The TNBC cell lines MDA-MB-231 and HCC-1937 were obtained from the Cell Bank of the Representative Culture Preservation Committee of the Chinese Academy of Sciences. All the cells were cultured in DMEM supplemented with 10% FBS (HyClone; Cytiva) at 37°C in 5% CO₂ and subcultured every 2–3 days. siRNA or negative control RNA were obtained from Shanghai GenePharma Co. Ltd. The cells were seeded in six-well plates at a density of 3×10⁵ cells/well. The transfections were performed using Transfection Reagent (Polyplus Transfection SA) according to the manufacturer's instructions. The transfection efficiency was confirmed via quantitative PCR. For the spheroid formation assay, the cells were transfected with siRNA targeting SOX2 or OCT4 for 24 h prior to treatment with bufalin (0.5 µM).

The MDA-MB-231 and HCC-1937 cells were plated in 96-well plates at a density of 3×10³ cells/well and incubated at 37°C for 24, 48 or 72 h. At the end of the incubation period, CCK-8 reagent (Beyotime Institute of Biotechnology) was added to each well and incubated at 37°C for 1 h according to the manufacturer's instructions. After staining, the absorbance was measured at 570 nm (Multiskan Spectrum; Thermo Fisher Scientific, Inc.).

Following incubation with 0.5 µM bufalin for 48 h, the MDA-MB-231 and HCC-1937 cells were harvested for PI staining or Annexin V-PI staining according to the manufacturer's instructions (Beyotime Institute of Biotechnology). Data acquisition and analysis were performed using a FACSCanto II Flow Cytometer (BD Biosciences). A total of 1×10⁴ cells were scanned in each analysis. Each experiment was repeated at least three times.

Following treatment with 0.5 µM bufalin or vehicle, MDA-MB-231 and HCC-1937 cells were reseeded in 12-well plates at a density of 10,000 cells/well and cultured at 37°C to form natural colonies. After 7 days, the cells were washed with PBS 3 times and fixed with 4% paraformaldehyde for 20 min at room temperature. The fixed colonies were stained with 20% crystal violet solution at room temperature for 10 min and captured.

The cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology). Equal amounts of proteins (25 µg) were separated using 10% SDS-PAGE, transferred onto nitrocellulose membranes (Beyotime Institute of Biotechnology) and blocked with 5% non-fat milk for 1 h at room temperature. After incubation with an antibody specific for poly (ADP-ribose) polymerase (anti-PARP; 1:1,000; cat. no. 9532, Cell Signaling Technology, Inc.) for 2 h at room temperature, the blots were incubated with anti-rabbit secondary antibody (1:10,000; cat. no. 3900, Cell Signaling Technology, Inc.) for 2 h at room temperature and then detected by enhanced chemiluminescence (Beyotime Institute of Biotechnology). β-actin was used as a loading control.

MDA-MB-231 cells (2×10⁶) were suspended in 100 µl PBS and injected into the lower flank of 14 4–6-weeks old nude mice housed in a room at 23–28°C and approximately 70% humidity, with an alternating 12 h light (from 7 a.m.)/dark cycle (from 7 p.m.) and free access to sterilized food and water. All experiment operations complied with laboratory animal ethics requirements approved by IACUC. Tumor diameter was measured with calipers. When the tumor diameter reached ~5 mm, bufalin (T1719; Shanghai Topscience Co., Ltd.) was injected into the tumor at a dose of 1 mg/kg three times per week (n=7), whereas control group animals were injected with an equal volume of DMSO (n=7). After 2 weeks, the tumors were weighed with an electronic balance. The animal experimental protocols were approved by the Ethics Committee of the University of Traditional Chinese Medicine.

The xenograted tumors were fixed in 10% formalin at room temperature for 48 h and embedded in paraffin. Then, the paraffin blocks were cut into 4-mm sections and deparaffinized. Routine IHC staining for Ki-67 (1:100; Cell Signaling Technology, Inc.) was performed on the slidesandstaining for SOX2 (1:100; Proteintech, Inc.) or OCT4 (1:100; Proteintech, Inc.) was performed on the TNBC tissue microarray (Shanghai Outdo Biotech, Inc). Detection was performed using Ventana's UltraView diaminobenzidine (DAB) detection kit (P0202; Beyotime Institute of Biotechnology). Apoptotic cells were identified by TUNEL colorimetric staining according to the manufacturer's instructions (Roche Applied Science, Inc.). The tissue microarray (HBreD050Bc01), including 40 TNBC patient tissue samples was purchased from Shanghai Outdo Biotech Co., Ltd., and subjected to IHC staining

Approximately 500 viable single cells were plated on ultra-low attachment 96-well plates (Thermo Fisher Scientific, Inc.) and cultured in cell growth medium (Thermo Fisher Scientific, Inc.) with or without bufalin at a dose of 0.5 µM for 7 days. The number of spheroids was counted under an inverted microscope (IX51; Olympus Corporation) at a magnification of ×100.

