction and Targeted Therapy (2022)7:258


Carcinoma of the Skin, Small
Cell Bronchial Carcinomas
NCT04202809 Phase 2 Resectable Stage III NSCLC Concurrent RT Durvalumab Recruiting University April 2024
Hospital, Essen
NCT03237377 Phase 2 Stage III Resectable NSCLC Concurrent Thoracic radiation: Active, not recruiting Sidney Kimmel September 2022
45 Gy in 25 fractions Comprehensive
Table 2. continued
ClinicalTrials.gov Trial Phase Condition or disease Sequence RT IO Results Sponsors Estimated/actual
identifier study
completion date

Durvalumab or Cancer Center at


durvalumab plus Johns Hopkins
tremelimumab
NCT04214262 Phase 3 Stage I-IIA NSCLC Induction/ SBRT Atezolizumab Recruiting National Cancer May 1, 2028
Consolidation Institute (NCI)
Atezoli-zumab
+ SBRT
NCT03871153 Phase 2 Stage III NSCLC Concurrent 45-61.2 Gy/25-30 f Durvalumab Active, not recruiting Greg Durm, MD April 2023
NCT03141359 Phase 2 Locally advanced NSCLC IO, RT IMRT/SBRT Durvalumab Recruiting Atrium Health May 2026
NCT04902040 Phase 1 Advanced Bladder RT, IO RT Atezolizumab, Recruiting M.D. Anderson June 1, 2025
Phase 2 Carcinoma, Advanced avelumab, Cancer Center
NSCLC, Advanced Malignant durvalumab,
Solid Neoplasm, Advanced nivolumab and

Signal Transduction and Targeted Therapy (2022)7:258


Melanoma, Advanced Merkel pembrolizumab
Cell Carcinoma, Advanced
Renal Cell Carcinoma
NCT05198830 Phase 2 Stage III Non- RT, IO RT Durvalumab Not yet recruiting National Cancer May 1, 2024
Squamous NSCLC Institute (NCI)
NCT04786093 Phase 2 Advanced NSCLC Concurrent SBRT/Personalized Durvalumab Recruiting University of Texas May 2027
Ultra-fractionated Southwestern
Stereotactic Medical Center
Radiotherapy
NCT04364776 Not Stage III Unresectable NSCLC IO, RT RT Durvalumab Recruiting IRCCS Policlinico April 15, 2024
Applicable S. Matteo
NCT04892953 Phase 2 Stage III NSCLC RT, IO RT Durvalumab Not yet recruiting M.D. Anderson September
Zhang et al.

Cancer Center 30, 2022


NCT02492867 Not Locally Advanced NSCLC RT, IO Response-driven Durvalumab Active, not recruiting University of November 2024
Applicable Adaptive Radiation Michigan Rogel
Therapy Cancer Center
NCT04238169 Phase 2 Stage IV NSCLC Concurrent SBRT Toripalimab Recruiting Xinqiao Hospital of December
Chongqing 31, 2023
NCT04597671 Phase 3 Stage III NSCLC Concurrent Low-dose prophylactic Durvalumab Recruiting Association NVALT December 2032
cranial irradiation Studies
NCT03337698 Phase 1 Metastatic NSCLC Concurrent RT Atezolizumab Recruiting Hoffmann-La Roche August 1, 2025
Phase 2
NCT04092283 Phase 3 Unresectable Stage III NSCLC IO, RT, IO RT Durvalumab Recruiting National Cancer October 31, 2028
Institute (NCI)
NCT05259319 Phase 1 Metastatic NSCLC, Metastatic Concurrent or SBRT Atezolizumab and Not yet recruiting Centre Georges February 28, 2030
Bladder Cancer, Metastatic sequential tiragolumab Francois Leclerc
Renal Cell Carcinoma,
Metastatic Head and
Neck Cancer
NCT03915678 Phase 2 Advanced solid tumors Concurrent SBRT Atezolizumab Recruiting Institut Bergonié March 2025
NCT03509012 Phase 1 Squamous Cell of Head and Concurrent RT Durvalumab/ Active, not recruiting AstraZeneca December
Neck Carcinoma, NSCLC tremelimumab 29, 2023
Radiotherapy combined with immunotherapy: the dawn of cancer treatment

