Suspension culture strategies to enrich colon cancer stem cells
- Authors:
- Published online on: February 6, 2023 https://doi.org/10.3892/ol.2023.13702
- Article Number: 116
-
Copyright: © Zhou et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Colon cancer is a highly aggressive malignancy of the digestive system that carries a poor prognosis; according to the results of the National Center for Health Statistics, the number of new cases of colon cancer is 104,610, and the number of deaths is 53,200 in the United States (1). It constitutes the third leading cause of cancer diagnosis and mortality in both males and females, despite the reduced mortality rates in females in recent decades, and reduced overall incidence and mortality rates due to colon cancer screening (2). Traditional surgery, radiotherapy and chemotherapy primarily target tumor cells in the proliferative phase, but have limited killing effects on cells in the non-proliferative phase, which leads to metastasis, recurrence, and drug resistance (3). Cancer stem cells (CSCs) may be responsible for these poor clinical outcomes (4,5).
CSCs are a self-renewing subpopulation of neoplastic cells that promote tumorigenesis, proliferation and growth (6,7). Originally discovered in leukemia, CSCs have been identified and isolated from solid tumors, such as those of the prostate, brain, colorectum, pancreas and breast (8). CSC isolation and identification methods include fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS) and serum-free medium (SFM) cultures (9). FACS separates CSCs via flow cytometry using cell surface markers; however, it is inefficient, expensive, and can injure cells. MACS relies on the labeling of cells with monoclonal antibodies tagged with magnetic beads, which are then sorted using a strong magnetic field; however, this technique produces small-scale cell yields of low purity that are inadequate to meet experimental requirements. CSCs survive and proliferate in serum-free suspension culture environment, but cancer cells cannot (10,11). Therefore, the SFM suspension culture method can effectively expand and enrich CSCs. Advantages include high yields, improved cell viability, ease of operation and low cost (9,12).
In the study of CSCs derived from breast cancer (13), prostate cancer (14), and endometrial cancer (15), researchers have applied the method of SFM suspension culture to enrich CSCs. The obtained CSCs were further sorted and purified for subsequent research, or directly used in the follow-up experiments, allowing studies on CSCs to be carried out successfully.
In the present study of colon CSCs, SFM suspension culture protocols were used to effectively obtain CSCs. In order to maintain the integrity of signaling pathways in CSCs, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were added to the culture medium. However, the concentration to be used for the supplementation of these two growth factors remains a controversial issue; some researchers have used 10 ng/ml EGF + 10 ng/ml bFGF in SFM suspension culture (16,17), whereas others have used 20 ng/ml EGF + 10 ng/ml bFGF (18,19), and others still have used only a single growth factor (20). The selection of the concentrations of the two growth factors is not very rigorous. Another important aspect is that the culture time required for enriching CSCs by SFM suspension culture is controversial. Certain groups have opted for a culture time of 7–10 days (21–24), whereas others have reported using cultures of up to 14 days (25,26), or even >28 days (27). Insufficient time for suspension culture may lead to the incomplete formation of cell spheroids. Conversely, a prolonged culture time will weaken cell stemness and induce abnormal differentiation of spheroid cells.
Although the abovementioned culture strategies can enrich CSCs, the efficiency of their enrichment remains to be studied, particularly as variable serum-free suspension culture strategies produce dissimilar enrichment efficiencies. Insulin can participate in the metabolism and stemness of CSCs through EGF/EGF receptor pathways, making CSCs more malignant (28). Previous studies reported adding insulin to SFM to promote the proliferation of CSCs and maintain stemness (27,29). The currently preferred effective SFM suspension culture strategy is to add insulin, cell culture additive B27 and other basic nutrients to DMEM/F12, followed by the addition of EGF and bFGF (30). However, to our knowledge, there have been no systematic studies into the effects of culture medium composition, EGF and bFGF concentrations, or culture time on CSC enrichment. Consequently, the aim of the present study was to determine the optimal EGF and bFGF composition strategy and culture time for the serum-free suspension method. An optimized, efficient and reliable enrichment strategy that generates high yields of CSCs may facilitate preclinical drug development and other areas of basic and clinical research.
Materials and methods
Materials
The human colon cancer DLD-1 cell line was obtained from the American Type Culture Collection. DMEM/F12 and RPMI 1640 media were purchased from HyClone (Cytiva). EGF and bFGF were purchased from PeproTech, Inc. Fetal bovine serum (FBS) and bovine serum albumin (BSA) were purchased from Amresco, LLC. Insulin was purchased from MilliporeSigma. SFM supplement factor B27 was purchased from Invitrogen (Thermo Fisher Scientific, Inc.). CD133-phycoerythrin (PE; cat. no. 130-098-826), CD44-FITC (130-095-195) were purchased from Miltenyi Biotec GmbH. Corresponding isotype control antibodies Mouse IgG1κ Isotype Control PE (12-4714-41) and FITC (cat. no. 11-4714-41) were purchased from eBioscience. PrimeScript™ RT Master Mix kits were purchased from Takara Biotechnology Co., Ltd. Phosphate-buffered saline (PBS), crystal violet dye and trypsin were purchased from Wuhan Boster Biological Technology, Ltd. PCR primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). The BD FACSCanto II Flow Cytometry Instrument was purchased from ACEA Bioscience, Inc. (Agilent Technologies, Inc.), and the PCR thermocycler was purchased from Roche Diagnostics. Sevoflurane was purchased from MilliporeSigma. BALB/C-Nu/Nu mice were purchased from Beijing HFK Bioscience Co., Ltd.
