Article https://doi.org/10.1038/s41467-024-51105-2

Toripalimab plus chemotherapy and

radiotherapy for treatment-naive advanced

esophageal squamous cell carcinoma: a

single-arm phase 2 trial

Lei Wu 1,7, Baisen Li1,7, Gang Wan1,7, Yi Wang1

, Jie Zhu1

, Long Liang1

,

Xuefeng Leng2

, Wenwu He2

, Lin Peng2

, Yongtao Han2

, Shuya He3

,

Dongsheng Wang3

, Yehan Zhou4

, Liang Yi5

, Wencheng Zhang 5

,

Qingsong Pang5

, Wei Zhang1

, Tao Li1

, Jinyi Lang1

, Yang Liu 4,8 ,

Bangrong Cao 6,8 & Qifeng Wang 1,8

This single-arm phase 2 trial (ChiCTR2100046715) examined previously

untreated patients with advanced esophageal squamous cell carcinoma (ESCC)

who received four cycles of paclitaxel with carboplatin every 3 weeks. Toripalimab was infused intravenously every 3 weeks for 12 months, or until disease

progression or intolerable toxicity. Radiotherapy that encompassed the primary lesions and metastases commenced in the third cycle. The median

progression-free survival time was 9.8 months (95% confidence interval [CI]:

6.8–not estimable) in the intent-to-treat population, failing to meet the prespecified primary endpoints. Secondary endpoints included an objective

response rate of 45.5%, a disease control rate of 57.6%, and a median duration of

response of 11.5 months (interquartile range, 6.4–15.0). The 1-year progressionfree survival and overall survival rates were 41.9% (95% CI: 27.7–63.5) and 69.7%

(95% CI: 55.7–87.3), respectively. Lymphopenia was the most frequent grade ≥3

adverse event (82%), and an esophageal fistula developed in three patients

(9.1%). No treatment-related deaths occurred. In prespecified exploratory biomarker analysis, higher densities of CD8 + T cells, CD11c+ dendritic cells, and

CD68+ macrophages correlated with improved tumor response and prognosis.

Radiotherapy supplementation to first-line chemo-immunotherapy for

treatment-naive advanced ESCC demonstrated some antitumor activity and

manageable safety profiles, warranting further randomized controlled trials.

Esophageal cancer is associated with significant mortality, particularly

in Asia, where esophageal squamous cell carcinoma (ESCC) is the most

common histological subtype, accounting for approximately 90% of all

cases. At the point of diagnosis, over two-thirds of patients already

exhibit metastatic or locally advanced disease1

. The National

Comprehensive Cancer Network guidelines specify the use of chemoimmunotherapy as the first-line treatment for advanced esophageal

cancer2

. This recommendation was based on several large-scale phase

III trials that reported substantial prolongation of progression-free

survival (PFS) (5.7–7.3 months) and overall survival (OS)

Received: 3 March 2024

Accepted: 30 July 2024

Check for updates

A full list of affiliations appears at the end of the paper. e-mail: liuyanglyon@uestc.edu.cn; caobangrong@uestc.edu.cn; wangqifeng@scszlyy.org.cn

Nature Communications | (2024) 15:7116 1

1234567890():,; 1234567890():,;

(12.6–17.2 months) compared to those using chemotherapy alone3–9

.

However, despite progress in therapeutic outcomes, the average

6-month PFS remains unsatisfactory, suggesting the development of

drug resistance and disease progression during maintenance

immunotherapy.

For cases with advanced esophageal cancer who have not

received previous treatment, radiotherapy targeting both the primary

and metastatic lesions is a vital therapeutic option. This intervention

significantly improves dysphagia and nutritional status when directed

at the primary lesion. It also alleviates pain and other symptoms, while

inducing an abscopal effect in metastatic cases. Two retrospective

studies indicated that compared to the use of chemotherapy alone, the

combination of chemotherapy with radiotherapy modestly enhances

the objective response rate and prognosis10,11. However, in the current

landscape where chemo-immunotherapy is the standard for advanced

esophageal cancer, radiotherapy is predominantly considered a salvage second-line treatment. Notably, reports on the use of radiotherapy with chemo-immunotherapy in cases of local recurrence and/

or distant metastasis after first-line treatment failure have demonstrated clinical benefits and acceptable safety12,13. Furthermore, some

studies have primarily focused on patients with metachronous oligometastatic esophageal cancer, considering radical radiotherapy

exclusively for metastatic lesions14,15. In a recent prospective trial

involving patients with a controlled esophageal primary lesion, the

group receiving local treatment—mainly consisting of stereotactic

body radiotherapy (SBRT) for metastatic lesions together with systemic therapy (chemotherapy or chemo-immunotherapy)—exhibited a

median PFS of 15.3 months, compared to 6.4 months for systemic

therapy alone15. Nonetheless, the safety and efficacy of this approach

as a primary treatment strategy in treatment-naive advanced ESCC

have yet to be substantiated by extensive research data, although

multiple ongoing prospective clinical studies are anticipated to publish their findings soon16–18. Consequently, the optimal treatment

approach for treatment-naive advanced ESCC and the role of radiotherapy within this regimen remains a subject of debate. This

encompasses the decision on when to integrate radiotherapy with

chemo-immunotherapy and the consideration of the simultaneous

irradiation of primary and metastatic lesions.

Herein, we conduct a single-arm trial to assess the safety and

efficacy of combining radiotherapy with chemo-immunotherapy as a

first-line treatment for advanced ESCC. The addition of radiotherapy to

first-line chemo-immunotherapy results in a median PFS of 9.8 months

and demonstrates a manageable safety profile. Despite the primary

endpoint (median PFS) was not met, this research highlights the feasibility of administering this combined treatment regimen to patients

with treatment-naive advanced ESCC.

