A fibroblast activation protein degrader enhances cisplatin sensitivity in non‑small cell lung cancer

Introduction

Non-small cell lung cancer (NSCLC) accounts for ~85% of all lung cancers (1,2). NSCLC is often classified into three main histological subtypes: Adenocarcinoma, squamous cell carcinoma and large cell carcinoma (1). Although smoking is the main risk factor for NSCLC (3), the incidence of NSCLC is also increasing among non-smokers who are exposure to tobacco (1). The standard treatment of NSCLC consists of surgery, chemotherapy, radiation therapy, targeted therapy and immunotherapy (4). Chemotherapy is the standard treatment modality for early-stage NSCLC (4). Similarly, favorable curative effects have been achieved through advances in radiotherapy techniques (5). Moreover, the most common targeted therapeutic strategy is vascular endothelial growth factor (6–8) or epidermal growth factor receptor inhibition (9,10). However, issues such as drug resistance, systemic toxicity and high costs remain major obstacles for the adoption of these strategies.

For decades, platinum-based chemotherapeutics have dominated the treatment of NSCLC (11–13). Among them, cisplatin has become the most commonly used first-line drug because of its excellent efficacy (14). Unfortunately, chemoresistance often develops during treatment, which seriously limits the clinical utility of cisplatin (14). Usually, combination treatment is a powerful means to overcome this resistance (15).

Meanwhile, fibroblast activation protein (FAP) a type-II transmembrane serine protease, expressed almost exclusively to pathological conditions including fibrosis, arthritis and cancer. Across most cancer types, FAP plays critical roles in tumor proliferation, tumor invasion, angiogenesis and drug resistance, and elevated FAP is associated with worse clinical outcomes (16–18). Previous research demonstrated that treatment strategies targeting FAP in combination with other approaches such as chemotherapy, radiotherapy and immunotherapy could strengthen treatment efficacy (17,18). However, the role of FAP degradation in enhancing cisplatin sensitivity of NSCLC is unclear. Thus, the present study investigated the effect of FAP degradation on the therapeutic effect of cisplatin against NSCLC.

Materials and methods

Reagents

Cisplatin (cat. no. HY-17394; CAS no. 15663-27-1) was purchased from MedChemExpress. Human anti-CD26 polyclonal antibody (cat. no. 29403-1-AP), human anti-FAP polyclonal antibody (cat. no. 11779-1-AP), human anti-caspase-3 polyclonal antibody (cat. no. 19677-1-AP), HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibody (cat. no. SA00001-2) and human anti-cleaved caspase 3 polyclonal antibody (cat. no. 25128-1-AP) were purchased from Proteintech Group, Inc. Pomalidomide-PEG2-FAP2286: FAP-D was obtained from Shanghai Apeptide Co., Ltd. Protein extraction kit (cat. no. R0018M), Cell-Counting Kit-8 (CCK-8; cat. no. c0038), radioimmunoprecipitation assay buffers (cat. no. P0013B) and bicinchoninic acid (BCA) protein concentration assay kit (cat. no. P0012) were purchased from Beyotime Institute of Biotechnology. NCI-H1299 cells (cat. no. SCSP-589) were purchased from National Collection of Authenticated Cell Cultures. All other reagents, chemicals and cell lines were local.

Molecular docking

Molecular docking was performed using AutoDock Vina 1.2.0 (19). Concerning the docking parameters of the receptor, the center coordinates of the docking box (center X, center Y and center Z) were set to (38.034, 0.53 and 69.455). The number of grid points in each direction of XYZ was set to 60×60×60, the docking accuracy exhaustiveness was 25 and the output binding conformation was set to 100. The docking results were visualized using PyMOL (version 2.5.0) (20) and LigPlot+(version 2.3) (21). The structure of human FAP alpha was obtained from Protein Data Bank (https://www.rcsb.org/structure/1Z68).

Protein degradation

NCI-H1299 cells (5×105 cells/ml) were cultured in 96-well plates. FAP-D was added to each well (Final concentrations: 0.01, 0.05, 0.1, 0.25, 0.5, 1, 5, 10 and 20 µM), and cells were cultured at 37°C for 12 h. Then, proteins were extracted from cells for western blotting. The protocols assessing the effects of FAP-D on protein degradation in H460 cells and effects of time on protein degradation were similar.

