Delivery of gefitinib loaded nanoparticles for effectively inhibiting prostate cancer progression

Abstract

Androgen deprivation therapy is administered to suppress the growth of prostate cancer (PCa). However, some cells continue to proliferate independent of hormones, leading to the development of castration-resistant prostate cancer (CRPC). Overexpression of the epidermal growth factor receptor (EGFR) has been observed in CRPC and is associated with an unfavorable prognosis. Gefitinib (GEF) is an EGFR inhibitor used to treat patients with CRPC. Nevertheless, some clinical studies have reported that gefitinib does not result in prostate-specific antigen (PSA) or objectively measurable CRPC reactions. This lack of response may be attributed to the limited solubility in water, high side effects, low tumor aggregation, and insufficient tumor-specific reactions of GEF. In order to tackle these obstacles, we present a practical and efficient approach to administer GEF, encompassing the utilization of biocompatible nanostructures as a vehicle for drug delivery to augment its bioaccessibility and curative potency. Despite their small particle size, poly(D,L-lactide-co-glycolide) acid nanoparticles (PLGA NPs) exhibit a high drug-loading capacity, low toxicity, biocompatibility, biodegradability, and minimal immunogenicity. The drug delivery efficiency can be improved by employing GEF@PLGA NPs, which could also enhance drug cytotoxicity and impede the advancement of prostate cancer. Moreover, through experiments in vivo, it has been verified that GEF@PLGA NPs exhibit selective accumulation in the tumor and effectively restrain tumor growth. Therefore, the GEF@PLGA NPs hold great promise for the treatment of PCa.

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Introduction

Prostate cancer (PCa) is a commonly occurring malignancy and ranks as the second primary cause of mortality in men residing in Western countries.¹ Even with the most effective androgen receptor (AR) signaling inhibitors, patients with advanced prostate cancer rarely achieve complete remission and ultimately develop castration resistant prostate cancer (CRPC).² Costimulation by multiple growth factors, including epidermal growth factor (EGF), is essential for the induction of in vitro prostate epithelial cell proliferation, which cannot be achieved by androgens alone.^3,4 The binding of EGF to its receptor triggers conformational alterations in the receptor structure and enhances the functionality of related tyrosine kinases, leading to heightened biologic activity such as cell differentiation or proliferation.⁵ Numerous malignancies, including prostate cancer, show abnormal expression of the EGF receptor (EGFR), either through mutation or overexpression.^5–7

EGFR is a highly desirable molecular target for suppression due to its overexpression in numerous cancerous cells,⁸ and it is tightly regulated in healthy cells. Several EGFR inhibitors are currently undergoing clinical investigation. Gefitinib (GEF) is an orally ingested molecule derived from quinazoline that effectively hampers EGFR tyrosine kinase activity. In preclinical models and initial clinical trials, the hindrance of EGFR exhibited potent antitumor properties.^9–11 However, certain clinical investigations have revealed that gefitinib does not lead to prostate-specific antigen (PSA) or objectively measurable CRPC responses.^12–14 In the meantime, the coadministration of GEF alongside other medications failed to manifest notable antitumor efficacy in PCa.^14–18

Limited solubility in water, low aggregation in tumors, inadequate tumor-specific reactions, and poor pharmacokinetics along with insufficient drug delivery caused by single GEF greatly diminish its therapeutic effectiveness and hampers its clinical utilization.^19–22

In order to overcome the challenges associated with drug delivery, nanoparticle-based drug delivery has emerged as a promising strategy. Various nanoparticle platforms have been devised to enhance drug delivery.²³ The utilization of nanoparticles as carriers for nanodrugs to address a range of ailments is well-documented.^24,25 Nanoparticles present numerous benefits including superior drug encapsulation efficiency, enhanced drug stability over an extended period, improved pharmacokinetics and pharmacodynamics of drugs, decreased toxicity of drugs, elevated drug effectiveness, and enhanced safety when loaded into nanoparticles. Additionally, nanoparticles facilitate optimal drug delivery by enabling enhanced permeability and retention effects, targeted delivery, and controlled and sustained release of drugs.²⁶ Among these, the poly(D,L-lactide-co-glycolide) acid (PLGA) polymer has gained significant attention due to its low toxicity, biocompatibility, biodegradability, and low immunogenicity.^23,27 The clinic extensively utilizes PLGA, with the FDA approving over 20 PLGA-based medications for human administration.²⁸ These particles act as carriers of therapeutic compounds for treating breast and prostate cancer, neurologic disorders, and various other diseases. Furthermore, PLGA finds widespread applications for delivering miRNAs and drugs through the use of nanoparticles.^28,29 Considering these advantages, we employed PLGA as the polymeric matrix and developed a technique to encapsulate GEF within PLGA-nanoparticles, thereby increasing the hydrophilicity of GEF.

