The human colon cancer cell line HCT-116 (Cell bank of the Chinese Academy of Science, Beijing, China) was cultured in McCoy’s 5A (Life Technologies Inc., Carlsbad, CA, USA). The human colon cancer cell lines SW-480 and HCT-116 with luciferase tdT (HCT116-Luc-tdT) (Cell bank of the Chinese Academy of Science, Beijing, China) were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, Life Technologies Inc., Carlsbad, CA, USA). The human colon cancer cell line DLD-1 (Cell bank of the Chinese Academy of Science, Beijing, China) was cultured in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies Inc., Carlsbad, CA, USA). All mediums supplemented with 10% fetal bovine serum (FBS, Life Technologies Inc., CA USA), penicillin (100 units/ml), and streptomycin (100 mg/ml) (Life Technologies Inc., CA, USA). All cells were cultured at 5% CO₂, 37°C in humidifier incubator.

AF was purchased from MedChemExpress Inc. (CAS No.: 34031-32-8, Monmouth Junction, NJ, USA); ICG-001 was purchased from MedChemExpress Inc. (CAS No.: 780757-88-2, Monmouth Junction, NJ, USA); Interleukin-6 (IL-6) was purchased from Life Technologies Inc. (PHC0063, Carlsbad, CA, USA). For the cell co-treatment, the cells were incubated with AF and ICG-001 premixed under the dose ratio of 1:10 for 24 h; for the experiments regarding the IL-6 activation, the cells were post-treated with 50 ng/ml IL-6 dissolved in culture medium for 15 min after the drug treatment; for the mice co-treatment, mice were treated with AF and ICG-001 premixed under the dose ratio of 1:2.

A 96-well cell culture plate was coated with Matrigel (BD Biosciences, MA, USA), and 1×10⁴ cells resuspended with drugs (2 μM AF, 20 μM ICG-001, 2 μM AF + 20 μM ICG-001) in culture medium were seeded into the plate and cultured in 37°C for 30 min. Then the culture mediums with 10% Matrigel were added into the plate and cultured for 4 days (33, 34). Then the diameters of the cell spheroid were measured, and spheroids were stained with Calcein-AM (CA1630, Solarbio, Beijing, China) to detect the living cells. The images were taken by microscope camera system (CKX41, OLYMPUS, Monolith, Japan).

CCK-8 assay (TransGen Biotech, Beijing, China) was used to measure cell viability. Briefly, 1,000 cells/well were co-incubated with different doses of AF, ICG-001, and the combination of both drugs in a 96-well cell culture plates for 24 h. Later, the value of OD_450nm of each well was obtained and recorded with a microplate reader by using CCK-8 assay. Cell viability was calculated based on surviving cell number (%treated/untreated); surviving cell number of control group corresponded to 100%. The experiments were carried out in triplicates, and the half inhibition rate (IC₅₀) values were calculated. The combination index (CI) was also calculated to examine the interaction between AF and ICG-001 by Compusyn software (version 1.0, Inc., Paramus, NJ, 07652 USA). CI values 1, <1, and >1 indicated an additive effect, synergism, or antagonism, respectively.

A scratch and wound healing assay was performed to measure cell migration. Cells (3 × 10⁵ cells/well) were plated into six-well cell culture plates. Cells were grown to confluence and were scratched crossly with sterile 1,000 μl pipette tips. After scratching, cells were washed twice and co-incubated with drugs (2 μM AF, 20 μM ICG-001, 2 μM AF + 20 μM ICG-001) for 24 h. The width of wound in each well was recorded by a microscope camera system (CKX41, OLYMPUS, Monolith, Japan), and the change rate of the wound width of each well was calculated.

Twenty-four-well inserts (Costar, New York, NY, USA) coated with Matrigel were used for cell invasion assays. Briefly, cells were diluted into 1 × 10⁴ cells/ml, resuspended in 100 μl serum-free medium with drugs (1 μM AF, 10 μM ICG-001, 1 μM AF + 10 μM ICG-001), and seeded into the upper chamber, while the lower chamber was filled with the medium with 10% FBS. Cells attaching to the bottom side of the upper chamber were stained with 0.1% crystal violet (Solarbio, Beijing, China) and counted under microscope 24 h later.

Cells were plated 1,000 cells/well in 6-well plates with drugs (1 μM AF, 10 μM ICG-001, 1 μM AF + 10 μM ICG-001) and further cultured at 37°C, 5% CO₂ for 10 days. Colony formation analysis was done by staining with 0.1% crystal violet (Solarbio, Beijing, China), and cell proliferating states were recorded by photography and counted manually.

