Synthesis of a novel specific nectin-4 tumor-targeted peptide optical molecular probe and its preliminary study in lung cancer

Abstract

Nectin-4/PVRL-4 is overexpressed in tumors, driving proliferation and invasion. It’s a key target for solid tumor therapy. Current tumor imaging methods are inadequate. Studying nectin-4 in lung cancer helps identify diagnostic biomarkers and develop radionuclide probes, intraoperative visualization,and new chemotherapies. High nectin-4 expression in lung cancer tissues was confirmed using TCGA data and IHC. Three optical probes targeting nectin-4 were synthesized. Optimal binding conditions were explored, and binding rates were assessed. Animal experiments showed the probes’ specificity and biodistribution. The probes effectively targeted nectin-4, with Cy5.5-nectin-4 PEP 02 showing the highest binding rate. In vivo, the probe targeted lung cancer tissues, indicating potential for intraoperative navigation. Nectin-4 is a promising target for lung cancer and intraoperative navigation, guiding further research.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-24074-9.

Introduction

Lung cancer has the highest incidence and mortality rates among malignancies, and is often diagnosed at an advanced stage¹. Imaging assessment, as a non-invasive examination method, is crucial in the diagnosis and evaluation of malignancies. Recent advancements in molecular imaging technology have significantly improved functional imaging, particularly in the study of tumor heterogeneity at the molecular level, offering early diagnostic value². Near-infrared optical imaging is an emerging molecular imaging technique following PET/CT, primarily utilizing fluorescent dyes to label tumor surface-specific recognition molecules and distinguishing tumor cells from normal cells at the molecular level using fluorescence microscopes and near-infrared fluorescence optical imagers³. However, this technique is limited by the expression of most tumor surface antigens not only in tumor tissues but also in normal human tissues. Therefore, identifying tumor-specific recognition molecules is crucial not only for tumor-specific imaging but also for targeted tumor therapy.

Nectin are a family of Ca2 + -independent immunoglobulin superfamily molecules, including nectin-1 to nectin-4. Among them, nectin-1 to nectin-3 are primarily distributed in normal human tissues, while nectin-4 is overexpressed in tumor tissues^4,5. The nectin-4 receptor is primarily located on the cell membrane surface, promoting epithelial-mesenchymal transition (EMT) by activating the AKT signaling pathway, thereby facilitating tumor proliferation, invasion, and migration⁶. Nectin-4 is a promising therapeutic target in multiple solid tumors, with corresponding antibody–drug conjugates (ADCs) already in clinical use for urothelial carcinoma⁷. Although nectin-4 expression has been extensively studied in breast and pancreatic cancers, research on its role in lung cancer, which has a relatively high incidence, is limited. Thus, nectin-4 has significant potential in the diagnosis and treatment of lung cancer. Peptide-drug conjugates (PDCs), compared to ADCs, have smaller molecular weights, lower autoimmune reactions, and are easier to synthesize and purify, making them promising candidates for new targeted drug development. Nectin-4 PEP 01, Nectin-4 PEP 02, and Nectin-4 PEP 03 are all derived from the extracellular domain of the Nectin-4 protein. These small-molecule peptides, each comprising 14 amino acids, specifically bind to the Nectin-4 receptor. In a study relevant to ovarian cancer, 27 peptides spanning the Nectin-4 extracellular domain were screened. The analysis revealed that three peptides—Nectin-4 PEP 01 (localized within the IgV/IgC1 domain), Nectin-4 PEP 02 (localized within the IgC1 domain), and Nectin-4 PEP 03 (localized within the IgC2 domain)—participate in cell adhesion and exhibited potent cell adhesion activity⁸. We used the fluorescent dye Cy5.5 to label these peptides to form novel near-infrared optical probes, studied their distribution in lung cancer cells and tissues through visualization, and verified their specific binding to lung cancer both in vitro and in vivo.

This study provides a real-time and visual monitoring method for tumor heterogeneity imaging and tumor efficacy assessment, offers insights into subsequent radionuclide probe development and PDC drug research, and provides new ideas for intraoperative navigation.

Methods

Materials

Cy5.5-Nectin-4 PEP 01, Cy5.5-Nectin-4 PEP 02, and Cy5.5-Nectin-4 PEP 03 were synthesized by Tanzhen Biotechnology Co., Ltd., Nanchang, China. The nectin-4 rabbit anti-human monoclonal antibody (dilution 1:3000) was purchased from Abcam, and the ready-to-use immunohistochemistry SP supersensitive kit (goat) was purchased from R&B and used strictly according to the manufacturer’s instructions. Immunohistochemistry instruments were purchased from LEICA, Germany. Reagents for cell experiments were obtained from Gibco, USA. The confocal microscope model was Zeiss LSM 880. The flow cytometer was purchased from BD, USA. HPLC was purchased from Agilent Technologies (Santa Clara, CA, USA). Products were separated and purified using a high-performance liquid chromatography C18 column (Zorbax SB-C18 4.6 × 250 mm, 5 μm). The HPLC conditions were as follows: The HPLC analysis was performed on a C18 column (250 mm × 4.6 mm, 5 μm) using a mobile phase consisting of water with 0.1% trifluoroacetic acid (TFA) (A) and acetonitrile (B). The column was first equilibrated with an isocratic mixture of A and B (75:25, v/v) for at least 5 min until baseline and pressure stability were achieved. The column temperature was maintained at 30 °C with a constant flow rate of 1.0 mL/min, and detection was carried out at 220 nm. For analysis, the sample (0.2 mg) was dissolved and diluted to 1 mL with water, and 20μL of the resulting solution was injected. Separation was achieved using the following gradient program: 75% A at 0 min, linearly changing to 55% A at 20 min.

