Alpelisib and metformin were ordered from LC Laboratories (Woburn, MA, USA) and Sigma-Aldrich (St Louis, MO, USA) respectively. Primary antibodies against phosphorylated ERK1/2 (Thr202/Tyr204) (p-ERK1/2, Cat: 9101), ERK1/2(Cat: 9102), phosphorylated Akt (Ser473) (p-Akt, Cat: 4060), Akt(Cat: 9272), phosphorylated Stat3 (Tyr705) (p-Stat3, Cat: 9145), Stat3 (Cat: 4904), Cyclin D1(Cat: 55506), c-Myc (Cat: 9402), phosphorylated ErbB2 (Tyr1221/1222) (p-ErbB2, Cat: 2243), ErbB2 (Cat: 2242), β-Catenin (Cat: 8480), Active β-Catenin (Cat: 19807), phosphorylated mTOR (Ser2448) (p-mTOR, Cat: 5536), mTOR (Cat: 2983), phosphorylated S6K (Thr389) (p-S6K, Cat: 9234), S6K (Cat: 9202), Nanog (Cat: 4903), KLF4 (Cat: 12173), Sox2 (Cat: 23064), AMPK (Cat: 2532), p-AMPK (Cat: 2535) along with Anti-mouse/rabbit HRP-linked secondary antibodies, were purchased from Cell Signaling Technology (Danvers, MA, USA). The antibodies against GAPDH (Cat: sc-47724) and LGR5 (Cat: sc-517295) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The SK-BR-3 and BT-474 breast cancer cell lines were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA). The cells were cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS), 100 μg/mL penicillin, and 100 μg/mL streptomycin. They were maintained in a humidified incubator at 37 °C with 5% CO2.

Cell Proliferation was determined by Cell Counting Kit-8 (CCK-8) assay. The cells were seeded into 96-well plates (1 × 10³ cells/well) for 24 h and then treated with metformin or alpelisib, or in combination, at the indicated concentrations for 5 days. The 5-day time point was selected based on preliminary studies and established protocols to capture the sustained anti-proliferative effects of the treatments. At each endpoint, 10 μl of CCK-8 reagent was added into each well and was then incubated for two hours at 37 °C. The colorimetric absorbance at 450 nm was measured with an ELISA microplate reader. Six parallel replicates per experiment were analyzed for each experimental sample. Representative data from three repeats were presented.

Cells were plated (600 cells/well) in 6-well plates for 24 h, followed by treatment with alpelisib or metformin at indicated concentrations for 14 days. Then, the colonies were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Colonies were counted using ImageJ software. Representative data from three sets of repeats were presented.

The treated cells from each group were collected by trypsinization, and the resulting single-cell suspensions were fixed in 70% ethanol overnight at −20 °C. The fixed cells were washed with PBS and incubated for 30 minutes at 37 °C in a solution containing 0.05% Triton X-100 in PBS, RNase A (100 μg/mL), and propidium iodide (PI, 50 µg/mL). Cell cycle phase distribution was analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). The percentages of cells in each phase were determined using ModFit software. Representative data from three independent experiments are presented.

Cells were seeded into ultra-low attachment 24-well plates (Corning) at a density of 1, 000 cells per well. The cells were treated with the indicated doses of alpelisib, metformin, or their combination and incubated in DMEM/F-12 medium supplemented with 10 μg/mL insulin (Sigma, St. Louis, MO, USA), 1 μg/mL hydrocortisone (Sigma), 1× B27 (Thermo Fisher Scientific, Waltham, MA, USA), 20 ng/mL EGF (Stemcell Technologies, Vancouver, BC, Canada), 20 ng/mL bFGF (Stemcell Technologies), and 4 μg/mL heparin (Stemcell Technologies) for seven days to assess primary sphere formation. Spheres with diameters >25 μm were counted and imaged. For secondary sphere formation, single-cell suspensions, generated from the primary spheres using trypsinization, were replated under the same conditions to evaluate secondary sphere formation. Sphere counting and imaging were performed following the same protocol. Primary and secondary sphere formation was analyzed in triplicate.

