
==== Front
Transl Oncol
Transl Oncol
Translational Oncology
1936-5233
Neoplasia Press

S1936-5233(24)00238-9
10.1016/j.tranon.2024.102111
102111
Original Research
EphA2 specific chimeric antigen receptor engineered T cells for the treatment of prostate cancer
Zhang Miaomiao abc
Wang Haiting ac
Wang Meng ad
Zhang Haoliang abc
Li Huizhong bd
Ma Ping bc
Zheng Junnian de
Wang Gang wangg@xzhmu.edu.cn
ade⁎
Li Shibao sdjnshlb@xzhmu.edu.cn
abc⁎
a Cancer Institute, Xuzhou Medical University, Xuzhou, Jiangsu, China
b Department of Laboratory Medicine, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu, China
c Medical Technology School of Xuzhou Medical University, Xuzhou, Jiangsu, China
d Jiangsu Center for the Collaboration and Innovation of Cancer Biotherapy, Cancer Institute, Xuzhou Medical University, Xuzhou, Jiangsu, China
e Center of Clinical Oncology, Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu, China
⁎ Corresponding authors. wangg@xzhmu.edu.cnsdjnshlb@xzhmu.edu.cn
09 9 2024
12 2024
09 9 2024
50 10211122 1 2024
27 6 2024
25 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Highlights

• EphA2 was highly expressed on the surface of PC3 and DU145 cells.

• EphA2 CAR-T cells efficiently controlled the growth of prostate cancer in an antigen-dependent manner in vitro and in vivo.

• Tumor cells could induce the proliferation of CAR-T cells and the release of high levels of cytokines of IFN-γ in vitro.

• EphA2 is a potential target for prostate cancer therapy.

Erythropoietin-producing hepatocyte receptor A2 (EphA2) is an attractive target for immunotherapy due to its high expression in a variety of solid tumors including prostate cancer. Among various types of immunotherapeutics, chimeric antigen receptor T (CAR-T) cell therapy has made promising progress in hematological and solid tumors. Here, we detected the expression of EphA2 in prostate cancer cells and developed a second-generation CAR targeting EphA2 with CD28 as a co-stimulatory receptor to explore its tumor suppressive potential for prostate cancer in vitro and in vivo. EphA2 was highly expressed on the surface of PC3 and DU145 cells. EphA2 CART cells effectively inhibited prostate cancer growth in an antigen-dependent manner in vitro and in vivo. In addition, tumor cells could stimulate the proliferation of CAR-T cells and the release of cytokine IFN-γ in vitro. These findings shed light on EphA2 as a potential target for prostate cancer, promising EphA2 specific CAR-T cells for the treatment of prostate cancer.

Keywords

Chimeric antigen receptor (CAR)
Prostate cancer
EphA2
Immunotherapy
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pmcIntroduction

In recent years, the incidence of prostate cancer (PCa) has been at the forefront of male cancer incidence and shows an increasing trend year by year [1,2], which also makes prostate cancer the fifth leading cause of cancer-related death in men [3]. Patients with early-stage prostate cancer rarely die after surgery, radiotherapy and hormone therapy, and the 5-year survival rate is nearly 100 % [4]. However, due to the lack of specific symptoms in the early stage of prostate cancer, most patients are diagnosed in the middle and advanced stages and can only be treated with androgen deprivation therapy (ADT), but eventually almost all cases progress to Castration-resistant prostate cancer (CRPC) [5]. This type of prostate cancer patients will then progress to refractory prostate cancer with dual resistance to androgen antagonists and chemotherapy drugs after chemotherapy, seriously affecting the quality of life and reducing the survival rate of patients. Therefore, there is an urgent clinical need for a therapy for this refractory prostate cancer that provides durable disease control and long-term survival benefits.

Chimeric antigen receptor (CAR) T cell therapy has achieved great breakthroughs in the treatment of hematological tumors [6,7]. Among them, the second-generation CAR is the most effective in practical application and has relatively few side effects in human trials. At present, it is mainly used in the treatment of B cell leukemia and lymphoma. Five CAR-T cell products have been approved for clinical treatment by the US Food and Drug Administration (FDA). The European Medical Agency (EMA) has also approved two treatments for patients with relapsed or refractory diffuse large B-cell lymphoma [8,9]. However, due to factors such as the secretion of aberrant chemokines and the tumor suppressive immune microenvironment, CAR-T cell therapy has not shown comparable effects in the hematological tumors [[10], [11], [12]]. But in the treatment of some solid tumors, CAR T therapy has also made breakthrough progress, which could be partly attributed to the selection of effective targets.