Ualcan data analysis method

Analyze the expression level of nectin-4 in clinical samples of lung adenocarcinoma patients based on the TCGA database. Search for nectin-4 (synonym PVRL4) in the TCGA database, set as follows: gene symbol: PVRL4, TCGA dataset: lung adenocarcinoma. Analyze the expression of PVRL4/Detect-4 mRNA in lung adenocarcinoma and explore its correlation with patient gender, age, race, grading, and lymph node metastasis.

Immunohistochemistry experiment

Material source tissue specimens and clinical data

67 pathological specimens of lung adenocarcinoma patients were obtained from the Pathology Department of Jinhua Hospital affiliated with Zhejiang University School of Medicine. Informed consent was obtained from all subjects and/or their legal guardian(s). This project has been approved by the Medical Ethics Committee of Jinhua Hospital Affiliated to Zhejiang University School of Medicine (Ethics Approval [2025] No. 105). I confirm that all methods were carried out in accordance with the relevant guidelines. All programs are conducted in accordance with the ethical standards set forth in the 1964 Helsinki Declaration and its subsequent amendments. I confirm that all experiments were conducted in accordance with relevant guidelines and regulations. I confirm that the author follows the ARRIVE guidelines. None of the patients received preoperative radiotherapy or chemotherapy. Corresponding paraffin-embedded specimens of adjacent normal lung tissues were also collected from these 67 patients. All lung adenocarcinoma patients were confirmed through postoperative pathological examination. All slides were reviewed, and a definitive diagnosis was made by two pathologists.

Experimental steps and interpretation methods

Immunohistochemistry steps: After dewaxing the sections, antigen retrieval, peroxidase blocking, primary antibody incubation, secondary antibody incubation, streptavidin–biotin-peroxidase solution treatment, and DAB coloration were performed. Finally, hematoxylin counterstaining and dehydration and transparency treatment were conducted, followed by sealing the slices for observation.

Immunohistochemistry interpretation: nectin-4 positivity was indicated by a yellowish-brown color. A 13-point scoring method was used to calculate the number of positive and total cells in each field of view⁹. Staining intensity was classified into four grades, and a semi-quantitative method was used to calculate the positive cell rate. The final score was the product of these two indicators.

Statistical methods

SPSS 19.0 statistical software was used for data analysis. Measurement data were expressed as mean ± standard deviation. Comparisons between groups were performed using the χ² test. P < 0.05 was considered statistically significant.

Cell culture

The lung adenocarcinoma cell lines A549 and PC9 were purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and are adherent cells. PC9 cells were cultured in DMEM medium supplemented with 1% double antibiotics (100 pg/ml streptomycin and 100 U/ml penicillin) and 10% fetal bovine serum. A549 cells were cultured in RPMI-1640 medium with the same supplements. The BEAS-2b cell line (human bronchial epithelial cells) was purchased from Zhejiang Bodi Biotechnology Co., Ltd. (Hangzhou, China). Cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin. Both cell lines were incubated in a cell culture incubator at 37 °C with 5% CO2 and saturated humidity.

Animal model establishment

BALB/c nude mice (20–25 g, 4–6 weeks old; purchased from the Animal Center of Zhejiang University School of Medicine) were used. Approximately 5 × 106 PC9 tumor cells mixed with 100 μL of PBS were injected subcutaneously into one flank of each mouse. Tumors were allowed to grow for 4–5 weeks until their volume reached approximately 1 cm3, at which point they were used for experiments.

Cell binding experiments

Concentration and time gradient experiments

PC9 and A549 cells were seeded in confocal dishes. When the cells covered approximately 80% of the dish bottom, they were washed three times with PBS, fixed with paraformaldehyde, stained with DAPI (1 ml PBS + 0.1 μl DAPI), and then incubated with the peptide solutions. The experiments were divided into concentration and time gradient groups. For the concentration gradient group, 500 μl of each of the three probe solutions at concentrations of 2 μM, 5 μM, 10 μM, and 20 μM were added to A549 cells, and 5 μM, 7.5 μM, 15 μM, and 30 μM to PC9 cells. All dishes were then incubated in an incubator for 1 h, followed by the addition of a fluorescence quencher, and observation under a confocal microscope. For the time gradient group, 500 μl of peptide solutions at a concentration of 20 μM for A549 cells and 30 μM for PC9 cells were added, followed by fluorescence quenching and incubation in an incubator for 10 min, 30 min, 60 min, and 90 min before observation under a confocal microscope. The quencher, Antifade PVP Mounting Medium (P0123, Beyotime Biotechnology, China), and 1–2 drops were applied per well.These experiments were repeated three times, and parallel wells were included in each experiment.

Cell line nectin-4 target validation

Cell line confocal experiment

Inoculate PC9, A549, and Beas-2b cells into confocal culture dishes. Cover the confocal dish with 80% cell volume, wash three times with PBS, fix with paraformaldehyde, stain with DAPI (1 ml PBS + 0.1 μ l DAPI), and then incubate in 30 μ M Cy5.5 Nectin-4 PEP02 peptide solution for 60 min. Then add fluorescence quencher and observe under confocal microscope. These experiments were repeated three times. Each experiment includes time and a parallel well.