The objective of this investigation was to explore the antitumor impact of GEF employing diverse modes of drug delivery and to develop new GEF nanodrugs with tumor-specific responses, controlled release in vivo and high tumor-suppressive properties. We utilized natural GEF and GEF encapsulated PLGA nanoparticles (referred to as GEF@PLGA NPs) in in vitro and in vivo experiments to examine their effectiveness in PCa models (Scheme 1).

Scheme 1 Illustration of the preparation of GEF@PLGA NPs and anti-tumor mechanism in vivo. Synthesis route of GEF@PLGA NPs; they induce PCa cell apoptosis via vein injection. Tumor cells take up GEF@PLGA, the drug is released, and the apoptosis program is initiated to treat prostate cancer.

Results

Characterization of the GEF@PLGA NPs

PLGA is used as a drug carrier in cancer therapy with excellent biocompatibility and redox sensitivity. We used PLGA nanoparticles (PLGA NPs) as the polymer matrix to prepare gefitinib (GEF) nanoparticles (GEF@PLGA NPs) wrapped with PLGA NPs, and improved the water solubility and absorption rate of GEF. We first verifies that the stability of GEF@PLGA NPs is good in RPMI-1640 medium at the macro-level through the Tindall effect (Fig. 1A). Subsequently, we determined the physical properties of GEF@PLGA NPs based on their particle size and particle size distribution of GEF@PLGA NPs using dynamic light scattering (DLS). The size of the PLGA NPs is 61.10 ± 1.97 nm and its PDI is 0.82 ± 0.007, besides, the zeta potential was measured as −25.5 ± 1.5 mV (Fig. 1B–D). The sizes of GEF@PLGA NPs decreased slightly compared to that of PLGA NPs, with a PDI of 0.268 ± 0.012 and a zeta potential of about −2.1 ± 0.84 mV (Fig. 1E–G). TEM images of the nanoparticles confirmed that both nanoparticles have clear spherical structures, consistent with the DLS results (Fig. 1C and F). The particle size of GEF@PLGA NPs in PBS did not show significant changes over time. In the presence of 10% FBS, there was a slight decrease in the particle size of GEF@PLGA. However, the average particle size remained between 40 and 60 nm for the entire 72 hour period (Fig. 1H). Further evaluation of the drug loading capacity and loading efficiency revealed values of 23.35 ± 2.64% and 5.21 ± 0.59%, respectively. To further understand the nanoparticle releasing behavior in the tumor microenvironment, we conducted additional investigations on particle size and cumulative drug release in both pH 5.0 (acidic tumor microenvironment) and pH 7.4 buffer (physiological conditions) (Fig. 1I). The in vitro release curves indicated that the drug release rate of GEF@PLGA NPs was faster under acidic conditions compared to that under alkaline conditions. Specifically, 23.85% and 58.77% of GEF were released by GEF@PLGA NPs within 168 hours under pH 7.4 and 5.0 conditions, respectively. The injection of nanocarriers is necessary for drug delivery into the bloodstream, necessitating the assessment of their stability and compatibility with blood components. In order to accomplish this, we performed additional assessments on the hemolysis rate in varying concentration scenarios. The findings consistently revealed a hemolysis rate of less than 5% within the range of concentrations tested (Fig. S1A and B†), providing evidence of a high level of blood safety. These findings suggest that the acidic environment in tumors fosters the liberation of drugs from GEF@PLGA NPs, exhibiting a favorable profile for blood safety. It can be deduced that GEF@PLGA NPs have the potential to augment drug release specifically at the site of the tumor, thereby bestowing advantageous effects in combating cancer.

Fig. 1 Characterization of GEF@PLGA NPs. A. The stability of PLGA NPs and GEF@PLGA NPs in RPMI 1640 medium. B. The size distribution of PLGA NPs. C. TEM image of PLGA NPs. D. Zeta potential of PLGA NPs. E. The size distribution of GEF@PLGA NPs. F. TEM image of GEF@PLGA NPs. G. Zeta potential of GEF@PLGA NPs. H. The stability of GEF@PLGA NPs in saline or saline supplied with 10% FBS for 168 h. I. The cumulative release profile of GEF@PLGA NPs at different pH values.

