Abstract

Introduction: Cancer stem cells (CSCs) represent a distinct group of cells within cancerous tissue that possess the ability to initiate tumorigenesis and exhibit potency, self-renewal, and drug resistance. The study of CSCs often encounters challenges in obtaining these cells of interest or generating a sufficient quantity for downstream analysis. Nevertheless, it is feasible to enrich CSCs in vitro by subjecting them to conditions that stimulate their CSC properties, such as prolonged exposure to drugs or radiation, or by promoting their self-renewal capability through spheroid culture. Spheroids are a specific type of cell culture that organizes cells into a three-dimensional structure, closely mimicking the in vivo environment. These spheroids consist of a heterogeneous cell population, including CSCs or tumor-propagating cells responsible for tumor growth and maintenance. In our study, we cultured spheroids derived from single cells as well as multicellular aggregates to enrich CSCs based on their self-renewal capability and the structural organization provided by the three-dimensional context.

Methods: Comparing the spheroid cultures with the parental adherent monolayer cells, we observed higher expression of CSC markers, pluripotent genes, and adipogenic differentiation in both multicellular spheroids (MCS) and single cell-derived spheroids (SCDS) of the two tested cell lines.

Results: The spheroids exhibited progressive growth in size throughout the culture period. When comparing the two methods, SCDS demonstrated greater expression of surface markers and all three pluripotent genes associated with CSCs. Furthermore, when assessing drug resistance potential and the expression of the ABCG2 drug efflux gene, only 5637 SCDS displayed increased resistance to cisplatin and upregulation of ABCG2.

Conclusion: In conclusion, both the MCS and SCDS methods effectively enriched the population of bladder CSCs in the 5637 and HT-1376 bladder cancer cell lines. However, the SCDS method demonstrated a higher upregulation of CSC markers and pluripotent gene expression compared to MCS. It is worth noting that spheroid culture and CSC enrichment are not mutually exclusive and can coexist with increased chemotherapy resistance and upregulation of ABCG2 drug efflux gene expression. Moreover, the drug efflux capability may vary depending on the specific cell line and clonal lineage. These strategies can serve as valuable models for CSC enrichment, the study of cancer cell behavior, disease modeling, and personalized chemotherapy investigations.

Introduction

According to the Global Cancer Observatory by the World Health Organization, bladder cancer is the fifth most common cancer worldwide and the most common cancer of the urinary system1, 2, 3. It was the 6th most common cancer in men and the 9th leading cause of cancer death in men globally in 2020. Due to the very high rate of relapse (more than 50%) after treatment4, 5, the long survival rate, and the need for costly life-long routine surveillance and therapy, urothelial cell carcinoma (UCC) has the highest per-patient total cost from diagnosis to death when compared to other common cancers, such as breast, colorectal, lung and prostate cancer6. The recurrence of cancers, including bladder cancer, is often related to the presence of a type of cell called the cancer stem cell (CSC), due to the cell’s capability to metastasize, repair its DNA damage and efflux chemotherapy drugs out of the cell2, 3, 7.

CSCs are a small population of cancer cells within a tumor bulk that carries stemness state-transitioning plasticity, “potency” and “unlimited self-renewal” capability8, 9, 10. The concept of CSCs began with a study on acute myeloid leukemia (AML) that was published in 1994, whereby a subpopulation of AML cells obtained from a patient were able to propagate tumorigenesis in severe combined immunodeficient (SCID) mice after transplantation11. This finding drives a domino effect on the research revolving around CSCs, as later in 2003, the discovery of cancer stem cells in solid tumors, namely, breast cancer and brain cancer, was also published12, 13. In addition, CSCs were discovered in various other cancers, such as melanoma14, osteosarcoma15, prostate cancer16, ovarian cancer17, gastric cancer18, lung cancer19, 20, 21, and bladder cancer2, 3, 5, 7.