Western blot

After cell lysis in RIPA buffer (Beyotime, Shanghai, China) and quantification of total protein concentration using a commercial bicinchoninic acid (BCA) method kit, protein aliquots (30 μg per lane) were electrophoresed on 10% SDS-PAGE gels and transferred to PVDF membranes. The membranes underwent blocking with 5% skim milk in TBST for 1 h at room temperature and overnight incubation at 4 °C with primary antibodies (1:1000 dilution). The primary antibodies used in this study were as follows: anti-Nectin-4(ab192033,Abcam) and anti-GAPDH(60004-1-Ig, Proteintech). Protein bands were then detected using enhanced chemiluminescence (ECL) substrate (Lot RPN5787, GE Healthcare, PA, USA) and imaged with a BioRad ChemiDoc XRS + system (CA, USA).

Competitive inhibition experiment

A549 and PC9 cells were seeded in 20 mm NEST confocal dishes and grown to the logarithmic phase. The medium was discarded, and the cells were washed three times with PBS. Then, 500 μl of probe solutions at concentrations of 0 μM and 150 μM were added to each dish and incubated in an incubator for 1 h. The cells were washed three times with PBS, fixed with 200 μl of 4% paraformaldehyde for 15 min, and washed again with PBS. Subsequently, 500 μl of a solution containing 15 μM of the corresponding fluorescently labeled peptide was added to each dish and incubated for 1 h. The cells were washed three times with PBS, stained with DAPI for 5 min, and observed under a confocal microscope.

Flow cytometry

Cells were seeded in 6-well plates and grown to 80% confluency. They were then fixed with 200 μl of 4% paraformaldehyde per well for 15 min, washed three times with PBS, and incubated with Cy5.5-Nectin-4 PEP 01, 02, or 03 at concentrations of 2 μM, 5 μM, 10 μM, and 20 μM for the concentration gradient group or at 20 μM for different time periods (10 min, 30 min, 60 min, and 90 min) for the time gradient group. The cells were prepared into cell suspensions, centrifuged, and analyzed using a BD flow cytometer to detect Per-CP channel fluorescence distribution.The experiment was repeated three times. FlowJo V10 software was used for binding rate analysis.

In vivo fluorescence experiments

Animal model establishment

BALB/c nude mice (20–25 g, 4–6 weeks old) were used. Approximately 5 × 106 PC9/A549 tumor cells mixed with 100 μL of PBS were injected subcutaneously into one flank of each mouse. Tumors were allowed to grow for 4–5 weeks until their volume reached approximately 1 cm3.

In vivo imaging

Tumor-bearing mice were randomly divided into three major groups, with each major group further divided into experimental and inhibition subgroups, with five mice per subgroup. In vivo imaging was performed using a live imaging system (excitation wavelength: 687 nm, emission wavelength: 712 nm). Mice were anesthetized with isoflurane before imaging. All images were acquired under the same lighting settings (excitation mode, exposure time, and field of view). In the experimental groups, 100 μL of Cy5.5-Nectin-4 PEP 01, 02, or 03 at a concentration of 30 μM was injected into the tail veins of tumor-bearing mice, followed by imaging at different time points (30 min, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h). In the inhibition groups, an excessive amount of the corresponding Nectin-4 PEP 01, 02, or 03 (100 μL at a concentration of 300 μM) was injected into the tail veins of tumor-bearing mice 30 min before probe injection, followed by the same imaging procedure as in the experimental groups. Regions of interest (ROIs) with similar areas were drawn around the tumors, and fluorescence intensities (C/mm²) were measured. After the in vivo fluorescence imaging experiments, mice were euthanized by cervical dislocation, and the tumor-bearing mice were dissected to collect subcutaneous tumors and major organs or tissues (spleen, blood, kidneys, stomach, intestines, liver, heart, lungs, skin, muscles, bones, and head) for studying the biodistribution of each probe in vivo, and the fluorescence intensity (C/mm²) of each sample was measured. The animal study has been approved by the Animal Welfare and Ethics Committee of Jinhua Hospital Affiliated to Zhejiang University School of Medicine.

Statistical methods

SPSS 19.0 statistical software was used for data analysis. Measurement data were expressed as mean ± standard deviation. Comparisons between groups were performed using the χ² test. P < 0.05 was considered statistically significant.

Results

Synthesis and identification of fluorescent molecular probes

Peptides were synthesized using solid-phase synthesis (Supplemental Fig. 1), as shown in (Supplemental Fig. 2).The yield of the conjugation process ranged from 40%-50%. The purity of the products, separated by high-performance liquid chromatography (HPLC), reached above 95% (Supplemental Fig. 3), and the molecular weights were identified by mass spectrometry (MS) (Supplemental Fig. 4).

Ualcan online analysis results

Nectin-4 expression was significantly higher in LUAD tissues than in normal tissues, with a statistically significant difference (P = 1E-12) (Supplemental Fig. 5G). Clinical (Supplemental Fig. 6) and pathological (Supplemental Fig. 5H–I) data from 574 LUAD patients in the Ualcan database were analyzed, revealing no statistically significant differences in nectin-4 expression across different smoking habits, races, genders, ages, tumor stages, or lymph node metastasis status (P > 0.05).

Fig. 6.