Erythropoietin-producing hepatocyte (Eph) receptors are the largest family of protein tyrosine kinases (PTKs), and erythropoietin-producing hepatocyte receptor A2 (EphA2) is persistently overexpressed with altered function in a variety of human cancers, such as PCa [13,14]. Recently, targeted therapies for PTKs, such as Epidermal Growth Factor Receptor (EGFR) and Human epidermal growth factor receptor 2 (HER2/neu), have proven successful in clinical applications for cancer therapy. However, the use of PTKs for PCa has been less reported than for other cancers [[15], [16], [17]]. EphA2 is widely expressed in epithelial cells and endothelial cells of normal blood vessels, and its phosphorylated form is rare. The unphosphorylated form of EphA2 is relatively highly expressed in prostate cancer, breast cancer, endometrial cancer, gliofibroma, ovarian cancer, bladder cancer, and renal cancer compared to normal tissues. Among these cancers, high levels of EphA2 predicted metastasis and lower survival [[18], [19], [20]]. Research has found that overexpression of EphA2 can enhance the proliferation and invasion ability of LNCaP cells, which is associated with aggressive features such as Gleason score, tPSA, and clinical progression. Specifically, EphA2 expression in prostate cancer tissue significantly increases with disease staging and lymph node metastasis [[21], [22], [23]]. Taddei et al. reported that EphA2 affects the metastatic growth and clonal potential of prostate cancer cells, indicating that EphA2 may be an important target in the treatment of prostate cancer [24].

EphA2 has been studied as a target for CAR-T cell therapy in a small number of tumors, but has not been reported in prostate cancer. Chow KK et al. showed that EphA2 specific CAR-T cells can recognize and kill EphA2-positive glioma cells and glioma initiating cells in vitro, and induce tumor regression in severe combined immunodeficiency (SCID) glioblastoma xenograft mouse model [25]. Shi et al. [26] showed that EphA2 CAR-T cells dose-dependently killed ESCC cells and promote cytokine production in vitro, opening up a new avenue to treat ESCC. Therefore, CAR-T targeting EphA2 may be a promising therapeutic strategy for prostate cancer.

Materials and methods

Clinical samples and cell lines

PC3, DU145, LNCaP and 293T cells were purchased from the Cell Bank of Chinese Academy of Sciences and cultured in F12, MEM, RPMI-1640 and IMDM media containing 10 % FBS and 1 % penicillin and streptomycin respectively. Peripheral blood mononuclear cells (PBMC) were isolated from the whole blood of healthy donors who had signed informed consent form in the Affiliated Hospital of XuZhou Medical University using Ficoll reagent, and then placed in L500, containing 10 % FBS, 1 % P/S, 1 ng/ml human IL-7 and 1 ng/ml human IL-15 (Puxin Bio, China), then activated with CD3/CD28 Dynabeads (Gibco, Life Technologies) for 48 h to obtain T cells. These cells were cultured in a sterile incubator at 37 °C with 5 % CO2.

Generation of EphA2 specific CAR-T cells

EphA2-scFv was synthesized by GENEWIZ. T4 ligase ligates the EphA2 scFv fragment with the pR-28ζ vector, which contains the CD8α transmembrane region, CD28 and CD3ζ intracellular signaling domains, to obtain EphA2 CAR. 293T cells were co-transfected with EphA2 CAR plasmid and helper plasmid using GeneJuice® Transfection Reagent (Merck &millipore, USA), and the supernatant was collected after 48 h and 72 h, centrifuged at 2000 rpm for 5 min, and then aliquoted into EP tubes and stored at −80 °C for subsequent infection of T cells. PBMC cells were isolated from whole blood using Ficoll reagent and cultured in L500 medium supplemented with 1 % penicillin/streptomycin, 10 % serum and 10 ng/ml rhIL-7 and 5 ng/ml rhIL-15. PBMC cells were activated with 1 μg/ml CD3/CD28 antibody for 48 h, then resuspended with viral solution and transferred to a RetroNectin (Takara Bio Inc.) coated low-adsorption plate, centrifuged at 1500 g 30 °C for 2 h, and replaced with L500 growth medium, and the CAR positivity was measured after 48 h. Uninfected activated T cells are used as control cells.