Results

Participants and treatment

From June 30, 2021 to September 30, 2022, we assessed 56 patients for

eligibility; among them, 33 (median age: 59 years; range: 43–74; 29

men) were enrolled (Fig. 1). The most common site for oligometastasis

was distant lymph nodes (63.6%), followed by the lungs (15.2%), and

bones (9.1%) (Table 1). We observed that 27 (81.8%) patients had a total

of 32 oligometastatic lesions, of which 12 were in distant organs and 15

in non-regional lymph nodes, whereas six patients (18.2%) had only

regional lymph node metastases (cTanyN3M0). Treatment was permanently stopped prior to the commencement of radiotherapy in five

patients for the following reasons: informed consent withdrawal

(n = 1), supraventricular arrhythmia (n = 1), esophageal fistulae (n = 2),

and disease progression (liver metastasis; n = 1). Among the 28 patients

who started radiotherapy, one withdrew consent after completing

three sessions of radiotherapy, and another refused radiotherapy for

liver metastasis after completing radiotherapy for the primary lesion. A

total of 26 patients (78.8% of the enrolled individuals) completed

radiotherapy of all lesions (both esophageal and metastatic) and four

chemotherapy cycles. Among these patients, 14 (42.4%) completed the

planned 1-year treatment with toripalimab, with a median of 10 treatment cycles (interquartile range [IQR]: 4.5–14). The primary reasons

for the early discontinuation of toripalimab included disease progression (n = 10), adverse events (AEs) (n = 4), and informed consent

withdrawal (n = 5). Detailed information is available in Supplementary

Table 1.

Efficacy outcomes in patients who completed radiotherapy of all

lesions

We evaluated the efficacy of the treatment regimen in the 26 patients

who completed radiotherapy of all lesions (esophageal and metastatic)

and four chemotherapy cycles. Assessment at 3 months after completing the radiotherapy revealed partial response (PR) in 15 patients

(57.7%), stable disease (SD) in four patients (15.4%), and disease progression in seven patients (26.9%). Notably, the objective response rate

(ORR) was 57.7% (15/26; 95% confidence interval [CI]: 36.9–76.7%),

whereas the disease control rate (DCR) was 73.1% (19/26, 95% CI:

52.2–88.4%) (Supplementary Table 2). The 1-year PFS and OS rates

were 50.0% (95% CI: 34–73.4%) and 76.9% (95% CI: 62.3–94.9%),

respectively. The median PFS was 12.8 months (95% CI:

8.0 months–not estimable) (Fig. 2A) and the median OS was not

attained (Fig. 2B).

Efficacy outcomes in intent-to-treat (ITT) patients

Analysis of the best overall response showed reductions in the sizes of

target lesions after treatment compared to baseline in 27 of the 33

enrolled patients (Fig. 3A). Responses included complete response

(CR) in seven cases, PR in 13, and SD in seven cases. Four patients

exhibited increases in the target lesion size, including one patient with

disease progression, while two patients could not be evaluated. Three

months after the completion of radiotherapy, an efficacy evaluation

was conducted on the ITT population. The results showed an ORR of

Fig. 1 | Trial profile. A total of 56 patients were screened, among them 23 were

excluded and 33 patients were included. Five patients, after completing at least one

cycle of chemo-immunotherapy, did not receive radiotherapy due to adverse

effects, tumor progression, or withdrawal of informed consent. Two patients discontinued the study treatment as they did not complete the entire course of

radiotherapy as per the study protocol.

Article https://doi.org/10.1038/s41467-024-51105-2

Nature Communications | (2024) 15:7116 2

45.5% (15/33; 95% CI: 28.1–63.7%) and a DCR of 57.6% (19/33; 95% CI:

39.2–74.5%) (Supplementary Table 2). A summary of the tumor

responses observed following two cycles of chemo-immunotherapy is

provided in Supplementary Table 3.

Overall, the median follow-up was 22.2 (range: 16.3–28.1) months

and the median duration of response (DoR) was 11.5 (IQR, 6.4–15.0)

months. Among the 33 patients, 21 experienced recurrences (64%), and

16 died because of the disease (48%). A summary of the information on

recurrence (pattern, site, and reasons for death) is provided in Supplementary Table 4. Responses and outcomes are summarized in

Fig. 3B. The 1-year PFS and OS rates were 41.9% (95% CI: 27.7–63.5%)

and 69.7% (95% CI: 55.7–87.3%), respectively. The median PFS was 9.8

months (95% CI: 6.83 months–not estimable; primary endpoint was

not met) (Fig. 2C), while the median OS was 16.5 months (95% CI:

13.2 months–not estimable) (Fig. 2D). Subsequent treatments after

recurrence are provided in Supplementary Table 5.

Safety

All 33 cases experienced treatment-related AEs (TRAEs) of varying

grades (Table 2). No unexpected AEs or treatment-related deaths

occurred. Commonly observed TRAEs included myelosuppression,

weight loss, and anorexia, and commonly observed grade 3 or higher

AEs were lymphopenia (27/33, 82%), neutropenia (9/33, 27%), and

leukopenia (8/33, 24%). More specifically, radiotherapy-related AEs

mainly included radiation esophagitis (24/28, 86%), radiation dermatitis (21/28, 75%), esophageal/epigastric pain (18/28, 64%), and radiation pneumonitis (6/28, 22%) (Table 3). The most frequent immunerelated AEs were hypertriglyceridemia (21/33, 64%), hypothyroidism

(18/33, 54%), and rash (4/33, 12%). Three patients (9.1%) developed

grade 3 esophageal fistula; two immediately after two cycles of chemoimmunotherapy, and one at 4 months after the completion of radiotherapy. All three patients who developed esophageal fistula had T4

tumors (tumor length >5 cm).

Biomarkers for treatment response and outcomes

We conducted a prespecified exploratory analysis of biomarkers to

assess treatment response and outcomes. Baseline tumor biopsies

from 76.9% (20/26) of patients were available for multiple immunofluorescence (mIF) analysis. Representative mIF images showed higher

infiltration of immune cells in three patients who achieved PR (Fig. 4A,

upper panel) compared to the corresponding in three patients who

achieved SD (Fig. 4A, lower panel). Patients with clinical PR exhibited

significantly greater densities of CD68+ macrophages (p = 0.019)

compared to those with SD (Fig. 4B). Additionally, there was a trend

toward increased densities of CD8 + T cells and CD11c+ dendritic cells

(DCs) in the PR group. Furthermore, higher densities of overall PDL1+

cells, including PDL1+ tumor cells, PDL1+ DCs, and PDL1+ macrophages, were observed in the PR group compared to those in the SD

group, with a statistically significant difference found in PDL1+ DCs

(p = 0.037) (Fig. 4B). Importantly, infiltration of PDL1+ DCs and PDL1+

macrophages in the stromal compartment positively correlated with

better treatment response (Supplementary Fig. 1A).

Higher infiltration of CD8 + T cells (PFS, p = 0.012; OS, p = 0.005),

CD11c+ DCs (PFS, p = 0.039; OS, p = 0.026), and CD68+ macrophages

(PFS, p = 0.008; OS, p = 0.04) correlated with PFS and OS (Fig. 4C).

