Self-assembled RNA nanocarrier-mediated chemotherapy combined with molecular targeting in the treatment of esophageal squamous cell carcinoma

Background Esophageal cancer is the fifth most common cancer affecting men in China. The primary treatment options are surgery and traditional radio-chemotherapy; no effective targeted therapy exists yet. Self-assembled RNA nanocarriers are highly stable, easily functionally modified, and have weak off-tumor targeting effects. Thus, they are among the most preferred carriers for mediating the targeted delivery of anti-tumor drugs. miR-375 was found to be significantly down-regulated in esophageal squamous cell carcinoma (ESCC) tissues and its overexpression effectively inhibits the proliferation, migration, and invasion of ESCC cells. Moreover, epidermal growth factor receptor (EGFR) was overexpressed in ESCC cells, and accumulation of RNA nanoparticles in ESCC tumors was enhanced by EGFR-specific aptamer (EGFRapt) modification. Results Herein, a novel four-way junction RNA nanocarrier, 4WJ-EGFRapt-miR-375-PTX simultaneously loaded with miR-375, PTX and decorated with EGFRapt, was developed. In vitro analysis demonstrated that 4WJ-EGFRapt-miR-375-PTX possesses strong thermal and pH stabilities. EGFRapt decoration facilitated tumor cell endocytosis and promoted deep penetration into 3D-ESCC spheroids. Xenograft mouse model for ESCC confirmed that 4WJ-EGFRapt-miR-375-PTX was selectively distributed in tumor sites via EGFRapt-mediating active targeting and targeted co-delivery of miR-375 and PTX exhibited more effective therapeutic efficacy with low systemic toxicity. Conclusion This strategy may provide a practical approach for targeted therapy of ESCC. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12951-021-01135-5.


Background
The latest statistics showed that there were approximately 19.29 million new cases of cancer and 9.96 million new cancer-related deaths worldwide in 2020. Among these were 600,000 new cases and 548,000 deaths related to esophageal cancer [1]. The incidence and mortality of esophageal cancer in China rank first in the world, with the predominant subtype being squamous cell carcinoma, which accounts for more than 90% of the cases. Currently, the treatment of esophageal cancer remains a comprehensive approach based on surgery and traditional radio-chemotherapy, with chemotherapy drugs primarily used to treat advanced stages of cancer [2]. However, the lack of targeting ability of chemotherapeutic drugs and the serious side effects produced greatly limit their therapeutic effects. Therefore, current research is focused on developing new targeted treatment strategies against esophageal squamous cell carcinoma (ESCC).
In recent years, nanomedicine has played an increasingly important role in the precision diagnosis and treatment of tumors. The main advantages of this application include enhancing the carrier-mediated targeted distribution of drugs in tumors [3,4], reducing the toxic and side effects of chemotherapy [5], and producing a more significant synergistic tumor inhibitory effect via combined drug delivery [6,7]. Nevertheless, existing nanocarriers (such as inorganic nanomaterials [8], polymer nanomaterials [9], carbon nanomaterials [10], and cationic liposomes [11]) display a notably significant nontumor tissue distribution effect, with a considerably lower level of drug accumulation at the tumor site than in organs and tissues such as the liver, kidney, and spleen. Owing to the low efficiency of targeted drug delivery, it is difficult to avoid toxic and side effects. Guo et al. revealed in 1998 that the packaging RNA (pRNA) of bacteriophage phi29 can be transformed-through self-assembly technology-into a dimer, trimer, or hexamer using a concise RNA structure [12]. Studies have confirmed that, in addition to possessing the characteristics of a strong enzyme [13], thermal stability [14,15], and easy functional modification properties [16,17], the RNA nanocarriers display a lower off-targeting effect than cationic liposomes due to their high degree of anionicity. When RNA nanocarriers are intravenously administered to subcutaneous xenotransplantation or metastatic tumor-bearing mice, they can specifically target cancer cells with little or no accumulation in normal organs or tissues, thereby exerting a better anti-tumor effect [13,18,19].
From our previous studies, we determined, through high-throughput sequencing, that miR-1973, miR-1246, and miR-375 were markedly under-expressed in ESCC tissues, and that their overexpression could significantly inhibit the proliferation, migration, and invasion of ESCC KYSE-150 cells. Among these, miR-375 was the most effective, highlighting its potential as a molecular target for the treatment of ESCC. Moreover, ESCC cells can overexpress the epidermal growth factor receptor (EGFR). The aptamer modification of EGFR (EGFR apt ) can not only effectively increase the accumulation and tumor penetration ability of the RNA nanocarrier 4WJ in ESCC cells, but also significantly increase the distribution of carriers in mouse xenograft ESCC tissues. With these characteristics and the RNA four-way junction (4WJ) as a premise, the objective of the present study was to construct the aptamer-modified nano-drug 4WJ-EGFR apt -miR-375-PTX, which was simultaneously loaded with miR-375 and paclitaxel (PTX). We determined through experimental analyses that the nano-drug possesses strong enzyme and thermal stability. Cytological analysis of small animal ESCC tumor-bearing models have confirmed that the modification of EGFR apt , as well as the synergistic delivery of miR-375 and PTX, can exert a more effective inhibitory effect on ESCC, thus providing a potential strategy for targeted therapy against this cancer type.

