VNBRCA1 stands for the Vietnamese breast cancer cell line no. 1, which was isolated and characterized from Vietnamese breast cancer patients and was described and used by our laboratory in previous studies20. Cancer cells were cultured in DMEM (Lonza, Switzerland) supplemented with 10% FBS (Thermo Fisher Scientific, USA) and 1% antimycotic-antibiotic (HyClone, USA) for enrichment in a T-25 flask (SPL Life Science, Korea Republic). All flasks were incubated at 37°C and 5% CO₂, and the medium was freshly changed after 48 to 72 hours. After reaching 80-90% confluency, cells were harvested by Deattachment reagent (Regenmedlab, Viet Nam) to perform subculture.

Cells were harvested by Deattachment reagent as described above. Approximately 1×10⁷ VNBRCA1 cells were stained with 10 µL of anti-CD44 (BD Biosciences, USA) and 10 µL of anti-CD24 (Sigma-Aldrich, USA) in phosphate-buffered saline (supplemented with 2% BSA) at 4°C in the dark for 20 minutes. Then, all cells were sorted based on fluorescent-bound antigen signals using the BD FACSMelody Cell Sorter (BD Biosciences, USA). The cell population that is strongly positive for CD44 and negatively or dimly expressed CD24 is considered breast cancer stem cells (BCSCs). They are marked as BCSCs hereafter in this article. BCSCs were cultured in M171 medium (Thermo Fisher Scientific, USA) supplemented with 1% MEGS (Thermo Fisher Scientific, USA) at 37°C and 5% CO₂.

Cytotoxicity was determined using the AlamarBlue assay21. BCSCs were seeded at a density of 3,000 cells per well in a 96-well plate (SPL Life Science, Korea Republic). Each well was filled with 100 µL of M171. After 24 hours of incubation, the medium was withdrawn and replaced with new medium containing CoCl₂ at different concentrations. Medium without CoCl₂ was used as a negative control. The percentage of cell viability was calculated based on the following formula:

Rate of viable cells (%)=OD value of treated group- OD value of blankOD value of untreated group- OD value of blankx 100%

Breast cancer stem-like cells were seeded at a density of 3,000 cells per well in a 96-well plate. They were then pretreated with different concentrations of CoCl₂ for various periods of time. Subsequently, the M171 medium was withdrawn and replaced with new medium containing cisplatin (Sigma-Aldrich, USA) at concentrations of 50, 100, 150, 200, and 250 µM, respectively. BCSCs were incubated at 37°C for the next 24 hours, and cell viability was assessed via the AlamarBlue assay as described above.

RNA isolation was performed using the easy-BLUE^TM Total RNA Extraction Kit (iNtRON Biotechnology, South Korea). The expression levels of target genes were determined by real-time quantitative RT-PCR (reverse transcriptase polymerase chain reaction) with specific primers (listed in Table 1). All primers were purchased from PHUSA Biochem Co., Viet Nam. RNA samples were divided into qPCR tubes (Thermo Fisher Scientific, USA) with appropriate amounts and supplemented with the necessary components for a standard PCR reaction, following the instructions in the Luna Universal One-Step RT-qPCR Kit (New England Biolabs, USA). Thermal cycling was performed using the Replex system (Eppendorf, USA). Gene expression levels were semi-quantitated using the Livak formula, with β-actin used for normalization.

Table 1

Genes	Primer sequence (5'-3')	Referrence
β-actin F	CTGGAACGGTGAAGGTGACA	22
β-actin R	AAGGGACTTCCTGTAACAATGCA
HIF-1 α F	TGGTGACATGATTTACATTTCTGA	23
HIF-1 α R	AAGGCCATTTCTGTGTGTAAGC
LC3B F	TGTCCGACTTATTCGAGAGCAGCA	24
LC3B R	TTCACCAACAGGAAGAAGGCCTGA
Beclin-1 F	ATGCAGGTGAGCTTCGTGTG	25
Beclin-1 R	CTGGGCTGTGGTAAGTAATGGA
ABCC10 F	CGGGTTAAGCTTGTGACAGAGC	26
ABCC10 R	AACACCTTGGTGGCAGTGAGCT
AMPK F	CAGGCATATGGTGGTCCATAGAG	27
AMPK R	TCATGGGATCCACCTGCAGC
PI3K F	TGTGTGGGACTTATTGAGGTG	28
PI3K R	ACACAGTATCCAGCACATGAAC
AKT F	TCTATGGCGCTGAGATTGTG	29
AKT R	CTTAATGTGCCCGTCCTTGT

Abbreviations: F: Forward, R: Reverse

All assays were performed in triplicate. Raw data were collected using Microsoft Excel 365 and analyzed with GraphPad Prism 9.0 software. All data are displayed as mean ± standard error of the mean (SEM). Differences between treatments were examined using the one-way ANOVA method, with a p-value < 0.05 considered statistically significant.

Figure 1

Abbreviations: BCSCs: Breast cancer stem cells, VNBCRA1: Vietnamese breast cancer cell line No. 1

The hypoxic environment caused by adding CoCl₂ strongly induced autophagy in BCSCs. In fact, in the presence of CoCl₂, LC3B, a central marker of autophagy induction, exhibits increased gene expression in a concentration-dependent manner. As CoCl₂ concentration rises, LC3B gene expression increases from a 1.45 to a 4.09-fold change compared to untreated cells. The expression of the Beclin-1 gene, another autophagy marker, also increases at CoCl₂ concentrations of 50, 75, and 100 µM (1.49, 2.28, and 1.64-fold change, respectively), but it shows no significant change at 25 µM (p > 0.05). Overall, both genes display increased expression with higher CoCl₂ concentrations, although these changes are not statistically significant (p > 0.05) (Figure 2A). In general, genes involved in autophagy regulation exhibit elevated expression levels when exposed to various CoCl₂ concentrations (Figure 2B). The AMPK gene shows the greatest expression increase at 50 µM CoCl₂ (3.14-fold change), while PI3K and AKT genes reach their peak expression at 75 µM CoCl₂ (2.69 and 2.14-fold change, respectively). After reaching peak expression, the levels of these genes gradually decline with further increases in CoCl₂ concentration.

The potential for drug resistance in BCSCs was assessed using a cytotoxicity assay with the anti-tumor drug cisplatin. The drug resistance of hypoxic BCSCs to cisplatin was evaluated either in the presence or absence of the autophagy inhibitor 3-MA in the culture medium. In this assay, BCSCs were first induced to become hypoxic by culturing them in a medium containing CoCl₂ (ranging from 25 to 100 μM) for 24 hours. Subsequently, these hypoxic BCSCs were cultured in a fresh medium containing 60 μM cisplatin for the next 24 hours.

The AlamarBlue assay results indicated that the viability of hypoxic BCSCs increased with the concentration of cobalt (II) chloride, although the increase was not significant. Concurrent treatment with 60 μM cisplatin and 5 mM autophagy inhibitor 3-MA also enhanced the viability of the BCSCs population in relation to the concentration of CoCl₂.

The results showed that, whether treated with or without 3-MA, the cell viability of BCSCs remained similar and peaked at 75 μM CoCl₂ (Figure 3).

Figure 4

Treatment with CoCl₂ results in an upregulation of the drug resistance gene ABCC10. The most significant increase in expression, observed as a 9.17-fold change, occurs at a concentration of 75 μM CoCl₂ compared to breast cancer cells not exposed to CoCl₂ (see Figure 4).