9MW2821 was provided by Mabwell (Shanghai) Biotechnology Co., Ltd. LY294002 was purchased from Selleck Chemicals (cat. no. S1105). Antibodies from Cell Signaling Technology, Inc. used in the present study were as follows: anti-PARP antibody (cat. no. 9542), anti-cleaved caspase-9 antibody (cat. no. 9502), anti-cleaved caspase-3 antibody (cat. no. 9662), anti-phospho-AKT antibody (Ser473; cat. no. 4060), anti-phospho-mTOR antibody (Ser2448; cat. no. 2971), anti-phospho-p70s6k antibody (Ser371; cat. no. 9208), anti-phospho-4EBP1 antibody (Thr45; cat. no. 2855), anti-LC3 antibody (cat. no. 3868) and anti-SQSTM1 antibody (cat. no. 8025). All of the primary antibodies were derived from rabbits and the dilution ratio was 1:1,000. HRP-conjugated anti-rabbit IgG secondary antibody (1:3,000; cat. no. 7074; Cell Signaling Technology, Inc.) and anti-β-actin antibody (rabbit; 1:5,000; cat. no. GB15003-100; Wuhan Servicebio Technology Co., Ltd.) were also used.

The human pancreatic cancer cell lines BxPC-3, YAPC and PANC-1 were purchased from the Shanghai Typical Cultures Depository of the Chinese Academy of Sciences. PANC-1 cells were cultivated in DMEM media (cat. no. MA 0212; Dalian Meilun Biology Technology Co., Ltd.), while BxPC-3 and YAPC cells were cultured in RPMI-1640 media (cat. no. MA 0215; Dalian Meilun Biology Technology Co., Ltd.). Cell lines were maintained in an environment with 5% carbon dioxide, suitable humidity, and a temperature of 37°C. It was confirmed that all cell lines are mycoplasma-free through routine mycoplasma testing.

Cells were resuspended in ice-cold PBS. Then 100 µl (1×10⁶ cells/ml) of the cell suspension and 2 µg/ml of anti-Nectin-4 antibody were added to a centrifuge tube. The suspension was incubated for 30 min at 4°C while being protected from light. The cells were washed three times and fluorescent-conjugated secondary antibody (1:1,000; cat. no. 398004; BioLegend, Inc) was added to the appropriate tubes. The suspension was incubated at 4°C in the dark for 30 min. After washing the cells three times, they were resuspended in PBS for subsequent analysis. Cells were analyzed by flow cytometry (CytoFLEX; Beckman Coulter, Inc.) using the software CytExpert 2.4 (Beckman Coulter, Inc.).

After the indicated co-incubation time, 100 µl of MTT solution (cat. no. MB4698; Dalian Meilun Biology Technology Co., Ltd.) was added to the cells and incubated at 37°C for 4 h. The methylated product was dissolved with DMSO, and the plates were gently shaken at room temperature for 5 min. An enzyme marker was used to detect the absorbance at 490 nm.

The internalization of ADCs and the subsequent autophagic flow were investigated using confocal fluorescence imaging. Using an Alexa Fluor^® 488 Protein Labelling Kit (cat. no. A10235; Thermo Fisher Scientific, Inc.), the ADCs were labeled with Alexa Fluor^® 488 and incubated for the specified amount of time. ADCs were then applied to YAPC and BxPC-3 cells for specified durations. Autophagosomes and lysosomes were identified using a Cyto-ID Autophagy Detection Kit (cat. no. ENZO-51031K200; Enzo Life Sciences, Inc.). The cells were observed using a Carl Zeiss LSM710 (Carl Zeiss AG) confocal microscope.