Flow cytometry

Flow cytometry were performed using the BD FACS Canto II flow cytometer and data analysis was performed using FlowJo v10. Flow cytometry was used to detect the expression of EphA2 on the surface of tumor cells stained with PE-conjugated EphA2 antibody (356804, Biogend, USA). PE-conjugated recombinant Protein L (11044-H07E-P, Sino Biological, China) was used to detect the positive rate of EphA2 CAR-T cells, and CFSE (C34554, Invitrogen, USA) staining was used to assess the proliferation of T cells. The cytokine release of T cells co-cultured with prostate cancer cells at a 1:5 ratio is detected with PE anti-human IFN-γ (502509, Biolegend, USA).

Cytotoxicity test

The cytotoxicity test is performed by two methods, the Real-Time Cell Analysis (RTCA) and flow cytometry. In the former, 50 μl of L500 medium is added to each well to determine the baseline, and then add 10,000 prostate cancer cells to each well. During the logarithmic growth phase of tumor cells, CAR-T cells are added at a certain target ratio at an appropriate time, and each well is supplemented with fluid to 200 μl. Subsequently, we observed the index of tumor cells and ended the experiment when the index was almost zero. In the second, tumor cells are first plated onto the culture plate, and CAR-T cells are added at a ratio of 1:5 after the tumor cells are adherent. After 72 h of incubation, all cells were collected for CD3 staining, and the percentage of T cells and tumor cells was detected by flow cytometry.

Cytokine production assay

Tumor cells and effector cells (control T or CAR-T) were seeded into 24-well plate at 1:5. After 24 h of co-culture, T cells were harvested for CAR staining, fixation, membrane permeabilization, IFN-γ antibody staining and flow cytometry.

Cell proliferation test

EphA2 CAR-T cells were starved overnight in serum-free L500 medium, incubated with 1.5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) solution for 10 min in the dark, stained by adding the same volume of FBS at 37 °C, and finally washed with PBS contained 2 % serum. CFSE intensity of effector cells was measured by flow cytometry at 0 h and 72 h. Experimental data were analyzed using FlowJo software.

Xenograft mouse model

All animal operations were approved and implemented strictly in accordance with the regulations of Animal Care and Use Committee of Xuzhou Medical University (AAALAC accredited). Four-week-old male immunodeficient NOD-Prkdc(em26Cd52)il2rg(em26Cd22)/Nju(NCG) mice were purchased from GemPharmatech Co. Ltd. and housed in a specific pathogen-free (SPF) facility at Xuzhou Medical University. DU145 cells were inoculated into the right lower flank of mice to construct prostate xenograft model. Body weight and tumor size of the mice were measured every 4 days. When tumor volume reaches 100–150 mm3, EphA2 CAR-T cells or control T cells were infused via tail vein to evaluate the therapeutic effect of EphA2 CAR-T cells. Peripheral blood were collected weekly through the tail vein to detect the proliferation of CAR-T cells during treatment. At the end of the experiment, the mice were euthanized, and the spleen and tumor tissues were ground to detect the content of T cells and CAR-T cells. Meanwhile, the infiltration of T cells in tumor tissue was evaluated through immunohistochemistry. This animal study has been approved by the Ethics Committee of Xuzhou Medical University (L20210226419).

Results

Overexpression of EphA2 in prostate cancer tissues and cells

To clarify the choice of target, we retrieved and displayed the RNA expression levels of EphA2 in different tumors and the protein expression in prostate cancer cells. The database results showed high mRNA expression of EphA2 in prostate cancer tissues (Fig. 1A). Flow cytometry showed EphA2 is highly expressed in prostate cancer cell lines of PC3 and DU145 cells and slightly higher expression in LNCaP cells compared to isotype control (Fig. 1B). Based on the above database and validation results, EphA2 was selected as a target for CAR-T cell therapy for prostate cancer in the present study.Fig. 1 EphA2 expression in prostate cancer cells and tissues. A. RNA expression levels of EphA2 in prostate cancer patients. B. Flow cytometry analysis of EphA2 protein expression on the surface of PC3, DU145 and LNCaP prostate cancer cells.