21
22
Table 2. continued
ClinicalTrials.gov Trial Phase Condition or disease Sequence RT IO Results Sponsors Estimated/actual
identifier study
completion date

CTLA-4
NCT03168464 Phase 1 NSCLC Metastatic Concurrent 6 Gy x 5 fractions Ipilimumab/ Recruiting Weill Medical December
Phase 2 Nivolumab College of Cornell 30, 2022
University
NCT04929041 Phase 2 Stage IV NSCLC Concurrent SBRT Ipilimumab/ Recruiting National Cancer December
Phase 3 Nivolumab/ Institute (NCI) 31, 2027
Pembrolizumab
NCT03275597 Phase 1 NSCLC Stage IV IO, RT SBRT Durvalumab and Active, not recruiting University of July 2025
tremelimumab Wisconsin, Madison
NCT03391869 Phase 3 Stage IV NSCLC IO, RT Local consolidation Nivolumab and Recruiting M.D. Anderson December
therapy ipilimumab Cancer Center 31, 2022
NCT02888743 Phase 2 Metastatic Colorectal IO, RT high dose radiation Tremelimumab and Active, not recruiting National Cancer December
or NSCLC therapy/low dose durvalumab Institute (NCI) 31, 2022
radiation therapy
NCT03223155 Phase 1 Stage IV NSCLC Concurrent or SBRT Ipilimumab/ Recruiting University of December 2024
sequential Nivolumab Chicago
NCT03237377 Phase 2 Stage III Resectable NSCLC Concurrent Thoracic radiation: Durvalumab or Active, not recruiting Sidney Kimmel September 2022
45 Gy in 25 fractions durvalumab plus Comprehensive
Zhang et al.
Radiotherapy combined with immunotherapy: the dawn of cancer treatment

tremelimumab Cancer Center at


Johns Hopkins
NCT04013542 Phase 1 Stage II–III NSCLC Concurrent RT Ipilimumab and Recruiting M.D. Anderson February 1, 2022
nivolumab Cancer Center
NCT02221739 Phase 1 Metastatic NSCLC Concurrent IMRT or 3-D CRT Ipilimumab Completed NYU October 27, 2015
Phase 2 Langone Health
NCT03509012 Phase 1 Squamous Cell of Head and Concurrent RT Durvalumab/ Active, not recruiting AstraZeneca December
Neck Carcinoma, NSCLC tremelimumab 29, 2023
TIGIT inhibitor
NCT05034055 Phase 2 Metastatic NSCLC RT, IO SBRT Atezolizumab/ Not yet recruiting Yonsei University December 2023
tiragolumab
NCT05259319 Phase 1 Metastatic NSCLC, Metastatic Concurrent or SBRT Atezolizumab and Not yet recruiting Centre Georges February 28, 2030
Bladder Cancer, Metastatic sequential tiragolumab Francois Leclerc
Renal Cell Carcinoma,
Metastatic Head and
Neck Cancer
Cytikines
NCT03705403 Phase 2 NSCLC Stage IV Concurrent SBRT L19-IL2 Recruiting Maastricht December 1, 2023
Metastatic Disease University
NCT03224871 Early Metastatic NSCLC Concurrent Hypo-fractionated Intralesional IL-2, Completed University of January 10, 2020
Phase 1 Radiotherapy nivolumab, California, Davis

Signal Transduction and Targeted Therapy (2022)7:258


pembrolizumab
Vaccines
NCT00006470 Phase 2 NSCLC IO, RT RT Monoclonal antibody Completed Radiation Therapy December 2004
11D10 anti-idiotype Oncology Group
vaccine or
Table 2. continued
ClinicalTrials.gov Trial Phase Condition or disease Sequence RT IO Results Sponsors Estimated/actual
identifier study
completion date

monoclonal antibody
3H1 anti-idiotype
vaccine
NCT00828009 Phase 2 Unresectable Stage IIIA and RT, IO RT Tecemotide Completed ECOG-ACRIN May 22, 2019
IIIB Non-Squamous NSCLC Cancer
Research Group
Others
NCT05269485 Phase 1 Stage III NSCLC High-dose fractionated IT Recruiting Anhui Provincial June 1, 2023
Phase 2 radiotherapy: Hospital
60–68 Gy/15-17f; low-
dose fractionated
radiotherapy: 48 Gy/
15-12f