Experimental group design
SFM cultures were prepared by combining DMEM/F12 with 10% BSA, 5 mg/ml insulin, B27 cell culture additive (1:50), 100 U/ml streptomycin, 0.1 mg/ml penicillin and variable concentrations of growth factors. A total of nine experimental groups were prepared using different combinations of EGF and bFGF: Group 1 (G1), 5 ng/ml EGF; G2, 5 ng/ml bFGF; G3, 5 ng/ml EGF + 5 ng/ml bFGF; G4, 10 ng/ml EGF; G5, 10 ng/ml bFGF; G6, 10 ng/ml EGF + 10 ng/ml bFGF; G7, 20 ng/ml EGF; G8, 20 ng/ml bFGF; and G9, 20 ng/ml EGF + 20 ng/ml bFGF.
DLD-1 monolayer cell culture
After thawing, DLD-1 cells adhered in a monolayer in RPMI 1640 medium containing 10% FBS. The medium was changed every 2 days. When cells reached the logarithmic growth phase, they were digested with EDTA-containing trypsin for 4 min at 37°C, followed by the addition of 4 ml RPMI-1640 and transfer to a centrifuge tube. After centrifugation at 111.8 × g for 5 min with 4°C, the supernatant was discarded, and cells were resuspended in new medium. Finally, cells were cultivated in a new culture dish according to the number of cells. All cell cultures were maintained in a humidified incubator at 37°C and 5% CO2.
Suspension culture of DLD-1 spherical cells
Adherent DLD-1 cells were cultured to the logarithmic growth phase, and then cultured in DMEM/F12 for 24 h at 37°C. Cultures underwent trypsin digestion for 4 min to create single-cell suspensions in DMEM/F12, followed by cell counting. A volume of 20 ml of the G1-G9 culture media preparations were added to super slip plastic culture flasks, followed by the addition of 3×104 cells to each experimental group flask. Cultures were then incubated with the addition of 2 ml of the corresponding group medium daily at 37°C and 5% CO2. Follow-up tests were performed at days 10, 20 and 30.
Flow cytometric analysis
Suspended cell spheroids were cultured to days 10, 20 and 30, and then were subjected trypsin digestion and quantification. Single-cell suspensions were obtained in PBS. Aliquots of 5×105 cells were rinsed twice with PBS, followed by the addition of staining buffer blocking solution (pH 7.2, containing 2% FBS and 0.4% BSA) and storage at 4°C for 1 h. The suspension was centrifuged (251.55 × g for 5 min with 4°C) and the supernatant was discarded. Subsequently, the cells were treated with the addition of 25 µl staining buffer and 1.25 µl of either anti-CD133 antibodies, anti-CD44 antibodies, or a mixture of both anti-CD133 and anti-CD44 antibodies (all 1:50), and protected from light at 4°C for 30 min. Finally, the supernatant was discarded after centrifugation (251.55 × g for 5 min with 4°C). CSCs were then suspended in 500 µl PBS and detected via flow cytometry. The data were analyzed using FlowJo software version 10.0 (BD Bioscience, ACEA Bioscience, Inc.).
RT-quantitative (q)PCR analysis
RNA was extracted from spheroids with chloroform and precipitated with absolute ethanol at days 10, 20 and 30. mRNA was then reverse-transcribed into cDNA (37°C for 15 min, 85°C for 5 min, 4°C for 5 min). GAPDH was used as an internal reference. PrimeScript™ RT Master Mix kit and TB Green® Premix Ex Taq™ (Tli RNaseH Plus; cat. no. RR420A; both from Takara Biotechnology Co., Ltd.) were used according to the manufacturer's instructions to detect the mRNA expression level in each sample using the 2−ΔΔCq method (31). PCR was executed according to the follows: 1 cycle of pre-incubation at 95°C for 10 min, followed by 40 cycles of amplification for 10 sec at 95°C, 10 sec at 60°C, 10 sec at 72°C, then 1 cycle of melting at 95°C for 10 sec, at 65°C for 60 sec, at 97°C for 1 sec, and cooling at 50°C for 30 sec. The primer sequences used are presented in Table I.
 | Table I.Primer sequences of spheroid cell genes analyzed via reverse transcription-quantitative PCR. |
Sphere-forming assay
A total of 100 spheroid cells were collected from each group and cultured in 24-well plates with 1 ml of the corresponding group medium for 14 days at 37°C. The number of spheroids in each group was then counted in three fields of view under a light microscope at ×100 magnification for statistical analysis. Count three times for each sample.