Anatomical images of organs and tissues in the PC9 transplant tumor model experimental group and blocking group (A). Showcasing the in vivo biodistribution study of the probes in the experimental group (B–F) and the blocking group (C–G) at 48 h post-injection. These images depict various organs and tissues, including 1-tumor, 2-spleen, 3-kidney, 4-stomach, 5-intestine, 6-liver, 7-cardiac, 8-lung, 9-muscle, 10-bone, 11-skin and 12-brain. For quantitative analysis of the probe biodistribution, the region of interest (ROI) function of IVIS Living Image 4.3.1 software was utilized to calculate the uptake intensity of each organ for the three probe groups (H). Additionally, the fluorescence intensity of tumor tissues and various organs in non-blocked and blocked tumor-bearing mice was determined (I–K).

Fig. 5.

Anatomical images of organs and tissues in the A549 transplant tumor model experimental group and blocking group (A). In vivo biodistribution study of the probes in the experimental group (B–F) and the blocking group (C–G) at 48 h post-injection. Anatomical images of organs and tissues (1-tumor; 2-spleen; 3-blood; 4-kidney; 5-stomach; 6- intestine; 7-liver; 8-cardiac; 9-lung; 10-muscle; 11-bone; 12-brain) 48 h after probe injection. To quantitatively analyze the biodistribution of the probes, the uptake intensity of each organ for the three probe groups was quantitatively calculated using the region of interest (ROI) function of IVIS Living Image 4.3.1 software (H). Additionally, the fluorescence intensity of tumor tissues and various organs in non-blocked and blocked tumor-bearing mice is presented (I–K).

Immunohistochemistry results of 67 lung adenocarcinoma specimens from our center

All patients underwent surgical treatment at our hospital between September 2018 and December 2019. The patient age range was 40–77 years, with a median age of 63 years. The postoperative pathological diagnosis for all patients was lung adenocarcinoma. There were 30 male patients and 37 female patients. Postoperative pathology confirmed lymph node metastasis in 11 cases and no lymph node metastasis in 56 cases. Immunohistochemical (IHC) analysis revealed negative results for all 67 adjacent lung tissue samples from these patients (Supplemental Fig. 5A). Among the 52 lung cancer specimens, varying degrees of nectin-4 positivity (IHC score 1–4) were observed in 47 cases, while 15 cases were negative (Supplemental Fig. 5B–F). The difference was statistically significant (P = 0.000). No correlation was found between nectin-4 expression and patient gender, age, tumor stage, or lymph node metastasis (Supplemental Table 1).

Molecular probe cellular uptake experiments

1. Concentration Gradient Experiment: Within the concentration range of 2–20 μM, the uptake of Cy5.5-Nectin-4 PEP 01 (Supplemental Fig. 7A), Cy5.5-Nectin-4 PEP 02 (Fig. 1A), and Cy5.5-Nectin-4 PEP 03 (Supplemental Fig. 8A) by A549 cells gradually increased with increasing concentration. For PC9 cells, the uptake of Cy5.5-Nectin-4 PEP 01 (Supplemental Fig. 7B), Cy5.5-Nectin-4 PEP 02 (Fig. 1B), and Cy5.5-Nectin-4 PEP 03 (Supplemental Fig. 8B) within the concentration range of 5–30 μM showed a positive correlation with concentration.

2. Time Gradient Experiment: In the time gradient experiment, the binding ability of A549 and PC9 cells to Cy5.5-Nectin-4 PEP 01 (Supplemental Fig. 9), Cy5.5-Nectin-4 PEP 02 (Fig. 2), and Cy5.5-Nectin-4 PEP 03 (Supplemental Fig. 10) within the incubation time range of 10–90 min increased gradually with increasing incubation time, and the cellular uptake of these three peptides also increased accordingly.

3. Competitive Inhibition Experiment.

   The fluorescence intensity of A549 and PC9 cells significantly decreased after adding tenfold excess of the inhibitor. The uptake of Cy5.5-Nectin-4 PEP 01 (Supplemental Fig. 7C-D), Cy5.5-Nectin-4 PEP 02 (Fig. 1C-D), and Cy5.5-Nectin-4 PEP 03 (Supplemental Fig. 8C-D) by the cells could be inhibited by excess corresponding unlabeled peptides, suggesting that these three peptides specifically bind to the nectin-4 target on the tumor cell membrane.

Fig. 1.

The fluorescence uptake intensity of A549 cells was measured at various concentration gradients, specifically 2 μM, 5 μM, 10 μM, and 20 μM (A). Similarly, the fluorescence uptake intensity of PC9 cells was assessed at concentration gradients of 5 μM, 7.5 μM, 15 μM, and 30 μM (B). The effect of excess Nectin-4 PEP 02 on the uptake of Cy5.5-Nectin-4 PEP 02 by A549 cells was assessed (C). The effect of excess Nectin-4 PEP 02 on the uptake of Cy5.5-Nectin-4 PEP 02 by PC9 cells was assessed (D).

Fig. 2.

The fluorescence uptake intensity of A549 cells was measured across different time gradients, specifically 10 min, 20 min, 60 min, and 90 min (A). Likewise, the fluorescence uptake intensity of PC9 cells was assessed at similar time gradients of 10 min, 20 min, 60 min, and 90 min (B).