The cells were lysed using RIPA buffer (cat. no. P0013D; Beyotime Institute of Biotechnology). The total protein concentration was measured after the supernatant was collected using the BCA Protein Assay Kit (cat. no. P0012; Beyotime Biotechnology). Loading buffer was added and the proteins were heated for 8 min at 100°C to denature the proteins. SDS-PAGE gels (8-15%) were loaded with equal volumes of proteins (20 µg per lane) and then underwent electrophoretic separation. The proteins were subsequently transferred to a PVDF membrane. Next, the membrane was blocked with 5% BSA (cat. no. MB4219; Dalian Meilun Biology Technology Co., Ltd.) for 2 h at room temperature, and incubated overnight with the primary antibody at 4°C, followed by the secondary antibody for 2 h at room temperature. Using an ECL kit (cat. no. MA0186; Dalian Meilun Biology Technology Co., Ltd.), the membrane protein bands were observed. Imaging was performed using a gel imager from Bio-Rad Laboratories, Inc. Densitometric analysis was carried out using Image LAB 5.2 software, and the grayscale values of the bands were calculated using ImageJ (National Institutes of Health).

Apoptosis was identified by using the Annexin V-FITC/PI Apoptosis Detection Kit (cat. no. MA0220; Dalian Meilun Biology Technology Co., Ltd.). Cells were processed and stained according to the manufacturer's protocol. After 1×10⁵ cells were centrifuged, the supernatant was discarded. 195 µl of Annexin V-FITC binding buffer was added and the cells were gently resuspended. Then, 5 µl of Annexin V-FITC and 10 µl of propidium iodide staining solution were added and gently mixed. Incubate the samples in the dark for 15 min before performing the detection on the instrument. Using a flow cytometer, cells were found and examined (CytoFLEX; Beckman Coulter, Inc.) using the software CytExpert 2.4 (Beckman Coulter, Inc.).

The autophagic structure of BxPC-3 cells was determined using transmission electron microscopy. When the cell density was ~70%, the cells were digested with EDTA-free digestive enzymes. After centrifugation (300 × g, 3 min, 20°C), the supernatant was discarded and the cells were fixed using 2.5% glutaraldehyde fixative. The samples were kept at 4°C. The embedding, sectioning and subsequent image acquisition work were entrusted to Wuhan Servicebio Technology Co., Ltd. The samples were recorded using a transmission electron microscope at magnifications of ×1,500 and ×6,000.

BALB/c nude male mice (n=25, 18 g, 6-weeks old) were purchased from the Shanghai Model Organisms Center. Male mice based the stable hormone level making it easier to determine the antitumor efficacy of ADCs. All animals were kept in the Experimental Animal Center of Fudan University under a 12/12 h light/dark cycle (17). The temperature in the animal room was controlled between 20-26°C. The mice were given full access to regular food and water during the study. BxPC3 cells suspended in PBS were subcutaneously injected at a density of 1×10⁷ cells per mouse to establish mouse xenograft tumor model. A total of 5 groups of mice were randomly selected, with 5 mice in each group. The treatments were as follows: Intraperitoneal injection of PBS; single dose intravenous injection of Nectin-4-MMAE (3 mg/kg); intraperitoneal injection of chloroquine (50 mg/kg; cat. no. C6628; MilliporeSigma) once a day; combination of Nectin-4-MMAE and CQ; and twice a week intravenous injection of the positive control drug gemcitabine (50 mg/kg). Tumor volume in mice was calculated by measuring length and width (volume=0.5 × length × width²). After 21 days of drug administration, the experimental animals were euthanized by cervical dislocation operation. The researchers observed the mice directly for undulating movements of the chest to determine respiratory arrest. At the same time, the left side of the mice's chest was gently touched with a finger to determine whether the heartbeat had stopped by touch. When the mice's vital signs completely stopped and there was no sign of recovery after continuous observation, the mice's death status was formally confirmed, and then the subsequent experimental process steps were carried out. Sections of excised tumor tissue were prepared for H&E staining.