Uptake kinetics of GEF@PLGA NPs in vitro

The evaluation of nanoparticle function relied heavily on the efficiency of cellular uptake. To observe the uptake and distribution of nanoparticles within cells, a green fluorescent probe called coumarin 6 (C6) was used to replace GEF, as nanoparticles lack fluorescence. This was achieved through the utilization of CLSM and flow cytometry. Following incubation with C6 loaded PLGA NPs (C6@PLGA NPs), the 22RV1 cells displayed a significant increase in green fluorescence (Fig. 2A and B). The intensity of fluorescence also increased over time. The qualitative analysis conducted using CLSM yielded similar results (Fig. S2†). These findings indicated that tumor cells efficiently and rapidly took up the GEF@PLGA NPs.

Fig. 2 Uptake kinetics of PLGA NPs in vitro. A. The cellular uptake of 22RV1 cells after incubation with PLGA NPs for 0, 0.5, 1, 2, 4 and 8 h was analyzed by flow cytometry. B. Mean fluorescence intensity of PLGA NPs internalized by 22RV1 cells for 0, 0.5, 1, 2, 4 and 8 h. C. Confocal microscopy images and quantitative analysis of 22RV1 cells incubated with PLGA NPs for 0.5, 1, 2, 4 and 8 h (scale bar = 100 μm). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

CLSM was then employed to determine if the C6@PLGA NPs were able to escape from the lysosomes within 22RV1 cells, a CRPC cell line. Based on the findings depicted in Fig. 2C, after incubating with the nanoparticles for different times, the green fluorescence of the nanoparticles strongly coincided with the red fluorescence producing a yellow fluorescence, and then the nanoparticles were released by the lysosomes. This indicates that the majority of the nanoparticles were primarily situated within the lysosomes at the 2-hour mark. Nonetheless, with longer incubation periods of 4 and 8 hours, the green fluorescence gradually detached itself from the red fluorescence, leading to a significant reduction in yellow fluorescence (Fig. 2C). These results indicate that the C6@PLGA NPs were able to successfully evade the lysosomes and enter the cytoplasm. In conclusion, these observations verify that the PLGA NPs possess the ability to escape from the lysosomes, thereby safeguarding them against degradation and removal.

Anticancer efficiency in vitro

After exposing 22RV1 cells to varying concentrations of PLGA NPs, no significant reduction in cell viability was observed (Fig. 3A and B). However, the viability of 22RV1 cells noticeably decreased after exposure to varying concentrations of free GEF and GEF@PLGA NPs for durations of 24 and 48 hours, exhibiting a dose-dependent behaviour (Fig. 3A and B). The IC50 values of free GEF and GEF@PLGA NPs were 8.64 vs. 5.27 μg mL⁻¹ at 24 hours and 6.89 vs. 5.30 μg mL⁻¹ at 48 hours. Additionally, to explore the induction of apoptosis by GEF@PLGA NPs, a 24-hour treatment of 22RV1 cells with GEF and GEF@PLGA NPs was performed. Flow cytometry analysis of Annexin V-FITC/PI labeled apoptotic cells revealed similar levels of apoptosis in the PLGA NPs group compared to the control group, suggesting that PLGA NPs do not induce apoptosis (Fig. 3C and D). The apoptosis results of immunofluorescence also confirmed the above results (Fig. S3†). In contrast, treatment with GEF and GEF@PLGA NPs for 24 hours resulted in significant apoptosis in 22RV1 cells, with apoptotic rates of 20.70% ± 0.84% (GEF) and 42.28% ± 0.77% (GEF@PLGA NPs) (Fig. 3C and D). Western blot analysis of apoptosis-related markers including cleaved-caspase 3, cleaved-caspase 9, and cleaved-PARP confirmed these findings, with GEF@PLGA NPs significantly increasing the protein levels of these markers (Fig. 3E). Therefore, it can be concluded that GEF@PLGA NPs exhibit greater cytotoxicity towards 22RV1 cells compared to free GEF. Following this, the intracellular biological function of GEF@PLGA NPs was evaluated. Cell migration and invasion assays demonstrated that GEF@PLGA NPs effectively suppressed the migration and invasion capacities of 22RV1 cells, surpassing the effects of free GEF (Fig. 3F and I). Therefore, the findings strongly demonstrate that PLGA NPs have the potential to boost the in vitro anti-cancer efficacy of GEF.

