2.1 Cell culture

Human cervical cancer HeLa cells were purchased from Procell Biotech Co., Ltd (Wuhan, China), and human gastric cancer MGC803, ovarian cancer A2780 cells were maintained in our laboratory. 3 cell lines were cultured in roswell park memorial institute (RPMI-1640) complete medium (Gibco, Thermo Fisher Scientifc, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Sijiqin biotech., HangZhou, China). They were incubated at 37 °C with 5% CO₂, and the culture medium was changed every 2–3 days. The passaging of cells was performed based on their growth status, and a portion of cultured cells was cryopreserved for future use. The trypsin used in the experiments was purchased from Gibco.

2.2 In vivo phage display library screening

For this study, 4–5-week-old female BALB/c nude mice, weighing between 18–20 g, were bought from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China) and housed in a specific pathogen-free (SPF) room at a controlled temperature of 22 ± 2 °C, with access to food and water ad libitum.

Cervical cancer HeLa cells (1 × 10⁷ cells) were subcutaneously injected into the murine dorsal area, and the tumor development was closely monitored on a weekly basis. Then, 4–6 weeks post-injection, when the tumor volume reached approximately 1.0 cm³, the Ph.D.-C7C™ procedure was conducted using a phage display library Kit (#E8110S, New England Biolabs, MA, USA) following the kit’s instructions, and the generated Ph.D.-C7C™ peptide library (1012 pfu) was injected into the mice’s tail veins. After a 20-minute incubation, the mice were intraperitoneally anesthetized with 10% chloral hydrate (0.03 mL/10 g). Following anesthesia, their chest cavity was opened to expose the heart and perfused the right ventricle with 20 mL of warm normal saline. Tumor, liver, kidney, brain and heart tissues were then extracted and placed on ice. Next, 50% of each tissue was fixed in 4% paraformaldehyde for immunohistochemical and affinity enzyme-linked immunosorbent assay (ELISA) phage analyses. The remaining tissues were weighed, homogenized for phage recovery, and placed in 1.5 mL EP tubes. They were then rinsed three times with 0.1% phosphate buffer solution-tween (PBST) (1 mL each time), followed by a 10-minute centrifugation and removal of the supernatant. Tissues were then homogenized in E. coli ER2738 (New England Biolabs, USA) solution, incubated for 20 minutes at room temperature, and incubated in an LB liquid medium. Infection was allowed to proceed for 30 minutes. Simultaneously, we prepared and purified the phage amplification stock solution, which was used for the second screening, consisting of three additional rounds of in vivo screening.

2.3 Immunohistochemical phage staining

Phage distribution was assessed through immunohistochemical staining using an anti-M13 monoclonal antibody. After tissue fixation with 4% paraformaldehyde, the tissues were embedded in paraffin, sectioned, deparaffinized and rehydrated using xylene. The sections were maintained in an incubator, followed by three 3-minute rinses in phosphate-buffered saline (PBS). For staining, the S-P immunohistochemical staining kit (kit-5003, Meixin Biotechnology Development Co., Ltd, Fuzhou, China) was used following the manufacturer’s instructions. Briefly, the tissue sections underwent a 10-minute incubation in reagent A (endogenous peroxidase blocker, 50 μL) at room temperature, followed by three subsequent 3-minute PBS rinses. They were then incubated for 10 minutes with reagent B (normal goat serum, 50 μL) at room temperature. Next, the sections were treated overnight (ON) with horseradish peroxidase (HRP)-conjugated mouse anti-M13 monoclonal antibody (1:100 dilution) at 4 °C, followed by three subsequent 3-minute PBS rinses, after which the sections were exposed to reagent C (Biotin-labeled sheep anti-mouse/rabbit IgG, 50 μL) for 20 minutes at room temperature, followed by three 3-minute PBS rinses. They were then treated for 10 minutes with reagent D (Streptomyces avidin protein-peroxidase, 50 μL) at room temperature, followed by three subsequent 3-minute PBS rinses. Lastly, 5-minute staining was performed using 3,3′-diaminobenzidine-tetrahydrochloride, and distilled water was used to promptly terminate the reaction. The sections were counterstained with hematoxylin for 30 seconds, followed by treatment with hydrochloric acid and rinsed with tap water for 5 minutes. Neutral gum was used for sealing, and the sections were allowed to air-dry. The mean staining absorbance value was calculated using Image-Pro Plus 6.0 analysis (microscope (CarlZeiss, Oberkochen, BW, Germany) equipped with a ImagePro Plus 6.0, Purchased by Cancer Research Institute, Hengyang Medical School, University of South China).