Signal Transduction and Targeted Therapy (2022)7:258


NCT04654520 Not Stage IV NSCLC Concurrent IMRT IT Not yet recruiting Guizhou Medical May 31, 2022
Applicable University
NCT04650490 Phase 2 Brain Metastases NSCLC IO, RT or RT, IO SBRT IT Not yet recruiting Duke University March 2025
NCT03827577 Phase 3 Oligometastatic NSCLC RT, IO SBRT IT Recruiting Azienda September 2022
Ospedaliera
Universitaria
Integrata Verona
NCT02839265 Phase 2 Advanced NSCLC Concurrent SBRT FLT3 Ligand Therapy Active, not recruiting Albert Einstein October 5, 2022
(CDX-301) College of Medicine
NCT04491084 Phase 1 Advanced NSCLC Concurrent SBRT FLT3 ligand (CDX- Recruiting Albert Einstein August 31, 2023
Phase 2 301), anti-CD40 College of Medicine
antibody (CDX-1140)
Zhang et al.

NCT00879866 Phase 1 NSCLC Stage IIIb With RT, IO 5 ×4 Gy Selectikine (EMD Completed Merck KGaA, September 2012
Malignant Pleural Effusion or 521873) Darmstadt,
Stage IV With Disease Germany
Control
NCT04081688 Phase 1 Refractory NSCLC Stage IV SBRT Atezolizumab/ Recruiting Rutgers, The State June 30, 2023
varlilumab University of
New Jersey
Radiotherapy combined with immunotherapy: the dawn of cancer treatment