However, research on CSCs is often restricted by the scarcity of the desired sample, as CSCs make up only a very small population of a tumor bulk22, 23 due to the feedback mechanism of self-renewal and stochastic differentiation to maintain homeostasis of an organ, or in this case, a malignant tumor, to ensure a feasible amount of functional progeny cells8. Because of this, some researchers aim to enrich the CSC population by various means and methods, such as isolation of CSC-related cell surface markers18, 21, 24, side population (SP) cells25, 26, chemotherapy- and radiotherapy-resistant cells22, 27, 28, 29, 30 and spheroid cultivation10, 31, 32. While some researchers opt for single-cell analysis for the study of CSCs in various cancers, including bladder cancer5, 33, 34, this method is not extensively accessible and is costly.

Isolation and enrichment of CSCs through the spheroid culture method is an effective approach to studying CSCs in solid tumors because spheroid culture provides 3D organization and cell-to-cell interactions that mimic the in vivo tumor structure35, 36, 37, 38. The formation of a 3D organization of cells in spheroids can be achieved through various methods depending on the aim of research or specimen origin. Cell-derived tumorspheres can be established through two approaches: multicellular tumor spheroids (MCTSs) and single-cell-derived tumor spheroids (SCDSs). MCTS is generated by culturing cells as multicellular aggregates, whereas SCDS is cultured as isolated single cells. Both methods can be performed with or without a scaffold, which provides structure and support that mimics the extracellular membrane in vivo 36, 37.

The application of 3D spheroid culture as a means for drug sensitivity testing for cancer has also been widely used, as the organization of cells in a 3D cluster allows for a better model in drug sensitivity testing and drug response analysis when compared with 2D monolayer culture. As an organization of cells in a 3D structure provides a niche to support the activation of EMT and the pluripotent pathway, which increases the number of CSCs39, this could also indirectly increase the drug resistance capability of the spheroid, as CSCs are known to be resilient to chemotherapy drugs by the activation of several pathways, such as the ABCG2 drug efflux pathway40.

In this study, spheroids were grown from either multicellular aggregates or single cells to mimic the cell‒cell organization of in vivo tumors and the self-renewal capability of CSCs, to provide methods for CSC enrichment and to test whether the culture of cells in a 3D organization and the enrichment of CSCs will result in a model system that is suitable for in vitro analysis of drug resistance in bladder cancer (Figure 1).

× Figure 1 . Illustration of the overview methodology for the Multicellular Spheroid (MCS) and Single Cell-Derived Spheroid (SCDS) development from 5637 and HT-1376 Bladder Cancer Cell Lines . Image created using biorender.com. Figure 1 . Illustration of the overview methodology for the Multicellular Spheroid (MCS) and Single Cell-Derived Spheroid (SCDS) development from 5637 and HT-1376 Bladder Cancer Cell Lines . Image created using biorender.com. Methods Cell lines and culture media

The bladder cancer cell lines 5637 and HT-1376 were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). The parental adherent monolayer 5637 and HT-1376 cell lines were maintained in RPMI 1640 complete media consisting of Roswell Park Memorial Institute (RPMI) 1640 basal media (Corning, USA) supplemented with 25 mM HEPES (Gibco, Thermo Fisher Scientific, USA), 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, USA) and 1% penicillin‒streptomycin in an incubator with 5% CO2 at 37°C. Spheroids were cultured in RPMI 1640 spheroid media consisting of RPMI 1640 basal medium supplemented with 1x B27 supplement (Gibco, Thermo Fisher Scientific, USA), 0.4% FBS (Gibco, Thermo Fisher Scientific, USA), 20 ng/ml basic fibroblast growth factor (bFGF) (R&D Systems, USA), 20 ng/ml epidermal growth factor (EGF) (R&D Systems, USA), 4 µg/ml insulin (Gibco, Thermo Fisher Scientific, USA) and 1% penicillin‒streptomycin (Corning, USA).