Colony formation assay
Spheroid cells were trypsinized, suspended and counted. A total of 500 spheroid cells were collected from each group and seeded in 6-well plates with 5 ml RPMI-1640 medium containing 10% FBS for 7 days at 37°C. Cells were fixed with 75% ethanol solution at 37°C for 15 min and stained crystal violet alcohol solution (Shangbao Biological Company) at 37°C for 30 min. Colonies (>3 cells) counted manually under a light microscope at 40× magnification for statistical analysis. The experiments was performed three times.
Subcutaneous tumorigenesis in nude mice
All animal experiments were approved by the Ethical Committee of Huazhong University of Science and Technology (approval no. S255). A total of 81 female nude mice (age, 4–6 weeks; weight, 15–21 g) were randomly divided into 27 groups (n=3 mice/group), and housed in a specific pathogen-free environment (18–26°C; humidity, 40–50%, 12/12-h light/dark cycle and free access to food and water) for 1 week to facilitate adaption to the environment. Spheroid cells were trypsinized, collected and counted. Suspensions of 1×105 cells in 200 µl physiological saline were prepared for later use. According to the grouping of nude mice, the corresponding cells were injected subcutaneously using a 1-ml syringe, and the injection tracks were clamped for 20 sec to prevent leakage. Nude mice were observed daily to evaluate their general condition and changes in body weight. After 30 days, sevoflurane (5%) was used to anesthetize the nude mice via breathing inhalation, then nude mice were sacrificed via cervical dislocation; after confirming the onset of rigor mortis, the tumors were harvested and measured, and tumor volumes were calculated using the following formula: Volume=length × width2 × 0.5.
Statistical analysis
SPSS 25.0 (IBM Corp) was used for statistical analysis, and GraphPad Prism 7 (GraphPad Software, Inc.) was used for graphing of results. The normal distributions of data were presented as the mean ± SD, and one-way analysis of variance and Tukey's post hoc tests were used to compare statistical differences between each group. P<0.05 was considered to indicate a statistically significant difference. All experiments were performed ≥3 times.
Results
Proliferation of adherent cells and suspension culture spheroids
Adherent cells were arranged individually and grew close to the bottom of the culture flasks. Cells in each group began to assume a spheroid morphology on day 3, and tended to be suspended in the culture medium as a single oval unit at day 7, after which the volume of cell spheroids gradually increased. There were no intergroup differences in spheroid volume (Fig. 1). The suspension cultures started to produce stable spheroids after the seventh day of culture.
Flow cytometry
Flow cytometry revealed the percentages of CD133+, CD44+ and CD133+CD44+ double-positive spheroid cells of the nine groups at days 10, 20 and 30 of culture (Table II). The percentages of CD133+ and CD44+ cells were highest in G9 at 30 days, and were significantly elevated compared with the other combinations (F=123.554 and 99.528, respectively, P<0.001). The percentage of CD133+ cells in the G9 group at day 30 was 2.558±0.085%, which was similar to the results for G7 at day 10, 20 and 30, and G8 at day 10 and 30 (P>0.999, P=0.686, P>0.999, P=0.282 and 0.818, respectively); however, the percentage of CD44+ cells was 72.990±2.188%, which was higher compared with in any other group. The percentage of CD133+CD44+ double-positive spheroid cells in G9 at day 30 was second only to G3 at day 10 and G6 at day 20 and G9 at day 30, and G9 at day 30 was significantly higher compared with other combinations (except G3 at day 10 and G6 at day 20) (F=57.897, P<0.001). The difference between G3 at day 10 and G6 at day 20 was not statistically significant (P=0.574). These results suggested that the G9 suspension-cultured spheroids at 30 days of culture exhibited more prominent CSC characteristics than those grown under the other experimental conditions.
RT-qPCR
RT-qPCR detected the mRNA expression of stemness genes [Krüppel-like factor 4 (KLF4), leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5), CD44, CD133], epithelial-mesenchymal transition (EMT) genes (E-cadherin, Vimentin) and Wnt/β-catenin pathway genes (Wnt-3a; Fig. 2). The mRNA expression levels of KLF4 (Fig. 2A), Lgr5 (Fig. 2B), CD44 (Fig. 2C), CD133 (Fig. 2D), Vimentin (Fig. 2E) and Wnt-3a (Fig. 2F) were significantly altered between the culture groups (F=22.682, 25.401, 3.272, 7.852, 13.331 and 17.445, respectively, P<0.001). with the highest expression values most commonly observed in G9 at day 30. Comparing the spheroids of each group with G9 at day 30, CD44 expression in G2 at day 20, G3 at days 20 and 30, G5 at day 20, G7 at day 30, and G9 at days 10 and 20 was similar (P=0.218, 0.830, 0.069, 0.162, 0.123, 0.060 and 0.133, respectively; Fig. 2C). There were no significant differences in CD133 expression between G9 at day 30 and G3 at day 20, G8 at days 20, G8 at days 30 (P=0.876, 0.304 and 0.664, respectively; Fig. 2D). There were no significant differences in Vimentin expression between G3 at day 30 and G9 at day 30 (P=0.350; Fig. 2E). Wnt-3a mRNA expression was similar between G9 at day 30 and G2, 3, 4 and 6 at day 20 (P=0.339, 0.235, 0.09 and 0.198; Fig. 2F). E-cadherin expression was lowest in the G9 group at day 30 (F=10.851; P<0.001), but was not significantly different compared with 15 other culture conditions (Fig. 2G). Increased expression of Wnt-3a indicated activation of the Wnt/β-catenin signaling pathway, whereas high expression of Vimentin and low expression of E-cadherin indicated that EMT had occurred, and was accompanied with substantially increased expression of stemness genes. In summary, these results indicated that cell spheroids exhibited the characteristics of CSCs. Comprehensive analysis of the data showed that G9 at day 30 exhibited the most upregulated expression of stemness genes and the lowest expression of E-cadherin, indicative of the most characteristic gene expression of CSCs (32,33).