Fig. 1

Preparation of EphA2 CAR-T cells

Using EphA2 as the target antigen, we synthesized EphA2 scFv sequence, which was tandem with the CD8α hinge region, transmembrane region, CD28 costimulatory domain and CD3ζ intracellular signal transduction domain to form the EphA2 CAR plasmid (Fig. 2A and B). As confirmed by enzyme digestion identification (Fig. 2C) and plasmid sequencing (Fig. 2D), the pRv-EphA2 scFv-28ζ plasmid was successfully constructed. 293T cells were co-transfected with EphA2 CAR plasmid and helper plasmid, and the supernatant (i.e., EphA2 CAR virus solution) was collected to infect CD3/CD28 activated PBMC cells to obtain EphA2 CAR-T cells. The expression of EphA2 CAR on the surface of T cells was measured by flow cytometry (Fig. 2E and F). The results showed that the positive rate of EphA2 CAR-T cells was >75 %.Fig. 2 Construction and identification of the EphA2 CAR. A. The sequence diagram of the CAR structures, LTR: long terminal repeats, EphA2 scFv: EphA2 single-chain variable fragment. B. EphA2 CAR vector plasmid map. C. Map of enzyme digestion identification of the pRv-EphA2 scFv-28ζ plasmid. D. Flow cytometry was used to detect the expression of EphA2 scFv on the surface of T cells, reflecting the positive rate of CAR-T cells. E. Statistical plots of the CAR positive rate.

Fig. 2

EphA2 redirected CAR-T cells efficiently kill prostate cancer cells

To test the ability of EphA2 CAR-T cells to kill prostate cancer cells PC3, DU145, and LNCaP, we incubated the three tumor cells with Control T and EphA2 CAR-T cells according to a certain effect-target ratio (E:T), and observed the changes of tumor cell index by RTCA in real time. The results showed that, compared with tumor cells alone, control T cells had no significant effect on the proliferation of three tumor cells, that is, control T did not exert a killing effect on prostate cancer cells. EphA2 CAR-T cells significantly inhibited the proliferation of PC3, DU145 and LNCaP tumor cells, inducing almost complete elimination of tumor cells within 4–5 days (Fig. 3A–C). Meanwhile, after 3–5 days of co-culture, the ratio of surviving CAR-T cells to tumor cells was analyzed by flow cytometry. The results showed that the two tumor cells in control T group could effectively proliferate and had high survival rate. EphA2 CAR-T group had less residual tumor cells, and the killing effect was dose-dependent, which was enhanced with the increase of E:T (Fig. 3D and E). The above results suggest that EphA2 CAR-T cells have obvious killing effects on prostate cancer cells.Fig. 3 Cytotoxicity of EphA2 CAR-T cells against prostate cancer cells. A, B. RTCA verified the killing of control T and EphA2 CAR-T cells against PC3 (Figure A) and DU145 (Figure B). C, D. Flow cytometry detected the final proportion of tumor cells co-cultured with EphA2 CAR-T and control T cells, and calculated the rate of killing. The data are represented as the mean ± SEM (*** P < 0.001, n = 3).

Fig. 3

EphA2 expressed prostate cancer cells promote the proliferation of EphA2 CAR-T cells

When CAR-T cells are co-incubated with tumor cells expressing the target antigen, tumor cells will stimulate the proliferation of CAR-T cells while CAR-T cells exert killing effect, and the proliferation of CAR-T cells can reflect the specificity and targeting of killing. Therefore, we stained control T and CAR-T cells with CFSE and incubated with tumor cells at target ratio 1:1 for 48 h, then the CFSE fluorescence intensity of T cells was measured by flow cytometry. The fluorescence intensity decreased with the proliferation of T cell. As shown in Fig. 4A and B, the fluorescence intensity of control T cells stimulated by PC3 and DU145 cells was no different from that of T cells alone, while that of EphA2 CAR-T cells was significantly weaker than that of CAR T cells alone. This indicates that EphA2 CAR-T cells kill tumor cells and tumor cells can stimulate the proliferation of CAR-T cells.Fig. 4 Proliferative levels of EphA2 CAR-T cells stimulated with tumor cells. A, B. The CFSE fluorescence intensity of control T and EphA2 CAR-T cells co-incubated with PC3 cells (Figure A) and DU145 cells (Figure B) was detected by flow cytometry.