23
Radiotherapy combined with immunotherapy: the dawn of cancer treatment
Zhang et al.
24
oligometastatic NSCLC have shown that the use of RT to all sites of delayed administration of checkpoint inhibitors after RT so that
disease is associated with a significant improvement in OS and newly recruited T cells can destroy tumor cells, both at the primary
PFS.393,394 Data from a phase III trial aiming to determine the site and systemically after being presented with novel tumor
efficacy of iRT in metastatic prostate cancer showed that results antigens. The PACIFIC trial has proved that durvalumab after
are negative when RT was delivered to a single bony lesion.395 chemoradiotherapy improved PFS significantly and the new
Subgroup analyses of this trial and other clinical trials also support results reported that estimated 4-year OS rates were 49.6% versus
IO may achieve better efficacy when patients with lower disease 36.3% for durvalumab versus placebo, and 4-year PFS rates were
burden and a reduction in tumor burden by comprehensive (but 35.3% versus 19.5% respectively.42,45 The PACIFIC trial has laid a
not single-site) RT may potentiate IO. Furthermore, a phase 2 trial framework of adjuvant administration of durvalumab after
of pembrolizumab therapy after locally ablative therapy (surgery chemoradiotherapy for patients who are suffering from NSCLC
or SBRT) for patients with oligometastatic NSCLC which treated all in stage III. Similarly, other clinical trials also confirmed the safety
metastatic sites demonstrated that FPS increased by 12 months and efficacy of pembrolizumab and nivolumab after chemora-
compared with the historical median of 6.6 months.396 Thus, diotherapy.398,406 However, the HOPE-005/CRIMSON, a multi-
multiple target RT might be required to optimize responses to iRT. center, retrospective, real-world cohort study of 275 patients
Of note, a phase I study estimated multi-site SBRT followed by receiving concurrent chemoradiotherapy in advanced NSCLC, 204
pembrolizumab for metastatic solid tumors, including NSCLC.367 of whom received durvalumab consolidation therapy showed that
This trial enrolled 79 patients receiving SBRT to 2 to 4 metastases 81.8% of patients who received durvalumab after concurrent
and metastases >65 mL were partially irradiated. After completion chemoradiotherapy had pneumonitis and 59.5% were asympto-
of SBRT, patients started to administer pembrolizumab within matic pneumonitis.407 Another real-world study of durvalumab
7 days and it lasted at least one cycle. The RECIST-based overall consolidation after chemoradiotherapy in stage III NSCLC sug-
ORR was 13.2% and the mOS and mPFS were 9.6 months (95% CI, gested that the incidence of grade 3 radiation pneumonitis was
6.5 months to undetermined) and 3.1 months (95% CI, 2.9 to 14.3 % in the durvalumab group versus 2.5 % in the observation
3.4 months), respectively. Their results are similar to data from group.408 These data remind that the adverse effect cannot be
KEYNOTE-028 (9–33% response rate), which evaluated the safety ignored and it needs to be further improved for PACIFIC mode.
and efficacy of pembrolizumab alone in patients with PD-L1- Analysis of PACIFIC study and a retrospective analysis of clinical
positive advanced solid tumors.397 Nevertheless, investigators data suggest that patient outcome seems to be better if IO is
should consider these data carefully because the tumor areas given concurrently or begun soon after RT, as compared with
excluded from the SBRT program are exposed to low doses of RT starting IO later after the RT.42,409 The update data from PACIFIC
and there is a lack of PD-L1 status for patients with various study show that there is a significant improvement of OS and FPS
cancers. both within 14 days and after RT, but the advantage is more
obvious within 14 days when combined IO with RT.45 However, a
Selection of immunotherapy modality retrospective study proved that patients who received
Although hypofractionated RT has shown advances for inducing IO ≥ 21 days after the onset of SBRT had a longer OS than those
antitumor immunity, it is not known which immune checkpoint who received IO within 21 days after the onset of SBRT.410 But the
inhibitor to use in combination with RT. There are an increasing data from this study should be considered carefully because it
number of studies which have already confirmed the efficacy of RT included many confounding factors. Thus, the optimal timing of
combined with ICIs including pembrolizumab, nivolumab, durva- iRT still needs to be explored through large randomized clinical
lumab, and atezolizumab.42,398–401 Considering that the most trials.
widely used ICI is PD-1 and CTLA-4 blockades, a retrospective Sequential administration of IO followed by RT have been
analysis of two single-institution prospective trials reported that affirmed, many studies started to investigate whether we could
the FPS of anti-PD1 combined with SBRT for metastatic NSCLC was use concurrent administration to achieve immunotherapy for-
significantly better than anti-CTLA4 combined with SBRT, although ward. Researchers hypothesized that adding IO concurrently with
there was no statistically significant difference in efficacy.365 The concurrent chemoradiotherapy may improve the efficacy without
PFS was 76% for anti-CTLA4 vs 94% anti-PD1 at 3 months, 52% vs additive toxicity so they designed a phase II trial of concurrent
87% at 6 months, 31% vs 80% at 12 months, and 23% vs 63% at administration.411 This phase II study was conducted in two parts.
18 months (p = 0.02). This tentative exploration requires further Part 1 involved administration of conventionally fractionated
data, and this conclusion is restricted to metastatic NSCLC. There chemoradiotherapy followed by consolidation chemotherapy
are few of studies to support and more clinical trials are needed (atezolizumab [two cycles] and maintenance atezolizumab up to
for other form of tumors. Notably, in the view of multi-site RT, we 1 year). Part 2 involved administration of concurrent chemor-
should consider whether we can use one or multiple ICIs. A adiotherapy with atezolizumab followed by the same consolida-
randomized, multicenter, phase II clinical study assessing the tion and maintenance therapies as in part 1. The results showed
primary safety of two kinds of ICIs as consolidation therapy after that immune-related adverse events of grade 3 or higher had an
concurrent chemoradiotherapy for patients with stage III, unre- incidence rate of 20% to 30%, and pneumonitis of grade 2 or
sectable NSCLC. This trial enrolled 105 patients with unresectable higher had an incidence rate of 10% to 16%. The median FPS of
stage IIIA/ IIIB NSCLC and they were divided equally into to group part 1/2 was 18.6 months and 13.2 months, respectively. There-
(nivolumab group and nivolumab + ipilimumab group) after fore, safety and efficacy of combining IO with chemoradiotherapy
concurrent chemoradiotherapy. Safety analysis of the first 50 concurrently have been confirmed. Since the PACIFIC trial
patients showed that after concurrent chemoradiotherapy, the indicated that there was a trend of PFS being longer in the
incidence of grade 3 adverse events is higher in nivolumab + 14 days group, investigators designed a phase 2, nonrandomized
ipilimumab treatment, leading to higher drug withdrawal rate KEYNOTE-799 clinical trial aiming for improve outcomes and
than that in nivolumab monotherapy group. Therefore, this area safety of pembrolizumab with concurrent chemoradiotherapy.46
requires further exploration from additional clinical trials. Patients corresponding to the inclusion criteria were selected by
investigators to enter the following cohort: in cohort A, patients
The optimal timing for iRT with squamous cell cancer/non-squamous cell cancer received
In addition to the selection of RT and immunotherapy molality, paclitaxel 200 mg/m2 combined with carboplatin (AUC = 6), and
one of the most important matters is the optimal timing for after one cycle were switched to paclitaxel 45 mg/m2 combined
combination “concurrent” or “sequential”.402–405 Generally, the with carboplatin (AUC = 2), lasting for 6 weeks. Two cycles of
data available to date seem to justify either simultaneous or pembrolizumab therapy (once every three weeks) and standard