CCK-8 assay

H1299 cells (1×105 cells/ml) were cultured in 96-well plates. FAP-D was added to each well (final concentrations: 0.5, 1, 5, 10 and 20 µM), and cells were cultured at 37°C for 24 h. Then, the culture medium was discarded, and cell viability was determined according to the standard CCK-8 assay protocol using the EnVision microplate reader (Revvity, Inc.). For half-maximal inhibitory concentration (IC50) assessment, FAP-D (0 or 5 µM) was added to each well, and cells were cultured at 37°C for 12 h. The culture medium was discarded, and cisplatin was added to each well (final concentrations: 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, and 500 µM), after which cells were cultured at 37°C for 12 h. After discarding the culture medium, cell viability was determined according to the standard CCK-8 assay protocol using the EnVision microplate reader. Volume of CCK-8 used was 20 µl and the solutions were incubated at 37°C for 2 h. The absorbance at 450 nm of solutions cultured without drugs was defined as 100%. The protocols of effects of FAP-D and cisplatin + FAP-D on cell viabilities of H460 cells were similar. Unless otherwise stated, all experiments were repeated three times.

Wound-healing assay

H1299 or H460 cells were cultured in serum-free medium without/with cisplatin (5 µM, 24 h) or cisplatin (5 µM) + FAP-D (5 µM; cells were cultured with FAP-D for 12 h and then with cisplatin for 12 h). Cells were then cultured to confluence and scratched with a cell scraper. The debris was removed via washes with the culture medium. The cells were subsequently cultured in an incubator for 24 h. The images were acquired by microscopy and analyzed using ImageJ software (version 1.52a; National Institutes of Health).

Western blotting

The samples were prepared according as follows: cells were lysed using the radioimmunoprecipitation assay buffers and the solutions were centrifuged (14,000 × g 10 min). The protein concentrations were obtained using BCA assay. The samples were separated through 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (transfer current set to 250 mA). The membranes were blocked in 5% milk at room temperature for 1 h, and then were incubated with primary antibodies (FAP antibody: Dilution, 1:1,000; CD26 antibody: Dilution, 1:1,500; caspase-3 antibody: Dilution, 1:1,000; and cleaved caspase 3 antibody: Dilution, 1:1,000) at 4°C. Membranes were then washed with Tris-buffered saline with Tween-20 (0.05%) buffer and incubated with horseradish peroxidase-modified secondary antibodies (1:10,000) at room temperature for 1 h. Lastly, the membranes were imaged through Amersham™ ImageQuant™ 800 western blot imaging systems. The densitometric analysis were performed from ImageJ software (1.52a; National Institutes of Health)

Animal experiments

The animal experiments were approved by the institutional animal ethics committee of Kunming Medical University (approval no. kmmu20241355; Kunming, China). BALB/c mice were purchased from the Department of Experimental Zoology, Kunming Medical University (Kunming, China). All clinical procedures were conducted in accordance with the relevant provisions of the Declaration of Helsinki. The studies were additionally conducted in accordance with local legislation and institutional requirements. Body weights of mice were ~18–19 g (healthy, 5–6 weeks old). Mice were fed at 25°C in the standard animal experiment lab and the humidity were maintained at 53%. The feeds and water were replaced every 3 days. The light/dark cycle was set as 12 h light/12 h dark. For biosafety experiments, BALB/c mice were randomly divided into two groups (n=5/group): PBS (control) group and FAP-D group. Mice in the FAP-D group were first anesthetized with 3% isoflurane via inhalation and then intraperitoneally injected with FAP-D (3 mg/kg, once daily for 7 days). Mice in the control group were first anesthetized with 3% isoflurane via inhalation and then intraperitoneally injected with phosphate-buffered saline (PBS). After treatment, mice were sacrificed via spinal dislocation, and the major organs were collected for analysis. For intervention experiments, nude BALB/c mice were randomly divided into three groups (n=6/group): PBS (control) group, cisplatin group, and cisplatin + FAP-D group. The mice were first subcutaneously injected with H1299 cells (5×106 cells, 100 µl, in the right sides of the mice back) and monitored for 10 days to permit tumor growth. After tumor formation, mice in the cisplatin group were first anesthetized with 3% isoflurane via inhalation and then intratumorally injected with cisplatin (20 mg/kg, once every 3 days, 3 times). Then, the body weight and volume were recorded every 3 days. After treatment, mice were sacrificed via spinal dislocation, and the tumors were weighed and then sliced or mashed for further experiments. Mice in the cisplatin + FAP-D group were first anesthetized with 3% isoflurane via inhalation and then intratumorally injected with FAP-D (1.5 mg/kg, once every 3 days, three times), followed by an intraperitoneal injection of cisplatin (20 mg/kg, once every 3 days, three times) after 12 h. Mice were euthanized after 21 days or upon reaching tumor volume ≥2.000 mm3 or >20% body weight loss, with tumors processed for analysis. The other protocols were similar.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9.0 (Dotmatics) and Origin 9 software (OriginLab Corporation). All data are determined from at least three independent experiments and presented as the mean ± standard deviation (SD). Statistical significance was assessed by ANOVA followed by Dunnett post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