Sphere-forming assay
Self-renewal ability is a landmark feature of CSCs, which is closely associated with cancer occurrence, development, recurrence and treatment resistance (34). The purpose of the sphere-forming assay is to test the self-renewal ability of spheroid cells. The sphere count was significantly different between the different conditions (F=19.147, P<0.001; Fig. 2H), with the highest yield of spheroids in the G9 condition from day 30. Of note, the difference between G6 and G9 from day 30 was not statistically significant (P=0.116). These results suggested that G9 spheroid cells cultured for 30 days exhibited the most efficient self-renewal capacity.
Colony formation assay
To test in vitro tumorigenicity, colony formation assays were performed for each experimental group (Fig. S1). Spheroid cells can redifferentiate into adherent cells. The number of colonies was significantly different between the different conditions (F=60.767, P<0.001), with the largest number of colonies observed for G9 cells cultured for 30 days. However, there was no statistically significant difference between G9 from day 30 compared with G6 from day 30 or G7 from day 20 (P=0.119 and 0.131; Fig. 3). Therefore, the results suggested that G9 spheroid cells cultured for 30 days exhibited the most prominent in vitro tumorigenic capacity.
Subcutaneous tumorigenesis in nude mice
In vivo tumorigenesis was analyzed by using the tumor cell xenograft formation assay (Fig. S2). G9 at day 30 promoted the largest tumor volume, which was significantly increased compared with other combinations (F=12.539, P<0.001; Fig. 4). However, there were no statistically significant differences between G9 from day 30 compared with G6 from days 20 and 30, G7 from day 30 or G9 from day 20 (P=0.892, 0.940. 0.053 and 0.997, respectively). It is therefore proposed that G9 spheroid cells cultured for 30 days exhibited the most potent in vivo tumorigenicity.
Discussion
Increasing evidence suggests that targeting and selective killing of CSCs to prevent tumor metastasis, recurrence and drug resistance may improve the prognosis of colon cancer (3,35). Annett and Robson (36) reported that therapeutic candidates that specifically target CSCs are now entering clinical trials. Gupta et al (37) reported that monoclonal antibodies that target surface markers on colon CSCs effectively treat colorectal cancer in preclinical in vivo models. Jahanafrooz et al (3) proposed that the combined targeting of CSCs and the tumor microenvironment (TME) may potentially lead to successful colon cancer eradication. Therefore, it is proposed that targeted therapy directed against colon CSCs will open novel opportunities for colon cancer treatment. However, a prerequisite for all supporting research is the procurement of sufficient and viable colon CSCs.
Acquisition and identification of colon CSCs is essential to targeted therapy research. Obtaining large quantities of high-purity colon CSCs has become a major challenge, due to their scarcity and difficult detection (38). FACS and MACS are traditional methods of obtaining CSCs, but are limited by low cell yields, reduced cellular viability and cost. By incorporating technological advances, suspension culture has become a complementary method to FACS or MACS, and has been widely recognized as a means to circumvent the aforementioned disadvantages (12). Spheroid cells enriched by SFM suspension culture have prominent characteristics of CSCs, and can either be used directly or purified via FACS or MACS prior to CSC-related research (39). CD133, CD44 (40), high aldehyde dehydrogenase activity (41) and Lgr5 (42,43) are markers of colon CSCs. Combined surface markers can accurately identify and facilitate the isolation of CSCs from spheroid cells (43).