Elevated numbers of PDL1+ cells showed a tendency toward better OS

(p = 0.055) but not PFS (p = 0.68) (Fig. 4C). Moreover, higher densities

of PDL1+ macrophages in the stroma were associated with improved

PFS and OS (Supplementary Fig. 1B).

To further investigate whether peripheral cytokines could predict

treatment response and patient outcomes, sera from 25 patients were

analyzed at both baseline and during therapy (20 baseline samples and

20 treatment samples, with 15 paired samples; Supplementary Fig. 2A).

The results indicated that levels of the eight tested cytokines were

similar between the baseline and on-treatment groups (Supplementary

Fig. 2B). Specifically, on-treatment levels of interferon-gamma (IFN-γ)

were significantly higher in patients who achieved PR compared to those

who achieved SD (Fig. 5A, p = 0.026). While there was a tendency for

higher baseline levels of IFN-γ and IL-10 in the PR group, no significant

differences were observed for interleukin (IL)−2, IL-4, IL-6, IL-17, IL-37, or

tumor necrosis factor-alpha (TNF-α) (Fig. 5A, Supplementary Fig. 3).

Moreover, higher baseline levels of IFN-γ (p < 0.001) and lower ontreatment levels of IL-6 (p = 0.033) were associated with improved PFS

(Fig. 5B). Furthermore, higher baseline levels of IL-4 (p < 0.001),

baseline IL-10 (p = 0.041), baseline IFN-γ (p < 0.001), on-treatment IFNγ (p = 0.007), baseline TNF-α (p = 0.005), and baseline IL-17 (p = 0.012)

were significantly associated with better OS (Fig. 5B). No associations

Table 1 | Baseline patient characteristics

Characteristics Total (n = 33)

Age, years; median (IQR) 59 (55–69)

Sex

Male 29 (87.9%)

Female 4 (12.1%)

Smoking history

Yes 25 (75.8%)

No 8 (24.2%)

Alcohol history

Yes 24 (72.7%)

No 9 (27.3%)

Bodyweight loss

<10% 30 (90.9%)

≥10% 3 (9.1%)

ECOG performance status

0 15 (45.5%)

1 18 (54.5%)

Tumor location

Upper 6 (18.2%)

Middle 12 (36.4%)

Lower 15 (45.5%)

Primary tumor length (cm)

≤5 7 (21.2%)

>5 26 (78.8%)

Clinical T stage

T3 26 (78.8%)

T4 7 (21.2%)

Clinical N stage

N2 14 (42.4%)

N3 19 (57.6%)

Clinical M stage

M0 6 (18.2%)

M1 27 (81.8%)

Clinical TNM stage

IV 33 (100%)

Site of metastases

Distant lymph nodes 22 (66.7%)

Lung 6 (18.2%)

Liver 1 (3.0%)

Bone 3 (9.1%)

Spleen 1 (3.0%)

Adrenal gland 1 (3.0%)

Data are presented as n (%). ECOG Eastern Cooperative Oncology Group, IQR interquartile range.

Article https://doi.org/10.1038/s41467-024-51105-2

Nature Communications | (2024) 15:7116 3

with PFS or OS were found for other serum cytokine levels (Supplementary Fig. 4).

Discussion

In the regimen of combining chemotherapy with immunotherapy for

advanced esophageal cancer, the role of radiotherapy remains

unknown. This phase 2 trial reported the safety, efficacy, and identification of candidate biomarkers for radiotherapy outcomes combined

with chemo-immunotherapy in treatment-naive advanced ESCC cases.

Our results revealed that radiotherapy targeting the primary esophageal and metastatic lesions is promisingly effective and has manageable toxicity when combined with chemo-immunotherapy.

In clinical practice, chemo-immunotherapy remains the preferred

approach for advanced esophageal cancer3,19. However, adding radiotherapy to primary and metastatic lesions has been shown, in certain

retrospective studies, to enhance local tumor control, potentially

delaying disease progression and prolonging survival5,6,20–22. Despite

this, chemo-immunotherapy remains the standard treatment, and

evidence supporting the safety and therapeutic benefits of adding

radiotherapy is currently lacking in prospective clinical research,

especially regarding the potential additive toxicity of combined radioand immunotherapy. In the present study, we observed a median PFS

of 9.8 months and a 1-year PFS rate of 41.9% in the ITT population.

Although the results did not meet the pre-specified primary endpoints,

they compared favorably to the median PFS reported in previous studies, such as the KEYNOTE-590 (6.3 months), JUPITER-06 (5.7 months),

and Checkmate-648 (5.8 months) trials3,4,7

. Moreover, our

Kaplan–Meier estimates of PFS indicated a plateau after 1 year, whereas

the curves for OS appeared to plateau after approximately 16 months,

suggesting long-term survival benefits in cases of advanced ESCC following treatment with radiotherapy plus chemo-immunotherapy.

Notably, most of our studied patients had synchronous oligometastatic

disease, with some patients exhibiting locally advanced N3 disease.

Increasing evidence suggests that higher numbers of individuals with

oligometastatic disease can attain long-term survival using local

treatment together with systemic therapy than those with multiple

metastases23–25. However, longer-term follow-up studies together with

prospective randomized controlled trials are required to verify these

findings. Additionally, there is no definitive evidence regarding the

optimal duration of immune checkpoint inhibitor (ICI) therapy in

patients with advanced esophageal cancer. A 2-year immunotherapy

maintenance period may pose greater toxic side effects for patients

who do not respond to the treatment. Therefore, we explored whether

short-course ICI therapy (1-year immunotherapy maintenance) combined with systemic therapy and radiotherapy could enhance efficacy

while reducing drug toxicity and financial burden for patients.

Patients with advanced esophageal cancer include those with

synchronous metastasis, metachronous metastasis, and locoregional

recurrence. Thus, in studies on advanced esophageal cancer combining radiotherapy with chemo-immunotherapy, the enrolled patients

exhibited substantial heterogeneity. In the ESO-SHANGHAI 13 study15,

approximately 90% of the patients developed metachronous oligometastatic disease after curative treatment, with radiotherapy limited

to metastatic lesions. Thus, the efficacy and safety of simultaneously

combining radiotherapy with systemic treatment for the primary

lesion remains unclear. The ongoing EC-CRT-003 trial is enrolling

patients with treatment-naive stage IVb ESCC17, and is considering

adding thoracic radiotherapy (45–50 Gy/25–28 f) after 4–6 cycles of

standard chemo-immunotherapy, without irradiating metastatic

lesions. Similar clinical studies are in progress26–31. Collectively, these

study designs reveal considerable debate over the optimal timing and

target area of radiotherapy intervention in chemo-immunotherapy for

advanced esophageal cancer. This debate centers on the following

issues: first, whether radiotherapy is added during first-line systemic

treatment in treatment-naive patients, after completing primary lesion

treatment, or as a second-line treatment for tumor recurrence and

Fig. 2 | Kaplan−Meier estimates of survival. A, B PFS and OS in the efficacy-evaluable population (n = 26). C, D PFS and OS in the ITT population (n = 33). The gray shaded

area represents the 95% CI. Source data are provided as a Source Data file. CI confidence interval, PFS progression-free survival, OS overall survival, ITT intention-to-treat.