Results and discussion
Detection and functional verification of miRNA MicroRNAs (miRNAs) are regulatory small non-coding RNAs of approximately 22 nt produced by virtually all the cells in the body [20]. They are important, highly conserved, non-coding small single-stranded RNAs, which play a key regulatory role in the occurrence and development of tumors [21,22]. We analyzed the differential expression of miRNAs in 5-paired ESCC and normal esophageal tissues using high-throughput sequencing and observed that the expression of miR-1973, miR-1246, and miR-375 was significantly low in ESCC (Additional file 1: Fig. S1). The results of cytological analyses revealed that miR-375 was the most effective in inhibiting proliferation (Fig. 1a), migration (Fig. 1b), invasion (Fig. 1c), and in promoting apoptosis in ESCC cells (Fig. 1d), indicating miR-375 as a potential therapeutic target for ESCC. Currently, clinical trials involving certain miRNA analogs (e.g., miR-34 and miR-16) have produced positive results [23,24], further highlighting the potential clinical application of miR-375.

EGFR aptamer modification promotes the accumulation of RNA nanocarriers in esophageal squamous cell carcinoma cells and 3D tumor microspheres
Normal cells in the body undergo a series of pathological changes (e.g., gene mutations or changes in expression levels) during the process of canceration. For example, EGFR, a member of the epidermal growth factor receptor family, is overexpressed in several solid tumors, and its overexpression is closely related to tumor proliferation, angiogenesis, invasion, and metastasis [25]. Thus, EGFR is one of the preferred genes used in designing and developing tumor-targeted therapies such as cetuximab [26] and gefitinib [27]. By analyzing the TCGA database, we found that EGFR is highly expressed in ESCC (Additional file 1: Fig. S2), and this expression was further confirmed by testing 140 ESCC tissue samples (Additional file 1: Fig. S3a, b). KYSE-150 cells (Additional file 1: Fig. S4) also demonstrated a high expression of EGFR, indicating that EGFR can be used as a candidate in therapy against ESCC.
Therefore, we aimed to use EGFR as the candidate to construct a novel targeted nano-delivery system that is simultaneously loaded with miR-375 and chemotherapeutics, as a treatment against ESCC. The selection of suitable molecules that mediate targeting is the key to the construction of effective delivery systems. It is wellknown that the ligands which mediate the targeted delivery of nanomedicine into tumors mainly include monoclonal antibodies [28], peptides [29], folic acid [30], and nucleic acid aptamers [31]. Aptamers are random oligonucleotide sequences that bind with high affinity and specificity to their target molecules, which are generated via screening using the systematic evolution of ligands by exponential enrichment (SELEX) technology. In addition to a high affinity and specificity comparable to those of antibodies, aptamers also have a wide range of targets, low molecular weight, low toxicity, non-immunogenicity, and high tissue permeability, contrary to antibodies [32]. They are currently one of the preferred candidates for mediating targeted nano-drug delivery. As mentioned previously, RNA nanoparticles have multiple advantages as drug delivery carriers. In the present study, the selected delivery carrier was 4WJ, which not only possesses the above characteristics but can also substantially increase the loading of chemotherapeutic drugs, as well as display higher thermodynamic stability than 3WJ [33]. Therefore, a EGFR apt -modified 4WJ carrier (4WJ-EGFR apt ) was developed (Additional file 1: Fig. S5), and the data showed that EGFR apt effectively promoted the accumulation of 4WJ in KYSE-150 cells (Additional file 1: Fig. S6a, b). The mouse ESCC tumor-bearing model also showed that EGFR apt significantly promoted the distribution of 4WJ within the tumor site (Additional file 1: Fig.  S7a, b), further indicating that EGFR apt modification can mediate the targeted delivery of 4WJ-based nano-drugs to the ESCC sites.

