2.1 Tumor xenograft mouse model

All animals’ procedures were approved by Institutional Animal Care and Use Committee of Research Center for Drug Safety Evaluation of Hainan province (China) and all animal experiments took place at Research Center for Drug Safety Evaluation of Hainan, Hainan Medical University. Male nude BALB/c mice were used to establish the TE- 1 xenograft model. TE- 1 cells were harvested and adjusted a cell density of 1 × 107 per mL with PBS, and then 0.1 mL of cell suspension was injected subcutaneously in the right armpit area of each mouse. The diameters of length and width were measured to calculate tumor size. The formula: V = 0.5 × (length × width2) mm3 was used to calculate tumor volume (mm3). When the tumor volume grew to 100–300 mm3, atotal of 23 mice with weights in the range of 18–22 g were selected randomly and subdivided into control (n = 8), 5-fluorouracil (5-FU) + cisplatin (DDP) (n = 8) and anlotinib (n = 7) groups. Anlotinib was given intragastrically in a dose of 3 mg/kg; 5-FU and DDP were administered into the caudal vein in doses of 5 mg/kg and 20 mg/kg respectively on day 0. Sterile saline was used as the control. The drugs were administered every 3 days for 5 times. The body weight and tumor sizes were measured before each administration and in the end of the experiment. On day 15, all mice were sacrificed by inhalation of carbon dioxide. Tumor tissues were isolated and weighed, and then the ratio of tumor weight to body weight was calculated.

2.2 Cell culture

The human-derived TE- 1 cell line and normal esophageal epithelial cell line (HET‑1A) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The TE- 1 cells were cultured in 1640 basal medium (Gibco, Waltham, MA, USA, Cat No: 8118131) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA, Cat No: 42F7180K); the HET‑1A cells were cultured in complete growth medium. The cells were cultured in a humidified atmosphere of 5% CO2 at 37 ℃. Cells in the logarithmic growth phase were utilized for study.

2.3 MTT assay

TE-1 and HET‑1A cells were meticulously seeded into individual wells of a 96-well plate, with a standardized count of 8000 cells per well to ensure consistency across experiments. Anlotinib, obtained from Sigma-Aldrich (St. Louis, MO, USA), was initially dissolved in dimethyl sulfoxide (DMSO) and then further diluted in basal medium to achieve a range of concentrations spanning from 0.01 to 10 μM. Following the preparation of the drug solutions, the cells were subjected to treatment with each concentration of anlotinib for a predetermined duration of 24 h to allow for adequate drug exposure. Subsequently, 20 μL of MTT solution (5 mg/mL, purchased from aladdin, Cat No: G1724034) was carefully added to each well, followed by an additional incubation period of 4 h. After completion of the incubation, the supernatant containing excess MTT solution was meticulously aspirated, and 150 μL of DMSO was added to each well in a light-protected environment to dissolve the formazan crystals formed by viable cells. The plate was then placed on a plate agitator and shaken for 10 min to ensure thorough dissolution. Finally, the optical density of the resulting solution was measured at 550 nm using a microplate reader to quantify the cellular metabolic activity indicative of cell viability. Each experiment was rigorously replicated three times to ensure the reliability and reproducibility of the results. The percentage of cell viability was calculated by averaging the values obtained from the three independent replications, providing a robust assessment of the cellular response to anlotinib treatment.

2.4 Flow cytometry

The TE-1 cell density was adjusted to 1 × 105 per mL, and then samples were placed in a 6-well plate with 1 × 105 cells per well. Cells were cultured for 48 h and treated with anlotinib in doses of 5 μM and 10 μM for another 24 h. Cells were digested with 0.25% trypsin without EDTA and then centrifuged (3000 rpm, 20 ℃, 3 min). The cells were washed 3 times for 2 min each time and suspended in 0.5 mL of PBS.

Cell apoptosis assay: A commercial annexin V-FITC/propidium iodide (PI) apoptosis detection kit (BD, Cat No: 556547) was used to analyze cell apoptosis. Cells were washed twice with cold PBS, and then were suspended and adjust to 1 × 106 per mL with buffer. 100 μL cell suspension was added with 5 μL of annexin V-FITC and 5 μL of PI and then incubated for 15 min at 25 ℃ in darkness according to the instruction of manufacturer. Cell apotosis was analyzed by flow cytometry within 1 h.

Cell cycle assay: The cells were fixed with 75% alcohol at 4 ℃ for 12 hand then centrifuged (1000 rpm, 20 ℃, 3 min). After discarding supernatant, the cells were washed and then supplemented with PBS to adjust the cell density to 1 × 105 per mL. The cells were incubated with 0.5 mL of PI (BD, Cat No: 550825) at 25 ℃ for 30 min in darkness. Cell cycle was analyzed by flow cytometry within 1h. All experiments were replicated three times.