Signal Transduction and Targeted Therapy (2022)7:258


Radiotherapy combined with immunotherapy: the dawn of cancer treatment
Zhang et al.
25
radiotherapy (total dose was 60 Gy) were synchronized. In cohort patients with tumors leave much for further study and we expect
B, patients with non-squamous cell cancer received pemetrexed the results of ongoing clinical trials.
(500 mg/m2) combined with cisplatin (75 mg/m2) and pembroli-
zumab (200 mg), combined with radiotherapy (60 Gy) in cycle 2 The toxicity of iRT
and 3. The results showed that ORR was 70.5% (79 of 112; 95% CI, Although iRT has made a breakthrough in both preclinical studies
61.2–78.8%) in cohort A and 70.6% (72 of 102; 95% CI, 60.7–79.2%) and clinical trials, it also raises the question of the toxicity of this
in cohort B. Another coprimary end points, incidence of grade 3 to combination strategy. It is well-documented that normal tissue
5 pneumonitis, was 8.0% in cohort A and 6.9% in cohort B which injuries are the main limiting factor of the dose for RT alone. In
were both less than 8.0% and meet expectation.46 A phase 1 trial view of the irradiated sites, these radiation-induced injuries
compared combined nivolumab and ipilimumab with sequential include may include brain injury, heart disease, lung, and liver
or concurrent multi-site SBRT directly in patients with stage IV injury as well as damage to the corresponding sites.425–431 The
NSCLC.412 Their results showed that the median FPS of most severe injury caused by RT is radiation-induced lung injury
concurrent/sequential was 18.6 months and 13.2 months, respec- (RILI). RILI manifests as lung tissue damage and comprises two
tively. Moreover, the concurrent group was no more toxic than injury types: radiation pneumonitis and radiation pulmonary
sequential and there were no dose-limiting toxicity results in fibrosis.428 Generally, radiation pneumonitis occurs in 6 months
concurrent group but two patients had dose-limiting toxicity after RT and radiation pulmonary fibrosis occurs >1 year following
results in the sequential group. Thus, the data from this study RT. The underlying mechanisms of RILI may include many signal
proved that multi-site SBRT combined with nivolumab and pathways such as TGF-β/Smad, HMGB1/TLR4, and Nrf2/ARE
ipilimumab concurrently had good safety and considerable signaling pathway as well as dysregulation of cytokines which
efficacy. Similarly, a multicentric retrospective study from AIRO are initiated from the DNA damage and ROS generation caused by
(Italian Association of Radiotherapy and Clinical Oncology) for RT. Another serious injury is radiation-induced heart disease
patients with brain metastases from NSCLC reported that patients (RIHD). It includes cardiomyopathy, conduction system abnorm-
with the interval between SBRT and IO ≤ 7 days had a longer alities, coronary artery disease and the like. Similar to RILI, the
survival compared with the interval between SBRT and pathogenesis of RIHD is associated with the production of
IO > 7 days.413 This study data supports the delivery of SBRT and cytokines induced by endothelial injury and oxidative stress.426
IO within a short time frame given that concurrent therapy is In addition, liver injury caused by RT cannot be neglected.
shown to be more effective without having an impact on toxicity. Radiation-induced liver injury, which is different from RILI and
In conclusion, these data from clinical trials and retrospective RIHD, usually occurs during RT for some upper abdominal
studies indicate that concurrent iRT may be more effective than malignant tumors. In the clinic, radiation-induced liver injury can
sequential one. Nevertheless, due to relatively less clinical data, be classified into classic and non-classic liver disease.432 DNA
more studies are needed to investigate the optimal timing. damage and reactive free radical generation are two major factors
New opinions have been provided that neoadjuvant RT to trigger liver injury caused by RT. There are various processes
increases response rates and provides local control during which are involved in radiation-induced liver injury and they
neoadjuvant systemic treatment, before definitive surgery.414 finally cause liver cell apoptosis or necrosis.430 In addition to the
Therefore, iRT may improve treatment responses and complete direct damage of RT, mechanisms of RT‐induced normal tissue