Construction and characterization of 4WJ-EGFR apt -miR-375-PTX
To test whether 4WJ-EGFR apt could mediate the targeted delivery of miR-375 and PTX, thereby achieving better therapeutic effects against ESCC, we constructed the EGFR apt -modified drug delivery system 4WJ-EGFR apt -miR-375-PTX, which was simultaneously loaded with miR-375 and 24 molecules of PTX (Fig. 2a). PTX-N 3 was synthesized (Additional file 1: Fig. S8) and conjugated to different RNA oligomers (Additional file 1: Fig. S9). The nanoparticles were assembled by mixing equimolar concentrations of four RNA-6 PTX oligomers and preliminary identified by native PAGE (Fig. 2b) and UV absorption (Fig. 2c). The atomic force microscopy (AFM) images (Fig. 2d, Additional file 1: Fig. S10) and the results obtained from dynamic light scattering (DLS) showed the average particle size of the nanocarriers to be approximately 10 nm, whereas that of 4WJ-EGFR apt -miR-375-PTX was approximately 13.4 ± 0.25 nm (Fig. 2e). The zeta potential on the surface of all nanocarriers was negative, and that of 4WJ-EGFR apt -miR-375-PTX was about − 9.1 ± 2.5 mV (Fig. 2f). Surface anionicity is another key advantage of RNA nanocarriers that can effectively reduce the non-target cell-binding effect of nano-drugs.

Stability of the nano-drug and its release from 4WJ-EGFR apt -miR-375-PTX
Once inside the body, the stability of the nanomedicine structure and-more importantly-rapid RNA degradation, are critical factors to achieve optimal pharmacokinetics and pharmacodynamics, as well as low toxic and side effects. However, studies have reported that RNA nanocarriers with special structures such as 4WJ possess superior enzyme and thermal stability. Moreover, the loading of the drug will also affect the stability of the carrier. For example, thermal stability is significantly reduced after loading the 3WJ carrier with 10 molecules of PTX [33]. Here, we tested the thermal, pH, and enzyme stability of 4WJ-EGFR apt -miR-375-PTX. The results revealed that 4WJ-EGFR apt -miR-375-PTX possessed good thermal stability and its melting temperature was 57.5 ± 3.9 °C (Fig. 3a). However, its stability was slightly lower than that of 4WJ, 4WJ-EGFR apt , 4WJ-miR-375, 4WJ-EGFR apt -miR-375, 4WJ-PTX, and 4WJ-EGFR apt -PTX (Fig. 3b, Additional file 1: Fig. S11). No significant degradation was observed after incubating 4WJ-EGFR apt -miR-375-PTX with RNase for up to 24 h (Fig. 3c). RNA nanoparticles of 4WJ in this study consists of fully modified RNA oligonucleotides at their 2' position, e.g. 2'F, 2'OMe, which prevent them from RNase and plasma degradation, and this was identical with the previous report [34]. The stability of 4WJ-EGFR apt -miR-375-PTX in PBS with different pH was also tested, and the results suggested that the stability was not affected by pH variation (Fig. 3d). Drug release is another key factor that affects drug efficacy. A release assay showed that PTX was gradually released from 4WJ-EGFR apt -miR-375-PTX after incubation with 50% FBS, and significant release occurred after 12 h (Fig. 3e). The release of RNA nano-loaded PTX occurs mainly through the action of esterase which breaks the ester bonds.