2.5 Wound closure assays

In our experiment, we followed rigorous steps for wound closure analysis. Firstly, we seeded 4 × 105 cells in each well of a 6-well plate, ensuring they were in logarithmic growth phase with seeding efficiency ranging between 80 to 90%. During the critical growth phase of cells, wounds were precisely mechanically created using a 10 μL pipette tip, which is crucial for standardizing the wound creation process and ensuring consistency across all samples. Post-wound creation, cells were immediately treated with three different doses of Alectinib (2.5 μM, 5 μM, and 10 μM). Throughout the experiment timeline, specifically at 24 and 48 h post-treatment, meticulous documentation was maintained. High-resolution images of the wounds were captured at designated time points using appropriate imaging equipment. Subsequently, precise analysis was performed using Image J software, including accurate measurements of the wound area to determine the percentage of wound closure. This step was repeated thrice to ensure robustness and reliability of our results. Through repetition of experiments, our aim was to reduce experimental variability and enhance the statistical power of the results.

2.6 Transwell assays

After TE-1 cells were treated with anlotinib for 48h, the cells were digested and resuspended in a concentration of 2.5 × 105/mL with basal 1640 medium. 200 μL suspension was added to the upper chambers with 8.0 µm membrane pores and cells were allowed to migrate toward the bottom chambers, which contained 500 μL basal 1640 medium with 10% FBS for 24 h. Then the cells on the underside of the filter membrane were fixed in 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet (Solarbio, Cat No: G1063) for 20 min. The migrated cells were photographed and counted under microscope, 5 views were chosen with 4 views from surrounding and 1 view from center of filter membrane and the migrated cell counts were used for analysis.

2.7 Colony formation assays

After treated with anlotinib in doses of 2.5, 5, 10 μM for 48 h, TE- 1 cells were seeded with a concentration of 150 cells/well and cultured a humidified atmosphere of 5% CO2 at 37 ℃. Cells culture was terminated when the cell colony can be seen with unaided eyes. Subsequently, the cell colonies were fixed in 4% paraformaldehyde for 20 min and then stained with 0.1% crystal violet for 20 min at room temperature. The stained colonies were then imaged and counted for further analysis.

2.8 Transcriptome assay

TE- 1 cells were treated with anlotinib (3, 10 μM) or 5-FU (3 μM) + DDP (3 μM) for 24 h. DMSO was used as a control. Total RNA was extracted by using a Trizol reagent kit (Invitrogen, Carlsbad, CA, USA, Cat No: G3013). The mRNA was enriched with oligo (dT) beads and then cleaved into short fragments, which were reverse transcribed into cDNA with random primers. Second-strand cDNA was synthesized with RNase H, DNA polymerase I, dNTP, and buffer. The cDNA fragments were then processed by purification with a QiaQuick PCR extraction kit (Qiagen, Venlo, The Netherlands, Cat No: G3322), end repair, poly (A) addition, and ligation to illumine sequencing adapters. The ligation products were selected according to their size by agarose gel electrophoresis, PCR amplified, and sequenced. Finally, the PCR products were subjected to an Illumina HiSeq2500 system from Gene Denovo Biotechnology Co.(Guangzhou, China).

2.9 Western blot assays

After treated with anlotinib in a dose of 10 μM or 5-fluorouracil (5-FU, 3 μM) + cisplatin (DDP, 3 μM) for 12 h, the TE- 1 cell was lysed in RIPA lysis buffer (Servicebio, Cat No: G2002) to extract protein. Then the protein was quantified and measured by using a BCA kit (Servicebio, Cat No: G2026) and SDS-PAGE (Servicebio, Cat No: G2003). 10 μg proteins per well were loaded on to the gel for immunoblotting. Polyvinylidene difluoride membranes (Servicebio, Cat No: G6015-0.45) were blocked with 5% nofat milk in TBST buffer, incubated with primary antibodies (1:1000) targeting Bax (Servicebio, Cat No: GB11690), Bcl-2 (Servicebio, Cat No: GB113375), P21(Servicebio, Cat No: GB113721), cyclin-dependent protein kinase 1(CDK1, Servicebio, Cat No:GB11398), cyclin A1(Servicebio, Cat No: GB113964), cyclin B1(Servicebio, Cat No: GB112098), survivin (Servicebio, Cat No: GB11177), Akt(Servicebio, Cat No: GB111114), phosphorylated-Akt (p-Akt, Servicebio, Cat No: AF0908), and PI3K (Servicebio, Cat No: GB112375) at 4°Covernight, and then incubated HRP-conjugated secondary antibody (1:5000) for 30 min at 25 ℃. The signals were visualized by using an ECLkit (Servicebio, Cat No: G2014). Image Pro software was employed to calculate the intensity of the signals in each sample.

2.10 Statistical Methods

All data are expressed as the mean ± the SD. SPSS 23.0 software was employed to perform the statistical analysis. Intergroup comparisons were studied by using attest or one-way ANOVA; P values less than 0.05 were considered statistically significant.