Background
Esophageal cancer is one of the most common gastrointestinal malignancies in the world, with a high recurrence rate and morbidity, and has become a global public health problem [
1]. Esophageal squamous cell carcinoma (ESCC) is the predominant histological classification in China [
2]. Although surgery is the most effective treatment for esophageal cancer, most patients are not candidates for direct esophagectomy [
3,
4]. For these patients, radiotherapy is an important method to control local recurrence and optimize the surgical strategy [
5,
6]. However, local residual lesions often exist, and local control in these patients is poor; the 5-year survival rate of patients that receive radiotherapy alone is only 20.9% [
7]. Therefore, identifying new targets to enhance the efficacy of radiotherapy is urgently needed.
Exosomes are vesicular structures produced by multivesicular bodies (MVBs) with diameters ranging from 30 to 150 nm. Exosomes can carry genetic material and biological information between cancer cells and the microenvironment, and play a key role in the establishment of intercellular communication [
8,
9]. The released exosomes bind to the receptor on the receptor cell membrane mainly through membrane surface ligands, activating receptor-mediated signal transduction, direct endocytosis of the receptor cell to bring the exosomes into the cell, and direct fusion of the exosome membrane with the cell membrane to bring the contents into the cell, and can change their physiological state [
10,
11].
Non-targeting effects can reflect the phenomenon that radiation may indirectly affect non-irradiated cells. In this case, bystander cells receiving stress signals from irradiated cells may exhibit the same properties as irradiated cells. Related studies have shown that radiation can affect the abundance of exosome contents in irradiated tumor cells and share the induction effect of radiation on non-irradiated cells, which may affect the function of recipient cells [
12,
13]. A growing number of studies have reported that exosomes contain a variety of biological information, which can be used for biological processes such as cell proliferation [
14], angiogenesis [
15], invasion or metastasis [
16], therapeutic resistance [
17], epithelial-mesenchymal transformation, and immune regulation [
18], thereby inducing carcinogenesis and even promoting malignant behavior in cancer cells [
19,
20].
High mobility group box 1 (HMGB1) is a nonhistone protein that extensively binds to chromosomes in mammalian cells and acts as a molecular DNA chaperone during DNA replication, transcription, recombination and repair [
21]. After binding with DNA, HMGB1 regulates gene transcription and maintains the stability of nucleosome structures [
22]. When tumor cells are stimulated by physical, chemical, biological and other factors, HMGB1 can be rapidly transferred to the cytoplasm. The C-terminal amino acid residue of HMGB1 is acetylated, and HMGB1 is actively or passively secreted to the extracellular form by triggering lysophosphatidylcholine. These HMGB1-containing exosomes can bind to receptors to stimulate biological effects on target cells [
23,
24]. However, the relationship between exosomal HMGB1 and radioresistance in ESCC has not been elucidated.
In this study, we investigated whether plasma exosomal HMGB1 in patients with ESCC was an indicator of the radiotherapy response and the role of irradiation (IR)-induced exosomal HMGB1 in radioresistance.
Materials and methods
Clinical serum samples
The study enrolled 21 patients who were admitted to Department of Radiotherapy, the Fourth Hospital of Hebei Medical University (Shijiazhuang, China) from May 2020 to May 2021. Each patient was originally diagnosed with ESCC, and none of them received any prior antitumor treatment, including chemotherapy, radiotherapy, targeted therapy, immunotherapy, or surgical resection. Twenty-four healthy blood donors were recruited from the Physical Examination Center of our hospital. Approximately 1 ml of peripheral blood (EDTA-K2 anticoagulant) was collected from each participant and stored at − 80 °C until use. Treatment response was determined by the RECIST 1.1 (Response Evaluation Criteria in Solid Tumors). This study was approved by the Ethics Committee of the Fourth Hospital of Hebei Medical University.
ELISA
Plasma concentrations of HMGB1 were measured by using an ELISA kit from CUSABIO (CSB-E08223h, Wuhan, China) according to the manufacturer’s instructions.
Cell culture and X-ray IR
Human ESCC lines (KYSE150, TE1, KYSE450, KYSE410, KYSE30 and ECA109) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C and 5% CO2. All cells were obtained from the Research Center of the Fourth Hospital of Hebei Medical University.
ESCC cells were IR by a 6-MV Siemens linear accelerator (Siemens, Buffalo Grove, IL, USA) at room temperature. The total dose was 6/8/10 Gy, and the dose rate was 300 MU/min. The source-skin distance (SSD) was 100 cm, and cells were then collected at specific times for further study.
