Human CRC cell lines, including HCT116 and LoVo, were cultured in RPMI-1640 medium (Gibco Laboratories, Grand Island, NY, USA) supplemented with 10% fetal calf serum (Gibco Laboratories) at 37°C in a 5% CO₂ humidified atmosphere.

Cisplatin was purchased from Qilu Pharmaceutical (Jinan, China). Bufalin was purchased from Sigma (St. Louis, MO, USA). CD44 (60224-1-IG), CD133 (18470-1-IG), OCT4 (11263-1-AP), SOX2 (11064-1-AP), and NANOG (14295-1-AP) primary antibodies were purchased from Proteintech (Chicago, IL, USA). GAPDH (#2118) and ABCG2 (#42078) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Cells were seeded in a 96-well plate at a density of 1×10⁴ cells/well. Cell viability assays used the Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). Cell viability was evaluated by determining the absorbance of each well at 450 nm using a plate reader (Bio-Rad, Hercules, CA, USA). Each sample was analyzed in sextuplicate, and experiments were repeated thrice.

The Annexin V-FITC/PI Apoptosis Detection kit (Becton-Dickinson, Franklin Lakes, NJ, USA) was used to investigate apoptosis. Tumorsphere cells were dissociated into single cells and were then stained with Annexin and PI separately. The apoptosis ratio was assessed by flow cytometry using the FACSCalibur system (Becton-Dickinson).

The DNA-binding dye Hoechst 33342 was used to evaluate the SP ratio. Dissociated sphere cells were stained with Hoechst 33342 for 10 min and were tested through dual-wavelength analysis using flow cytometry (Hoechst red 675/20; Hoechst blue 424/44). SP cells were shown to have a characteristic tail, which differentiated them from other cells of the population.

The protein expression of stemness markers, such as CD133 and CD44, was detected using flow cytometry. Dissociated sphere cells were incubated with primary antibodies, including CD44 and CD133 antibodies, at 4°C for 1 h. They were then washed with phosphate-buffered saline (PBS) twice and incubated with Alexa Fluor 488 conjugated anti-rabbit secondary antibodies (R37116) and Alexa Fluor 488 conjugated anti-mouse secondary antibodies (A-21202) (Invitrogen, Carlsbad, CA, USA) at 4°C for 30 min in the dark. The fluorescence values of CD133 and CD44 were determined using flow cytometry and analyzed using the FlowJo 7.6 software (Treestar, Inc., Ashland, OR, USA).

Dissociated sphere cells were seeded on cover slips pre-coated with 0.01% polylysine at a density of 1,000 cells per well in a 48-well chamber. After 24 h, the cells were treated with cisplatin and bufalin for 48 h. The cells were then treated in turn with 4% paraformaldehyde for 20 min, 0.1% Triton X-100 for 10 min, 5% bovine serum albumin (BSA) for 60 min, and primary antibodies overnight at 4°C. Next, the cells were washed thrice using PBS and incubated with Alexa Fluor 488 conjugated anti-mouse secondary antibodies, Alexa Fluor 488 conjugated anti-rabbit secondary antibodies, and Alexa Fluor 555 conjugated anti-rabbit secondary antibodies (A-31572) (Invitrogen) for 1 h. The cells were then observed using a fluorescence microscope (Leica, Wetzlar, Germany).

Secondary tumorspheres treated with cisplatin and bufalin were collected through centrifugation and concentration. Subsequently, tumorspheres were lysed with M-PER Mammalian Protein Extraction reagent with protease inhibitor cocktail (100X) (Sangon Biotech, China) and 1 mM PMSF. The lysate was centrifuged at 4°C at 12,000 g for 15 min, and the supernatant was used for western blotting. The protein concentration was measured using the Bradford Coomassie Blue G-250 method. Protein (40 µg) was mixed with 5X SDS sample buffer and was denatured by boiling for 10 min. The denatured protein was loaded onto 10% polyacrylamide SDS gels (PAGE-SDS) and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked in 5% BSA for 2 h followed by incubation with primary antibodies overnight at 4°C. After washing thrice for 10 min in TBST, membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature (RT). Subsequently, the membranes were washed thrice for 10 min in TBST and were visualized using the ECL Western Blotting Detection system (Millipore). The ratio of the optical densities of the bands was measured using a gel image analysis system (Bio-Rad) and normalized to GAPDH.

HCT116 and LoVo cells were separately seeded in ultra-low attachment 24-well plates (Corning, Corning, NY, USA) with DMEM/F-12 (12660012, Gibco) culture media, B27 (17504044, Gibco), 20 ng/ml EGF (PHG0311, Gibco), and 20 ng/ml bFGF (13256029, Gibco) at a density of 200 cells/well. The medium was replaced by half every 3 days. After 14 days, tumorspheres were counted and photographs were obtained through microscopy.