Article https://doi.org/10.1038/s41467-024-51105-2

Nature Communications | (2024) 15:7116 4

metastasis; and second, whether radiotherapy targets only the primary

lesion, metastatic lesions, or both.

Our study enrolled treatment-naive patients with advanced esophageal cancer, with 81% of the patients being oligometastatic, indicating a higher tumor burden at initial treatment compared to the ESOSHANGHAI 13 study15. Therefore, in our study, radiotherapy was conducted concurrently after two cycles of systemic treatment to reduce

the tumor burden and shrink the radiotherapy target volume, thus

mitigating radiotherapy toxicity. During the third cycle of chemoimmunotherapy, concurrent radiotherapy was performed because of

the apparent synergistic mechanism of radiotherapy and chemoimmunotherapy. Radiotherapy of the primary lesion can increase

the production of tumor antigens, thus, enhancing antigen presentation via DCs32. Moreover, SBRT for metastatic lesions may achieve

systemic antitumor immunity through local activation33. Notably, our

radiotherapy covered both primary and metastatic lesions. An opinion

piece reported that targeting all lesions could enhance the likelihood

of successfully initiating an antitumor immune response, overcome

the problem of tumor heterogeneity, and enhance the destruction of

drug-resistant subclones34. However, in our study, the primary endpoint was not met in the ITT population, which may be related to

the sandwich treatment strategy we used. Unless radiotherapy and

chemo-immunotherapy are fully synchronized during the initial

treatment, various circumstances, including disease progression and

treatment side effects, may prevent the administration of radiotherapy

following chemo-immunotherapy in advanced esophageal cancer, as

observed in this study. The results from the efficacy-evaluable group

indicate that patients who can tolerate and respond to chemoimmunotherapy are more suitable candidates for the addition of local

radiotherapy.

Fig. 3 | Tumor responses. A Best percentage changes in target lesion sizes from

baseline (n = 31). Dashed lines at +20% and −30% represent thresholds for disease

progression and partial response, respectively, according to the RECIST 1.1 criteria.

Among the total 33 patients, One patient withdrew informed consent after one

cycle of chemo-immunotherapy. One patient experienced severe cardiac adverse

events after two cycles of chemo-immunotherapy. Both patients refused further

assessment. Therefore, only 31 patients had a baseline and at least one postbaseline radiologic assessment. B Onset of response, duration of response, and

outcome (n = 33). Source data are provided as a Source Data file. RECIST Response

Evaluation Criteria in Solid Tumors.

Article https://doi.org/10.1038/s41467-024-51105-2

Nature Communications | (2024) 15:7116 5

An increasing body of evidence now indicates that the interaction

between the tumor microenvironment and cancer cells is a key factor

in tumor progression35. Our analyses using multiple immunohistochemistry/immunofluorescence (mIHC/mIF) techniques indicate

that higher densities of CD8 + T cells, CD11c+ DCs, and CD68+ macrophages correlate with improved tumor response and prognosis.

This effect is attributed to the enhanced tumor immune response from

the adequate infiltration of these immune cells in the immune

microenvironment, a relationship well-established in prior studies36–38.

However, there have been conflicting reports on the predictive value

of PD-L1 status in esophageal cancer3,4,7,36. This variation in findings

may arise because the studies investigating this have typically used

immunohistochemistry to detect PD-L1 expression, with the combined

positive score (CPS) being the most commonly used. However, CPS

only includes tumor-associated immune cells, such as macrophages

and lymphocytes, which are in close proximity to the tumor cells.

Conversely, our study examined PD-L1 expression in a broader range

of cells, including tumor cells, macrophages, and dendritic cells. Our

results show that a high expression of PD-L1 on tumor and immune

cells is associated with better tumor outcomes. This finding suggests

that comprehensive PD-L1 detection across all relevant cells may more

accurately predict a patient’s treatment response. Moreover, our

findings are in concordance with existing studies4–6 that highlight the

beneficial predictive value of PD-L1 in cancer therapy.

Further, our findings demonstrate a significant correlation

between elevated baseline serum IFN-γ levels and reduced ontreatment IL-6 levels and an extended PFS in patients with advanced

ESCC. A previous investigation established a correlation between

reduced IL-6 levels and improved prognosis in advanced melanoma39,

and attributed this to the role of IL-6 in accelerating tumor progression

through the inhibition of cancer cell apoptosis and the promotion of

angiogenesis. In contrast, IFN-γ activates antitumor immune cells and

suppresses immunoregulatory cells in immune antigens40. Therefore,

IFN-γ and IL-6 could serve as reliable predictors of response to combined immunotherapy and chemoradiotherapy in advanced esophageal cancer cases. IL-4, IL-10, and TNF-α play multifaceted roles in

cancer immunity, activating antitumor immune cells and facilitating

tumor immune suppression and escape. IL-10, IL-17, and TNF-α, known

for their immunosuppressive properties, promote tumor growth and

are associated with poor prognosis, as corroborated by several clinical

studies, whereas IL-4 is considered an enhancer of immune cell antitumor activity41–44. Our results showed that higher baseline levels of IL4, IL-10, and IFN-γ, as well as elevated treatment levels of IFN-γ, TNF-α,

and IL-17, were associated with improved OS. This result contrasts with

previous findings and highlights the complexity and variability of the

local tumor immune microenvironment. As different cancers harbor

distinct immunosuppressive cells and cytokines within their tumor

microenvironment, and various ICIs employ unique mechanisms of

action, the relationship between cytokines and immunotherapy efficacy warrants further exploration.