Inhibitory effect of 4WJ-EGFR apt -miR-375-PTX on esophageal squamous cell carcinoma cells in vitro
To test whether EGFR apt can improve the efficiency of 4WJ-targeted delivery of miR-375 and PTX, we assessed the ability of EGFR apt -conjugated nanomedicine to bind and integrate KYSE-150 cells through cytological experiments. Confocal analysis revealed that EGFR apt significantly enhanced the accumulation of 4WJ-miR-375-PTX in KYSE-150 cells (Fig. 4a, 4b). Cell proliferation assays showed that both 4WJ-miR-375 and 4WJ-EGFR apt -miR-375 inhibited the proliferation of KYSE-150 cells. 4WJ-PTX, 4WJ-EGFR apt -PTX, and 4WJ-miR-375-PTX significantly suppressed the proliferation of KYSE-150 cells; however, this inhibitory effect was weaker than those of PTX and 4WJ-EGFR apt -miR-375-PTX at low concentrations (120 nM PTX, 5 nM nanoparticles) (Fig. 4c). This inhibitory effect tended to be consistent with PTX and 4WJ-EGFR apt -miR-375-PTX as their concentrations increased (Additional file 1: Fig. S12). This may be related to the cell uptake and drug release kinetics. Studies have demonstrated that PTX exert the anti-tumor efficiency by inducing apoptosis and suppressing proliferation, migration and invasion. To elucidate the potential mechanism of synergetic effect of miR-375 and PTX on ESCC inhibition, apoptosis (Bax, Bcl2 and caspase-3), cell cycle (Cyclin A2, Cyclin B1 and Cyclin D1), migration and invasion-related proteins (E-cadherin) were determined. Data ( Fig. 4d) suggested that the improved anti-ESCC effect of 4WJ-EGFR apt -miR-375-PTX is due to apoptosis, cell cycle and epithelial-mesenchymal transition arrest induced by miR-375.
Anti-tumor drugs must reach the tumor site and penetrate the tissue to exert their inhibitory effects. However, the features of tumor microenvironment, which include high peritumoral and low intratumoral blood vessel density, and a dense interstitial structure in the center of the tumor, lead to poor permeability of nanomedicines [35]. Most of the nanocarriers, therefore, are unable to consistently deliver the drug throughout the entire tumor tissue. This eventually results in uneven drug distribution, and, consequently, poor therapeutic efficacy. Therefore, enhancing the tumor permeability has become one of the most important strategies to improve the therapeutic efficacy of nanodrugs. Studies have confirmed that aptamers exhibit strong tissue permeability and their modification can significantly enhance the penetration of nanoparticles into tumors [36,37]. In the present study, 3D-tumor spheroids  of KYSE-150 cells were established and confocal analysis confirmed that the permeability of 4WJ-miR-375-PTX was dramatically enhanced by EGFR apt decoration (Fig. 4e), and thus, 4WJ-miR-375-PTX exerted the strongest inhibitory effect on the growth of spheroids (Fig. 4f).
In vivo biodistribution and anti-esophageal squamous cell carcinoma activity of 4WJ-EGFR apt -miR-375-PTX The above data (Additional file 1: Fig. S7a, b) have confirmed that EGFR apt modification can promote the targeted distribution of 4WJ in ESCC tissues. Therefore, we further investigated whether miR-375 and PTX affected the biodistribution of the nanoparticles. 4WJ-miR-375-PTX and 4WJ-EGFR apt -miR-375-PTX were separately injected intravenously into KYSE-150 tumorbearing mice. Live imaging revealed that the distribution of 4WJ-EGFR apt -miR-375-PTX in the tumor tissues was much higher than that of 4WJ-miR-375-PTX (Fig. 5a, b), which supports the application of 4WJ-EGFR apt -miR-375-PTX in ESCC treatment.

In vivo toxicity analysis
Potential toxic and side effects are important indicators to evaluate the application value of drugs in clinical transformation. PTX is one of the most widely used chemotherapeutic agents and displays a positive therapeutic effect during the treatment of ESCC. However, in its conventional formulation, PTX would cause strong toxic and side effects, including hypersensitivity reactions, myelosuppression, neurotoxicity, cardiotoxicity, hepatotoxicity, and alopecia [38]. We analyzed the following changes in mice treated five times with PTX and other nanodrugs: body weight, histopathology, blood biochemical indicators (hepatotoxicity indicators such as aspartate  transaminase (AST), alanine transaminase (ALT), and albumin (ALB); kidney toxicity indicators such as blood urea nitrogen (BUN), and creatinine (CREA); and cardiotoxicity indicators such as lactate dehydrogenase (LDH), creatine kinase (CK), creatine kinase myocardial band (CK-MB)). Although no significant change was recorded in the body weight (Additional file 1: Fig. S14) and histopathology (Additional file 1: Fig. S15) of the mice in each group, analysis of the biochemical indicators showed that the hepatotoxicity indicator (ALT) (Fig. 7a, *p < 0.05) and cardiotoxicity indicator (LDH) (Fig. 7b, *p < 0.05) of mice in the PTX and group were significantly higher than those in other groups. Additionally, various indicators in 4WJ-EGFR apt -miR-375-PTX group were improved compared with the 4WJ-miR-375-PTX group (Fig. 7a-c), indicating that EGFR apt modification can reduce the toxicity and side effects of nano-drugs.