Exosome isolation and identification
When the degree of ESCC cell fusion reached 80%, the medium was replaced with exosome-free fetal bovine serum, and exosomes (Exos) were extracted from the supernatant 48 h later. In addition, the IR group was administered 8 Gy X-ray IR and cultured for 48 h, after which the supernatant was collected to extract exosomes (IR-Exos). Exosomes were carefully extracted from plasma or cell culture supernatant using the exoEasy Maxi Kit (cat. no. 76,064) as described previously [
25] and according to the instructions of the manufacturer. All steps were performed at 4 °C. The exosomes were resuspended in sterile PBS and stored at − 80 °C for subsequent experiments. According to the manufacturer’s instructions, a bicinchoninic acid assay (BCA) (Solarbio, Beijing, China) was used to determine the protein concentration of exosomes.
Exosomes were characterized by transmission electron microscopy (TEM), and their morphology was observed. Nanoparticle tracking analysis (NTA) was used to analyze the particle size and collect video images of exosomes. Western blotting was used to measure exosome surface markers (CD9, TSG101, HSP70, and Calnexin).
Immunofluorescence staining
KYSE150 and ECA109 cells were inoculated on 48-well slides and cultured overnight. The cells were fixed with 4% paraformaldehyde and then permeabilized with Triton X-100 (5%), and nonspecific binding was blocked with a BSA solution. The cells were treated with primary antibodies and incubated overnight at 4 °C. After the unbound primary antibody was removed, fluorescent secondary antibodies (dilution 1:200; cat. nos. SA00013-2 and SA00013-3; Proteintech) were added and incubated at 37 °C for 1 h. DAPI (Solarbio, Beijing, China) was added to the slides for nuclear staining, and glycerol was used to seal the slides. The cells were observed under an ECLIPSE Ti2 confocal laser scanning microscope. The primary antibodies used were anti-HMGB1 (dilution 1:500; cat. no. ab79823; Abcam) and anti-γH2AX (dilution 1:500; cat. no. ab81299; Abcam).
Exos and IR-Exos were fluorescently labeled according to the manufacturer’s instructions. Then, 4 mg/ml DIL solution (Med Chem Express (MCE) Princeton, NJ, USA) was added to PBS containing exosomes and incubates. Exosomes were extracted to remove excess dye. These DIL-labeled exosomes were cocultured with ESCC cells for 24 h, and then the cells were washed with PBS and fixed in 4% paraformaldehyde. Confocal laser microscopy was used to observe the uptake of DIL-labeled Exos and IR-Exos by ESCC cells in both groups. Finally, a laser confocal microscope (Nikon A1, Japan) was used for photography.
Cell transfection
The HMGB1 knockdown lentivirus was purchased from Gene Chem Co., Ltd. (Shanghai, China). The cells (5 × 105/well) were cultured in 6-well plates until they reached 50% confluence, and then the overexpression lentivirus and the negative control (NC) lentivirus were added and incubated for 24 h according to the instructions. The transfection efficiency was observed by fluorescence microscopy after 72 h. The cells were screened with 1 µg/mL puromycin, subcultured and maintained with 0.25 µg/mL puromycin. After the cells were IR, HMGB1 knockdown exosomes (IR-shHMGB1-Exos) and negative control exosomes (IR-NC-Exos) were extracted from the supernatant after 48 h of culture in exosome-free fetal bovine serum.
Real-time quantitative polymerase chain reaction (RT‒qPCR)
RNA extraction and RT‒qPCR was performed as described in a previous study [
26]. The primers for RT‒qPCR was as follows:
HMGB1:
5′-AATACGAAAAGGATATT GCT-3′ (forward),
5′-GCGCTAGAACCAACTTAT-3′ (reverse);
and GAPDH: 5′-CGCTGAGTACGTCGTGGAGTC-3′ (forward),
5′-GCTGATGATCTTGAGGCTGTTGTC-3′ (reverse).
Cell counting Kit-8 (CCK-8) assay
KYSE150 and ECA109 cells were treated with exosomes (20 µg/ml) for 24 h, counted and diluted to 2 × 104 cells/ml. A 100 µl cell suspension was inoculated in each well of 96-well plates with 5 replicates per sample. When the cells had adhered, they were IR. Then, 10 µl/well cell counting kit-8 reagent (Med Chem Express (MCE) Princeton, NJ, USA) was added at 24 h, 48 h, 72 h, and 96 h. The absorbance was measured at 450 nm by Multiskan 2 h later. The experiment was repeated three times before analysis.
KYSE150 and ECA109 cells were IR with 0, 2, 4, 6 and 8 Gy, and the appropriate cells were treated with exosomes (20 µg/ml) for 24 h and plated on 6-well plates with 3 replicates in each group. After 14 days, the cells were fixed with paraformaldehyde (4%) and stained with crystal violet (0.1%). A colony count (> 50 cells) was then performed. The results were analyzed by Graph Pad Prism 8.0 (Graph Pad Software, Inc., La Jolla, CA, USA) to calculate survival curves (SF), and the multitarget single-hit model was fitted to the data using the formula [SF = 1 − (1 − e−D/D0) N].