A key issue in combining first-line radiotherapy with chemoimmunotherapy is safety. The present findings corroborate those of

earlier clinical trials combining ICIs with radiotherapy for esophageal

cancer36,45. In this study, we did not observe any unexpected safety

signals, and most TRAEs were of grades 1–2. Grade 3 or higher TRAEs

predominantly included myelosuppression, with no treatmentrelated deaths. Of note, 17 (51.5%) patients in this trial were still

alive at the time of cut-off. Importantly, among the three cases of

esophageal fistula, two occurred after two cycles of chemoradiotherapy and prior to radiotherapy. Retrospective analysis indicated that the incidence of esophageal perforation or fistula in

patients with the T4 stage was as high as 30.1%. Hence, there may be a

link between the occurrence of esophageal fistula and the clinical

stages of the primary tumors, as all three patients presented with

cT4 stage with tumor length >5 cm. In summary, the precise influence

of the addition of PD-1 inhibitors with chemoradiotherapy on fistula

risk requires further investigation.

This study had several limitations. First, it was a single-arm study

conducted at a single center with a small sample size, which may have

led to selection bias, thus, limiting the generalizability of the findings.

Second, although all patients received the necessary imaging and

multidisciplinary evaluation before enrollment, not all metastatic

lesions were pathologically confirmed. Third, this study investigated

biomarkers; however, the limited sample size precludes definitive

conclusions. These results may inform the design of future large-scale

trials. To enhance the reliability of our findings, we are currently participating in a multi-center, randomized, controlled, phase III clinical

trial to examine the effect of first-line radiotherapy combined with

chemo-immunotherapy in 100 patients (ClinicalTrials.gov identifier:

ChiCTR2300070300).

Table 2 | Treatment-related adverse events in all patients

Total (n = 33)

Grade 1 Grade 2 Grade 3 Grade 4

Treatment-related adverse events, n (%)

Lymphopenia 0 2 (6%) 18 (55%) 9 (27%)

Leukopenia 1 (3%) 14 (42%) 7 (21%) 1 (3%)

Neutropenia 6 (18%) 7 (21%) 8 (24%) 1 (3%)

Thrombocytopenia 14 (42%) 6 (18%) 1 (3%) 0

Anemia 22 (67%) 5 (15%) 3 (9%) 1 (3%)

Elevated triglyceride 17 (52%) 2 (6%) 2 (6%) 0

Bilirubin elevation 6 (18%) 2 (6%) 0 0

Hypoproteinemia 31 (94%) 2 (6%) 0 0

Creatinine increased 3 (9%) 0 0 0

AST elevation 17 (52%) 1 (3%) 0 0

ALP elevation 10 (30%) 1 (3%) 0 0

Gamma-glutamyl transferase elevation

9 (27%) 1 (3%) 2 (6%) 0

ALP elevation 7 (21%) 0 1 (3%) 0

Hypothyroidism 14 (42%) 4 (12%) 0 0

Anorexia 3 (9%) 8 (24%) 0 0

Fatigue 2 (6%) 1 (3%) 0 0

Nausea or vomiting 3 (9%) 5 (15%) 2 (6%) 0

Constipation 4 (12%) 3 (9%) 0 0

Rash 2 (6%) 1 (3%) 1 (3%) 0

Arrhythmia 1 (3%) 1 (3%) 1 (3%) 0

Weight loss 7 (21%) 5 (15%) 0 0

Fever 1 (3%) 0 0 0

Diarrhea 1 (3%) 1 (3%) 0 0

Esophageal fistula 0 0 3 (9.1%) 0

ALP alkaline phosphatase, ALT alanine aminotransferase, AST aspartate aminotransferase, GGT

gamma-glutamyl transferase.

Table 3 | Radiotherapy-related adverse events

Total (n = 28)

Grade 1 Grade 2 Grade 3 Grade 4

Radiotherapy-related adverse events, n (%)

Esophagitis 9 (32%) 12 (43%) 3 (11%) 0

Radiation dermatitis 21 (75%) 0 0 0

Cough 1 (4%) 8 (29%) 0 0

Pneumonitis 0 3 (11%) 3 (11%) 0

Esophageal/epigastric pain 12 (43%) 6 (21%) 0 0

Article https://doi.org/10.1038/s41467-024-51105-2

Nature Communications | (2024) 15:7116 6

CD8 CD4 CD68 CD11c PD-L1 panCK DAPI

0.067

100

300

1000

3000

PR SD

0.64

100

300

1000

3000

PR SD

CD8+ cells (counts/mm2

)

CD4+ cells (counts/mm2

)

0.067

300

1000

3000

10000

PR SD

CD11c+ cells (counts/mm2

)

0.019

300

1000

3000

PR SD

CD68 cells (counts/mm2

)

0.11

100

1000

10000

PR SD

Response

PDL+ cells (counts/mm2

)

0.18

10

100

1000

PR SD

Response

PDL1+CK (counts/mm2

)

0.037

30

100

300

1000

3000

PR SD

Response

PDL1+DC (counts/mm2

)

0.097

30

100

300

1000

3000

PR SD

Response

PDL1+CD68 (counts/mm2

)

+++ +

+

p = 0.012 0.00

0.25

0.50

0.75

1.00

0 5 10 15 20 25

+

+

CD8+ low

CD8+ high

+++ + +

p = 0.039 0.00

0.25

0.50

0.75

1.00

0 5 10 15 20 25

+

+

CD11c+ low

CD11c+ high

+++ +

+

p = 0.008

0.00

0.25

0.50

0.75

1.00

0 5 10 15 20 25

+

+

CD68+ low

CD68+ high

+

++

+ +

p = 0.68

0.00

0.25

0.50

0.75

1.00

0 5 10 15 20 25

+

+

PDL1+ low

PDL1+ high

+

++++++ + +++

p = 0.005 0.00

0.25

0.50

0.75

1.00

0 5 10 15 20 25

Time (months)

+

+

CD8+ low

CD8+ high

++

+

+++++ + ++

p = 0.026 0.00

0.25

0.50

0.75

1.00

0 5 10 15 20 25

Time (months)

+

+

CD11c+ low

CD11c+ high

+ +

+

+++++ + ++

p = 0.04 0.00

0.25

0.50

0.75

1.00

0 5 10 15 20 25

Time (months)

+

+

CD68+ low

CD68+ high

+

+

+++++ + + ++

p = 0.055 0.00

0.25

0.50

0.75

1.00

0 5 10 15 20 25

Time (months)