Currently, the most prevalent strategy for enriching CSCs with SFM suspension culture utilizes serum-free suspension medium with various concentrations and combinations of EGF and bFGF, and incubation times ranging from 7 to 15 days (15,44,45). Suspension culture time is crucial for the expression of CSC characteristics. Excessive culture time not only increases costs and the risk of culture contamination, but also increases the risk of in vitro culture variation and attenuation of CSC characteristics (46,47). Insufficient culture time produces enriched spheroid cells with diminished CSC characteristics (48). Culture times of either 7 or 14 days are favored in current suspension culture strategies, but there is no empirical validation that these are optimal time intervals (21,22). The present study observed that spheroid cell volume and stability did not change with increasing culture time after the seventh day of culture. However, PCR and flow cytometry results showed that the expression of various genes in spheroid cells were higher at 30 days compared with at 10 or 20 days. Moreover, the results of the sphere-forming and colony formation assays demonstrated that spheroid cells at 30 days exhibited more evident CSC characteristics. It is therefore proposed that CSC characteristics gradually increase through proliferation and accumulation of spheroid cells in suspension culture with increased culture time.
Colon CSCs simulate a CSC niche, where the TME maintains the CSC population and modulates the occurrence and development of cancer (49–51). Ahmed et al (52) reported that the chemoresistance, metastasis and recurrence of CSCs are regulated by the TME. Lenos et al (49) proposed that CSCs are regulated by cytokines, chemokines and stem cell factors in the TME. Consequently, the TME determines CSC characteristics and capabilities. In vitro serum-free suspension culture simulates the TME, as EGF and bFGF are among the most important factors affecting the TME; during CSC enrichment, EGF regulates proliferation, apoptosis and EMT via the EGF signaling pathway (28,53), whereas bFGF promotes self-renewal and maintains stemness (34,54). Varying concentrations of EGF and bFGF have been used previously to enrich CSCs in suspension culture, but to our knowledge, the optimization of growth factor concentrations and culture times has not been addressed. The present study comprehensively analyzed multiple experimental conditions, and found that G9 spheroids after 30 days exhibited the most proficient self-renewal, in vivo and in vitro tumorigenicity, and characteristic expression of stemness-associated genes compared with the other groups. Although it is possible that higher concentrations of EGF and bFGF may be more effective and require investigation in future studies, it was determined that a 30-day incubation using 20 ng/ml EGF + 20 ng/ml bFGF medium supplement was the most conducive method for CSC enrichment compared with other strategies.
Next, the mechanisms underlying the effects of EGF and bFGF combination on CSC enrichment were explored. Activation of the Wnt/β-catenin pathway promotes EMT, and Wnt signaling contributes to the maintenance of CSC populations (55). In addition, EMT serves important roles in CSC metastasis, tumorigenesis, self-renewal and drug resistance (56–58). The Wnt/β-catenin pathway and EMT should be considered when identifying or isolating CSCs. The present study showed that Wnt/β-catenin pathway activation and EMT occurred in all groups of spheroid cells. It is worth mentioning that the expression levels of Wnt/β-catenin pathway-(Wnt-3a) and EMT-associated (Vimentin) genes were highest and the expression of E-cadherin was lowest in the G9 condition (20 ng/ml EGF + 20 ng/ml bFGF) after 30 days of culture compared with the other groups. It is proposed that the results observed for G9 after 30 days of culture are at least partly explained by the activation of ETM and the Wnt/β-catenin pathway due to the combination of EGF and bFGF.
Nonetheless, the present study has several shortcomings. The suspension culture method was not applied to a variety of cell lines and primary cells, and the study did not evaluate enrichment after 30 days. In addition, the underlying molecular mechanisms have not been explored completely. Such issues require further investigation.
In summary, the present study reported that SFM suspension cultures can effectively enrich tumor stem cells using the DLD-1 cell line, and that a 30-day culture using medium supplemented with a combination of 20 ng/ml EGF + 20 ng/ml bFGF presented the best enrichment efficiency due to activation of EMT and the Wnt/β-catenin pathway.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