Multicellular Spheroid (MCS) Culture

5637 and HT-1376 cells were cultured in RPMI complete media as monolayers until 80% confluence. Cells were then dissociated by adding 0.25% trypsin-EDTA (Gibco, Thermo Fisher Scientific, USA) for 5 minutes at 37°C to obtain a single-cell suspension. A suspension of 200,000 cells/ml was prepared using RPMI spheroid media in a 50 ml tube and transferred to a trough. Twenty-five microliters of the suspension was pipetted using a multichannel pipette onto a sterile petri dish as droplets containing 5000 cells. The petri dish was then inverted upside down and cultured for 2 days to form MCSs. The MCSs were transferred to 24-well ultralow attachment (ULA) plates containing 500 µL of RPMI 1640 spheroid media and cultured for 8 days with media replenishment every 3 days. The MCS morphology and diameter were observed and measured at 4 different timepoints (day 2, day 5, day 7 and day 10) using an inverted phase contrast microscope (Olympus IX51) and were then harvested after day 10 of culture, which consisted of 2 days in the hanging drop and 8 days in the ULA plate. MCSs were collected in a 15 ml tube and centrifuged at 1500 rpm for 5 minutes. The supernatant was decanted, and the cell pellet containing MCSs was gently mixed with 0.25% trypsin-EDTA and shaken at 300 rpm at 37°C to dissociate the MCSs and obtain a single-cell suspension to be used in downstream experiments.

Single Cell-Derived Spheroid (SCDS) Culture

5637 and HT-1376 cells were cultured in RPMI complete media as a monolayer until 80% confluence. Cells were then dissociated by adding 0.25% trypsin-EDTA (Gibco, Thermo Fisher Scientific, USA) for 5 minutes at 37°C to obtain a single-cell suspension. At 4°C (on ice), 20,000 cells from the single-cell suspension were added to a 1.5 ml Eppendorf tube. Additional media was added if required until the volume reached 7.5 µl. The cells were mixed with 7.5 µl of Matrigel™ (Corning, USA) to establish a 15 µl cell:Matrigel mixture at a 50:50 ratio containing 20,000 cells. The cell:Matrigel mixture was pipetted onto a 24-well plate as a dome-shaped droplet and kept in a 37°C incubator for 30 minutes for the Matrigel™ to solidify. Then, 500 µl of spheroid media was added and cultured for 10 days with media replenishment every 3 days. The SCDS morphology and diameter were observed and measured at 4 different timepoints (day 2, day 5, day 7 and day 10) using an inverted phase contrast microscope (Olympus IX51) and harvested after day 10 of culture. SCDS was collected in a 15 ml tube and centrifuged at 1500 rpm for 5 minutes. The supernatant was decanted, and the cell pellet containing MCSs was gently mixed with 0.25% trypsin-EDTA and shaken at 300 rpm at 37°C to dissociate the MCSs and obtain a single-cell suspension to be used in downstream experiments.

Bladder cancer stem cell-related surface marker analysis by flow cytometry

The stem-like characteristics of the MCSs and SCDSs were determined by flow cytometry analysis of CD24, CD44 and CD133. A total of 2 x 105 cells harvested from MCSs and SCDSs were added to 15 ml tubes labeled with CD24/CD44, CD44/CD133, CD24/CD133, APC, FITC, PE and unstained. Cells were washed one time with 1x sterile PBS and centrifuged at 1500 rpm for 5 minutes, and the supernatant was decanted. Cells were then resuspended in 200 µl FACS staining buffer and stained with 2 µl of anti-CD24-PE antibody (BD Biosciences, USA), anti-CD44-FITC antibody (BD Biosciences, USA) and anti-CD133-APC (Miltenyi Biotec, USA) antibody according to the tube label for 40 minutes in the dark at 4°C. Cells were then washed one time with 1x sterile PBS, resuspended in 200 µl FACS staining buffer and further analyzed using a flow cytometer. The expression of CD24, CD44 and CD133 in MCS and SCDS cells was compared to that in parental cells.

Quantitative Real-Time PCR (qRT‒PCR) analysis of Pluripotent Transcriptional Factor of SOX2, NANOG and POU5F1 and ABCG2 Drug Efflux gene expression