2.4 Phage monoclonal affinity to cervical cancer HeLa cells

Phage ELISA was employed for this experiment. Briefly, the cells were seeded in a 96-well plate (1 × 10⁴ cells/well, Corning) and allowed to adhere for 24 hours. Subsequently, they were rinsed with PBS and incubated in serum-free RPMI-1640 for 2 hours at 37 °C. The cells were then fixed for 20 minutes in 4% paraformaldehyde (PFA) and permeabilized with 0.5% (vol/vol) Triton X-100 in PBS for 10 minutes at room temperature, followed by additional rinsing with 0.1% PBST. The cells were blocked for 1 hour at 37 °C using 2% (wt/vol) bovine serum albumin (BSA) suspended in PBS (2% PBS-BSA) and incubated for 2 hours with randomly selected amplified phage clones (1 × 10¹² pfu/well) at 37 °C, followed by four washes with 0.1% PBST. Next, an HRP-conjugated anti-M13 monoclonal antibody (200 μL/well, 2% BSA-PBS, 1:5000) was added for 1 hour at 37 °C, and color development was initiated using TMB (3,3′,5,5′-Tetramethylbenzidine, Beyotime, China). The reaction was terminated with 0.5 M H₂SO₄ solution (50 μL/well). Lastly, absorbance was measured at 450 nm using an automated ELISA plate reader, and three measurements were taken for each treatment. Selectivity was assessed by comparing the positive phage optical density (OD) to the control phage OD, with the criterion being P/N (the positive phage OD divided by the control phage OD) >2.1.

2.5 Sequence analysis

The ELISA-identified phage-positive clones were amplified, and DNA extraction was performed using the QIAprep Spin M13 Kit (#27704, Qiagen, Dusseldorf, NRW, Germany), following the kit’s instructions. DNA sequencing of the phage clones was conducted using Sangon Biotech (Shanghai) Co., Ltd (Shanghai, China), and then we predicted the amino acid sequences of the identified peptides based on the DNA sequence.

2.6 Peptide synthesis and conjugation

CCSP and control (NCSP) peptides with 5-Carboxyfluorescein (5-FAM) labeling were bought from Chinapeptides Co., Ltd, China, and the resulting peptides were referred to as 5-FAM-CCSP and 5-FAM-NCSP, respectively. Product purification was performed using high-performance liquid chromatography (HPLC), and confirmation was achieved through mass spectrometry (MS).

2.7 Immunofluorescent cytochemical staining

Immunofluorescent cytochemical staining was conducted to validate the targeting of cervical cancer HeLa cells by the CCSP peptides. HeLa, MGC803 and A2780 cells were cultured in the logarithmic growth phase for experimental use. Following pancreatic enzymatic digestion and resuspension, the cells (2.5 × 10⁵ cells/mL) were plated in 6-well plates and incubated in 1 mL of complete medium, with an autoclaved cover slide placed in each well. After cell adherence, the cells were rinsed thrice with PBS, and the old medium was replaced with 2 mL of a complete medium.

Experimental cells were treated with 2 μL (5.5 mmol/L) of 5-FAM-CCSP, while control cells received 2 μL (5.5 mmol/L) of 5-FAM-NCSP. Both sets of cells also received 2 μL of 50% dimethyl sulfoxide (DMSO) as a solvent agent. The cells were then incubated in darkness for 16 hours. After removing the media, the cells were gently rinsed five times with PBS. Then, the coverslips were carefully removed, and the slices were fixed in acetone for 15 minutes, followed by three PBS rinses and exposure to a fluorescence quenching agent (#3245, Solarbio, Beijing, China) for sealing. The slides were then observed, and images were captured using the Life AMAFD1000 fluorescent microscope. All experiments were performed three times.

2.8 Immunofluorescent histochemical staining

Human cervical cancer tissue specimens were used to confirm the affinity of the CCSP peptide for the tissues. Cervical cancer tissue microarrays (Number: CR1001#, comprising 9 CCAC, 12 CAC, 29 SCC, and 8 normal cervical tissues) were purchased from Alenabio Biotechnology Co., Ltd (Xi’an, China). The tissue slides were then deparaffinized, rehydrated, then subjected to antigen retrieval in 0.1 mol/L citrate solution at pH 6.0 and heated in a microwave for 6 minutes until boiling. After a 30-minute incubation, the slides were removed and rinsed twice in distilled water (ddH₂O) for 5 minutes each, followed by two additional rinses in PBS for 5 minutes each. After a 30-minute blocking step in BSA at 37 °C, they were incubated with 5-FAM-CCSP (100 μL, 2 mmol/L) or 5-FAM-NCSP (100 μL, 2 mmol/L) at 4 °C overnight, followed by three 5-minute rinses in 0.1% PBS. The slides were sealed with a fluorescence quenching agent (containing 4,6-diamino-2-phenyl indole (DAPI)) (Solarbio, Beijing, China) and observed under a fully-equipped Life AMAFD1000 fluorescent microscope. Lastly, the same tissue slides were subsequently used for H&E staining.

2.9 Statistical analysis

Data are shown as mean ± standard error of mean (SEM). Statistical analysis was performed using the SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) for Windows. Mean comparisons were conducted using one-way analysis of variance (ANOVA). A p-value less than 0.05 was considered for statistical significance.