This study was supported by the Foundation of the National Natural Science Foundation of China (grant no. 81402444), the Bureau of Science and Technology Nanchong City (grant no. 18SXHZ0460); the Department of Science and Technology of Sichuan province (grant no. 2017JY0170), the 2019 National Natural Science Foundation Pre-research of North Sichuan Medical College (grant no. CBY-19YZ06), the Scientific Research of the Affiliated Hospital of North Sichuan Medical College (grant no. 2019ZD007) and the Research and Development Project of North Sichuan Medical College (grant no. CBY17-A-YB53).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
GZ, XL and XZ conducted the cell and animal experiments, the analysis of experimental data, and the writing and revision of the manuscripts. WY and WL contributed to the cell experiments. YF and QX contributed to the subcutaneous tumor formation experiments in nude mice. JL, SJ and ZL made substantial contributions to the experimental design, and analysis and interpretation of data. ZL was involved in drafting the manuscript and revising it critically for important intellectual content. GZ and ZL confirmed the authenticity of all the raw data. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
The animal experiments were approved by the Ethical Committee of Huazhong University of Science and Technology (approval no S255; Wuhan, China).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
References
|
Siegel RL, Miller KD and Jemal A: Cancer statistics, 2020. CA Cancer J Clin. 70:7–30. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Katona BW and Weiss JM: Chemoprevention of colorectal cancer. Gastroenterology. 158:368–388. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Jahanafrooz Z, Mosafer J, Akbari M, Hashemzaei M, Mokhtarzadeh A and Baradaran B: Colon cancer therapy by focusing on colon cancer stem cells and their tumor microenvironment. J Cell Physiol. 235:4153–4166. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Agnoletto C, Corrà F, Minotti L, Baldassari F, Crudele F, Cook WJJ, Di Leva G, d'Adamo AP, Gasparini P and Volinia S: Heterogeneity in circulating tumor cells: The relevance of the stem-cell subset. Cancers (Basel). 11:4832019. View Article : Google Scholar : PubMed/NCBI | |
|
Dwivedi AR, Thakur A and Kumar V, Skvortsova I and Kumar V: Targeting cancer stem cells pathways for the effective treatment of cancer. Curr Drug Targets. 21:258–278. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Miyoshi N, Mizushima T, Doki Y and Mori M: Cancer stem cells in relation to treatment. JPN J Clin Oncol. 49:232–237. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Najafi M, Mortezaee K and Ahadi R: Cancer stem cell (a)symmetry & plasticity: Tumorigenesis and therapy relevance. Life Sci. 231:1165202019. View Article : Google Scholar : PubMed/NCBI | |
|
Najafi M, Farhood B and Mortezaee K: Cancer stem cells (CSCs) in cancer progression and therapy. J Cell Physiol. 234:8381–8395. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Cammareri P, Lombardo Y, Francipane MG, Bonventre S, Todaro M and Stassi G: Isolation and culture of colon cancer stem cells. Methods Cell Biol. 86:311–324. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Mather JP: In vitro models. Stem Cells. 30:95–99. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Zhu YT, Wang CY, Pang SY, Lei CY, Luo Y and Tan WL: A modified method by differential adhesion and serum-free culture medium for enrichment of cancer stem cells. J Cancer Res Ther. 14 (Suppl):S421–S426. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Rai N, Singh AK, Singh SK, Gaurishankar B, Kamble SC, Mishra P, Kotiya D, Barik S, Atri N and Gautam V: Recent technological advancements in stem cell research for targeted therapeutics. Drug Deliv Transl Res. 10:1147–1169. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Li W, Li Y, Cui Y, Li S, Zhu Y, Shang C, Song G, Liu Z, Xiu Z, Cong J, et al: Anti-tumour effects of a dual cancer-specific oncolytic adenovirus on breast cancer stem cells. J Cell Mol Med. 25:666–676. 2021. View Article : Google Scholar : PubMed/NCBI | |
|
Ying C, Xiao B, Qin Y, Wang BR, Liu XY, Wang RW, Fang L, Yan H, Zhou XM and Wang YG: GOLPH2-regulated oncolytic adenovirus, GD55, exerts strong killing effect on human prostate cancer stem-like cells in vitro and in vivo. Acta Pharmacol Sin. 39:405–414. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Laranjo M, Carvalho MJ, Serambeque B, Alves A, Marto CM, Silva I, Paiva A and Botelho MF: Obtaining cancer stem cell spheres from gynecological and breast cancer tumors. J Vis Exp. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Tian C, Lang T, Qiu J, Han K, Zhou L, Min D, Zhang Z and Qi D: SKP1 promotes YAP-mediated colorectal cancer stemness via suppressing RASSF1. Cancer Cell Int. 20:5792020. View Article : Google Scholar : PubMed/NCBI | |
|