Sectioning and H&E staining were both entrusted to Wuhan Servicebio Technology Co., Ltd. The tumor tissue was collected and fixed in a 4% paraformaldehyde solution for 24 h. Subsequently, paraffin embedding was carried out, and the tissue was sectioned into slices with a thickness of 3-4 µm. The slices were then deparaffinized in xylene and hydrated through a series of alcohol solutions with decreasing concentrations. Next, the slices were stained with H&E. After staining, the slices were dehydrated with anhydrous ethanol and made transparent with xylene. Finally, the treated slices were mounted and observed under the Whole Slide Scanning System (Olympus VS200).

Adobe Photoshop and Illustrator were used to create the graphs. GraphPad Prism 8.3 (Dotmatics) and Excel (version 2019; Microsoft Corporation) were used to analyze the data, and the results of three independent replicate experiments are shown as the mean ± SD. Groups were compared using a one-way ANOVA followed by Dunnett's or Tukey's post hoc tests and the unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Flow cytometry screening was performed on two Nectin-4 positive pancreatic cancer cell lines, BxPC-3 and YAPC, and 1 Nectin-4 negative cell line, PANC-1, for subsequent experiments (Fig. 1A). YAPC and BxPC-3 cells were cultured with 10 µl (2 µg/ml) of Nectin-4-MMAE labeled with Alexa Fluor^® 488. After 30-90 min of treatment, intracellular green fluorescence was observed using laser confocal microscopy, indicating that Alexa Fluor^® 488-labeled Nectin-4-MMAE bound to the cell surface receptors and enter the cell for internalization (Fig. 1B and C). Using an MTT assay, it was found that the cytotoxicity of BxPC-3 and YAPC cells increased significantly as the concentration of Nectin-4-MMAE increased after 72 h, while PANC-1 cell viability was not significantly affected (Fig. 1D). The cytotoxicity of ADCs is not potent enough, which might depend on the targets, the cell lines and the properties of ADCs.

To explore the mechanism of cell elimination, cells were analyzed for apoptosis by staining with Annexin V/PI after BxPC-3 and YAPC cells were treated with different concentrations of Nectin-4-MMAE for 72 h. As the concentration of Nectin-4-MMAE within BxPC-3 and YAPC cells rises, the overall number of both early apoptotic and late apoptotic cells similarly increased (Fig. 2A-D). The expression of apoptosis-related proteins was analyzed in YAPC and BxPC-3 cells by western blotting (Fig. 3A and B). As the concentration of Nectin-4-MMAE increased, there was a significant rise in apoptosis-related protein levels. This finding suggested that Nectin-4-MMAE caused cell death by cleaving PARP downstream and through apoptosis by activating the caspase cascade. The results suggested that Nectin-4-MMAE induced apoptosis and cytotoxicity in BxPC-3 and YAPC cells.

Autophagosomes in BxPC-3 cells after Nectin-4-MMAE treatment were observed using transmission electron microscopy (Fig. 4A). The autophagosomes had a double-membrane structure (marked by red arrows). This result suggested that Nectin-4-MMAE resulted in the production of intracellular autophagic vesicles. To further confirm the relationship between autophagy and ADCs, the cells were divided into three groups: The negative control group, the Nectin-4-MMAE group (15 µg/ml), and the positive control rapamycin group. BxPC-3 and YAPC cells were plated and incubated with the corresponding drugs for 12 h. The cells were stained with the autophagosome dye Cyto-ID and observed by confocal microscopy. It was found that the intensity of green fluorescence significantly increased in the Nectin-4-MMAE groups, which was consistent with the results of the positive control rapamycin group (Fig. 4B and C). In addition, the expression of the autophagy marker protein SQSTM1 decreased as the Nectin-4-MMAE concentration increased, and LC3 II expression increased as the Nectin-4-MMAE concentration increased, suggesting an increase in autophagy (Fig. 4D-F).