FAP degradation

FAP-D consisted of three components: Pomalidomide, the FAP-binding peptide FAP 2286, and a linker (Diethylene glycol, Fig. 1A). The results of high-performance liquid chromatography and mass spectrometry are presented in Figs. S1 and S2. Molecular docking exhibited that several hydrophobic interactions and hydrogen bonds were formed between FAP-D and FAP (Figs. 1A and S3). Specifically, Arg123, Tyr124 and Gln547 in FAP formed hydrogen bonds with oxygen atoms in FAP-D. Trp623, Leu571, Ala554, Val552, Ile367, Phe350, Phe579, Gly349, Phe351, Val352, Ala207 and Ile398 in FAP formed hydrophobic interactions with FAP-D. The binding energy between FAP-D and FAP was −10.484 kcal/mol. These results suggested the binding force between FAP-D and FAP was strong. Western blotting demonstrated that FAP degradation increased as the FAP-D concentration increased (Figs. 1B and S4). Conversely, CD26 was not degraded by FAP-D, supporting the selectivity of FAP-D. In addition, FAP was degraded by FAP-D within 12 h (Figs. 1C and S5). These findings indicate that FAP-D is an ideal FAP degrader.

Figure 1. (A) Chemical formula of FAP-D and the binding model between FAP-D and FAP (PDB: 1z68). (B) Western blotting of FAP (n=3) and CD26 expression in H1299 cells treated with different concentrations of FAP-D (0, 0.01, 0.05, 0.1, 0.25, 0.5, 1, 5, 10 and 20 µM) for 12 h. (C) Western blotting of FAP (n=3) and CD26 expression in H1299 cells treated with 5 µM FAP-D for 0, 3, 6, or 12 h. FAP, fibroblast activation protein; FAP-D, FAP degrader.

Enhancement of the cisplatin sensitivity of H1299 cells by FAP-D

Considering the degradation performance of FAP-D, its effect on the cisplatin sensitivity of H1299 cells was explored. First, the effects of FAP-D on H1299 cell viability were studied. As presented in Fig. 2A, the viability of H1299 cells was reduced from 100 to 80% in response to increasing FAP-D concentration, suggesting that FAP degradation has little effect on cell viability. Second, FAP degradation was found to reduce the IC50 of cisplatin in H1299 cells (1.36 µM vs. 5.71 µM, Fig. 2B), indicating enhanced cisplatin sensitivity. In addition, the mobility of H1299 cells treated with cisplatin + FAP-D was reduced (Fig. 2C and D). This result suggested that the extracellular matrix, which is responsible for proliferation and migration, was destroyed by FAP-D, leading to reduced cell viability. Lastly, the expression of cleaved caspase 3 (a major index of apoptosis) was increased by treatment with cisplatin + FAP-D (Figs. 2E and S6). These results reveal that the cisplatin sensitivity of H1299 cells was enhanced by FAP-D, resulting in strengthened apoptosis. Similarly, these results were confirmed through H460 cells (Figs. S7A-E and S8).

Figure 2. (A) Effect of different concentrations of FAP-D (24 h of treatment) on H1299 cell viability. (B) Effects of cisplatin or cisplatin + FAP-D concentrations (treatment for 24 h) on H1299 cell viabilities. (C) Images of wounded H1299 cells in the different groups. Conditions: cisplatin (5 µM, 24 h), FAP-D (5 µM, 24 h), and cisplatin + FAP-D (5 µM FAP-D for 12 h followed by 5 µM cisplatin for 12 h). (D) The statistical results from 2C. ANOVA test (n=3). (E) Western blotting of caspase 3 and cleaved caspase 3 (Cle. Cas 3, n=3) expression in different groups of H1299 cells. Cells were treated with cisplatin (1, 10, or 20 µM, 24 h) or cisplatin + FAP-D (5 µM FAP-D for 12 h followed by 1, 10, or 20 µM cisplatin for 12 h). FAP, fibroblast activation protein; FAP-D, FAP degrader.

Therapeutic effects of cisplatin + FAP-D in vivo

The in vivo therapeutic effects of cisplatin + FAP-D were then studied. First, the potential in vivo toxicity of FAP-D was investigated. As presented in Fig. 3, no damage was observed in the major organs (heart, liver, spleen, lungs and kidneys) of mice treated with FAP-D, indicating its favorable biocompatibility.

Figure 3. Images of heart, liver, spleen, lung and kidney tissue from mice treated without or with FAP-D. Scale bar, 50 µm. Magnification, ×100. FAP-D, fibroblast activation protein-degrader.