Protein was extracted from harvested cells or tumor and lung tissues from mice and quantified through bicinchoninic acid (BCA) analysis (Life Technologies Inc., CA, USA). Equal amounts of protein were loaded. The following primary antibodies were used for Western blot analysis: Phospho-Stat3 (Tyr705) (D3A7) (9145T, Cell Signaling Technology, Danvers, MA, USA, 1:2,000), Stat3 (9139T, Cell Signaling Technology, Danvers, MA, USA, 1:2,000), Bcl-xL (54H6) (2764T, Cell Signaling Technology, Danvers, MA, USA, 1:2,000), Caspase-3 (9662S, Cell Signaling Technology, Danvers, MA, USA, 1:1,000), cleaved-Caspase-3 (9661T, Cell Signaling Technology, Danvers, MA, USA, 1:1,000),α-Tubulin (66031-1-Ig, Proteintech, Rosemont, IL, USA, 1:2,000), and GAPDH (60004-1-lg, Proteintech, Rosemont, IL, USA, 1:2,000). After incubating with primary antibodies and washed five times, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) anti-mouse/rabbit IgG (SA00001–9, SA00001–1, Proteintech, Rosemont, IL, USA, 1:5,000), washed five times, and exposed under chemiluminescent imaging analysis system (Tanon 5200, China). Densitometry analysis was done by Image J (Version 1.50i, National Institutes of Health, USA).

Commercial STAT3 siRNA was obtained from Oligobio Inc. (Beijing, China) and used to target human STAT3 (Gene ID: 6774). Cells were transfected with siRNA (50 nM) for 24 h before drug treatment by using Lipofectamine™ 2000 Transfection Reagent (11668019, Life Technologies Inc., CA USA) according to the manufacturer’s instructions. Non-specific siRNA (NC siRNA, Oligobio Inc., Beijing, China) was used as a negative control. A western blot assay was done to confirm the selective silencing of STAT3 and then detect the expression of Bcl-xL, Caspase-3 and cleaved-Caspase-3 of STAT3-slienced cells treated with drugs compared with those of control cells.

Cells (5 × 10⁴ cells/ml) were plated on coverslips, treated with drugs (2 μM AF, 20 μM ICG-001, 2 μM AF + 20 μM ICG-001) for 24 h, fixed in 4% paraformaldehyde solution for 20 min at room temperature, and permeabilized in 0.5% Triton X-100 for 5 min. Then, the cells were washed twice, incubated with anti-Phospho-Stat3 antibodies for overnight at 4°C, then incubated with appropriate conjugated secondary antibodies for 1 h at 37°C. Then the coverslips were mounted on slides with DAPI (S2110, Solarbio, Beijing, China). The slides were visualized using a Nikon Confocal microscope, and pictures were taken with NIS-Element Viewer (version 5.21.0, Nikon Instruments Inc., Shanghai, China).

Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) assay was performed to measure cell apoptosis. Briefly, 5×10⁴ cells were plated on cell culture slides in 12-well plates. When cells were approximately 70% confluent, they were then treated with drugs (2 μM AF, 20 μM ICG-001, 2 μM AF + 20 μM ICG-001) for 24 h. After fixation in 4% paraformaldehyde for 1 h, samples were stained with TUNEL reagent (TransGen, Beijing, China) following the manufacturer’s instructions. Then images were captured with Nikon confocal microscope.

The animal study was reviewed and approved by the Animal Ethics Committee of the China Agricultural University (approval code AW12601202-2-1), according to the guidelines for Laboratory Animal Use and Care from the Chinese Center for Disease Control and Prevention and the Rules for Medical Laboratory Animals (1998) from the Chinese Ministry of Health, under protocol CAU20151001-1.

To establish a tumor xenograft mouse model, 1 × 10⁶ cells of HCT-116 were resuspended in 150 μl PBS and injected subcutaneously into 5-week-old male null-balb/c mice (Vital River, Beijing, China). Tumor sizes were measured with a digital caliper every 3 days. The volume of tumor size was calculated as follows: V = L×W²/2, of which W corresponded to the width and L to the length. When the length reached about 5 mm, mice were then randomly assigned into four groups and treated with drugs (AF 5 mg/kg, ICG-001 10 mg/kg, AF 5 mg/kg + ICG-001 10 mg/kg) or vehicle by intraperitoneal injection daily for another 14 days. At the end of treatment, all mice were sacrificed and xenograft tumors were harvested. To evaluate the drug toxicity, (1) mice body weight was measured every day during treatment; (2) the blood biochemistry indexes of mice were detected; (3) the lungs, livers, kidneys, spleens, and hearts were harvested to perform pathological section with HE staining at the end of treatment.