Conclusions
In this study, a novel 4WJ-based RNA nanoparticle which was co-loaded with PTX and ESCC-suppressive miR-375 selected by RNA sequencing and cytological function verification and decorated with EGFR apt (4WJ-EGFR apt -miR-375-PTX) was developed, and its anti-ESCC capacity was investigated. 4WJ-EGFR apt -miR-375-PTX possessed optimal thermal and pH stability. Moreover, PTX release was directly related to the esterase hydrolysis. In vitro analysis demonstrated that 4WJ-EGFR apt -miR-375-PTX could be internalized by KYSE-150 cells and penetrates into tumor spheroids with high efficiency, and thus, suppresses the proliferation of KYSE-150 cells more efficiently. More importantly, 4WJ-EGFR apt -miR-375-PTX was selectively accumulated in the tumor site via EPR effect and EGFR apt -mediated active targeting. The optimal therapeutic efficacy of 4WJ-EGFR apt -miR-375-PTX against KYSE-150-derived tumors, which was mediated by the enhanced PTX distribution in tumors and synergistic effect of miR-375 and PTX, was observed in vivo. Therefore, this delivery system offers a new targeted therapy strategy for the treatment of ESCC, with attractive prospects for its clinical application in transformation.

Migration and invasion assays
Migration and invasion of KYSE-150 cells were detected using transwell chambers. For migration, KYSE-150 cells (4 × 10 4 /well) were cultured in 6-well plate for 24 h, and transfected with miRNA mimics (miR-375, miR-4776-5p, miR-1973, miR-1246, miR-139) by lipofectamine 3000. After transfection for 6 h, cells were collected and resuspended with serum-free DMEM. Cells were then seeded (2 × 10 4 ) in the upper chamber of each insert and the medium with 10% FBS was placed in the lower chamber. After incubation for 48 h, cells on the upper layer of the membrane were discarded, the cells on the lower surface were fixed with methanal and stained with 0.1% crystal violet dye. The stained cells were then photographed and counted in three randomly selected fields. The invasion assays were performed using Matrigel-coated Transwell inserts with the same procedure as migration assay.  (Wuhan, China). After deparaffinization and rehydration, sections were treated for antigen retrieval for 5 min and blocked with 5% BSA for 1 h at room temperature. After 3 times washing, tissue sections were incubated with anti-EGFR antibody (1:150) for 3 h, and the EGFR expression was detected by the EnVision FLEX/ HRP (Dako Denmark A/S). The intensity and extent of EGFR expression was finally determined and quantified using the histochemical scoring system (H-score).

EGFR expression in ESCC tissue and KYSE-150 cells
To test the EGFR expression in EYSE-150 cells, cells (2 × 10 4 /well) were cultured on coverslips in 24-well plate for 24 h. After 3 times washing, cells were fixed with 4% paraformaldehyde for 20 min and blocked with 5% BSA for 30 min. After 3 times washing, cells were successively incubated with anti-EGFR antibody and Alexa 488 labeled goat anti-rabbit IgG. The expression of EGFR was finally observed by a confocal microscope (NIKON A1 +).

Synthesis of RNA oligomers and PTX-N3
RNA oligomers were obtained from ExonanoRNA Biomedicine (Foshan, China), rG, rG, rC, rU, 2'F rC and 2'F rU phosphoramidites were purchased from Huaren Science and Technology Co., Ltd. (Wuhu, China). 2'O-propargyl rC and rU were ordered from Chemgene. RNA oligomers were purified by desalting using Glen Pak purification cartridges and gel electrophoresis (Bio-Rad). PTX-N3 prodrug was synthesized according to previous report. In brief, paclitaxel, N,N′dicyclohexyl-carbodiimide, 4-(dimethylamino) pyridine and 6-azido-hexanoic acid were mixed and reacted in 10 mL dichloromethane with an equivalent ratio of 1:2:1:2. The reaction was carried out at 25 °C with stirring for 16 h. The crude product was yielded by filtration and rotary evaporation, and finally purified by silica gel chromatography.