3.1 Screening for a peptide that targets cervical cancer HeLa cells

We performed three rounds of screening on the Ph.D.-C7C™ peptide library to identify peptides targeting cervical cancer HeLa cells in an in vivo xenograft model. During each screening, phage concentration was found to be significantly increased in tumor tissues but decreased in other tissues (Table 1). Immunohistochemical staining revealed that phage content in murine tumor tissue was extensive, in contrast to the scarce expression in the liver and no expression in the heart, kidney, brain tissues. The mean OD values for tumor, liver, kidney, brain and heart tissues were 0.1159 ± 0.0220, 0.3028 ± 0.0524, 0.5026 ± 0.0456, 0.6058 ± 0.0468 and 0.6188 ± 0.0507, respectively (p < 0.05). These findings confirmed the widespread presence of phages in murine tumor tissues (Fig. 1).

Fig. 1.Immunohistochemical phage staining after three rounds of in vivo screening (magnification 200×). Phages were widely distributed among tumor cells, while minimal immunoreactivity was observed in control organs (liver, kidney, brain and heart).

To further confirm the specific interactions between phages and cervical cancer HeLa cells, we randomly selected 10 unique phage clones (blue plaques) from the 3rd selection series for cell-based ELISA examination. To assess selectivity, we compared individual phage interactions with cervical cancer HeLa cells to the original library locus coeruleus. The results showed that the phage OD ratio exceeded 2.1, indicating specific targeting of cervical cancer HeLa cells. Notably, 9 phage clones (phages: 1, 2, 3, 4, 5, 6, 8, 9, 10) exhibited the highest specific interaction abilities (Fig. 2).

Fig. 2.Assessment of the affinity between phage monoclonals and cervical cancer HeLa cells using cell-based ELISA. Nine phage clones (1, 2, 3, 4, 5, 6, 8, 9 and 10) exhibited the highest specific binding capacity.

Therefore, we randomly selected 10 phage clones from the third selection series for further analysis using cell-based ELISA. Initially, we determined the phage clone sequences and predicted the corresponding amino acid sequences from the acquired DNA sequences. Remarkably, all 10 clones displayed the same sequence with 100% identity, which was LLRSTGF. This sequence was designated as CCSP.

3.2 CCSP specifically interacts with cervical cancer HeLa cells in vitro

To assess the in vitro tumor-targeting properties of CCSP, immunofluorescent cytochemical staining was performed, and the results revealed that after 16-hour exposure to 5-FAM-CCSP, HeLa cells exhibited robust green fluorescence signal, which was observed in both the cell membrane and cytoplasm, with a stronger presence in the cell membrane. Conversely, when MGC803 and A2780 cells cells were treated with 5-FAM-CCSP, no fluorescence was observed, similar to the treatment with 5-FAM-NCSP (negative control peptides labeled with 5-FAM) and DMSO (solvent control) in both HeLa and MGC803, A2780 cells (Fig. 3). These findings indicate that CCSP specifically and strongly interacts with HeLa cells, with the binding sites predominantly located on the cell membrane and cytoplasm.

Fig. 3.Affinity and specificity of 5-FAM-CCSP for HeLa cells (magnification 200×). Strong green fluorescence signals were primarily localized in the cytoplasm and cell membrane of cervical cancer HeLa cells treated with 5-FAM-CCSP. In contrast, no fluorescence signals were observed in control cells (MGC803 and A2780). Additionally, no fluorescence was detected in cells treated with 5-FAM-NCSP (negative control peptides labeled with 5-FAM). FAM: 5-Carboxyfluorescein; CCSP: Cervical cancer specifically targeting peptide; NCSP: negative control peptide; DMSO: Dimethyl sulfoxide.

3.3 CCSP selectively associates with human CCAC specimens

We next evaluated the tumor affinity of 5-FAM-labeled CCSP in human cervical cancer specimens in vitro using immunofluorescent histochemical staining. The cervical cancer tissues were treated with either 5-FAM-CCSP or 5-FAM-NCSP at 4 °C in a light-free environment, and the results indicated that in 8 of 9 cases of CCAC, the green fluorescent signal was notably more intense after incubation with 5-FAM-CCSP. Within tumor cells, the fluorescence was primarily localized in the cell membrane and cytoplasm (Fig. 4, Table 2). Conversely, among the tested CACs, only 1 of 12 cases displayed weak positivity for CCSP. In SCC and normal cervical tissues, incubation with 5-FAM-CCSP did not result in fluorescence. Collectively, these findings strongly suggest that CCSP exhibits strong specificity for CCAC.

Fig. 4.Specific binding of 5-FAM-CCSP (5-Carboxyfluorescein cervical cancer specifically targeting peptide) to cervical cancer tissues, as demonstrated by immunofluorescent histochemical staining (magnification 200×). Strong green fluorescence was considered a positive result. Robust green fluorescence was observed from the cell membrane and cytoplasm of CCAC (cervical clear cell adenocarcinoma) tissue cells. Conversely, minimal fluorescence was detected in CAC (cervical adenocarcinoma), SCC (cervical squamous cell carcinomas), and NC (normal cervical) tissues. Cervical tissues treated with 5-FAM-NCSP (negative control peptides labeled with 5-FAM) exhibited no fluorescence.