Amino acid profile changes during enrichment of spheroid cells with cancer stem cell properties in MCF‐7 and MDA‐MB‐231 cell lines

Abstract Background Cancer stem cells (CSCs), subpopulations of cancer cells, are responsible for tumor progression, metastasis, and relapse. Changes in amino acid metabolism are linked to breast cancer recurrence and metastasis. Aims This study aimed to evaluate the changes in the amino acid profile in MCF‐7 and MDA‐MB‐231 cells during spheroid formation to discover the specific metabolic properties in CSCs. Methods MCF‐7 and MDA‐MB‐231 breast cancer cells were cultured as spheroids and evaluated to characterize their CSC properties. The characteristics of CSC were evaluated by examining the expression of CSC markers and conducting drug resistance assays. In addition, amino acid profile change during the enrichment of breast cancer stem cells in the spheroids was investigated by high‐performance liquid chromatography (HPLC). Results The results indicated that out of 20 different amino acids analyzed, 19 of them decreased during the spheroid formation process. Alanine, lysine, phenylalanine, threonine, and glycine showed significant reductions in the conditioned media of both cell lines in the spheroid form compared to the monolayer cells. Only one of the amino acids increased in MCF‐7 and MDA‐MB‐231 spheroids (histidine and serine, respectively). Conclusion Our results suggest that certain amino acids identified in this study can be used for a better understanding of the molecular mechanisms associated with breast cancer stem cell formation.

resides in a tumor. These cells are responsible for the metastasis and tumorigenesis properties of the cancer cells. [3][4][5][6] CSCs are categorized by their slow cell cycle, resistance to chemotherapy and oxidative stress, and rapid response to genotoxic damages. [7][8][9][10] Many studies have tried interfering with various metabolic pathways in tumors to eradicate cancer cells. [11][12][13][14] Compared to other metabolic pathways, the amino acid metabolic network is more complicated and deeply connected with other pathways. 15 Specifically, amino acid metabolism has a significant role in cancer cell behavior under oxidative, nutritional, and genotoxic stresses. 16 Thus, we can consider targeting amino acid metabolism as a potential therapeutic strategy for cancer patients.
Metabolic reprogramming in CSCs has a significant role in these cells adapting to changes in the tumor microenvironment and maintaining their unlimited self-renewal ability. 17,18 Therefore, we developed spheroids from MCF-7 and MDA-MB-231 breast cancer cell lines to investigate their amino acid contents after comprehensively characterizing their CSC properties. We used HPLC to compare the amino acid profiles of these CSC-enriched spheroids in contrast to the parental cells. 12500096, Gibco, UK) complemented with 10% fetal bovine serum (FBS) (Cat. No. A5256701, Gibco, UK) and 1% penicillin/streptomycin (P/S) (Cat. No. 15070-063, Gibco, UK). Cells were grown at 37 C in a humidified atmosphere of 95% air/5% CO 2 . Upon 80% confluency, cells were subcultured in a fresh medium using trypsin-EDTA.

| Sphere formation assay (SFA)
To assess the anchorage-independent colony formation capacity of CSCs, a soft agar assay was performed. In brief, 1% (g/mL) agarose powder (Cat. No. 16550100, Invitrogen) was mixed with distilled water and autoclaved for sterilization. Then, we added 2 mL of the agarose solution to the wells of 6-well plates (Cat. No. 220100, Sorfa, Malaysia). After solidification, single-cell suspensions of MCF-7 and MDA-MB-231 cells were thoroughly suspended and placed in agarose-coated 6-well plates at 5 Â 10 4 cells/well density in 2 mL of sphere formation media. The culture dishes were stored at 37 C in a CO 2 incubator. The culture medium contained DMEM/F12, 5% HS (human serum), and 1% P/S. Colonies were formed after 2 days of growth. Following 48 h of culture, the primary sphere formation was assayed under an inverted microscope (OPTIKA SN263975; Italy).
Spheres were defined as cell colonies >50 μm in diameter, showing a 3-dimensional structure and beclouded cell margins.

| Morphology assessment
Spheroid formation was recorded using an inverted microscope. The cell morphological changes subjected to drug sensitivity assays were also subjectively explored via microscopic evaluation.

| RNA extraction and real-time PCR analysis
After 48 h of incubating MCF-7 and MDA-MB-231 cells in monolayer and spheroid forms at 37 C in a humidified atmosphere of 95% air/5% CO 2 , RNAX-Plus (Cinaclon, Iran) was used to extract the total RNA content from cells according to the manufacturer's instructions.
Then, centrifugation was carried for 10 min at 600 g. The supernatants were collected and placed on ice for HPLC analysis of amino acids.
Substance quantification in the cell-free microenvironment was done using HPLC analysis with the YL9100 system (Young Lin, Korea).

| Statistical analysis
Values are reported as the mean ± standard deviation at 95% confidence intervals of a minimum of three separate experiments.

| MCF-7 and MDA-MB-231 spheroids exhibited higher drug resistance against conventional chemotherapy
We evaluated anticancer drug sensitivity using monolayer and spheroid cells cultured in different concentrations of doxorubicin.
We cultured MCF-7 and MDA-MB-231 cell lines in 96-well plates (in monolayer and spheroid forms) and assayed them after drug treatment. Overall spheroid cultures were more resistant to drug treatment than monolayer cells (Table 1). Microscopic imaging that we carried out on spheroids 24 h after treatment at different drug concentrations showed more rigid and compact spheroids ( Figure   2), indicating the possibility of CSC enrichment in the spheroid cultures is associated with higher drug resistance compared to monolayer cells. with CSC properties, such as survival, tumorigenesis, self-renewal, stemness, metastasis, and chemoresistance. 23 Studies have shown that amino acid uptake increases in the CSC population. 16,[24][25][26][27][28][29][30] In our study, out of the 20 different amino acids analyzed, 19 amino acids decreased during the spheroid formation cell proliferation. The serine metabolic pathway is essential for the support of TNBC proliferation and metastasis. 32 Our results indicated that amino acid metabolism plays a role in the phenotypic behavior and unique properties of CSCs.

| Enrichment of CD44
One of the limitations of this study was the inability of our device to measure proline and cysteine amino acids. Studies have shown that these two amino acids are related to the specific characteristics of cancer stem cells, such as self-renewal, sphere formation, and resistance. 33 In addition, mimicking the CSC tumor model by spheroid enrichment approach may only enrich a subset of the CSCs.
The change in the expression of amino acid transporters plays a role in reprogramming cancer cells, which is responsible for the formation of cancer stem cells. 34 Therefore, for future studies, examining the expression change of these transporters in spheroids and monolayer cells can support the findings obtained from the change of amino acid profiles during the formation of spheroids. In addition, it is necessary to characterize circulating amino acids in patients with breast cancer to indicate whether the in vitro effects of breast CSCs on extracellular amino acids still exist in vivo.

| CONCLUSION
We showed in this study that amino acid levels change during