Fig. 3 Anticancer efficiency of GEF@PLGA NPs in vitro. (A and B) In vitro cytotoxicity of 22RV1 cells treated with PLGA NPs, GEF and GEF@PLGA NPs at different concentrations for 24 h (A) and 48 h (B). C. Cell apoptosis of 22RV1 cells incubated with PBS, PLGA NPs, GEF and GEF@PLGA NPs measured using a flow cytometer. D. In vitro apoptotic rates of 22RV1 cells treated with PBS, PLGA NPs, GEF and GEF@PLGA NPs for 24 h. E. The western blot analysis was used to detect the changes of apoptosis indicator proteins of 22RV1 cells treated with PBS, PLGA NPs, GEF and GEF@PLGA NPs for 24 h. F–I. The representative images of migration (F) and invasion (G) assays and quantification of migratory (H) or invasive (I) cell counts were obtained using 22RV1 cells after treatment with PBS, PLGA NPs, GEF and GEF@PLGA NPs for 48 h. Scale bar: 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Biodistribution of the PLGA NPs in vivo

The utilization of nanoparticles with distinct sizes enables the accomplishment of passive targeting at tumor sites by capitalizing on the enhanced permeability and retention (EPR) effect. This occurrence prolongs the duration of circulation of nanoparticles in living organisms. To explore the capacity of GEF@PLGA NPs to specifically target mice with 22RV1 xenograft tumors, we utilized DiR as a drug imitator and labeling component for the PLGA NPs (Dir@PLGA NPs). To visualize the distribution of Dir@PLGA NPs in the mice, fluorescence imaging was conducted using IVIS after injecting the Dir@PLGA NPs via the tail.

As shown in Fig. 4A, the concentration of DiR@PLGA NPs increased gradually in organs with high blood perfusion over time, including the liver and spleen. It is worth mentioning that there was also notable accumulation at the tumor site, potentially due to the gradual infiltration of nanoparticles facilitated by the ERP effect. In contrast, free DiR accumulated predominantly in the liver and exhibited visibly lower levels in the tumor tissue. Imaging and quantitative analysis were conducted after 72 hours of injection to examine the major organs and tumor tissues. The fluorescence signal of free DiR was considerably weak in tumor tissues, predominantly localized in the liver and spleen. In contrast, the signal emitted by DiR@PLGA NPs exhibited substantial enhancement in the tumor tissues (Fig. 4B and C). These findings demonstrate that PLGA NPs possess a strong tumor aggregation ability, facilitating the effective enrichment of the therapeutic drug GEF in tumor tissues. Furthermore, PLGA NPs enhance drug accumulation and retention in tumor tissues. Consequently, these results highlight the significant potential of PLGA NPs as delivery vehicles for prostate cancer drugs.

Fig. 4 Biodistribution of the PLGA NPs in vivo. A. The biodistribution of DiR and DiR-NPs in 22RV1 tumor-bearing mice at different time periods after intravenous injection. B. DiR fluorescence images of primary organs and tumors extracted from mice at 72 h. C. Quantitative results for the fluorescence intensity in major organs and tumor accumulation. 20.8×: 20.8 fold. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Antitumor activity in vivo

Taking advantage of efficient cellular uptake, increased cytotoxicity, apoptosis, and nanoparticle accumulation, we verified the in vivo anti-tumor efficacy of GEF@PLGA NPs using a tumor-bearing 22RV1 mouse model. After administering various formulations, the mice exhibited diverse tumor growth patterns. The negative control group and PLGA NPs treatment group experienced rapid tumor growth, with tumor volumes surpassing 1000 mm³ after 12 days of treatment. However, the mice treated with free GEF and GEF@PLGA NPs showed inhibited tumor growth, with the GEF@PLGA NPs group exhibiting significantly higher tumor inhibition rates (Fig. 5A). Following the completion of the treatment, the tumors were surgically removed and weighed, yielding comparable findings of tumor volume progression and tumor weight (Fig. 5B and C). Additionally, no significant reduction in weight was observed among any of the experimental cohorts (Fig. 5D). Consequently, GEF@PLGA NPs exhibited enhanced effectiveness against tumor growth in comparison with the unbound GEF entities. Remarkably, the mice's body weights exhibited no fluctuations over the treatment duration across all groups, thereby confirming the dearth of profound toxic adverse effects instigated by the administered interventions.