Single cells were prepared and seeded into 96-well plates at a density of 200 cells/well. The medium was replaced every 2 days. After 10 days, cells were treated in turn with 4% paraformaldehyde for 20 min and crystal for 20 min, and were washed with PBS at least twice. The colonies were counted, and photographs were obtained through microscopy.

For the in vivo xenograft tumor growth assay, male nude mice [BALB/c nu/nu, 5-week-old, purchased from SLAC (Shanghai Laboratory Animal Center, Shanghai, China)] were used to prepare the in vivo tumor xenograft model. Two million cells in 0.1 ml of PBS were injected into the subcutaneous tissues of each mouse. After 2 weeks, mice were injected intraperitoneally with cisplatin (10 mg/kg body weight) and bufalin (1 mg/kg body weight) every 3 days for 4 weeks. Finally, the tumor-bearing mice were sacrificed and the tumors were excised and weighed.

All tumor xenograft bodies were formalin-fixed, embedded in paraffin, serially sectioned (5-µm thickness), and mounted on glass slides. The reagents in the subsequent process were purchased from Maixin Bio (Fuzhou, China). Sections were incubated for 10 min in peroxidase blocking agent, washed for 3 min thrice with PBS, blocked with rabbit serum for 60 min at RT, and incubated with antibodies at 4°C overnight. Subsequently, the sections were washed thrice with PBS, incubated with HRP-conjugated secondary antibodies for 10 min at RT, washed again with PBS, developed with diaminobenzidine solution, and counterstained with hematoxylin. Additionally, serial sections were stained with hematoxylin and eosin.

Data are presented as mean ± SD. All analyses were performed using the SPSS 17.0 software (IBM Corp., Armonk, NY, USA). A p-value of <0.05 was considered statistically significant.

Tumorsphere formation assay using a non-adhesive culture system is an important method for the identification of stemness in vitro (34,35). To evaluate the effect of cisplatin on the stemness of CRC cells, we tested the ability of tumorsphere formation of two CRC cell lines (HCT116 and LoVo). At the same time, colony formation assay was used to evaluate the effects of cisplatin on the proliferation of these two cell lines. In order to analyze the results of the two experiments, we used the same cisplatin concentrations and the same experiment duration (14 days).

As shown in Fig. 2A-C, with increasing cisplatin concentration (0–5 µM), the numbers and diameters of HCT116-PTSs^cis and LoVo-PTSs^cis increased. Therefore, cisplatin could increase tumorsphere formation of CRC cells in a dose-dependent manner within a certain concentration range. However, the numbers and diameters started to decrease when the cisplatin concentration reached 10 µM, and tumorspheres were not found when the cisplatin concentration reached 50 µM, which suggested that the anti-proliferation effects of higher concentrations of cisplatin (10–50 µM) were greater than the stemness effects.

Colony formation assay using the adhesive culture system was used to analyze the anti-proliferation effects of cisplatin in this study. We found that the efficiency of colony formation decreased with cisplatin treatment in a dose-dependent manner (Fig. 2D and E). When the cisplatin concentrations were 5 µM and 10 µM, the number of colonies decreased. The results of the colony formation assay and tumorsphere formation assay were opposite with cisplatin treatment at the same concentrations and experiment durations, which further supported the increasing stemness effects of cisplatin on CRC cells.

To determine the effects of bufalin on the stemness of CRC cells, we first tested the tumorsphere formation capacity of CRC cells treated with different concentrations of bufalin. We also analyzed the effects of bufalin on killing and proliferation inhibition using the colony formation assay. We found that bufalin could inhibit tumorsphere formation of HCT116 and LoVo cells in a dose-dependent manner (Fig. 3A-C). The trend of the colony formation assay results was similar to that of the tumorsphere formation assay results (Fig. 3D and E); however, 1 nM of bufalin inhibited tumorsphere formation but not colony formation, which suggested that the inhibition of tumorsphere formation effects of bufalin relied not only on anti-proliferation but also on anti-stemness.

In view of the anti-stemness role of bufalin, we speculated that it would be effective against cisplatin with regard to the stemness of CRC cells. Primary tumorsphere cells treated by cisplatin (5 µM), referred to as PTSCs^cis, were used for the secondary tumorsphere formation assay (Fig. 4A-C). When compared with the control, we found that cisplatin promoted the formation of secondary tumorspheres, while bufalin alone decreased the formation of secondary tumorspheres. On the other hand, the combination of cisplatin and bufalin could inhibit the numbers and diameters of secondary tumorspheres relative to the control and cisplatin groups. These results suggested that bufalin works against cisplatin with regard to the stemness of CRC cells.

After the secondary tumorsphere formation assay, the ratios of SP cells were tested using flow cytometry through Hoechst 33342 staining. As shown in Fig. 4D and E, the SP/total ratio increased in secondary tumorspheres treated with cisplatin relative to that of the control and decreased with the combination treatment or with bufalin alone. Moreover, Hoechst 33342-stained cells were photographed using a fluorescence microscope (Fig. 4F and G). The results of photography and flow cytometry corresponded with each other. These findings further confirmed the reversing effects of bufalin on an increase in stemness induced by cisplatin in CRC cells.