+

+

PDL1+ low

PDL1+ high

Progression−free survival Overall survival

n = 14

n = 6

n = 14

n = 6

n = 14

n = 6

n = 14

n = 6

n = 14

n = 6

n = 14

n = 6

n = 14

n = 6

n = 14

n = 6

A

B

C

Fig. 4 | Biomarkers in the tumor microenvironment. A Representative mIF

images of CD8, CD68, CD11c, and CD4 cells in the tumor immune microenvironment from patients achieving clinical PR (upper panel, n = 3 samples) and SD (lower

panel, n = 3 samples). Scale bars: 100 μm. B Immune cell infiltration levels in the

tumor tissues between patients achieving PR (n = 13 samples) and those achieving

SD (n = 7 samples) assessed by mIF. For box plots, the central line represents the

median value, the bottom and top of the box represent the values of the 25th and

75th percentile, and the lower and upper whiskers represent the minimum and

maximum value of the data, respectively. The p-values are derived from a two-tailed

Mann–Whitney test. C Progression-free and overall survival analyses for the tumor

infiltration levels of diverse immune cells. Patients were grouped into low- and highexpression of each variable as described in the Methods (n = 20 samples). The pvalues are derived from a two-tailed Log-rank test. Source data are provided as a

Source Data file. Abbreviations: mIF multiplex immunofluorescence, PR partial

response, SD stable disease.

Article https://doi.org/10.1038/s41467-024-51105-2

Nature Communications | (2024) 15:7116 7

In conclusion, in patients with treatment-naive, advanced ESCC,

first-line radiotherapy to both primary and metastatic lesions in combination with chemo-immunotherapy demonstrated some antitumor

activity with a manageable safety profile. Furthermore, our findings

provide insights into potential biomarkers for assessing clinical

effectiveness.

Methods

Study design and patients

This single-arm, open-label, phase 2 trial was conducted at Sichuan

Cancer Hospital (Chengdu, Sichuan Province, China). The study was

performed in compliance with the Declaration of Helsinki and Good

Clinical Practice guidelines, and was approved by the Institutional

Review Board of Sichuan Cancer Hospital, Sichuan, China (Ethics

number: SCCHEC-02-2021-021). An interim analysis, authorized by the

Institutional Review Board of Sichuan Cancer Hospital at a later stage,

included data on some secondary endpoints (ORR, DCR, toxicity) to

provide an early assessment of efficacy and to help identify and

address safety issues early. All patients provided written informed

consent before any procedure. No participation compensation was

provided. The trial was registered with chictr.org.cn

(ChiCTR2100046715) on May 27, 2021. Analyses were conducted as

planned in the preregistration. There were some minor deviations

from the preregistration regarding the radiotherapy scheme, followFig. 5 | Peripheral cytokines predict treatment response and patient survival.

A Serum levels of different cytokines at baseline or on-treatment between patients

achieving a PR (n = 14 samples) and those achieving SD (n = 6 samples). For box

plots, the central line represents the median value, the bottom and top of the box

represent the values of the 25th and 75th percentile, and the lower and upper

whiskers represent the minimum and maximum value of the data, respectively. The

p-values are derived from a two-tailed Mann–Whitney test. B Progression-free and

overall survival analyses for different periphery cytokines. Patients were grouped

into low- and high-expression of each variable as described in the Methods

(n = 20 samples). The p-values are derived from a two-tailed Log-rank test. Exact pvalues: baseline IFN-γ for PFS, p = 0.000044; baseline IFN-γ for OS, p = 0.0000075;

baseline IL4 for OS, p = 0.00099. Source data are provided as a Source Data file.

Abbreviations: IFN-γ interferon-gamma, IL interleukin, PR, partial response, TNF

tumor necrosis factor.

Article https://doi.org/10.1038/s41467-024-51105-2

Nature Communications | (2024) 15:7116 8

up, statistical analyses, and other aspects. These deviations are explicitly indicated at the end of the Supplementary Note (see section

“Summary of Amendments to Protocol”). The first patient was enrolled

on June 30, 2021, and the last patient on September 30, 2022.

Patients (aged 18–75 years) with histologically diagnosed unresectable, treatment-naive, stage IV ESCC based on the eighth Edition of

the American Joint Committee on Cancer (AJCC) tumor, node,

metastasis (TNM) staging system, with multiple lymph node metastases (N3) or distant oligometastases (M1) were eligible for inclusion.

Other inclusion criteria included having a minimum of one measurable

lesion, in terms of the Response Evaluation Criteria in Solid Tumors

(RECIST) version 1.1, Eastern Cooperative Oncology Group performance status (ECOG PS) score of 0–1, a life expectancy ≥6 months, and

sufficient bone marrow and organ function. The exclusion criteria

included a history of any other malignancy, metastases to the central

nervous system, a previous history of immunotherapy, a history of

autoimmune or interstitial lung disease, or serious comorbidities, such

as congestive heart failure or uncontrolled diabetes. The study protocol is presented as Supplementary Note in the Supplementary

information file.

Definition of oligometastasis

In our study, oligometastasis was defined as ≤5 metastatic lesions in ≤3

metastatic organs; notably, the involvement of a single non-regional

lymph node station was also considered an oligometastasis46. For

example, the presence of ≥1 lymph node metastases in the left axilla

was considered a single oligometastatic lesion.

Definition of regional lymph nodes and distant (non-regional)

lymph nodes

In this study, the eighth edition of the AJCC TNM staging system was

used47. The regional lymph nodes, irrespective of the primary tumor

site, are those within the esophageal drainage area, including the

coeliac axis nodes and paraesophageal nodes in the neck, but not

supraclavicular nodes. This includes nodes in regions 1 R/1 L/2 R/2 L/

4 R/4 L/7/8U/8 M/8 L/9 R/9 L/15/16/17/18/19/20. Specifically, lymph

nodes located above the upper boundary of Region 1 (above the apex

of the lung) or below the lower boundary of Region 20 (below the

coeliac artery) are considered distant (non-regional) lymph nodes.

Additionally, lymph nodes located in the anterior mediastinum,

supraclavicular region, axilla, and groin, beyond the previously defined

regions, are considered distant metastatic lymph nodes (M1).