The ALDEFLUOR assay kit (STEMCELL Technologies, Vancouver, Canada) was used to determine the percentage of ALDH1+ cells in SK-BR-3 cells following treatment. Cells were trypsinized and resuspended in assay buffer containing the ALDH substrate according to the manufacturer’s instructions. Negative controls were prepared by incubating a portion of the sample with the ALDH inhibitor, diethylaminobenzaldehyde (DEAB). All samples were incubated at 37 °C for 45 minutes, washed, and resuspended in assay buffer. ALDH activity was analyzed using a CytoFLEX flow cytometer, and data were processed with CytExpert software. The percentage of ALDH1-positive cells was quantified based on gating established with DEAB-treated controls.

SK-BR-3 and BT-474 cells were treated with alpelisib, metformin, or their combination at the indicated concentrations for 48 hours. The treatment duration was selected to capture peak, steady-state modulation of signaling pathway activity following drug exposure, particularly to metformin. With the cell lysate prepared from the treated cells, protein concentrations were determined using a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA), and equal amounts of protein (50 µg) were resolved on 8–15% SDS-PAGE gels before being transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in TBST for 2 hours at room temperature and incubated with specific primary antibodies overnight at 4 °C. After washing with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 hours at room temperature. Following final washes in TBST, protein bands were visualized using enhanced chemiluminescence (ECL) reagents (Thermo Scientific) and imaged with a FluorChemE imager. Western blot band intensities were analyzed using ImageJ software. The integrated density of each target band was measured, background-corrected, and normalized to its corresponding loading control. For phosphorylated proteins, levels were further normalized to their corresponding total protein levels to evaluate the degree of activation/phosphorylation. Data from three independent experiments are presented as fold-change relative to the control.

The synergistic effect between alpelisib and metformin was evaluated using CCK-8 assays as previously described (28). Cells were treated with alpelisib, metformin, or their combinations at the indicated concentrations for 5 days, followed by incubation with the CCK-8 reagent. Combination index (CI) values were calculated using the Chou–Talalay method with CompuSyn software (ComboSyn, Inc.; Paramus, NJ, USA) (29, 30). CI values of < 1, = 1, and > 1 represent synergistic, additive, and antagonistic effects, respectively.

A base layer was prepared by adding 100 µL of 5% Matrigel in complete media (on ice) to each well of a 48-well plate and allowing it to polymerize for 30 minutes at 37 °C. Subsequently, cells were suspended in pre-cooled DMEM/F12 media supplemented with 10% FBS, containing 2X the indicated concentrations of Alpelisib, Metformin, their combination or control, at a density of 8, 000 cells/mL. An equal volume of this cell suspension was mixed with 5% Matrigel on ice to generate the working cell solution. Then, 300 µL of this mixture was carefully layered atop the pre-formed base layer. The plates were incubated for 14 days in a humidified 37 °C incubator with 5% CO₂. After the incubation period, colonies were imaged, and those ≥25 µm in diameter were counted. Colony counts from triplicate wells were statistically analyzed using one-way ANOVA with Tukey’s post-hoc test.

Statistical analyses, including the determination of significance and IC₅₀ values, were performed using Prism 7 software (GraphPad, La Jolla, CA, USA). Statistical significance for data based on biological replicates from clonogenic, tumorsphere, and ALDEFLUOR assays was determined by one-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons. Statistical significance was indicated as follows: * p < 0.05; ** p < 0.01.

We first evaluated the effect of alpelisib as a single agent on the growth of SK-BR-3 and BT-474 breast cancer cells, both of which overexpress erbB2. Using a CCK-8 assay, we measured cell proliferation after 5 days of alpelisib treatment. As shown in Figure 1A, alpelisib inhibited cell viability in a dose-dependent manner, with BT-474 cells showing greater sensitivity (IC₅₀ = 0.329 μM) compared to SK-BR-3 cells (IC₅₀ = 8.022 μM). The results indicate that alpelisib inhibits the proliferation of both cell lines, with BT-474 cells showing greater sensitivity than SK-BR-3 cells. To further assess alpelisib-induced tumor inhibition effects, we performed colony formation assays. The results likewise demonstrated significant suppression in both lines (Figure 1B, C). BT-474 cells again exhibited greater sensitivity than SK-BR-3 cells, with effective inhibition observed at concentrations of 0.1–3 μM for BT-474 and 1–10 μM for SK-BR-3. Taken together, these findings demonstrate that alpelisib effectively inhibits proliferation and clonogenic potential in erbB2-overexpressing breast cancer cells, underscoring its therapeutic potential in this subtype.