Drug-treated cancer cells in the tumorsphere formation assay showed higher expression of stemness markers such as CD133, CD44, NANOG, OCT4, SOX2, and ABCG2 (36–39). Therefore, we tested the expression of these stemness markers in secondary tumorsphere cells using immunofluorescence, flow cytometry, and western blotting.

Initially, secondary tumorspheres were dissociated into single cells and were seeded in a 24-well plate with slides. When most cells adhered to the slides, immunofluorescence assay was used to detect the expression and locations of the stemness markers in the cells. As shown in Fig. 5A, the expression of CD133, CD44, NANOG, OCT4, SOX2, and ABCG2 increased in the secondary tumorsphere cells treated with cisplatin alone. However, bufalin and combination treatment inhibited their expression.

At the same time, the two colorectal CSC markers CD133 and CD44 of secondary tumorsphere cells were assessed using flow cytometry (Fig. 5B). Consistent with the immunofluorescence results, bufalin antagonized the effects of cisplatin with regard to the expression of CD133 and CD44.

Finally, secondary tumorspheres underwent protein extraction to test the expression of stemness markers using western blotting. As shown in Fig. 5C, secondary tumorsphere cells treated with cisplatin (both HCT116 and LoVo cell lines) displayed higher expression of CD133, CD44, NANOG, OCT4, SOX2, and ABCG2 proteins compared to the control. Bufalin decreased their protein expression alone. In addition, high expression of these proteins induced by cisplatin could be reversed by bufalin. These data further supported the effect of bufalin against cisplatin-induced stemness.

Studies have shown that acquired drug resistance is associated with increased expression of stemness markers induced by chemotherapeutic drugs (38,40,41). The results of this study also suggested that cisplatin increases the expression of stemness markers, while the effects of bufalin were the opposite. Therefore, we speculated that STSCs^cis had drug-resistant properties, while bufalin could inhibit this kind of acquired drug-resistance. To verify these speculations, we compared the sensitivity of STSCs^cis and parent cells to cisplatin. At the same time, we tested the synergistic effects of bufalin on the sensitivity of STSCs^cis to cisplatin. The STSCs^cis and their parent cells were seeded in 10% FBS RPMI-1640 medium at a density of 1×10⁴ cells/well, in a 96-well plate. Then, 5 nM bufalin and different concentrations of cisplatin were added for 48 h. The results of the cell viability assay using CCK8 are shown in Fig. 6A. The IC₅₀ (concentration that produces 50% inhibition) of cisplatin in HCT116-STSCs^cis was 18.06±1.43 µM, which was higher than that of HCT116 cells (IC₅₀=2.13±0.12 µM). Bufalin decreased the IC₅₀ of HCT116-STSCs^cis to 5.61±0.42 µM. Similarly, the IC₅₀ values of LoVo cells, LoVo-STSCs^cis, and LoVo-STSCs^cis treated with bufalin were 20.81±1.15, 2.13±0.19 and 4.9±0.23 µM, respectively.

Apoptosis assay involving flow cytometry was used to verify the results of the cell viability assay (Fig. 6B). STSCs^cis were treated with 5 mM cisplatin, 5 nM bufalin, and their combination for 48 h, and the apoptosis rate was calculated. We found that the apoptosis rate with the combination was much higher than the rates with cisplatin and bufalin alone. The results further suggested that bufalin reverses the acquired drug-resistance induced by cisplatin in CRC cells.

In vitro studies have shown that bufalin could inhibit stemness and increase the sensitivity of cisplatin in CRC cells. To investigate the anti-stemness effect of bufalin in vivo, a subcutaneous xenograft model of HCT116 cells in nude mice was used. HCT116 cells were subcutaneously injected into nude mice for 2 weeks, and then, the mice were treated with cisplatin alone or cisplatin + bufalin for 3 weeks. After sacrifice, the tumor tissues were weighed and paraffin-embedded tissue blocks were created for immunohistochemistry and H&E staining. As shown in Fig. 7A and B, inhibition of tumor growth was greater with the combination of cisplatin and bufalin than with cisplatin alone. The tumor tissue weights also showed the synergistic effects of bufalin on cisplatin. According to the results of H&E staining (Fig. 7C), tumors treated with the combination of cisplatin and bufalin showed more cell vacuolization and nuclear shrinkage than with cisplatin alone.

The expression of CD133, CD44, NANOG, OCT4, SOX2, and ABCG2 was assessed using immunohistochemistry to evaluate the effect of bufalin on stemness in vivo. Similar to the in vitro results, immunohistochemistry showed that cisplatin alone increased the protein expression of stemness markers (Fig. 7C), while the combination of cisplatin and bufalin decreased the protein expression of stemness markers. These results suggested that bufalin increased the sensitive of cisplatin in CRC cells through a reduction in stemness.