Fig. 5 Antitumor activity of GEF@PLGA NPs in vivo. A. Representative image of the tumor from a 22RV1 tumor-bearing mouse after intravenous injection with PBS, PLGA NPs, GEF and GEF@PLGA NPs. B. Weights of dissected tumors from the mice after treatment. C. Tumor growth inhibition of 22RV1 tumor-bearing mice after treatment. D. Body weights of 22RV1 tumor-bearing mice after treatment. E. H&E staining and IHC examination of Ki67, caspase 3, and cleaved-caspase 3 expressed in tumors from the treated mice. Scale bar: 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

In order to further examine the effectiveness of GEF@PLGA NPs in treating tumors, we conducted H&E and immunohistochemical staining on tumor sections at the conclusion of the treatment. The tumor sections from the control and PLGA NPs groups exhibited a structurally intact and tightly packed appearance, with no noticeable abnormalities in the cell nuclei (Fig. 5E). Conversely, the tumor tissues of the GEF and GEF@PLGA NPs groups demonstrated a loosely arranged structure and showed evident crinkling and fragmentation of the cell nuclei. The tumor sections from the control and PLGA NPs groups exhibited a structurally intact and tightly packed appearance, with no noticeable abnormalities in the cell nuclei (Fig. 5E). Tumor tissue immunohistochemistry revealed that the GEF@PLGA NPs group showed an increased apoptosis index of cleaved caspase 3 compared to the free GEF group, while the proliferation marker of Ki-67 was decreased. The tumor sections from the control and PLGA NPs groups exhibited a structurally intact and tightly packed appearance, with no noticeable abnormalities in the cell nuclei (Fig. 5E). These findings further supported the notion that GEF@PLGA NPs exhibit stronger cytotoxicity towards the tumor tissue compared to free GEF, demonstrating their superior antitumor effect.

To assess the potential impact on the vital organs of the mice, a thorough pathological examination was conducted. After performing H&E staining, no significant alterations in the cellular and tissue structures of any analyzed organ were detected. The tumor sections from the control and PLGA NPs groups exhibited a structurally intact and tightly packed appearance, with no noticeable abnormalities in the cell nuclei (Fig. S4†). Additionally, blood plasma analysis of the liver (AST and ALT) and kidneys (BUN and CRE) showed that they function fell within the normal range, signifying normal liver and kidney function in the mice (Fig. S5A–D†). It is worth noting that no evident toxic or side effects associated with the GEF@PLGA NPs were observed. These results not only confirm the high biosafety of GEF@PLGA NPs, but also highlight their potential for clinical applications.

To summarize, the utilization of GEF@PLGA NPs showcases improved drug targeting, diminished toxicity and adverse reactions, and enhanced efficacy in tumor treatment.

Discussion

Novel therapeutic strategies have emerged targeting EGFR and its signal transduction cascade as a result of increased EGFR expression in prostate cancer cells. These procedures involve monoclonal antibodies that particularly target the extracellular ligand-binding domain of the receptor, antisense oligonucleotides that inhibit the expression of EGFR ligands or the receptor itself, and inhibitors with low molecular weight that disrupt the tyrosine kinase activity of the receptor. Moreover, there are compounds with low molecular weight that can impede the downstream constituents of the signal transduction pathway.³

GEF, an effective inhibitor that selectively targets the tyrosine kinase of EGFR, displays the capability to reduce tyrosine kinase activity and obstruct the exchange of information among tumor cells linked with EGFR. As a result, this compound demonstrates its anti-tumor properties by impeding the survival, reproduction, and spread of malignant cells.^30,31 However, various clinical investigations have documented insignificant anti-tumor activity either with GEF or when combined with other therapeutic agents in relation to PCa.^12–18 The present GEF formulations available on the market are predominantly in tablet forms due to their slow oral absorption, widespread distribution in the body, and a corresponding rise in gastrointestinal adverse reactions upon dosage escalation. This deficiency in therapeutic response might be attributable to the restricted solubility of GEF in water, its considerable side effects, inadequate capacity for tumor aggregation, and an insubstantial tumor-specific reaction.^20,22,23,30

Nanoparticle systems are highly appealing platforms that exhibit a remarkable capacity for carrying an abundant load of drugs when compared to antibody conjugates.^32,33 The attributes of nanoparticle carriers offer an efficient resolution to the limitations encountered in traditional tumor treatment therapies, including non-specific accumulation, inadequate tumor targeting aptitude, and a diminished therapeutic impact.^34,35 It is precisely due to the rapid development of nanotechnology that more and more new materials have been discovered or synthesized for tumor treatment. For example, micro/nanorobots have become a research hotspot due to their self-propulsion and controllable navigation characteristics,³⁶ adaptive nanomaterials have also shown great prospects in cancer treatment due to their spatiotemporal controllable drug release characteristics.³⁷ In addition, some candidate materials related to the structure–property relationship of polymers are gradually being applied in the treatment of prostate cancer,³⁸ polyamino acids have functions such as immune regulation, anti-inflammatory and antioxidant activities, and promotion of cell apoptosis, and are often used as drug carriers to promote effective cancer treatment.³⁹ In addition, materials for some new forms of cell death have also begun to be developed, such as nanomaterials that induce immunogenic cell death.⁴⁰ Furthermore, the particles contain enclosed drugs, guaranteeing that the pharmaceutical characteristics of the drugs do not interfere with the dispersion of the nanoparticles themselves.^41,42