Total RNA was extracted from MCS and SCDS using a Nucleospin™ RNA plus extraction kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s recommendation, and the RNA concentration and purity were measured using Nanodrop and converted to cDNA using a Tetro™ cDNA Synthesis Kit (Meridian Life Science Inc., USA) according to the manufacturer’s recommendation. Real-time qPCR was performed to determine the expression of pluripotency genes (SOX2, NANOG, POU5F1) and a drug efflux gene (ABCG2) in 100 ng of cDNA from MCS and SCDS cells using a SensiFAST™ Probe Hi-ROX Kit and analyzed by an Applied Biosystems 7500 fast instrument (Meridian Life Science Inc., USA). Fluorescent-probed primers were obtained from Applied Biosystems (CA, USA): GAPDH (control) Hs02758991_g1, SOX2 Hs01053049_s1, NANOG Hs04399610_g1, POU5F1 Hs00999632_g1 and ABCG2 Hs01053790_m1. Ten microliters of 2x SensiFAST Probe Hi-ROX Mix, 1 µl of fluorescent-probed primers, 100 ng of cDNA, and H2O were added until the final reaction mix was 20 µl. Gene amplification was performed as follows: 95°C for 2 minutes (1 cycle), 95°C for 10 seconds and 60°C for 50 seconds (40 cycles). The mRNA values of SOX2, NANOG, POU5F1 and ABCG2 were normalized to the GAPDH (control) values in each sample by subtracting the mean Ct (cycle threshold) values of the control sample from the Ct values of the genes of interest (SOX2, NANOG, POU5F1, ABCG2). The expression of SOX2, NANOG, POU5F1 and ABCG2 in MCS and SCDS cells was compared to that in parental cells.

Adipogenic and osteogenic lineage differentiation in vitro analysis

The MCS and SCDS of both cell lines were induced to differentiate into adipogenic and osteogenic lineages using differentiation media (Stempro™, Thermo Fisher Scientific, USA). Briefly, cells harvested from MCS and SCDS were cultured in a 24-well plate in RPMI spheroid media until 80-90% confluence. The RPMI 1640 complete culture medium in three of the wells was replaced with differentiation media (Stempro™, Thermo Fisher Scientific, USA) to induce differentiation. Another three wells were maintained in RPMI 1640 complete culture medium as a control and cultured for 14 days (adipogenesis) and 21 days (osteogenesis), and the medium was replenished every 3 days. At the end of culture, cells were washed with 1x sterile PBS, fixed with 10% neutral buffered formalin and stained with Oil Red O staining for adipogenesis to observe the formation of lipid vesicles or Alizarin Red S staining for osteogenesis to detect calcium deposition. Cells were then washed with distilled water to remove any excess stain, added to 500 µl of 1x sterile PBS and observed under an inverted phase contrast microscope (Olympus IX51).

Adipogenesis and osteogenesis differentiation imaging analysis

After staining with the respective staining method, culture images of adipogenic differentiation and osteogenic differentiation of 5637 and HT-1376 parental, MCS, SCDS and UC-MSC (positive control for mesodermal lineage differentiation) were captured using an inverted phase contrast microscope (Olympus IX51). The percentage (%) of adipogenic and osteogenic differentiation color intensity was analyzed using ImageJ color threshold by using the formula: (color threshold of the fat droplet in Oil Red-O stain or calcium deposition in Alizarin Red stain/color threshold of whole image) x 100%.

Cell viability assay and cisplatin inhibitory concentration 50 (IC50)

Parental monolayer adherent bladder cancer cells of both the 5637 and HT-1376 cell lines were seeded in 96-well plates at a seeding density of 5000 cells/well in RPMI-1640 complete media with a final volume of 100 µl per well. Cells were incubated for 48 hours in a humidified 5% CO2 incubator at 37°C. After 48 hours, the media were replaced with media containing a gradually increasing concentration of cisplatin, and the cells were further incubated for 48 and 72 hours for treatment. After treatment, 10 µl of MTT reagent was added to each well and incubated again in a humidified 5% CO2 incubator at 37°C for 4 hours. After the incubation time, media containing MTT reagent was removed, and 100 µl DMSO was added to each well and incubated in a humidified 5% CO2 incubator at 37°C for 10 minutes to dissolve the formazan crystal formed. Absorbance in triplicate of each well was measured using FlourOmega at 570 nm and 620 nm, using a well without treatment as a blank. The percentage of cell viability was determined for each concentration of cisplatin, and the IC50 concentration was determined by plotting a graph of percentage viability against drug concentration.