Cell binding rate (Supplemental Fig. 11)

Under the same fluorescence concentration and incubation time conditions, A549 and PC9 cells showed high fluorescence binding under a confocal microscope. However, to quantitatively analyze the binding ability of the three peptides to lung cancer cells, we employed flow cytometry experiments. The results revealed that the binding rates of A549 and PC9 cells to the three probes increased with increasing concentration. When the concentration of Cy5.5-Nectin-4 PEP 01 was 2 μM, 5 μM, 10 μM, and 20 μM, the binding rates (%) of A549 cells were 8.03 ± 1.46, 15.03 ± 1.76, 39.80 ± 5.03, and 70.47 ± 1.94, respectively. For PC9 cells, the binding rates (%) were 6.07 ± 0.47, 23.17 ± 3.00, 49.70 ± 5.71, and 58.00 ± 3.63, respectively. Under the same concentration conditions, the binding rates (%) of Cy5.5-Nectin-4 PEP 02 to A549 cells were 17.47 ± 1.15, 26.73 ± 1.21, 61.87 ± 4.09, and 89.90 ± 3.10, respectively, and to PC9 cells were 6.67 ± 1.00, 19.23 ± 5.19, 37.13 ± 7.02, and 88.10 ± 2.59, respectively. The binding rates (%) of Cy5.5-Nectin-4 PEP 03 to A549 cells under this concentration gradient were 3.93 ± 1.52, 9.67 ± 1.11, 16.00 ± 2.02, and 22.40 ± 2.76, respectively, and to PC9 cells were 2.27 ± 0.87, 7.13 ± 1.46, 8.13 ± 2.64, and 19.93 ± 2.87, respectively. At the same concentration, the binding rates of A549 and PC9 cell lines to Cy5.5-Nectin-4 PEP 02 were significantly higher than those to Cy5.5-Nectin-4 PEP 01 and Cy5.5-Nectin-4 PEP 03. The binding rates of both cell lines to the three probes increased with increasing incubation time. When the incubation time was 10 min, 30 min, 60 min, and 90 min, the binding rates (%) of Cy5.5-Nectin-4 PEP 01 to A549 cells were 5.73 ± 0.85, 17.27 ± 1.89, 53.03 ± 2.70, and 60.00 ± 1.15, respectively, and to PC9 cells were 6.07 ± 0.47, 23.17 ± 3.00, 49.70 ± 5.71, and 58.00 ± 3.63, respectively. The binding rates (%) of Cy5.5-Nectin-4 PEP 02 to A549 cells were 16.73 ± 1.45, 52.13 ± 3.11, 67.87 ± 3.02, and 80.77 ± 1.69, respectively, and to PC9 cells were 8.57 ± 0.93, 42.87 ± 4.29, 63.97 ± 4.27, and 77.33 ± 1.62, respectively. The binding rates (%) of Cy5.5-Nectin-4 PEP 03 to A549 cells were 3.30 ± 0.92, 6.20 ± 1.10, 17.33 ± 1.52, and 21.10 ± 2.18, respectively, and to PC9 cells were 2.93 ± 0.71, 4.30 ± 0.82, 15.03 ± 2.18, and 16.93 ± 1.76, respectively. At the same time points, the binding rate of Cy5.5-Nectin-4 PEP 02 to A549 was higher than that of the other two probes, and its binding rate to PC9 was also higher. This indicates that Cy5.5-Nectin-4 PEP 02 has stronger targeting ability toward lung cancer cells and is more significantly taken up by them, suggesting that Cy5.5-Nectin-4 PEP 02 is more suitable for targeted tumor experiments and clinical diagnosis.

Cell line nectin-4 target validation results

Confocal microscopy analysis revealed peptide binding to A549 and PC9 cell lines, but not to Beas-2B cells (Supplementary Fig. 12A). Correspondingly, Western blot detection of Nectin-4 expression showed positive signals in A549 and PC9 cells, whereas negligible expression was observed in Beas-2B cells. GAPDH served as the loading control and was uniformly detected across all cell lines (Supplementary Fig. 12B).

In vivo fluorescence imaging

Each experimental group of nude mouse models bearing lung adenocarcinoma tumors was administered a single dose either 30 μM Cy5.5-Nectin-4 PEP 01, Cy5.5-Nectin-4 PEP 02,or Cy5.5-Nectin-4 PEP 03 peptide probe respectively via tail vein injection. The blocking group received a co-injection of polypeptide probes (30 μM) and corresponding unlabeled polypeptides (300 μM). Near-infrared fluorescence optical imaging was performed at multiple time points (0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h) post-probe injection (Figs. 3A-C, 4A-C). To determine the tumor tissue’s probe uptake capacity, the region of interest (ROI) function of the IVIS Living Image 4.3.1 software was utilized to quantitatively calculate the fluorescence intensity of the tumor tissue in tumor-bearing mice. The binding rate of the probes to tumor tissue peaked at 1 h, and the blocking group exhibited the same trend but with significantly reduced fluorescence intensity. By drawing ROIs of equal area at the tumor site, the fluorescence intensity of the tumor tissue in the experimental and blocking groups at different time points was obtained (Figs. 3D-F, 4D-F). Among them, Cy5.5-Nectin-4 PEP 02 showed the highest binding rate to tumor tissue. In the A549 experimental group, the fluorescence intensity of the tumor tissue changed over time (0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h) as follows: (2.15 ± 0.19)E+07, (2.65 ± 0.25)E+07, (2.55 ± 0.22)E+07, (1.73 ± 0.16)E+07, (1.81 ± 0.14)E+07, (1.4 ± 0.17)E+07, and (8.64 ± 0.20)E+06, respectively (Fig. 3G). In the blocking group, the fluorescence intensity changed as follows: (1.45 ± 0.20)E+07, (1.61 ± 0.20)E+07, (1.48 ± 0.10)E+07, (1.15 ± 0.17)E+07, (6.36 ± 0.11)E+06, (6.16 ± 0.15)E+06, and (5.79 ± 0.89)E+06, respectively. In the PC9 experimental group, the fluorescence intensity changed as follows: (3.23 ± 0.38)E+07, (3.53 ± 0.12)E+07, (3.35 ± 0.48)E+07, (2.33 ± 0.24)E+07, (1.48 ± 0.07)E+07, (1.14 ± 0.54)E+07, and (8.64 ± 0.12)E+06, respectively (Fig. 4G). In the blocking group, the fluorescence intensity changed as follows: (1.70 ± 0.17)E+07, (2.78 ± 0.49)E+07, (2.48 ± 0.38)E+07, (1.12 ± 0.16)E+07, (8.40 ± 0.09)E+06, (5.89 ± 0.04)E+06, and (6.40 ± 0.05)E+06, respectively.