Enzymatic stability assay
5 μM of 4WJ-EGFR apt -miR-375-PTX was incubated with RNase (0.1 μg/μL) at 37 °C for different times (0, 1, 3, 6, 12 and 24 h). The samples were then examined by 2% agarose gel electrophoresis and the percentage of intact nanoparticles (intensity of the band at a time point/intensity of the band at 0 h) was quantified by Image J.

Confocal microscopy imaging
To investigate the cellular binding and uptake efficiency, KYSE-150 cells were cultured in chamber slides (2 × 10 4 /well) for 24 h. Then, AF647-labeled RNA nanoparticles (400 nM) including 4WJ, 4WJ-EGFR apt , 4WJ-miR-375-PTX and 4WJ-EGFR apt -miR-375-PTX were respectively added and incubated at 37 °C for 12 and 24 h. Cells were then washed 3 times with PBS, fixed with 4% PFA for 10 min at room temperature followed by staining with DAPI. The AF647 signals in cells was examined and quantified using a confocal microscope (NIKON A1 +).
To study the penetration capacity of nanoparticles, 3D multicellular tumor spheroids were prepared by suspending KYSE-150 cells (1 × 10 3 ) with DMEM/Matrigel (1:1, v/v) in 35 mm culture dishes. After 10-14 days culturing, culture medium was removed and cells were washed with PBS for 3 times, then the AF647-labeled 4WJ-miR-375-PTX and 4WJ-EGFR apt -miR-375-PTX (800 nM) were respectively added and incubated with tumor spheroids for 12 and 24 h. Then the spheroids were fixed with 4% PFA, stained with DAPI and the AF647 signals were detected and quantified by confocal microscope.
Growth of 3D tumor spheroids was further tested by treatment with aforementioned nanoparticles and PTX every 5 days for 2 times. Then the spheroids were imaged and the size was calculated. , and E-cadherin (1:2000) overnight at 4 °C. After three times washing, membrane was incubated with secondary antibody for 1 h. Finally, the proteins were visualized using an enhanced chemiluminescence (ECL) detection reagent (Tanon).

Biodistribution of nanoparticles
To verify whether EGFR apt could enhance the distribution of nanoparticles in KYSE-150-derived tumor tissues, 5 nmol of AF647 labeled 4WJ, 4WJ-EGFR apt , 4WJ-miR-375-PTX and 4WJ-EGFR apt -miR-375-PTX were intravenously injected into KYSE-150-bearing BALB/c nude mice, live imaging was performed at 2, 4, 6 and 8 h after administration. Mice were sacrificed, the organs including livers, lungs, kidneys, spleens, hearts, and tumor tissues were collected, the distribution of nanoparticles was scanned and quantified by live imaging system (Bruker FX Pro).

Xenograft tumor model
Female BALB/c nude mice (6 − 8 weeks) were subcutaneously injected with KYSE-150 cells (1 × 10 7 /mouse). When tumors reached approximately 50 mm 3 in volume, the mice were then randomly divided to different groups for following studies.
Livers, lungs, hearts, kidneys, spleens, and tumors were removed, tumors were photographed, HE staining was performed to detect the pathological changes, IHC staining of Ki67 was carried out to analyze the proliferation of cancer cells.

Ki67 staining
Paraffin-embedded, 5 μm thick tumor tissues were dried at 60 °C for 2 h, deparaffinized in xylene and hydrated in decreasing alcohol series (100%, 95%, 85%, 70%) before Ki67 staining. Antigen retrieval was carried out by boiling the slides for 5 min. After 3-time washes with PBS, endogenous peroxidase was inactivated with 0.3% hydrogen peroxide at room temperature for 15 min. After 3-time washes, slides were blocked with 5% BSA for 1 h and then stained with Ki67 for 3 h at room temperature.
The sections were washed and incubated with HRPlabeled goat anti-rabbit IgG at room temperature for 30 min. After coloring with DAB, the counterstaining, dehydration, and vitrification were successively performed. Finally, the slides were mounted and the percentage of Ki67 positive cells was calculated.