Spheroid chemotherapy resistance assay

After 10 days of spheroid culture, the MCSs and SCDSs were retrieved and recultured in Matrigel™ (Corning, USA) at a 50:50 ratio of media:Matrigel for cisplatin treatment. Spheroids were embedded in Matrigel™ (Corning, USA) to ensure immobilization of spheroids. Initially, spheroids were harvested from the MCS and SCDS culture methods and added to the newly prepared media:Matrigel mixture. Fifteen microliters of the media:Matrigel mixture was then pipetted on a 24-well plate to form a dome-shaped droplet, with 2 domes per well, and kept in a 37°C incubator for 30 minutes for the Matrigel™ to solidify. Then, 500 µl of spheroid media was added to the well and cultured for 2 days to ensure that spheroids were able to survive in the new environment. After 2 days of culture, the spheroid media was replenished with new spheroid media containing the IC50 dose of cisplatin. The IC50 dose of cisplatin for 5637 was 1.1 µm and 2.75 µm for HT-1376 (IC50 dose obtained by MTT assay of parental adherent monolayer cells of both cell lines that were treated for 48 hours with cisplatin). Spheroids were treated for 48 hours with an IC50 dose of cisplatin. Spheroid morphology and diameter pretreatment and posttreatment were captured and recorded.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 9 version 9.4 (GraphPad Software, California, USA). The results obtained were tested using one-way ANOVA or t-test analysis to assess the statistical significance of differences between the control group and experimental groups. Differences were considered statistically significant when p ≤ 0.05, whereas p ≤ 0.0001 was considered highly significant.