Fig. 3.

Near-infrared fluorescence optical imaging revealed the rapid tumor-targeting ability of the probes in the A549 cell line. Probe uptake peaked at 1 h and gradually decreased over time. The blocking group showed a similar trend but with significantly reduced fluorescence intensity (A–C). By drawing regions of interest (ROIs) of equal area at the tumor site, the fluorescence intensity distribution in tumor tissues of the experimental and blocking groups at different time points was obtained, and fluorescence intensity-time curves for the three groups were plotted (D–F). Cy5.5-Nectin4 PEP 02 exhibited a higher peak fluorescence uptake (G).

Fig. 4.

Near-infrared fluorescence optical imaging demonstrated the rapid tumor-targeting capability of the probes in the PC9 cell line. The probe uptake peaked at 1 h and gradually decreased over time. The blocking group exhibited a similar trend but with notably weaker fluorescence intensity (A–C). By delineating regions of interest (ROIs) of equal area at the tumor site, the fluorescence intensity distribution within the tumor tissues of both the experimental and blocking groups at various time points was obtained. Fluorescence intensity-time curves for the three groups were plotted (D–F), revealing that Cy5.5-Nectin4 PEP 02 had a higher peak fluorescence uptake (G).

To investigate the biodistribution of the probes, A549 tumor-bearing mice were euthanized after 48 h of near-infrared fluorescence (NIRF) imaging (Fig. 5A). NIRF imaging was performed on major organs including tumors, spleens, blood, kidneys, stomachs, intestines, livers, cardiac, lungs, muscles, bones and brains from both the experimental and blocking groups (Fig. 5B-G). By analyzing the fluorescence intensity in the regions of interest (ROIs), the uptake capacity of the probes by the tissues and organs of the tumor-bearing mice was determined (Fig. 5I-K). The results were plotted to compare the quantitative values and ratios of fluorescence intensity in these organs between the experimental and blocking groups (Fig. 5H). Similarly, PC9 tumor-bearing mice were euthanized after 48 h of NIRF imaging (Fig. 6A). NIRF imaging was performed on major organs including tumors, spleens, kidneys, stomachs, intestines, livers, cardiac, lungs, muscles, bones, skin and brains from both groups (Fig. 6B-G). The analysis of fluorescence intensity in ROIs confirmed the probe uptake by the tissues and organs of the tumor-bearing mice (Fig. 6I-K). The results were plotted to compare the quantitative values and ratios of fluorescence intensity in these organs between the experimental and blocking groups (Fig. 6H). Notably, besides the tumors, the kidneys exhibited high fluorescence intensity, which is attributed to probe excretion through urine. The digestive organs (including the liver, stomach, and intestine) showed higher probe uptake than other major organs, suggesting that the probe is primarily metabolized via the hepatic biliary system and excreted from the body. After blocking, the probe uptake by the tumor tissue was significantly reduced, and there was a statistically significant difference in tumor fluorescence intensity between the experimental and blocking groups (P < 0.05), supporting the targeted specificity of the probe toward tumor tissue. In both A549 and PC9 models, the Cy5.5-Nectin4 PEP 02 group exhibited the highest tumor tissue fluorescence intensity, with statistically significant differences compared to the other two groups (P < 0.05).

Fluorescence imaging-guided tumor resection

Given the excellent properties of Cy5.5-Nectin4 PEP 02, it was further applied for fluorescence imaging-guided tumor resection. without the injection of Cy5.5-Nectin4 PEP 02, neither tumor nor normal tissues exhibited fluorescence. However, after 0.5 h of Cy5.5-Nectin4 PEP 02 injection, a significant fluorescence signal appeared in the tumor tissue, clearly distinguishing the tumor and its margins (Fig. 7A). Post-tumor resection, no new fluorescent tissue was observed during fluorescence imaging, indicating complete tumor removal (Fig. 7B). The fluorescence intensity of the tumor tissue was 16.42 times higher than that of the surrounding normal tissue. Immunohistochemical experiments on frozen tumor tissue sections revealed strong Nectin-4 positivity in the tumor tissue (Fig. 7D), corresponding to the fluorescence results (Fig. 7C). These findings suggest that Cy5.5-Nectin4 PEP 02 can be administered 30 min before surgery for fluorescence imaging-guided precise tumor resection.

Fig. 7.

Preoperative fluorescence specific imaging in PC9 transplant tumor model (A) . After complete excision of the tumor, the surgical margins in the operative area were negative with no fluorescent residue (B). Post-excision, tumor-specific imaging confirmed the removal (C).immunohistochemical staining of the tumor was strongly positive (D).