The utilization of PLGA NPs is a common strategy for targeted drug delivery in various nanoparticle systems, owing to their high biocompatibility and low toxicity.^43,44 Previous investigations have also employed PLGA NPs to target the delivery of paclitaxel in PCa.⁴⁵ In our investigation, we observed that the cytotoxic effects of GEF@PLGANPs were evident in both the 22RV1 PCa cell line and PCa xenografts. The utilization of PLGA NPs for the targeted delivery of GEF has the potential to enhance its anti-cancer effects in PCa. At the same time, GEF@PLGA NPs can also inhibit the migration and invasion of prostate cancer, indicating that although GEF@PLGA NPs improves the effectiveness of GEF, it may also have other effects. Further research is needed to study other potential capabilities of GEF@PLGA NPs.

A limitation of this study was that it did not further elucidate the molecular mechanisms that cause changes in apoptosis related factors, in order to discover more therapeutic targets for prostate cancer. Moreover, although GEF@PLGA NPs have a good therapeutic effect on tumors, there is no information whether they also have toxicity to healthy cells and hence there is a need to establish an improved method to make the nanoparticles safer and more effective biomaterials.

Conclusions

To summarize, we have presented evidence of GEF's potential as an anti-cancer agent in different modes of drug delivery in PCa models. For the first time, we have successfully synthesized PLGA nanoparticles loaded with GEF. These GEF@PLGA NPs exhibit characteristics such as high water-solubility, passive targeting of tumors, and favorable compatibility with blood. In vitro and in vivo experiments have confirmed the anti-cancer effects of GEF@PLGA NPs in PCa cells. Overall, GEF@PLGA NPs possess tremendous therapeutic potential for PCa treatment. To facilitate their clinical utilization, it is crucial to conduct further experiments and clinical trials in the future.

Ethical statement

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Sun Yat-Sen University and approved by the Animal Ethics Committee of Sun Yat-Sen University (approval no. SYSU-IACUC-2021-000041).

Author contributions

Hai Huang: conceptualization, project administration, writing – review & editing, and funding acquisition. Jun Wu: conceptualization, supervision, writing – review & editing, and funding acquisition. Kaiwen Li: project administration, data curation, and writing – review & editing. Zhi Xiong: conceptualization, data curation, methodology, software, supervision, validation, visualization, and writing – original draft. Tong Tong: investigation, resources, formal analysis, software, and writing – original draft. Zhaoxiang Xie: methodology, data curation, resources, and writing – original draft. Shunli Yu: investigation, methodology, and software. Ruilin Zhuang: investigation, visualization, software, and data curation. Qiang Jia: writing – review & editing. Shirong Peng: investigation and formal analysis. Bingheng Li: methodology, software, and validation. Junjia Xie: validation and visualization. All authors read and approved the final manuscript.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Key R&D Plan of China (no: 2022YFC3602904); National Natural Science Foundation of China (no: 81974395 and 82173036); Guangzhou Science and Technology Program City-University Joint Funding Project (no. 2023A03J0001); Guangdong Basic and Applied Basic Research Foundation (no: 2019A1515011437); Sun Yat-Sen University Clinical Research 5010 Program (no: 2019005); Beijing Bethune Charitable Foundation (no: mnzl202001); Guangzhou Science and Technology Key R&D Project (no: 202206010117); Beijing Xisike Clinical Oncology Research Foundation (no: Y-MSDZD2022-0760 and Y-tongshu2021/ms-0162); Yangcheng Scholars Research Project of Guangzhou (no. 20183197), and Guangzhou Science and Technology Plan (no. 201901010170). Support was also received from the open research funds from the Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital to Jun Wu, and the open research funds from the Sixth Affiliated Hospital of Guangzhou Medical University, Qingyuan People's Hospital by Hai Huang.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3bm01735d

‡ These authors contributed equally to this work.

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