Farace C, Pisano A, Griñan-Lison C, Solinas G, Jiménez G, Serra M, Carrillo E, Scognamillo F, Attene F, Montella A, et al: Deregulation of cancer-stem-cell-associated miRNAs in tissues and sera of colorectal cancer patients. Oncotarget. 11:116–130. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Chen Y, Wang MH, Zhu JY, Xie CF, Li XT, Wu JS, Geng SS, Han HY and Zhong CY: TAp63α targeting of Lgr5 mediates colorectal cancer stem cell properties and sulforaphane inhibition. Oncogenesis. 9:892020. View Article : Google Scholar : PubMed/NCBI | |
|
Chen Y, Wang X, Zhang Q, Zhu JY, Li Y, Xie CF, Li XT, Wu JS, Geng SS, Zhong CY and Han HY: (−)-Epigallocatechin-3-gallate inhibits colorectal cancer stem cells by suppressing Wnt/β-catenin pathway. Nutrients. 9:5722017. View Article : Google Scholar : PubMed/NCBI | |
|
Wen Y, Xiong X, Zaytseva YY, Napier DL, Vallee E, Li AT, Wang C, Weiss HL, Evers BM and Gao T: Downregulation of SREBP inhibits tumor growth and initiation by altering cellular metabolism in colon cancer. Cell Death Dis. 9:2652018. View Article : Google Scholar : PubMed/NCBI | |
|
Cho YH, Ro EJ, Yoon JS, Mizutani T, Kang DW, Park JC, Il Kim T, Clevers H and Choi KY: 5-FU promotes stemness of colorectal cancer via p53-mediated WNT/β-catenin pathway activation. Nat Commun. 11:53212020. View Article : Google Scholar : PubMed/NCBI | |
|
Guo H, Zhang B, Nairn AV, Nagy T, Moremen KW, Buckhaults P and Pierce M: O-Linked N-acetylglucosamine (O-GlcNAc) expression levels epigenetically regulate colon cancer tumorigenesis by affecting the cancer stem cell compartment via modulating expression of transcriptional factor MYBL1. J Biol Chem. 292:4123–4137. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
Park JH, Kim YH, Shim S, Kim A, Jang H, Lee SJ, Park S, Seo S, Jang WI, Lee SB and Kim MJ: Radiation-activated PI3K/AKT pathway promotes the induction of cancer stem-like cells via the upregulation of SOX2 in colorectal cancer. Cells. 10:1352021. View Article : Google Scholar : PubMed/NCBI | |
|
Guo P, Wang J, Gao W, Liu X, Wu S, Wan B, Xu L and Li Y: Salvianolic acid B reverses multidrug resistance in nude mice bearing human colon cancer stem cells. Mol Med Rep. 18:1323–1334. 2018.PubMed/NCBI | |
|
Wang L, Huang X, Zheng X, Wang X, Li S, Zhang L, Yang Z and Xia Z: Enrichment of prostate cancer stem-like cells from human prostate cancer cell lines by culture in serum-free medium and chemoradiotherapy. Int J Biol Sci. 9:472–479. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Sanches JGP, Song B, Zhang Q, Cui X, Yabasin IB, Ntim M, Li X, He J, Zhang Y, Mao J, et al: The role of KDM2B and EZH2 in regulating the stemness in colorectal cancer through the PI3K/AKT pathway. Front Oncol. 11:6372982021. View Article : Google Scholar : PubMed/NCBI | |
|
Mirzaei H, Salehi H, Sahebkar A, Avan A, Jaafari MR, Namdar A, Rezaei A and Mirzaei HR: Deciphering biological characteristics of tumorigenic subpopulations in human colorectal cancer reveals cellular plasticity. J Res Med Sci. 21:642016. View Article : Google Scholar : PubMed/NCBI | |
|
Chen M, Sharma A, Lin Y, Wu Y, He Q, Gu Y, Xu ZP, Monteiro M and Gu W: Insluin and epithelial growth factor (EGF) promote programmed death ligand 1(PD-L1) production and transport in colon cancer stem cells. BMC Cancer. 19:1532019. View Article : Google Scholar : PubMed/NCBI | |
|
Liu QQ, Li CM, Fu LN, Wang HL, Tan J, Wang YQ, Sun DF, Gao QY, Chen YX and Fang JY: Enterotoxigenic bacteroides fragilis induces the stemness in colorectal cancer via upregulating histone demethylase JMJD2B. Gut Microbes. 12:17889002020. View Article : Google Scholar : PubMed/NCBI | |
|
Shiraiwa K, Matsuse M, Nakazawa Y, Ogi T, Suzuki K, Saenko V, Xu S, Umezawa K, Yamashita S, Tsukamoto K and Mitsutake N: JAK/STAT3 and NF-κB signaling pathways regulate cancer stem-cell properties in anaplastic thyroid cancer cells. Thyroid. 29:674–682. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Leng Z, Tao K, Xia Q, Tan J, Yue Z, Chen J, Xi H, Li J and Zheng H: Krüppel-like factor 4 acts as an oncogene in colon cancer stem cell-enriched spheroid cells. PLoS One. 8:e560822013. View Article : Google Scholar : PubMed/NCBI | |
|
Munro MJ, Wickremesekera SK, Peng L, Tan ST and Itinteang T: Cancer stem cells in colorectal cancer: A review. J Clin Pathol. 71:110–116. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Kozovska Z, Gabrisova V and Kucerova L: Colon cancer: cancer stem cells markers, drug resistance and treatment. Biomed Pharmacother. 68:911–916. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Otte J, Dizdar L, Behrens B, Goering W, Knoefel WT, Wruck W, Stoecklein NH and Adjaye J: FGF signalling in the self-renewal of colon cancer organoids. Sci Rep. 9:173652019. View Article : Google Scholar : PubMed/NCBI | |
|
Castagnoli L, De Santis F, Volpari T, Vernieri C, Tagliabue E, Di Nicola M and Pupa SM: Cancer stem cells: Devil or savior-looking behind the scenes of immunotherapy failure. Cells. 9:5552020. View Article : Google Scholar : PubMed/NCBI | |