Total RNA was extracted from MB-231 and HCC-1937 cells using TRIzol^®(Invitrogen; Thermo Fisher Scientific, Inc.). For reverse transcription-quantitative PCR (RT-qPCR) analysis, 1 µg total RNA was reverse-transcribed (37°C for 1 h and 70°C for 10 min) by using Superscript III RT (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR was performed using the ABI PRISM 7300HT Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 1 cycle at 95°C for 5 min, followed by 40 cycles at 95°C for 15 sec (denaturation), 60°C for 30 sec (annealing), 72°C for 30 sec (extension) and 72°C for 5 min (final extension). The relative expression level of each gene was calculated using the 2^−∆∆Cq method (∆∆Cq=∆Cq_bufalin-∆Cq) (28). The primers used for SOX2, OCT4, c-Myc, β-catenin, Nanog and β-actin are listed in Table SI.

For statistical analysis, Student's t-test was used for parametric variables. One-way ANOVA followed by Dunnett's test was used for comparisons among multiple groups. All tests were performed three times, and P<0.05 was considered to indicate statistically significant differences.

To investigate the biological effect of bufalin in TNBC, in vitro assays were performed in MDA-MB-231 and HCC1937 cells using the dosages mentioned in previous studies (29,30). First, the CCK-8 assay was used to examine the effect of bufalin on cell proliferation. As shown in Fig. 1A, bufalin significantly inhibited the proliferation of both MDA-MB-231 and HCC1937 cells (P<0.05). Second, the antiproliferative effects of bufalin were further determined by colony formation assays, and the data revealed that the number and size of the colonies were markedly reduced by bufalin treatment (Fig. 1B). The effect of bufalin on the regulation of the tumor cell cycle was then assessed using FACS analysis. As shown in Fig. 1C and D, bufalin led to increased accumulation of cells in the G2/M phase of the cell cycle. Next, the apoptotic rates induced by bufalin were evaluated by flow cytometry, and our data demonstrated that bufalin triggered apoptosis of MDA-MB-231 and HCC1937 cells (Fig. 2A and B). Consistently, the induction of PARP, an apoptosis regulator, also confirmed this effect (Fig. 2C).

After confirming that bufalin inhibits cell proliferation and enhances apoptosis in vitro, it was examined whether bufalin can inhibit breast cancer growth in a xenograft TNBC model. As shown in Fig. 3A and B, bufalin significantly decreased the tumor volume and weight compared with vehicle control. Ki-67 and TUNEL staining were also performed on paraffin sections of tumor samples collected from xenografts. A reduction in Ki-67 expression and an increase in apoptosis were observed in tumors treated with bufalin compared with those treated with vehicle (Fig. 3C and D). These results further confirmed the therapeutic effect of bufalin in TNBC.

To explore whether bufalin attenuates the stemness of TNBC cells, a spheroid formation assay was performed. MDA-MB-231 and HCC1937 cells exhibited a reduced capacity to form spheroids when treated with 0.5 µM bufalin compared with control cells (Fig. 4A and B). Furthermore, the proportion of sphere-forming cells was determined by performing a limiting dilution analysis of cells incubated with or without bufalin, and the results demonstrated that the proportion of MDA-MB-231 and HCC1937 cells forming spheroids was significantly decreased by bufalin (P<0.05; Fig. 4C and D). These data indicated that bufalin effectively suppressed the self-renewal of TNBC stem cells.

To elucidate the mechanisms through which bufalin attenuates the stem cell characteristics of TNBC cells, RT-qPCR analysis was performed to analyze the expression level of stemness-related genes following bufalin treatment, as shown in Fig. 5A and B. The expression levels OCT4 and SOX2 were found to be significantly downregulated following treatment with bufalin (P<0.05). To further confirm whether bufalin attenuates the stemness characteristics of TNBC cells by inhibiting SOX2/OCT4 expression, MDA-MB-231 or HCC1937 cells were transfected with siRNAs targeting OCT4 and SOX2, followed by a spheroid formation assay. The results demonstrated that the ability of TNBC cells to form spheroids was not affected by bufalin after SOX2 or OCT4 expression interference (P<0.05), suggesting that bufalin may attenuate the stemness of TNBC cells via downregulating the stemness-associated factors SOX2/OCT4 (Fig. 5C-E). In addition, SOX2/OCT4 protein expression was examined in 40 tissue samples from patients with TNBC. As expected, most TNBC tissues exhibited upregulated SOX2 (27/40) and OCT4 (30/40) expression (Fig. 5F).