Fig. 4

Cytokine release of EphA2 CAR-T cells after co-culture with prostate cancer cells

CAR-T cells activate, proliferate and release cytokines IFN-γ, TNF-α, Granzyme A and Granzyme B in the presence of target antigen. We collected CAR-T cells co-cultured with tumor cells at a target ratio of 1:5, and detected the release of IFN-γ in CAR-T cells using intracellular cytokine assay kit. As shown in Fig. 5, PC3 and DU145 cells significantly increased the release level of IFN-γ in EphA2 CAR-T cells compared with control T, and LNCaP cells had no significant effect on the content of IFN-γ in EphA2 CAR-T cells.Fig. 5 Cytokine release levels of EphA2 CAR-T cells after co-incubation with tumor cells. The levels of IFN-γ released from control T and EphA2 CAR-T cells stimulated with PC3, DU145 or LNCaP cells were determined by flow cytometry (** P < 0.01, n = 3).

Fig. 5

Effect of EphA2 CAR-T cells in prostate cancer xenograft mice

After validating the effective killing of prostate cancer cells by EphA2 CAR-T cells in vitro, we constructed a prostate cancer xenograft model to explore whether there is an antitumor effect in vivo. 4 × 106 DU145 cells were inoculated into the right lower limb of male NCG mice to establish a subcutaneous transplantation tumor model. When the tumor volume reached 100–150 mm3, the mice were randomly divided into 3 groups: PBS, control T and EphA2 CAR-T, 6 in each group, and 5 × 106 T cells were injected through the tail vein. The treatment protocol is shown in Fig. 6A. Control T and EphA2 CAR-T cells are infused on days 29 and 65 after tumor cell infusion. After treatment, the body weight and tumor size of the mice were measured every 3 to 4 days, The volume of tumor (a × b2/2) was recorded and calculated, and the tumor growth curve was plotted with the average value.Fig. 6 Antitumor effect of EphA2 CAR-T cells in DU145 xenografted mice. A. Flow chart of DU145 subcutaneous transplantation treatment regimen for NCG mice. B. Tumor volume curves of DU145 mice after infusion with PBS, control T cells, and EphA2 CAR-T cells (* P < 0.05, *** P < 0.001, ns: no statistical difference, n = 6). C. Body weight curves of mice in the PBS, control T and EphA2 CAR-T group. D. Flow cytometry for the detection of T cells in the peripheral blood of DU145 mice. E. T cell content in tumor tissue of DU145 mice by flow cytometry. F. Immunohistochemical CD3 antibody staining assesses the infiltration of T cells in control T and EphA2 CAR-T tumor tissues (20 ×), Scale bar: 20 μm.

Fig. 6

As shown in Fig. 6B, the tumors of mice in PBS group continued to grow after tumor cell injection, the tumors of mice in the control group showed no tumor inhibition and continued to grow after T cell infusion, indicating that control T cells had no obvious effect on tumor growth in DU145 mice. In contrast to the PBS and control T groups, the tumor of mice treated with EphA2 CAR-T cells no longer grew and regress on day 12 after CAR-T infusion. On day 36 after the first CAR-T cell infusion, we infused the same dose of control T and EphA2 CAR-T cells as the first due to incomplete tumor regression in mice. After the second treatment, the tumors of PBS and control T mice continued to grow, and the tumors of CAR-T group shrank. At the experimental end point, EphA2 CAR-T cells significantly inhibited the growth of subcutaneous transplanted tumors in DU145 mice compared to the PBS and control T group (P < 0.05). Continuous weight monitoring of mice showed increased weight, illustrating that EphA2 CAR-T cells had no significant toxic side effects on mice (Fig. 6C). To assess the survival and expansion of EphA2 CAR-T cells in mice, we collected 30–50 μl of blood from the tail vein after T cell treatment, and measured the content of T cells and CAR-T cells in the peripheral blood of DU145 mice by flow cytometry. Mice were killed on day 55 of treatment, and the proportion of T cells and CAR-T cells in the spleen of control T and EphA2 CAR-T groups was detected. As shown in Fig. 6D and E, the T cells in the peripheral blood and tumor tissues of the mice in the CAR-T group were higher than those in the control group (P < 0.05). Immunohistochemistry showed that the tumor tissues in the control T and CAR-T group had significant T cell infiltration (Fig. 6F).