× Figure 2 . MCS and SCDS of 5637 and HT-1376 . ( A ) Culture image of MCS and SCDS of 5637 and HT-1376 cells at day 2, day 5, day 7 and day 10, taken at x10 magnification. ( B ) Bar chart of mean spheroid diameter (µm) of MCS and SCDS for 5637 and HT-1376 at day 2, day 5, day 7 and day 10. The bars are represented as mean +SD (n = 10). Statistical significance was measured with the two-way ANOVA. *p Figure 2 . MCS and SCDS of 5637 and HT-1376 . ( A ) Culture image of MCS and SCDS of 5637 and HT-1376 cells at day 2, day 5, day 7 and day 10, taken at x10 magnification. ( B ) Bar chart of mean spheroid diameter (µm) of MCS and SCDS for 5637 and HT-1376 at day 2, day 5, day 7 and day 10. The bars are represented as mean +SD (n = 10). Statistical significance was measured with the two-way ANOVA. *p × Figure 3 . Flow cytometry analysis of 5637 and HT-1376 Parental, MCS and SCDS CSC Surface Markers (CD133/CD24, CD24/CD44, CD44/CD133) expression . ( A ) Bar chart and dot plot of 5637 Parental, MCS and SCDS CSC Surface Markers (CD133/CD24, CD24/CD44, CD44/CD133) expression. ( B ) Bar chart and dot plot of HT-1376 Parental, MCS and SCDS CSC Surface Markers (CD133/CD24, CD24/CD44, CD44/CD133) expression. The bars are represented as mean +SD (n = 3). Statistical significance was measured with the two-way ANOVA. *p Figure 3 . Flow cytometry analysis of 5637 and HT-1376 Parental, MCS and SCDS CSC Surface Markers (CD133/CD24, CD24/CD44, CD44/CD133) expression . ( A ) Bar chart and dot plot of 5637 Parental, MCS and SCDS CSC Surface Markers (CD133/CD24, CD24/CD44, CD44/CD133) expression. ( B ) Bar chart and dot plot of HT-1376 Parental, MCS and SCDS CSC Surface Markers (CD133/CD24, CD24/CD44, CD44/CD133) expression. The bars are represented as mean +SD (n = 3). Statistical significance was measured with the two-way ANOVA. *p × Figure 4 . qRT-PCR analysis of 5637 and HT-1376 Parental, MCS and SCDS pluripotent genes ( SOX2, NANOG, POU5F1 ) expression . ( A ) Bar chart of 5637 Parental, MCS and SCDS relative fold gene expression of pluripotent genes ( SOX2, NANOG, POU5F1 ) by qRT-PCR. ( B ) Bar chart of HT-1376 Parental, MCS and SCDS relative fold gene expression of pluripotent genes ( SOX2, NANOG, POU5F1 ) by qRT-PCR. The bars are represented as mean +SD (n = 3). Statistical significance was measured with the two-way ANOVA. *p Figure 4 . qRT-PCR analysis of 5637 and HT-1376 Parental, MCS and SCDS pluripotent genes ( SOX2, NANOG, POU5F1 ) expression . ( A ) Bar chart of 5637 Parental, MCS and SCDS relative fold gene expression of pluripotent genes ( SOX2, NANOG, POU5F1 ) by qRT-PCR. ( B ) Bar chart of HT-1376 Parental, MCS and SCDS relative fold gene expression of pluripotent genes ( SOX2, NANOG, POU5F1 ) by qRT-PCR. The bars are represented as mean +SD (n = 3). Statistical significance was measured with the two-way ANOVA. *p × Figure 5 . Adipogenesis differentiation of parental cells of 5637 and HT-1376, MCS and SCDS of 5637 and HT-1376, and UC-MSC . ( A ) Oil Red-O staining of parental cells of 5637 and HT-1376, MCS and SCDS of 5637 and HT-1376, and UC-MSC taken at x20 magnification. ( B ) Table showing mean adipogenesis differentiation colour intensity (%) + standard error of 5637 and HT-1376 parental, MCS and SCDS. ( C ) Bar chart showing comparison in mean adipogenesis differentiation colour intensity (%) + standard error of 5637 and HT-1376 parental, MCS and SCDS. Figure 5 . Adipogenesis differentiation of parental cells of 5637 and HT-1376, MCS and SCDS of 5637 and HT-1376, and UC-MSC . ( A ) Oil Red-O staining of parental cells of 5637 and HT-1376, MCS and SCDS of 5637 and HT-1376, and UC-MSC taken at x20 magnification. ( B ) Table showing mean adipogenesis differentiation colour intensity (%) + standard error of 5637 and HT-1376 parental, MCS and SCDS. ( C ) Bar chart showing comparison in mean adipogenesis differentiation colour intensity (%) + standard error of 5637 and HT-1376 parental, MCS and SCDS. × Figure 6 . Osteogenesis differentiation of parental cells of 5637 and HT-1376, MCS and SCDS of 5637 and HT-1376, and UC-MSC . ( A ) Alizarin Red staining of parental cells of 5637 and HT-1376, MCS and SCDS of 5637 and HT-1376, and UC-MSC taken at x10 magnification. ( B ) Table showing mean osteogenesis differentiation colour intensity (%) + SD of 5637 and HT-1376 parental, MCS and SCDS. ( C ) Bar chart showing comparison in mean osteogenesis differentiation colour intensity (%) + SD of 5637 and HT-1376 parental, MCS and SCDS. Figure 6 . Osteogenesis differentiation of parental cells of 5637 and HT-1376, MCS and SCDS of 5637 and HT-1376, and UC-MSC . ( A ) Alizarin Red staining of parental cells of 5637 and HT-1376, MCS and SCDS of 5637 and HT-1376, and UC-MSC taken at x10 magnification. ( B ) Table showing mean osteogenesis differentiation colour intensity (%) + SD of 5637 and HT-1376 parental, MCS and SCDS. ( C ) Bar chart showing comparison in mean osteogenesis differentiation colour intensity (%) + SD of 5637 and HT-1376 parental, MCS and SCDS. × Figure 7 . qRT-PCR analysis of 5637 and HT-1376 Parental, MCS and SCDS ABCG2 drug resistant gene expression . ( A ) Bar chart of 5637 Parental, MCS and SCDS relative fold gene expression of ABCG2 drug resistant gene by qRT-PCR. ( B ) Bar chart of HT-1376 Parental, MCS and SCDS relative fold gene expression of ABCG2 drug resistant gene by qRT-PCR. The bars are represented as mean +SD (n = 3). Statistical significance was measured with the two-way ANOVA. *p Figure 7 . qRT-PCR analysis of 5637 and HT-1376 Parental, MCS and SCDS ABCG2 drug resistant gene expression . ( A ) Bar chart of 5637 Parental, MCS and SCDS relative fold gene expression of ABCG2 drug resistant gene by qRT-PCR. ( B ) Bar chart of HT-1376 Parental, MCS and SCDS relative fold gene expression of ABCG2 drug resistant gene by qRT-PCR. The bars are represented as mean +SD (n = 3). Statistical significance was measured with the two-way ANOVA. *p × Figure 8 . Cell viability assay and cisplatin inhibitory concentration 50 (IC 50 ) of 5637 and HT-1376 parental cells . ( A ) Line chart of 5637 parental cell line after 48 and 72 hours of cisplatin treatment. ( B ) Line chart of HT-1376 parental cell line after 48 and 72 hours of cisplatin treatment. Figure 8 . Cell viability assay and cisplatin inhibitory concentration 50 (IC 50 ) of 5637 and HT-1376 parental cells . ( A ) Line chart of 5637 parental cell line after 48 and 72 hours of cisplatin treatment. ( B ) Line chart of HT-1376 parental cell line after 48 and 72 hours of cisplatin treatment. × Figure 9 . IC 50 Cisplatin treatment on MCS and SCDS of 5637 and HT-1376 . ( A ) Culture image of MCS and SCDS of 5637 and HT-1376 pre-treatment and post-treatment with IC 50 dose of cisplatin, taken at x10 magnification. ( B ) Bar chart of mean spheroid diameter (µm) of MCS and SCDS for 5637 and HT-1376 pre-treatment and post-treatment with IC 50 dose of cisplatin. The bars are represented as mean +SD (n = 3). Statistical significance was measured with the t-test. *p Figure 9 . IC 50 Cisplatin treatment on MCS and SCDS of 5637 and HT-1376 . ( A ) Culture image of MCS and SCDS of 5637 and HT-1376 pre-treatment and post-treatment with IC 50 dose of cisplatin, taken at x10 magnification. ( B ) Bar chart of mean spheroid diameter (µm) of MCS and SCDS for 5637 and HT-1376 pre-treatment and post-treatment with IC 50 dose of cisplatin. The bars are represented as mean +SD (n = 3). Statistical significance was measured with the t-test. *p Results Multicellular Spheroid (MCS) and Single Cell-Derived Spheroid (SCDS) culture of 5637 and HT-1376 cells