To establish a pulmonary metastasis mouse model, 1 × 10⁶ cells of HCT116-Luc-tdT were injected into the tail vein of 5-week-old male null-balb/c mice (Vital River, Beijing, China). On the second day after injection, the mice were randomly assigned into four groups and treated with drugs (AF 5 mg/kg, ICG-001 10 mg/kg, AF 5 mg/kg + ICG-001 10 mg/kg) or vehicle by intraperitoneal injection daily for 14 days. The fluorescence intensity of living mouse lung and lungs collected from sacrificed mice at the end of treatment was measured by using in vivo imaging system (IVIS Spectrum, PerkinElmer Inc., USA). The lung tissues were also harvested to perform a pathological section with HE staining and to measure the metastatic area.

Xenograft tumor tissues collected from xenograft nude mice and lung tissues collected from the pulmonary metastasis mice were fixated with 10% (v/v) neutral-buffered formalin (Biosharp, Beijing, China). Then samples were embedded in paraffin wax and sectioned into 3 μm slides. After deparaffination and antigen retrieval with Sodium Citrate-Hydrochloric acid Buffer solution, sections were incubated with primary antibodies: Phospho-Stat3, Bcl-xL, and cleaved-Caspase-3 at 4°C overnight, followed by incubation of biotinylated secondary antibodies at 37°C for 1 h. Sections were stained with diaminobenzidine (Solarbio, Beijing, China) and counterstained with hematoxylin (ZSGB-BIO, Beijing, China). Images were captured with a bright field digital microscope.

Statistical analysis was performed by using GraphPad Prism5 software (version 5, GraphPad Software Inc., San Diego, CA, USA). A two-tailed unpaired t-test with Welch’s correction was applied when the variances of two groups were proved equal by the F test, and p values of 0.05 or less were the threshold for statistical significance.

According to the cell viability assay, AF and ICG-001 both suppressed the proliferation of colon cancer cell lines. The IC₅₀ of AF in HCT-116, SW-480, DLD-1 cell lines were 7.85, 9.68, 7.74 μM, respectively. The IC₅₀ of ICG-001 in HCT-116, SW-480, DLD-1 cell lines were 106.39, 264.66, 2,697.72 μM, respectively ( Supplementary Table S1 ). In addition, the CI value was calculated to describe the combination effect of drugs, and the CI value could reach <1 in all three cell lines ( Figures 1A, B ). For example, the CI value of HCT-116, SW-480, DLD-1 at 70% cell growth inhibited was 0.41769, 0.74747, and 0.86046, respectively ( Supplementary Table S1 ). Those results indicated the combination of these two drugs might synergistically inhibit the growth of colon cancer in vitro. Then we confirmed that combination application of AF and ICG-001 could significantly produce a more marked inhibitory effect on three colon cancer cell lines than treatment with AF or ICG-001 alone ( Figure 1C ).

The 3D cell culture models can be used to culture cancer stem cell for drug screening, and provide more in vivo–like results than in vitro (35). So, we arranged a 3D cell culture assay and detected the diameter of the cell spheroid after drug treatment ( Figure 1D ). Moreover, the living cells were stained with Calcein-AM to confirm the results ( Figure 1D ). Those results indicated that combination of AF and ICG-001 could synergistically inhibit the growth of cell spheroid.

To assess whether the combination therapy would suppress cell migration ability, clonogenic activity, and invasion ability, we performed the cell migration assay, colony forming assay, and cell invasion assay, respectively. Our results showed that the combination use of AF and ICG-001 could significantly inhibit cell migration ( Figure 2A ), colony formation ( Figure 2B ), and invasion ( Figure 2C ) of colon cancer cells compared with those in the control group or the individual groups.

The high-level p-STAT3 expression is frequently reported in colon cancer (26), so we suppose that the combination application of AF and ICG-001 may involve in regulation of STAT3-related pathways. And our results indicated that AF or ICG-001 alone could significantly suppress the expression of p-STAT3 than that in the control group, but the combination therapy exerted a greater inhibitory effect on p-STAT3 expression than the individual drug treatment ( Figure 3A ). In addition, the combination treatment significantly downregulated the expression of p-STAT3 with a concentration-dependent manner ( Figure 3B ). Then, this differential expression of p-STAT3 among different groups was confirmed by using immunofluorescence assay ( Figure 3C ). Furthermore, the expression of Bcl-xL, which is a downstream mediator of p-STAT3, also was significantly inhibited with a concentration-dependent manner in the combination group compared with that in other groups, while the expression level of cleaved-caspase 3 was significantly upregulated with a concentration-dependent manner in the combination group compared with that in other groups ( Figures 3A, B ). To confirm the apoptosis induced by the combination of AF and ICG-001, a TUNEL assay was utilized. As the results showed, the number of apoptotic cells was significantly higher in the combination group than those in AF or ICG-001 alone or the control group ( Figure 3D ).