BxPC-3 and YAPC cells were treated with 15 µg/ml Nectin-4-MMAE for different time intervals. Autophagosome formation was detected at 12 h and peaked at 24 h. Lysosome formation was detected in BxPC-3 and YAPC cells after 24 h. After 48 h, lysosomes were observed in BxPC-3 cells, while lysosomes disappeared in YAPC cells. Yellow fluorescence was observed in the merged image at 24 h and disappeared at 48 h, indicating that autophagosomes become autophagic lysosomes and were subsequently degraded. This result demonstrated the entire process of Nectin-4-MMAE-induced autophagic flux (Fig. 5A and B).

To improve understanding of how Nectin-4-MMAE induces autophagy, the phosphorylation levels of the upstream regulators of the classical Akt/mTOR autophagy pathway were further examined through western blotting. It was identified that as the quantity of Nectin-4-MMAE increased, phosphorylated (p)-Akt, p-mTOR, p-p70S6k and p-4EBP1 protein levels dramatically decreased (Fig. 5C-F). These results revealed that the Akt/mTOR signaling pathway was inactivated with increasing concentrations of Nectin-4-MMAE. Combining these results showed that autophagy was induced by inactivating the classical Akt/mTOR pathway, and Nectin-4-MMAE achieved the goal of eliminating tumor cells through autophagy and apoptosis.

Since autophagy demonstrated a crucial role in the efficacy of Nectin-4-MMAE, it was investigated if autophagy modulators could be combined to further improve efficacy. CQ (5 µM) and LY294002 (3 µM) autophagy inhibitors were chosen for testing with Nectin-4-MMAE. Cell survival was significantly lower in the combination treatment group than in the monotreatment group (Fig. 6A and B). In addition, flow cytometry results demonstrated a significant increase in the proportion of Annexin-V-positive BxPC-3 cells in the combination treatment group but not in the monotreatment group (Fig. 6C-F). Caspase-9 and PARP were more activated in the combination treatment group than in the monotreatment group, as evidence by cleaved protein bands (Fig. 7A and B). The results indicated that combining Nectin-4-directed ADCs with autophagy inhibitors increased apoptosis and produced greater cytotoxicity.

To further confirm the in vitro results, BxPC-3 xenograft tumor models were established to conduct in vivo research. Mice were randomly assigned to the negative control group, Nectin-4-MMAE group, CQ group, CQ and Nectin-4-MMAE group, and positive control drug gemcitabine group. After 21 days of drug administration, the maximum volume of the tumor was 904.74 mm³, maximum length was 13.84 mm and maximum width was 11.73 mm. The volume of tumors in the negative control group was 741.65±82.27 mm³, that in the gemcitabine group was 340.50±44.86 mm³, while the tumor volume in the Nectin-4-MMAE group was 269.38±31.98 mm³, and that in the group treated with the combination of Nectin-4-MMAE and CQ was 79.12±7.62 mm³. Mice treated with Nectin-4-MMAE showed significantly reduced in tumor weights and volumes compared with the negative control group. Mice treated with CQ and Nectin-4-MMAE exhibited significantly reduced tumor weights and volumes compared with the Nectin-4-MMAE alone group (Fig. 8A and B). Tumor tissue was collected from mice in each group, and histological changes were observed through H&E staining. The number of tumor cells was notably reduced in the Nectin-4-MMAE treatment groups, and particularly in the combined treatment group. The size of nucleoli was reduced, nuclear divisions decreased, the density of tumor cells was reduced, and the necrosis rate of the tumor increased (Fig. 8C). Furthermore, the expression levels of autophagy-related proteins in the tumor tissues of mice were detected. CQ is an inhibitor of the late stage of autophagy, which can inhibit the fusion of autophagosomes and lysosomes. The degradation of LC3 II was inhibited, resulting in its accumulation (Fig. 8D). The results suggested that autophagy inhibitors promoted the treatment efficacy of Nectin-4-MMAE in animal models, which was consistent with the results of our cellular experiments.