Given the critical role of cancer stem cells in tumor development, progression, and drug resistance (31), we evaluated the effect of alpelisib on the stem-like properties of BT-474 and SK-BR-3 cells using cell-line-specific assays. Tumorsphere formation assays, a classical method for studying the stem-like properties of BT-474 cells, were employed to assess the impact of alpelisib for this cell line. As shown in Figure 2A, alpelisib significantly inhibited both primary and secondary tumorsphere formation. Notably, the reduction in secondary sphere formation reflects a marked decrease in cancer stem cell self-renewal capacity, demonstrating a potent anti-stemness effect. SK-BR-3 cells are known to have limitations in forming typical tumorspheres in standard assays (32). In contrast, the ALDEFLUOR assay, which measures ALDH1 activity as a surrogate marker for cancer stem cells (33), is commonly used for SK-BR-3 cells. Therefore, we employed this assay to quantify the stemness in SK-BR-3 cells. The results from the ALDEFLUOR assays on alpelisib-treated SK-BR-3 cells indicate that alpelisib treatment led to a significant reduction in the percentage of ALDH1+ cells (Figure 2B), further validating its inhibitory effect on cancer stemness in SK-BR-3 cells. Together, these findings demonstrate that alpelisib effectively suppresses cancer stemness in both SK-BR-3 and BT-474 cells, highlighting its potential as a therapeutic agent targeting cancer stem cell populations.

Previously, we demonstrated that metformin, a first-line drug for diabetes, inhibits the proliferation and stemness of erbB2-overexpressing breast cancer cells (34). Our preliminary studies also showed that metformin effectively inhibited the proliferation of SK-BR-3 and BT-474 cells, which was associated with the phosphorylation and activation of AMPK (Supplementary Figure 1). Since a major side effect of alpelisib is a significant increase in blood sugar levels (10, 11), testing the effect of the alpelisib-metformin combination on these cancer cells represents a rational therapeutic strategy. We then evaluated the combination effect on the proliferation of SK-BR-3 and BT-474 cells using the CCK-8 assay, followed by synergistic analysis with the combination index (CI) using the Chou-Talalay method. In this experiment, SK-BR-3 cells were treated with 0, 3.3, 10, and 30 μM of alpelisib alone or in combination with 0, 3.3, 10, and 30 mM of metformin. BT-474 cells were treated with 0, 0.33, 1, and 3 μM of alpelisib alone or in combination with 0, 0.33, 1.1, and 3.3 mM of metformin. As shown in Figures 3A, B most of the CI values between alpelisib and metformin for each cell line were below 1.0, with the exception of one combination ratio in each cell line. These data suggest a synergistic activity of alpelisib and metformin in inhibiting proliferation in these cells. To test the potential synergistic effect of the metformin-alpelisib combination on the colony formation efficiency of breast cancer cells, SK-BR-3 cells were treated with 10 μM of alpelisib, 3 mM of metformin, or their combination, while BT-474 cells were treated with 1 μM of alpelisib, 3 mM of metformin, or their combination for 10 days, followed by colony quantification and analysis (Figures 3C-E). While each drug alone was able to inhibit colony formation, the combination of metformin with alpelisib resulted in a significant reduction in colony formation in both cell lines, indicating the enhanced efficacy of this combination.

Moreover, we examined the effect of the metformin-alpelisib combination on cell cycle progression in SK-BR-3 and BT-474 cells (Figure 4). To this end, SK-BR-3 cells were treated with 3 μM alpelisib, 5 mM metformin, or their combination, while BT-474 cells were treated with 0.33 μM alpelisib, 1 mM metformin, or their combination for 24 hours. Cell cycle analysis indicates that the combination of metformin and alpelisib induces enhanced inhibition of cell cycle progression in both cell lines, as evidenced by a significant reduction in the percentage of cells in the S phase compared to the results with the corresponding single agents. Taken together, these results demonstrate that the combination of metformin and alpelisib induces strong synergistic effects on the proliferation and colony formation of SK-BR-3 and BT-474 cells.