Treatments

Each chemo-immunotherapy cycle lasted for 3 weeks and consisted of

240 mg toripalimab and 135–175 mg/m2 paclitaxel plus carboplatin

(area under the curve, 4–6) on day 1. Concurrent radiotherapy was

initiated on the third chemo-immunotherapy cycle. Primary lesions

were treated with intensity-modulated radiotherapy at 50–50.4 Gy in

25–28 fractions 5 days/week. The gross tumor volume (GTV) included

the primary tumor (GTV-P), metastatic lymph nodes (GTV-N), and

metastatic lesions (GTV-M). The planning target volume was defined as

GTV with an additional 1–2 cm at the proximal and distal margins and a

radical margin of 0.5–1.0 cm. For distant lymph nodes, such as supraclavicular and retroperitoneal lymph nodes, conventionally fractionated radiotherapy (50–50.4 Gy in 25–28 fractions) was administered

at the physician’s discretion, in consideration of adjacent organs at risk,

such as the trachea, stomach, and intestines. SBRT was recommended

in cases with suitable oligometastatic lesions in the liver, lungs, or

bones, with consideration for the same factors. SBRT was administered

to all metastatic lesions at doses of 30–40 Gy in 3–5 fractions. The

delineation of the target area is shown in Supplementary Figs. 5, 6.

Upon completion of four chemo-immunotherapy cycles, chemotherapy was discontinued; toripalimab was continued at 240 mg

every 3 weeks for a maximum of 1 year or until the patient exhibited

disease progression or evidence of intolerable toxicity. Dose reduction

was permitted for paclitaxel and carboplatin but not for toripalimab.

Chemotherapy was suspended or deferred if grade ≥3 AEs occurred.

Follow-up and outcomes

Baseline computed tomography examination was performed within

14 days before treatment initiation. Tumor evaluations were conducted at 6-weekly intervals (± 7 days) during chemotherapy. Meanwhile, during chemoradiotherapy, tumor evaluations were conducted

once every 12 weeks (± 7 days) to the end of year 2, at 6-monthly

intervals during the 3rd and 4th years, and annually thereafter. Efficacy

assessments were performed in accordance with the RECIST 1.1 criteria. Laboratory analyses, including complete blood count, blood

chemical tests, electrocardiography, routine urine analysis and stool

examination, coagulation testing, and thyroid function testing, were

conducted once every 3 weeks. AEs were identified and monitored

using the National Cancer Institute Common Terminology Criteria for

Adverse Events version 5.0.

The primary endpoint was PFS, defined as the time between

beginning treatment and tumor progression, patient death, or the last

follow-up. Secondary endpoints included the ORR, DCR, DoR, 1- and

2-year OS rates, patient-reported health-related quality of life, and AEs.

Objective responses included CR and PR. Disease control represented

CR, PR, and SD, while DoR was determined as the interval between the

first objective response to the first documentation of progression or

all-cause death. OS was assessed from the initiation of therapy to allcause death. Exploratory outcomes included the relationship between

clinical outcomes with immune cell types in the tumor microenvironment, and biomarkers in peripheral blood (e.g., soluble PD-L1 and

cytokines). Two-year OS rates, quality of life, and soluble PD-L1 in

peripheral blood are not reported in this article because of data

immaturity; these results will be reported in future.

mIHC/mIF

Tumor biopsy sections were analyzed via mIHC/mIF analysis using the

Opal 7-color kit (NEL811001KT; Akoya Biosciences, Marlborough, MA,

USA), in accordance with the manufacturer’s instructions. Following

antigen retrieval in EDTA buffer (pH 9.0; 3 min, 125 °C) and cooling to

room temperature (RT), the sections were washed with ddH2O followed by TBST/0.5% Tween (repeated three times for 2 min each time).

Then, the slides were blocked with a blocking buffer at RT for 10 min

and treated with primary anti-PDL1 (ZA-0629, 1:50; ZSGB Biotech,

Beijing, China) at 37 °C for 60 min followed by rinsing in TBS. The

slides were incubated with an HRP-conjugated secondary antibody

(10 min, 37 °C) followed by TSA dye 620 (1:100) for 5 min after further

TBST washes. The same procedure was repeated for the other primary

antibodies; namely, anti-CD68 (ZM-0060, 1:100, dye480; ZSGB Biotech), CD8 (ZA-0508, 1:100, dye570; ZSGB Biotech), CD11c (45581,

1:400, dye520; Abcam, Cambridge, UK), CD4 (ZA-0509, 1:100, dye690;

ZSGB Biotech), and pan-cytokeratin (ZM-0067, 1:100, dye780; ZSGB

Biotech). Additional rounds of antigen retrieval were undertaken in

EDTA (pH 6.0) buffer using a pressure cooker for 2 min. DAPI was used

for nuclear staining (100 μL DAPI, 5 min, RT).

Whole slide images were scanned using the TissueFAXS SL system

(7.1.120; Tissue Gnostics, Vienna, Austria). Digital images were analyzed using the HALO™ software (v 3.5). Immune cell densities were

assessed as positively stained cell counts per mm2

. The cell density was

calculated in total, tumor, and stromal areas, respectively.

Periphery cytokines

Sera were obtained at baseline and during treatment (after two cycles

of chemo-immunotherapy and before radiotherapy). Inflammationrelated cytokines, namely IL-2, IL-4, IL-6, IL-10, IL-17, TNF-α, and IFN-γ,

were assessed using a magnetic beads kit (281004; Wellgrow, Beijing,

China). Briefly, 50 μL of the serum sample or reference standards was

Article https://doi.org/10.1038/s41467-024-51105-2

Nature Communications | (2024) 15:7116 9

added to 50 μL of capture beads suspension, mixed thoroughly, and

incubated at RT in the dark for 1 h. The supernatant was removed after

magnetic precipitation, and the beads were incubated with 100 μL of a

fluorescent-labeled antibody at RT for 1 h. After washing, the beads

were assessed using flow cytometry (BD FACSCanto™; BD Biosciences,

San Jose, CA, USA). List of antibody clones utilized for flow cytometry

are included in Supplementary Data 1. The flow cytometry gating

strategy is presented in Supplementary Fig. 7. The data were analyzed

using the FCAP ArrayTM Software (Version 3.0; BD Biosciences, Franklin

Lakes, NJ, USA). IL-37 levels were assessed using the human IL-37 ELISA

Kit (ab213798, Abcam), following the manufacturer’s instructions.

Statistical analysis

The necessary sample size was assessed based on an increase in

median PFS from 6.3 to 12 months with chemo-immunotherapy, as

previously described3

. This calculation assumed a significance level of

0.05 (one-sided), a statistical power of 80%, and a 20% dropout rate.

The follow-up duration was calculated from the time of enrollment to

the date of the last follow-up. To observe 16 PFS events, we calculated

that 32 patients were needed. This was based on an assumption of a

uniform accrual accomplished over a period of approximately

12 months, with an additional 12 months of follow-up subsequent to

the enrollment of the last patient. The data collection cut-off date was

October 12, 2023.