Discussion

Due to the existence of tumor heterogeneity and advancements in the understanding of tumor molecular mechanisms, several biomarkers have emerged as predictive, prognostic, and diagnostic markers for lung cancer, particularly for non-small cell lung cancer (NSCLC), such as the ALK fusion oncogene, EGFR mutations, and ROS1 gene rearrangements^10,11. Similar to other malignancies, precise and individualized diagnosis and treatment are crucial for improving survival in lung cancer. Unlike other members of the nectin family, nectin-4 is predominantly expressed in tumor cells and rarely in normal tissues. Its expression level positively correlates with tumor metastasis, adhesion, and intratumoral microvessel density. While some scholars believe that high nectin-4 expression in most solid tumors is negatively correlated with survival, Derycke et al. found no correlation between nectin-4 positivity (48.6%) in ovarian cancer and overall survival or recurrence time¹²). However, there is limited research on the association between nectin-4 and prognosis in lung cancer. Some studies suggest that nectin-4 levels are related to tumor initiation and progression, advocating for its consideration as a diagnostic and prognostic biomarker in tissues and serum and as a therapeutic target for malignancies^13,14. Similarly, in patients with lymph node-negative, early-stage, luminal-A breast cancer, elevated nectin-4 expression is associated with reduced metastasis-free survival, suggesting its potential use as a reliable indicator of distant metastasis and as a therapeutic target for these patients¹⁵. Research has shown that nectin-4 is a potential target for antibody–drug conjugate (ADC) therapy in nectin-4-expressing malignancies¹⁶. The novel ADC enfortumab vedotin (EV), which comprises a human nectin-4 antibody conjugated to the potent microtubule-disrupting agent MMAE, can bind with high affinity to cell surface-expressed nectin-4 and induce cell death in a dose-dependent manner in vitro. On December 18, 2019, the Food and Drug Administration (FDA) granted accelerated approval for EV for the treatment of patients with locally advanced or metastatic urothelial cancer¹⁷.Due to the lack of in-depth research on nectin-4 in lung cancer, we aimed to investigate its expression levels in lung cancer to provide new avenues for precise diagnosis and treatment.

Some studies have shown that nectin4 is a diagnostic and therapeutic target of lung cancer, and is highly expressed in lung cancer¹⁸. Nectin-4 demonstrated a total positive expression rate of approximately 55% in non-small cell lung cancer (NSCLC) tissues (strongly positive: 7%; moderately positive: 20%; weakly positive: 28%), significantly higher than in normal lung tissues (negative rate: 45%) (P < 0.05)¹⁴. Based on large-sample analysis from the TCGA database, nectin-4 is highly expressed in lung adenocarcinoma (LUAD) and may serve as a potential biological target, with no correlation to other clinical or pathological characteristics of LUAD patients¹⁹. Immunohistochemical (IHC) analysis of 67 LUAD samples from our center revealed significantly increased nectin-4 expression in tumor tissues (77.6% positivity), with no notable expression in normal tissues, and the difference was statistically significant. Analysis of clinical and pathological data showed no significant correlation between nectin-4 expression and patient gender, age, tumor stage, or lymph node metastasis, consistent with the large-sample statistics from the TCGA database. Challita-Eid’s large-sample IHC analysis of solid tumor specimens found nectin-4 positivity in 69% of specimens, with moderate to strong staining observed in 60% of bladder and 53% of breast tumor specimens, while nectin-4 expression in normal tissues was more limited¹⁶. A similar finding was observed in another large-sample study (n = 5673) of invasive breast cancer, gastric cancer, and pancreatic cancer, where high nectin-4 mRNA expression was associated with lymph node metastasis, tumor differentiation, histological subtype, Ki-67 proliferation index, and poor prognosis (R = 0.436, P < 0.001)²⁰. In LUAD, we did not observe a correlation between nectin-4 and tumor stage or lymph node metastasis, which may be attributed to our relatively small sample size and potential bias. Due to missing clinical and pathological data and the small sample size, we did not follow up or statistically analyze tumor histological subtypes, Ki-67 proliferation index, or survival, which will be addressed in future studies.