resection rates in the neoadjuvant setting for the better locally injury also involve a large number of immune cells and
control and systemic antitumor responses. Besides, there are immunological factors so it is pivotal for immune system in this
numerous ongoing clinical trials to optimize PACIFIC mode and complex dynamic process.
strategies for IO forward such as KEYLYNK-012, CheckMate 73L, Meanwhile, IO also brings concern about IO-related toxicities
and PACIFIC-2 trials.415–417 We expect the data from these trials such as checkpoint inhibitor pneumonitis, colitis, hepatitis,
and believe in that the optimal combination will be established in immune checkpoint inhibitors-related endocrinopathies, and
the future. dermatologic toxicity.433–443 Due to the mechanisms of action,
In addition, other drugs also show enormous potential in cancer IO especially ICIs, leads to unique toxicity which is different from
treatment and may promote the efficacy of iRT. Nanoparticles, as the toxicities of RT. ICIs can overcome the inhibition of immune
immunomodulators and radiosensitizers to overcome therapy cells induced by tumor cells, but it also destroys the status of
resistance and improve survival, play a crucial role in cancer immune homeostasis which may lead to autoimmunity and
treatment.418 For the special properties, nanoparticles are an ideal nonspecific inflammation. This damage can occur in almost all
carrier to enhance antitumor IO. It is reported that combining IO organs and bring adverse events. ICIs-associated pneumonitis has
with nanoparticles may promote the accumulation and retention been widely reported and it may be potentially lethal.444 The
of antibodies in the target cells.419 For instance, nanoparticles- incidence of ICIs-associated pneumonitis ranges from 3% to 5% in
based antigens and adjuvants delivery strategies can address the different tumor types.434 Although the mechanisms remain
issues of off-target side effect and low immunogenicity.420 unclear, there are three potential pathways which may account
Moreover, nanomaterials with heavy-metal showed a promising for ICIs-associated pneumonitis: generalized immune activation,
radiosensitization which can efficiently absorb, scatter, and emit pre-existing autoantibodies, and off-target effects.428 Another
radiation energy and redistribution cell cycle.421,422 Nanoparticles worrisome adverse event of IO is endocrine toxicities of ICIs
also can serve as delivery vehicles carrying the radiosensitivity involving the thyroid, pituitary, adrenal, and pancreas.437 The
drugs which shows a promising prospect.423 Of note, Wang et al. potential mechanisms of ICIs-associated endocrine toxicities are
reported that cisplatin nanoparticles can promote the abscopal similar to ICIs-associated pneumonitis. Hypophysitis is rare in the
effect induced by RT with PD1 inhibitors which breaks through the population who have not received ICIs but hypophysitis occurs up
efficiency limitation of iRT.424 to 10% in the patients who receive anti-CTLA-4 therapy.445
Collectively, the authors have discussed the optimal combina- Pituitary glands also expressed CTLA-4 and these may become the
tion modality for patients with metastatic NSCLC which may be targets of CTLA-4 antibody which explain the high incidence of
hypofractionationated radiotherapy combined with anti-PD1 at hypophysitis in the patients receiving CTLA-4 blockades. More-
the present and plenty of clinical trials have proved that the over, ICIs may lead to hypothyroidism, hypocortisolism and
efficacy of iRT is significantly better than mono immunotherapy. diabetes mellitus due to the toxicities of ICIs on respective organs.
The addition of radiotherapy increased the out-of-field response It is an unquestionably important one mechanistically to
rate of immunotherapy alone from 19.7% to 41.7%.376 However, evaluate the safety of iRT. For instance, radiation recall
the optimal combinations that improve clinical outcomes for pneumonitis, an entity described as pneumonitis localized to a

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Zhang et al.
26
previously irradiated field after exposure to a systemic agent, 3. Mole, R. H. Whole body irradiation; radiobiology or medicine? Br. J. Radiol. 26,
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