5637 and HT-1376 cells were tested for their capability to generate and maintain a 3D spheroid structure using Matrigel™ as a scaffold/matrix for SCDS and Ultra-Low Attachment Plate for culture without scaffold/matrix for the MCS. Both cell lines were able to form spheroids of different morphologies according to the culture method used.

According to Kenny et al. (2007), spheroids can be classified into four different morphological groups: round, mass, grape-like and stellate. For the 5637 cell line, in the MCS culture, the cells formed a grape-like cluster with loose aggregation and poor cell‒cell adhesion, resulting in a spheroid structure that was easily dissociated but tended to aggregate with neighboring spheroids. In the SCDS culture, 5637 cells formed a round sphere with a smooth border and very robust cell‒cell adhesion (Figure 2 A). However, for the HT-1376 cell line, in both MCS and SCDS culture, the cells formed a spheroid mass with an irregular border with strong cell‒cell adhesion (Figure 2 A).

In terms of spheroid size, the spheroid size for MCSs (both 5637 and HT-1376) was larger than that for SCDSs (both 5637 and HT-1376) at all time points (day 2, day 5, day 7 and day 10). The initial mean spheroid diameters for the MCS were 122.02 µm (5637) and 120.94 µm (HT-1376), while the initial mean spheroid diameters for the SCDS were 26.68 µm (5637) and 25.52 µm (HT-1376) (Figure 2 B). On day 10 of culture, the mean spheroid diameter for MCS was 454.42 µm (5637) and 239.54 µm (HT-1376), while the mean spheroid diameter for SCDS was 79.77 µm (5637) and 74.13 (HT-1376) (Figure 2 B). Both MCS and SCDS for 5637 and HT-1376 cells showed significant (p

CSC marker expression in the MCS and SCDS of 5637 and HT-1376 cells

The expression of CSC markers in the MCS and SCDS in 5637 and HT-1376 cells was determined using flow cytometry. Higher expression of all CSC marker combinations, CD24 and CD44, CD44 and CD133, and CD133 and CD24, was found in the MCS and SCDS of 5637 and HT-1376 cells compared to parental cells.