IL-6 can be an activator to induce the phosphorylation of STAT3, so in order to further explore the antitumor mechanism, we treated cancer cells with IL-6 after the combination therapy. The results showed that the p-STAT3 level was significantly regressed in the control group, AF alone group, and ICG-001 alone group, while the suppression of p-STAT3 in the combination group could not be rescued by IL-6 ( Figures 4A–C ). Simultaneously, the suppression of Bcl-xL and the promotion of cleaved-Caspase-3 could not also be rescued by IL-6 in the combination group ( Figures 4A–C ). Those indicated that the antitumor mechanism of the AF and ICG-001 combination may be through directly decreasing the level of p-STAT3. To confirm it, STAT3 were knocked down in CRC cell lines by siRNA silencing, and the western blot assay results showed this siRNA could effectively reduce the expression of STAT3 and p-STAT3 compared with the negative control groups ( Figures 4D–F ). Then the results showed the combination drug treatment could not induce the upregulation of cleaved-Caspase-3 and downregulation of Bcl-xL in STAT3 knockdown cells ( Figures 4D–F ). These indicated that the synergistical antitumor effect of the AF and ICG-001 combination could be exerted by inducing caspase-3-dependent apoptosis via directly inhibiting phosphorylation of STAT3 of colon cancer cells.

In order to investigate the combination effect of AF and ICG-001 in vivo, we built the tumor xenograft mice model. The results showed that combination therapy resulted in a significant reduction in tumor volume of mice compared with the vehicle and the individual treatment ( Figure 5A ). Further, at the end of the 14-day treatment, the tumors were harvested, then the IHC and western blot assay were carried on to explore the underlying mechanism. The results showed the expression level of p-STAT3 and Bcl-xL were significantly lower in the combination group than those in the individual or the control groups, and the expression level of cleaved-Caspase-3 in the combination group was significantly higher than that in the other groups ( Figures 5B, C ).

To evaluate the drug toxicity, the mice body weight, blood biochemistry, and the visceral pathology were detected. The results found that there were no statistically significant differences in the body weight among groups at the end of treatment ( Supplementary Figure S1A ); the level of the urea nitrogen in mice blood after drug treatment showed only a slight increase, but the glucose, alanine aminotransferase (ALT), aspartate transaminase (AST) levels did not significantly change ( Supplementary Figure S1B ); the pathology of lungs, livers, kidneys, spleens, and hearts of mice showed no significant change after treatment ( Supplementary Figure S1C ). These results indicated that ICG-001, AF, and combination treatment could not cause severe side effect on the experimental mice.

For the purpose of exploring the effect of the AF and ICG-001 combination in suppressing colon cancer metastasis, a pulmonary metastasis mice model was built. The fluorescence intensity of lungs was detected to assess the effect of combination therapy on mice bearing with pulmonary metastasis after treated with the same treatment of the tumor xenografted mice. As the results showed, the fluorescence intensity of lungs in combination group was significantly lower than those in the individual groups and the control group ( Figure 5D ). And to confirm the combination effect on pulmonary metastasis, the lung tissue samples were harvested to perform a pathological section with HE staining. And the results indicated that the lungs in the combination group showed significant less metastatic area than those in the individual groups and the control group ( Figure 5E ). Next, the underlying mechanism was also explored by harvesting the lung tissue to perform IHC and western blot assay. The results showed the expressions of p-STAT3 and Bcl-xL were significantly downregulated, and the expression of cleaved-Caspase-3 was significantly upregulated in the combination group compared with other groups ( Figures 5F, G ). Those results indicated the mechanism of antimetastasis effect induced by combination therapy was consistent with the antitumor effect in the tumor xenograft mice model.

In conclusion, our study demonstrated that the combination therapy of AF and ICG-001 could significantly suppress the proliferation and metastasis of colon cancer in vitro and in vivo via directly inhibiting phosphorylation of STAT3. The low level of p-STAT3 could downregulate the expression of Bcl-xL, and then induce Caspased-3-dependent apoptosis of colon cancer cells ( Figure 6 ).