After confirming the biocompatibility of FAP-D, the in vivo therapeutic effects of cisplatin + FAP-D were systematically examined. As illustrated in Fig. 4A, the tumor volumes of mice treated with cisplatin alone were smaller than those in control mice. Interestingly, tumor growth was inhibited in mice pre-treated with FAP-D, as confirmed by tumor weight (Fig. 4B). Moreover, H&E and TUNEL staining revealed that apoptosis was enhanced in the tumors of mice co-treated with FAP-D (Fig. 4C and D). In addition, no significant changes in body weights were observed (Fig. S9). These results reveal that apoptosis was enhanced by FAP-D in tumors.

Figure 4. (A) Left: tumors from mice in the control, cisplatin and cisplatin + FAP-D group. Right: the tumor volumes of mice in the different groups. (n=6). (B) Tumor weights of mice in the different groups (n=6/group; ANOVA test). (C) H&E staining results of tumors from mice in the different groups. Scale bar, 50 µm. (D) TUNEL staining results of tumors from mice in the different groups. Scale bar, 20 µm. Magnification, ×200. FAP-D, fibroblast activation protein-degrader.

Discussion

Cisplatin resistance has become a serious issue in NSCLC (22–24), which can be overcome by combination treatment strategies (15). In the past, a high response rate with modest side effects could be achieved in stage IIIB and IV NSCLC by the combination of gemcitabine and cisplatin (25). Additionally, cisplatin-etoposide combination chemotherapy achieved success (26). Meanwhile, combinations of traditional Chinese medicine and cisplatin have emerged as effective strategies (27,28). Moreover, the treatment efficacy of chemotherapy can be strengthened by targeting FAP (17,18). In light of the development of nanotechnology in recent years, novel nanoparticles co-loaded with cisplatin and oridonin were developed for NSCLC treatment (29). Unfortunately, treatment efficacy can be restricted by resistance or side effects from other drugs. Thus, an auxiliary medicine with low drug resistance is urgently needed for cisplatin-based combination regimens.

Protein degraders with the advantages of high selectivity, catalytic dosages and extremely low drug resistance (30,31) are ideal candidates for this purpose. In the present study, a FAP-D was pioneered instead of a FAP inhibitor to eliminate side effects. FAP was degraded by FAP-D with excellent selectivity (Fig. 1). As expected, the IC50 of cisplatin in H1299 cells was lowered by combined treatment with FAP-D (Fig. 2), suggesting enhanced cisplatin sensitivity. In addition, H1299/H460 cell apoptosis was strengthen by this combination strategy (Figs. 2 and S7) and FAP-D exhibited favorable biocompatibility (Fig. 3). In vivo, apoptosis in tumors was more strongly induced in mice co-treated with FAP-D (Fig. 4). Compared with previous results (32), the dosage of FAP-D used in the present study was lower, and the biocompatibility was excellent, highlighting the advantages of FAP-D.

In the present study, the therapeutic effects of cisplatin + FAP-D on H1299 cells and heterogeneous tumors were investigated systematically. The results demonstrated that the cisplatin sensitivity of H1299 cells and tumors was enhanced by co-treatment with FAP-D. The present findings revealed the great clinical utility of cisplatin + FAP-D for NSCLC treatment.

The present study establishes a link between FAP-D co-treatment and enhanced cisplatin sensitivity in NSCLC. However, several limitations exist: The therapeutic effects of such method on the organoids models are lack. Moreover, patient-derived tumor xenograft models are also worth exploring. Finally, the deep molecular mechanism of the role of the cisplatin + FAP-D needs to be investigated. All the aforementioned approaches represent important directions for our future investigations.

Supplementary Material

Supporting Data

Acknowledgements

The authors would like to thank Dr Joe Barber Jr. from Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the English text of a draft of this manuscript.

Funding

The present study was supported by the Doctoral Research Fund Project of the First Affiliated Hospital of Kunming Medical University (grant nos. 2022BS031 and 2023BS013), the Yunnan Provincial Department of Education Science Research Fund Projects (grant nos. 2025J0175 and 2025J0177), the Yunnan Fundamental Research Kunming Medical University Projects (grant nos. 202501AY070001-063 and 202301AY070001-205), the Key Clinical Specialty of Thoracic Surgery in Yunnan (grant no. 300067-3) and the Science and Technology Department of Yunnan (grant no. 202401AT070066).

Availability of data and materials

The data generated in the present study are included in the figures and/or tables of this article.

Authors' contributions

YZ, RL, XC and CZ conducted all experiments and analyzed the data. YZ, RL, XC, CZ, YB, YC, XT and TX assisted in data acquisition, data analysis and drafted the manuscript. XY and JY performed the MD assays and assisted in data analysis. YZ and XY designed the project and acquired funding. YZ, RL and XY confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

The animal experiments were permitted by the institutional animal ethics committee of Kunming Medical University (approval no. kmmu20241355; Kunming, China). All clinical procedures were conducted in accordance with the relevant provisions of the Declaration of Helsinki. The studies were conducted in accordance with the local legislation and institutional requirements.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References