The ORR, DCR, and DoR were assessed in the following two

populations: the ITT group, which included all participants, and the

efficacy-evaluable group, comprising patients who actually received

radiotherapy of all lesions and underwent at least one post-baseline

disease assessment. Safety was assessed in all cases where a minimum

of one dose of the study drug had been administered. OS, PFS, and the

corresponding 95% CIs were determined using the Kaplan–Meier

method. Survival outcomes were analyzed using Log-rank tests. Clinical and demographic features, together with TRAEs, were analyzed

using descriptive statistical methods.

Differences in tumor-infiltrating immune cell densities and periphery levels of cytokines between the PR and SD groups were compared using the Mann–Whitney test. For PFS and OS analyses, patients

were categorized into those having low- and high-levels of immune cell

densities based on the 30th percentile values: CD8+ cells, 210 cells/mm2

;

CD11c+ cells, 1318 cells/mm2

; CD68+ cells, 257 cells/mm2

; PDL1+ cells,

190 cells/mm2

. Similarly, cutoffs for peripheral cytokines were based on

the 30th percentile values of each variable.

Associations between biomarkers and PFS and OS were assessed

using the Log-rank test. Because of the exploratory nature of this

clinical study, no adjustments were made for multiple comparisons.

Statistical analyses were conducted using R software (v4.3.1; Vienna,

Austria), SPSS 22.0 (IBM Corp., Armonk, NY, USA), and SAS 9.4 (SAS

Institute; Cary, NC, USA). Differences were considered statistically

significant at p < 0.05.

Reporting summary

Further information on research design is available in the Nature

Portfolio Reporting Summary linked to this article.

Data availability

All data requests will undergo review by Sichuan Cancer Hospital and

the study sponsor, Shanghai Junshi Biosciences Co., Ltd., to assess any

potential intellectual property or confidentiality obligations. A proposal detailing the study objectives and statistical analysis plan will be

required for evaluation. Additional materials may also be requested

during the evaluation process. Data will be available upon request 12

months after the publication of this article. Detailed individual data are

available under restricted access for both legal and ethical concerns.

Requests for access to de-identified participant data from this study

can be submitted via email to wangqifeng@scszlyy.org.cn,

accompanied by a detailed proposal for approval. Please allow 1 month

for a response to the request. Access to the shared data will require

signing a data access agreement with the sponsor. The raw identifying

individual participant data are protected and are not available due to

data privacy laws. The study protocol is available as Supplementary

Note in the Supplementary Information file. The remaining data are

available within the Article, Supplementary Information, or Source

Data file. Source data are provided with this paper and are also available

in Figshare at: https://doi.org/10.6084/m9.figshare.26387656. Source

data are provided with this paper.

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Acknowledgements

The authors thank the participating patients and their families and the

study centers and investigators for their contributions to the study. The

authors also thank Dongmei Tang, Qin Ren, and Jianchao Lu for their

assistance in data collection and sample checking. This trial was supported by grants from the Science and Technology Department of

Sichuan Province (2023YFS0488 and 2023YFQ0055) and the Immunotherapy Research Fund of the Radiotherapy Oncology Branch of the

Chinese Medical Association. All investigators received no remuneration. Shanghai Junshi Biosciences provided toripalimab but had no role

in the study design or writing of the manuscript. The manuscript was

edited and proofread by Editage.

Author contributions

W.Q.F., C.B.R., and L.Y. were the principal investigators and participated

in trial design, study management, data and toxicity review, review of the

report, supervision of the study, and final approval of the report. W.L.,

Z.J., and L.L. contributed to the writing of the protocol, recruitment and

Article https://doi.org/10.1038/s41467-024-51105-2

Nature Communications | (2024) 15:7116 11

treatment of the patients, data and trial management, data analysis and

interpretation, and writing of manuscript. L.B.S., W.G, W.Y., and Z.W.

participated in the recruitment and treatment of the patients, data and

trial management, and report preparation. L.X.F., H.W.W., H.S.Y., Z.Y.H.,

Y.L., and W.D.S. were responsible for statistical analysis and interpretation as well as data review. Z.W.C., P.Q.S., L.T., P.L., H.Y.T., and L.J.Y.

contributed to patient accrual, toxicity review, and review of the completed report. All authors have reviewed and approved the final draft. All

authors had full access to all data in the study. The corresponding author

held final responsibility for the decision to submit the manuscript for

publication.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information The online version contains

supplementary material available at

https://doi.org/10.1038/s41467-024-51105-2.

Correspondence and requests for materials should be addressed to

Yang Liu, Bangrong Cao or Qifeng Wang.

Peer review information Nature Communications thanks Xiumei Ma and

the other, anonymous, reviewer(s) for their contribution to the peer

review of this work. A peer review file is available.

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© The Author(s) 2024

1

Department of Radiation Oncology, Sichuan Clinical Research Center for Cancer, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center,

Affiliated Cancer Hospital of University of Electronic Science and Technology of China, Chengdu, Sichuan 610041, China. 2

Department of Thoracic

Surgery, Sichuan Clinical Research Center for Cancer, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, Affiliated Cancer Hospital of

University of Electronic Science and Technology of China, Chengdu, Sichuan 610041, China. 3

Department of Clinical Laboratory, Sichuan Clinical

Research Center for Cancer, Sichuan Cancer Hospital & Institute, Sichuan Cancer Center, Affiliated Cancer Hospital of University of Electronic Science

and Technology of China, Chengdu, Sichuan 610041, China. 4

Department of Pathology, School of Medicine, Sichuan Cancer Hospital & Institute,

Sichuan Cancer Center, University of Electronic Science and Technology of China, Chengdu, Sichuan 610041, China. 5

Department of Radiation

Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention

and Therapy, Tianjin, Tianjin’s Clinical Research Center for Cancer, Tianjin, China. 6

Sichuan Key Laboratory of Radiation Oncology, Sichuan Cancer

Hospital & Institute, Sichuan Cancer Center, Affiliated Cancer Hospital of University of Electronic Science and Technology of China, Chengdu,

Sichuan 610041, China. 7

These authors contributed equally: Lei Wu, Baisen Li, Gang Wan. 8

These authors jointly supervised this work: Yang Liu,

Bangrong Cao, Qifeng Wang. e-mail: liuyanglyon@uestc.edu.cn; caobangrong@uestc.edu.cn; wangqifeng@scszlyy.org.cn

Article https://doi.org/10.1038/s41467-024-51105-2

Nature Communications | (2024) 15:7116 12