Discussion

In this study, we detected the expression of EphA2 in prostate cancer cell lines and constructed a CAR targeting EphA2. Then, EphA2 CAR-T cells and prostate cancer cells were co-cultured for killing experiments, and the release of cytokine IFN-γ from T cells during cell killing was detected. Meanwhile, we also observed that prostate cancer cells had significant effect on the proliferation of EphA2 CAR-T cells in vitro. The significant killing effect of EphA2 CAR-T cells on prostate cancer cells has been further validated in vivo experiments on DU145 tumor xenograft mice, and no significant side effects of EphA2 CAR T cell therapy on mice were found through animal behavioral observation and weight monitoring.

CAR-T cell therapy is a genetically engineered immunotherapy that introduces chimeric receptor with specific signaling binding domains into patient's T cells. Once CAR-T cells bind to the corresponding structures of target cells, CAR-T cells release cytotoxins to induce apoptosis and necrosis of tumor cells. In addition, CAR-T cells can also induce the killing effect of immune cells and enhance the immune response of patients. At present, CAR-T cell therapy has achieved remarkable results in hematological tumors. At present, CAR-T cell therapies have been approved for clinical treatment. However, it still faces challenges in the treatment of solid tumors. Recently, Qi C et al. conducted a clinical trial for gastric cancer using CAR-T cells targeting the novel antigen claudin18.2 and achieved significant therapeutic effects. The overall response rate (ORR) and disease control rate (DCR) of 37 subjects reached 48.6 % and 73.0 %, respectively. Therefore, the development of new targets for tumor is of great significance to overcome challenges.

EphA2 is a member of tyrosine kinase receptor family, which has been proved to be related to the occurrence and progression of various tumors. In prostate cancer, EphA2 expression levels are higher than normal tissues and are associated with tumor invasion, metastasis, and poor prognosis. Ashwin Sachdeva et al. have also reported that progression of prostate cancer is associated with atypical EphA2 activation. Therefore, EphA2 is expected to be a potential therapeutic target for prostate cancer.

Compared with traditional treatment methods, EphA2 CAR-T cell therapy has certain advantages. Firstly, EphA2 CAR-T cells have strong tumor specificity and can selectively kill prostate cancer cells, thereby reducing toxic side effects on normal tissues. Secondly, CAR-T cell therapy has sustainable anti-tumor effects and can even trigger immune memory, providing long-term defense. However, EphA2 CAR-T cell therapy also faces some challenges, such as difficulties in preparing and expanding CAR-T cells, and potential serious side effects such as cytokine release syndrome during treatment. At present, there are relatively few clinical studies of EphA2 CAR-T cell therapy in prostate cancer, but some encouraging results have been achieved in metastatic or non-metastatic glioblastoma, ependymoma, and bone tumor.

EphA2 CAR-T cell therapy, as a new immune therapy, has shown potential advantages in the treatment of prostate cancer. Although current research is still in early stages, EphA2 CAR-T cell therapy holds promise for improving the prognosis and survival rate of prostate cancer patients. This study provides preclinical evidence for EphA2 CAR-T cell therapy for prostate cancer.

CRediT authorship contribution statement

Miaomiao Zhang: Writing – original draft, Validation, Project administration, Methodology. Haiting Wang: Writing – original draft. Meng Wang: Methodology. Haoliang Zhang: Methodology, Investigation. Huizhong Li: Supervision, Methodology. Ping Ma: Supervision, Resources. Junnian Zheng: Resources. Gang Wang: Writing – review & editing, Validation, Supervision, Conceptualization. Shibao Li: Writing – review & editing, Supervision, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding

This work was supported by the grant from the grant from the 10.13039/501100010246 Postdoctoral Science Foundation of China (No. 2020M670076ZX ), the key medical talents of Xuzhou (No. XWRCHT20220065), Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX21_2653 and KYCX22_2970), the Key R&D projects of Xuzhou Science and Technology Bureau (KC22250), and the Xuzhou Health Commission Youth Medical Technology Innovation Project (XWKYHT20210581).
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