Given that both alpelisib and metformin may inhibit cancer cell stemness, we next determined the effect of the metformin-alpelisib combination on the stemness of SK-BR-3 and BT-474 cells. Similar to the experiment in Figure 2, we evaluated this effect on BT-474 cells using tumorsphere assays. As shown in Figure 5A, the combination of 3 mM metformin and 0.2 μM alpelisib induced a significant reduction in both primary and secondary tumorspheres in each cell line, compared to the results with the single agents. These results suggest that the combination enhances the inhibition of stemness in BT-474 cells. To evaluate the effect of the combination on the stemness of SK-BR-3 cells, the cells treated with 5 μM alpelisib and 3 mM metformin, either alone or in combination, were analyzed using the ALDEFLUOR assay (Figure 5B). The results showed that the percentage of ALDH1+ cells in the combination group was significantly decreased compared to the single agent groups. To further demonstrate that metformin-alpelisib combination induces enhanced stemness inhibition, we evaluated anchorage-independent growth, a key property of cancer stem cells (35), using 3D culture in semisolid Matrigel under low growth factor conditions. As shown in Figure 6, single-agent treatments reduced 3D colony formation in both cell lines. However, the combination of metformin and alpelisib resulted in significantly enhanced inhibition compared to monotherapies. Together, these results from tumorsphere, ALDEFLUOR, and anchorage-independent growth assays demonstrate that the metformin-alpelisib combination synergistically inhibits cancer stemness in HER2+ breast cancer cells.

To investigate the mechanism underlying the enhanced anti-tumor activity induced by the metformin-alpelisib combination, we analyzed key markers of the receptor tyrosine kinase (RTK) pathway and related oncogenic signals, followed by examination of stemness-related pathways. SK-BR-3 cells were treated with 3.3 μM alpelisib and 5 mM metformin, either alone or in combination, while BT-474 cells were treated with 1.1 μM alpelisib and 1.1 mM metformin, either alone or in combination. We examined critical effectors downstream of PI3K—Akt, mTOR, and S6K—which mediate survival and growth signals (36). Additionally, we evaluated ERK and STAT3, key regulators in the MAPK/ERK and JAK/STAT pathways respectively, both of which can cross-talk with the PI3K/Akt pathway (37, 38). Cyclin D1 and c-Myc, key factors downstream of multiple signaling pathways critical for regulating cell cycle and growth (39, 40), were also analyzed. Although erbB2 is an upstream RTK, its activation status was assessed given its regulation through interactions and feedback loops. As shown in Figure 7A, results from both cell lines demonstrated that the combination treatment induced enhanced inhibition of signaling in these pathways, as indicated by reduced protein levels of phosphorylated or active forms of the individual markers. The combination-induced suppression was particularly prominent for p-Akt, p-Stat3, c-Myc, p-erbB2, and p-ERK1/2. These results suggest that the metformin-alpelisib combination not only induces enhanced inhibition of signaling downstream of the PI3K pathway but also exerts a broader impact on the RTK network.

To further elucidate the mechanisms underlying the enhanced inhibition of cancer stemness, we investigated the effect of this regimen on stemness-associated signaling pathways and transcription factors. Beyond the Wnt/β-catenin pathway, a well-established regulator of cancer stemness (41), we analyzed the expression of core pluripotency factors, including Nanog, KLF4, LGR5, and Sox2, which are critical for maintaining self-renewal, tumorigenicity, and therapy resistance in breast cancer stem cells (42–45). As shown in Figure 7B, Western blot analysis revealed that both metformin and alpelisib monotherapies reduced the expression of active β-catenin and these stemness-related markers to varying degrees in both cell lines. Notably, the combination treatment resulted in markedly enhanced suppression of these factors compared to either agent alone. Specifically, in SK-BR-3 cells, the combination significantly decreased protein levels of active β-catenin, Nanog, KLF4, LGR5, and Sox2. A similar enhanced inhibitory trend was observed in BT-474 cells, particularly for active β-catenin, Nanog, and LGR5, although some cell line-specific variations in response were noted. Together with the c-Myc inhibition observed in Figure 7A, these findings indicate that the metformin-alpelisib combination synergistically targets both oncogenic signaling networks and stemness regulatory pathways, providing an integrated mechanistic basis for its enhanced anti-tumor effects.