Flow cytometry
Flow cytometry (FCM) was used to observe the effect of exosomal HMGB1 after IR on the cell cycle and apoptosis. The cells were collected according to the instructions after 48 h. The cells were stained with a propidium iodide solution (Multi Sciences Biotech Co., Ltd.) to detect cell cycle distribution. Annexin V and propidium iodide (BD Biosciences, San Jose, CA, USA) were used to measure the apoptosis rate.
Western blotting
The procedures were conducted as previously described [
27]. The membranes were scanned, and the relative grayscale value of each protein was examined by an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The density of the protein bands was semiquantified using ImageJ (National Institutes of Health, Bethesda, MD, USA). To observe the changes in the protein expression levels, we calculated the ratio of each protein to the corresponding GAPDH / β-actin level. Three separate experiments were conducted for each western blotting analysis. The antibodies used were the exosome panel (Calnexin, CD9, HSP70, TSG101; dilution 1:1000; cat. no. ab275018; Abcam), anti-HMGB1 (dilution 1:10,000; cat. no. ab79823; Abcam), anti-γH2AX (dilution 1:1,000; cat. no. ab81299; Abcam), anti-Cyclin B1 (dilution 1:50,000; cat. no. ab32053; Abcam), anti-CDK1 (dilution 1:10,000; cat. no. ab133327; Abcam), anti-Bax (dilution 1:5000; cat. no. 60267-1-Ig; Proteintech), anti-Bcl2 (dilution 1:2000; cat. no. ab182858; Abcam), anti-GAPDH (dilution 1:5000; cat. no. 60004-1-Ig; Proteintech), anti-β-Actin (dilution 1:5000; cat. no. 60009-1-Ig; Proteintech), anti-p-AKT (dilution 1:2000; cat. no. 66444-1-Ig; Proteintech), anti-AKT (dilution 1:1000; cat. no. 4685; Cell Signaling Technology, Inc.), anti-PI3K (dilution 1:500; cat. no. 20584-1-AP; Proteintech), anti-FOXO3A (dilution 1:1000; cat. no. 66428-1-Ig; Proteintech), and anti-p-FOXO3A (dilution 1:1000; cat. no. ab154786; Abcam).
Statistical analysis
The data are presented as the means ± standard error of measurement (SEM) of at least three independent experiments. Two groups were compared using Student’s t-test (normally distributed data) or the Mann-Whitney U test (nonnormally distributed data). Analysis of variance was used for comparisons between groups of continuous variables. ROC curve analysis was used to compare the predictive ability of plasma exosome HMGB1 levels in ESCC patients for immediate efficacy. A value of P < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad 8.0 Prism software (GraphPad Software, Inc., La Jolla, CA, USA).
Discussion
Radiotherapy is a well-established therapeutic method to shrink tumor mass and eliminate tumor cells by damaging tumor cells through DNA disruption, ROS production and the destruction of cell structural integrity [
28]. The radiosensitivity of different tumors is significantly different. However, resistance to IR is still a major obstacle in ESCC treatment [
29]. Thus, extensive efforts should be made to investigate the potential targets and eliminate radioresistance.
Emerging evidence has indicated that serum-based exosomes can serve as important biomarkers of lung cancer [
30], colorectal cancer [
31], glioma [
32], endometrial cancer [
33], liver cancer [
34], and prostate cancer [
35]. In this study, the expression level of HMGB1 in the plasma exosomes of ESCC patients was higher than that of healthy individuals, and the radioresistance of ESCC patients with high HMGB1 expression (n = 11) was significantly higher than that of ESCC patients with low HMGB1 expression (n = 10). Therefore, the expression level of HMGB1 in plasma exosomes can be used as a minimally invasive diagnostic marker to predict the radiosensitivity of ESCC. In addition, recent studies have shown that exposing various cells to environmental stress can cause tumor cells to increase the release of exosomes and change their molecular composition [
36]. IR can damage DNA and other structures in target cells, which is a stress condition that affects the biological behavior of tumor cells [
37]. IR-induced exosomes are released to participate in transcription, translocation and cell division [
38‐
40].
IR-induced the bystander effect (RIBE) in which exosomes can influence the function of recipient cells that have not been exposed to IR [
41,
42]. Currently, there is no consensus on the role of exosomes in cancer cell proliferation and radiosensitivity [
43,
44]. Recently, Yamana et al. found no change in the proliferation of OSCC cells treated with exosomes induced by radiation, but increased the radiation resistance of radiation sensitive cells [
43]. Payton et al. reported that exosomes derived from radiation resistant cells in breast cancer can increase cell viability and colony formation in recipient cells and increase resistance to chemotherapy and radiotherapy [
45]. In this study, we found that exosomes from irradiated donor cells could promote the proliferation and radioresistance of ESCC cells. In addition, ESCC cells treated with irradiated exosomes potentially have a higher ability for radiosensitivity regulation than that of ESCC cells treated with non-irradiated exosomes. These results suggest that IR-Exos may act specifically at sites where cells are irradiated and some damage occurs.