Near-infrared fluorescence (NIRF) optical imaging is a well-established molecular imaging modality that combines tumor-specific molecules to form visual probes, distinguishing the heterogeneity between tumor and normal cells, and can be used for early tumor diagnosis^21,22. It offers advantages over traditional molecular imaging techniques, which require high operational conditions, involve radioactivity, and have poor specificity²³. Peptide-based probes are easy to synthesize and exhibit high biocompatibility and safety, making them key advantages for clinical applications of peptide-based molecular probes^24,25. We utilized Cy5.5-labeled peptides to form molecular probes for in vitro and in vivo NIRF imaging of lung adenocarcinoma. This imaging method is an essential tool for studying the molecular mechanisms of tumor initiation and progression and for the development of anticancer drugs^26,27.By conjugating fluorescent dyes to bioactive molecules, without altering their original bioactivity, they can be visually monitored using various machines, enabling qualitative and quantitative studies²⁸. In this study, we successfully synthesized three peptides targeting the extracellular domain of nectin-4: Nectin-4 PEP 01, Nectin-4 PEP 02, and Nectin-4 PEP 03, and labeled them with Cy5.5 to form corresponding fluorescent molecular probes. HPLC analysis revealed purities ranging from 95.43% to 99.38%, and mass spectrometry confirmed the molecular weights of the peptides, meeting experimental requirements. To investigate the targeting ability of the molecular probes in lung adenocarcinoma, we selected lung adenocarcinoma cell lines A549 and PC9 for in vitro cellular uptake experiments to confirm the binding of nectin-4 in these cell lines. The experiments showed that both A549 and PC9 cells specifically bound to all three probes. In A549 cells, within the concentration range of 2–20 μM, the cellular uptake of the three probes gradually increased with increasing concentration. In PC9 cells, the uptake of the three probes positively correlated with concentration within the range of 5–30 μM. Among them, Cy5.5-Nectin-4 PEP 02 exhibited the strongest cellular binding ability at the same concentration and showed a time-dependent increase within the 10–90 min incubation period. The binding ability of Cy5.5-Nectin-4 PEP 02 in both A549 and PC9 cells was significantly higher than that of the other two probes, with binding rates of 89.90 ± 3.10% and 88.10 ± 2.59% to A549 and PC9 cells, respectively, at optimal concentrations. Furthermore, its binding could be inhibited by a tenfold excess of unlabeled corresponding peptides, consistent with competitive binding patterns. The tumor-targeting and specific binding abilities of the probes were validated in vivo experiments. NIRF imaging of A549 and PC9 tumor-bearing mouse models confirmed the tumor imaging efficacy of the three probes. The peak probe uptake by tumor tissues occurred 1 h post-injection, followed by a gradual decrease in fluorescence intensity. The fluorescence intensity-time curves showed that Cy5.5-Nectin4 PEP 02 had the highest uptake peak and could be competitively blocked. After 48 h, fluorescence measurements of dissected tissues revealed statistically significant differences in tumor tissue fluorescence uptake between the experimental and blocking groups, with the highest uptake observed in the Cy5.5-Necin4 PEP 02 group, which was statistically significant compared to the other two groups. These results indicate that Cy5.5-Nectin-4 PEP 02 specifically binds to tumor tissues and is excreted through urine and the digestive tract, with low uptake in normal tissues. Based on these findings, we designed fluorescence-mediated tumor resection experiments. Surgical margin assessment is crucial in surgery, as excessive resection may damage adjacent vital tissues and organs, leading to adverse functional outcomes^29–34. With the use of fluorescence-guided surgery (FGS), previously undetected malignancies can be illuminated in real-time during surgery.

In summary, our experiments revealed that the Cy5.5-labeled Nectin-4 PEP 02 molecular probe exhibits high targeting and specificity toward lung adenocarcinoma cells and tissues both in vitro and in vivo. The Cy5.5-Nectin-4 PEP 02 molecular probe has potential applications in the diagnosis and efficacy monitoring of lung cancer, visualizing tumor margins and detecting occult lesions during surgery, and demonstrates clinical potential for the future development of related prodrug-activated chemotherapeutics (PDC) or ADC molecular probes.

Conclusions

Nectin-4 is highly expressed in lung adenocarcinoma and minimally expressed in normal tissues, making it a promising biological target for lung adenocarcinoma. We synthesized molecular probes targeting nectin-4 and confirmed through in vitro and in vivo experiments that the Cy5.5-Nectin-4 PEP 02 molecular probe had the highest binding rate to lung cancer cells and tumor tissues. It can be used for early diagnosis and efficacy monitoring of lung cancer, accurately localizing tumor sites and margins, and assisting surgeons in making more precise decisions during surgery, thereby reducing positive surgical margins. Additionally, it shows potential for the future development of related PDC or ADC molecular probes.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Ting Jiang, Mengjie Dong and Zhijian Zheng carried out the studies, participated in collecting data, and drafted the manuscript. Qianqian Zhang and Bilian Zhu performed the statistical analysis and participated in its design. Binjing Jiang and Jianfei Fu participated in acquisition, analysis, Ke Dong and Yong Zheng interpretation of data and draft the manuscript. All authors read and approved the final manuscript.

Funding

Jinhua Science and Technology Project Social Development Key project (Project number: 2023–3-108); Jinhua Science and Technology Project Social Development public welfare Project (Project number: 2024–4-079); Youth Research Start-up Fund (Project No. JY2022-1–03); Basic Research Foundation (Project No. JY2023-6–11); Supported by Shenzhen High-level Hospital Construction Fund, and by Peking University Shenzhen Hospital Scientific Research Fund KYQD2024368; Jinhua Science and Technology Project Social Development Key project (Project number: 2022–3-077); Jinhua Science and Technology Project Social Development Key project (Project number: 2022–3-084).

Data availability

All data generated or analysed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing financial interest.

Ethics approval and consent to participate

Informed consent and ethical approval were obtained from 67 patients with lung adenocarcinoma from the Pathology Department of Jinhua Hospital affiliated with Zhejiang University School of Medicine. This project has been approved by the Medical Ethics Committee of Jinhua Hospital Affiliated to Zhejiang University School of Medicine (Ethics Approval No. 105, 2025). I confirm that all methods were performed in accordance with the relevant guidelines. All procedures were performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. In addition, the animal study has been approved by the Animal Welfare and Ethics Committee of Jinhua Hospital Affiliated to Zhejiang University School of Medicine (Approval No. AL-JHYY202416). I confirms that all experiments were performed in accordance with relevant named guidelines and regulations. I confirms that the authors complied with the ARRIVE guidelines.

Consent for publication

Not applicable.