The CD24+CD44+ coexpression in the 5637 MCS was 69.67%, the CD44+CD133+ coexpression was 0.32%, and the CD133+CD24+ coexpression was 0.73%. The SCDS for 5637 cells showed 80.2% CD24+CD44+ coexpression, 0.28% CD44+CD133+ coexpression and 0.19% CD133+CD24+ coexpression (Figure 3 A).

The HT-1376 MCS had CD24+CD44+ coexpression of 1.38%, CD44+CD133+ coexpression of 2.71% and CD133+CD24+ coexpression of 1.23%. The SCDS for HT-1376 cells showed CD24+CD44+ coexpression of 1.59%, CD44+CD133+ coexpression of 5.52% and CD133+CD24+ coexpression of 1.46% (Figure 3 B).

Pluripotency gene expression in the MCS and SCDS of 5637 and HT-1376 cells

Following the increase in the coexpression of CSC-related markers when investigated by flow cytometry analysis, further investigation was performed on the RNA collected from MCSs and SCDSs of both cell lines to determine the expression of stem cell transcription factors such as SOX2, NANOG and POU5F1. Analysis was performed using the qRT‒PCR method, and the relative expression of the genes in MCS and SCDS of both cell lines was compared with the expression of the genes in the parental monolayer adherent cells of each cell line. The expression of SOX2, NANOG and POU5F1 in both the MCS and SCDS of both cell lines was upregulated at the transcriptional level (Figure 4).

Both MCS and SCDS of 5637 cells showed upregulation of the pluripotent genes SOX2, NANOG and POU5F1 compared to parental cells. For SOX2, the relative fold gene expression was 6.73 in 5637 MCS cells and 17.02 in 5637 SCDS cells (p > 0.05 and p NANOG relative fold gene expression for 5637 MCS and SCDS was 3.19 and 7.01 (p POU5F1, 5637 MCS and SCDS relative fold gene expression was 3.56 and 6.35 (p Figure 4 A).

In HT-1376 cells, both the MCS and SCDS of HT-1376 cells showed upregulation of all 3 pluripotent genes compared to parental cells. The SOX2 relative fold gene expression was 74.03 in HT-1376 MCS cells and 92.84 in HT-1376 SCDS cells (p NANOG relative fold gene expression for HT-1376 MCS and SCDS was 20.68 and 28.84 (p POU5F1, HT-1376 MCS and SCDS relative fold gene expression were 70.35 and 71.18 (p Figure 4 B).

Osteogenic and adipogenic differentiation capabilities of the MCS and SCDS of 5637 and HT-1376 cells

Osteogenic and adipogenic differentiation culture was performed on the MCS and SCDS of 5637 and HT-1376 cells to investigate the lineage differentiation capability of cancer stem cells. Following culture, the cells were stained to visualize lipid droplets and calcium deposition (Figure 5 A and Figure 6 A). ImageJ was used to evaluate the percentage area of differentiated cells.

In adipogenic differentiation, 5637 MCS and SCDS showed a lower percentage of differentiation (5.97% ± 1.93 and 5.36% ± 0.74, respectively) when compared to the parental monolayer adherent cells (6.01% ± 0.59) (p > 0.05). However, HT-1376 MCSs showed a lower percentage of differentiation, 7.52% ± 2.32 (p > 0.05), while SCDS were higher, 9.17% ± 4.57 (p > 0.05), compared to parental cells (8.17% ± 3.15 (Figure 5 B and C)).

In osteogenic differentiation, the MCSs and SCDSs of both 5637 and HT-1376 cells showed a higher percentage of differentiation (26.09% + 5.70, 21.74% + 4.64, 50.11% + 12.80, and 41.04% + 13.43, respectively) than the parental cells (19.80% + 2.62 and 36.13% + 8.84, respectively) (p > 0.05) (Figure 6 B and C).

ABCG2 drug resistance gene expression in the MCS and SCDS of 5637 and HT-1376 cells

To analyze the drug resistance capability of the MCS and SCDS of both 5637 and HT-137