Our previous study reported that HMGB1 expression was frequently upregulated in ESCC tissue and negatively related to improved clinical long-term outcomes; HMGB1 expression promotes DNA damage repair and confers radioresistance [
26]. In this study, we confirmed that HMGB1 was released by exosomes into the extracellular space after IR and was absorbed by other cells to promote DNA damage repair, leading to radioresistance. Tang et al. revealed that HMGB1 could be released from stressed cells in the form of exosomes and was an important component of the tumor microenvironment after chemotherapy or radiotherapy [
46]. Moreover, exosome-derived HMGB1 can significantly promote proliferation [
47,
48], metastasis [
49], immune escape [
46], and radioresistance. Our results were consistent with previous studies. The results indicated that exosomes from irradiated donor cells could enhance the vitality of ESCC cells and significantly increase their radioresistance. In addition, HMGB1 knockdown could reduce the level of HMGB1 released by ESCC cells through exosomes after IR and reverse radioresistance.
The PI3K/AKT pathway is a commonly mutated oncogenic pathway in ESCC that acts as a key regulator of radioresistance [
50]. Exosomes released from gastric cancer cells can activate the PI3K/AKT and MAPK/ERK signaling pathways, which are closely related to radiological resistance and tumor progression and ultimately promote the proliferation of recipient gastric cancer cells [
51]. Furthermore, the PI3K/AKT/FOXO3A signaling pathway plays an important regulatory role in a variety of tumorigenic processes [
52]. FOXO3A is a transcription factor that is downstream of AKT and can be inhibited by AKT-mediated phosphorylation. Activation of this pathway reduces the transcription of key genes that mediate a variety of cellular processes, including proliferation, cell cycle arrest, apoptosis and DNA damage [
53,
54]. Interestingly, our study showed that IR-shHMGB1-Exos inhibited AKT phosphorylation in vitro, which indicated that IR-shHMGB1-Exos could reverse radioresistance in ESCC cells. These findings raised the possibility that targeting these signaling pathways may improve radioresistance.
Cell cycle dysregulation has been implicated in the proliferation of cancer cells, and irreversible cell cycle arrest may result in cellular senescence [
55,
56]. Our results indicated that the IR-shHMGB1-Exos group had significantly increased cell cycle arrest in the G2/M phase after IR. Moreover, IR-shHMGB1-Exos treatment increased the number of cells that were arrested in G2/M phase, which was associated with the downregulation of cell cycle-related proteins, such as cyclin B1 and CDK1. This cell cycle arrest may trigger radioresistance by IR-shHMGB1-Exos. In addition, we found that the level of apoptosis in the IR-shHMGB1-Exos group was significantly higher than that in the negative control group. We hypothesize that the loss of HMGB1 in IR-Exos can reverse the apoptosis inhibition mediated by IR-induced exosomes. Bax and Bcl2 are apoptosis-related proteins that are implicated in radiosensitivity. Bax increases the formation of oligomers that participate in apoptogenic molecule release and initiate intrinsic apoptosis [
57]; Bcl-2 proteins are a family of structurally related proteins that act as intrinsic pathway regulators of apoptosis [
58]. The western blotting results revealed that Bax expression was upregulated with the downregulation of Bcl2 expression in the IR-shHMGB1-Exos group. Therefore, IR-induced exosomal HMGB1 inhibits apoptosis in irradiated ESCC cells by regulating members of the proapoptotic Bcl-2 family. However, the exact mechanism of the pathways in the IR-shHMGB1-Exos group that lead to apoptosis remains unclear and requires further investigation.
DNA damage repair (DDR) is one of the major modulators that confers radiosensitivity to tumor cells [
59]. The γH2AX protein was shown to be an indicator of DNA double-strand breaks (DSBs) [
60]. Exosomes derived from tumor cells increased the radioresistance of adjacent tumor cells by inducing the repair of DSBs [
61]. HMGB1 binds to RAGE and TLR4 to promote the phosphorylation of PI3K/AKT and the expression of γH2AX [
24]. Our study identified that ESCC cells release exosomal HMGB1 after IR, which regulates the phosphorylation and translocation of ATM by activating the PI3K/AKT/FOXO3A signaling pathway in recipient ESCC cells. Exosomal HMGB1 enhanced DDR in recipient cells and made tumor cells more resistant to radiotherapy. In addition, γH2AX expression in the IR-shHMGB1-Exos group was lower than that in the IR-NC-Exos Group 2 h after 8 Gy IR. These findings suggest that IR-shHMGB1-Exos resulted in a defective DDR, which improved radiosensitivity.
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