|
Annett S and Robson T: Targeting cancer stem cells in the clinic: Current status and perspectives. Pharmacol Ther. 187:13–30. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Gupta R, Bhatt LK, Johnston TP and Prabhavalkar KS: Colon cancer stem cells: Potential target for the treatment of colorectal cancer. Cancer Biol Ther. 20:1068–1082. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Garza Treviño EN, González PD, Valencia Salgado CI and Martinez Garza A: Effects of pericytes and colon cancer stem cells in the tumor microenvironment. Cancer Cell Int. 19:1732019. View Article : Google Scholar : PubMed/NCBI | |
|
Fong CY, Peh GS, Gauthaman K and Bongso A: Separation of SSEA-4 and TRA-1-60 labelled undifferentiated human embryonic stem cells from a heterogeneous cell population using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Stem Cell Rev Rep. 5:72–80. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Tsunekuni K, Konno M, Haraguchi N, Koseki J, Asai A, Matsuoka K, Kobunai T, Takechi T, Doki Y, Mori M and Ishii H: CD44/CD133-positive colorectal cancer stem cells are sensitive to trifluridine exposure. Sci Rep. 9:148612019. View Article : Google Scholar : PubMed/NCBI | |
|
Toledo-Guzmán ME, Hernández MI, Gómez-Gallegos ÁA and Ortiz-Sánchez E: ALDH as a stem cell marker in solid tumors. Curr Stem Cell Res Ther. 14:375–388. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Fumagalli A, Oost KC, Kester L, Morgner J, Bornes L, Bruens L, Spaargaren L, Azkanaz M, Schelfhorst T, Beerling E, et al: Plasticity of Lgr5-negative cancer cells drives metastasis in colorectal cancer. Cell Stem Cell. 26:569–578.e7. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Leng Z, Xia Q, Chen J, Li Y, Xu J, Zhao E, Zheng H, Ai W and Dong J: Lgr5+CD44+EpCAM+ strictly defines cancer stem cells in human colorectal cancer. Cell Physiol Biochem. 46:860–872. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Olejniczak A, Szaryńska M and Kmieć Z: In vitro characterization of spheres derived from colorectal cancer cell lines. Int J Oncol. 52:599–612. 2018.PubMed/NCBI | |
|
Olejniczak-Kęder A, Szaryńska M, Wrońska A, Siedlecka-Kroplewska K and Kmieć Z: Effects of 5-FU and anti-EGFR antibody in combination with ASA on the spherical culture system of HCT116 and HT29 colorectal cancer cell lines. Int J Oncol. 55:223–242. 2019.PubMed/NCBI | |
|
Prasetyanti PR and Medema JP: Intra-tumor heterogeneity from a cancer stem cell perspective. Mol Cancer. 16:412017. View Article : Google Scholar : PubMed/NCBI | |
|
Kuşoğlu A and Biray Avcı Ç: Cancer stem cells: A brief review of the current status. Gene. 681:80–85. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
Kim B, Seo Y, Kwon JH, Shin Y, Kim S, Park SJ, Park JJ, Cheon JH, Kim WH and Il Kim T: IL-6 and IL-8, secreted by myofibroblasts in the tumor microenvironment, activate HES1 to expand the cancer stem cell population in early colorectal tumor. Mol Carcinog. 60:188–200. 2021. View Article : Google Scholar : PubMed/NCBI | |
|
Lenos KJ, Miedema DM, Lodestijn SC, Nijman LE, van den Bosch T, Romero Ros X, Lourenço FC, Lecca MC, van der Heijden M, van Neerven SM, et al: Stem cell functionality is microenvironmentally defined during tumour expansion and therapy response in colon cancer. Nat Cell Biol. 20:1193–1202. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Gur-Cohen S, Yang H, Baksh SC, Miao Y, Levorse J, Kataru RP, Liu X, de la Cruz-Racelis J, Mehrara BJ and Fuchs E: Stem cell-driven lymphatic remodeling coordinates tissue regeneration. Science. 366:1218–1225. 2019. View Article : Google Scholar : PubMed/NCBI | |
|
van der Heijden M and Vermeulen L: Stem cells in homeostasis and cancer of the gut. Mol Cancer. 18:662019. View Article : Google Scholar : PubMed/NCBI | |
|
Ahmed N, Escalona R, Leung D, Chan E and Kannourakis G: Tumour microenvironment and metabolic plasticity in cancer and cancer stem cells: Perspectives on metabolic and immune regulatory signatures in chemoresistant ovarian cancer stem cells. Semin Cancer Biol. 53:265–281. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Zhang X, Bandyopadhyay S, Araujo LP, Tong K, Flores J, Laubitz D, Zhao Y, Yap G, Wang J, Zou Q, et al: Elevating EGFR-MAPK program by a nonconventional Cdc42 enhances intestinal epithelial survival and regeneration. JCI Insight. 5:e1359232020. View Article : Google Scholar : PubMed/NCBI | |
|
Mossahebi-Mohammadi M, Quan M, Zhang JS and Li X: FGF signaling pathway: A key regulator of stem cell pluripotency. Front Cell Dev Biol. 8:792020. View Article : Google Scholar : PubMed/NCBI | |
|
Clara JA, Monge C, Yang Y and Takebe N: Targeting signalling pathways and the immune microenvironment of cancer stem cells-a clinical update. Nat Rev Clin Oncol. 17:204–232. 2020. View Article : Google Scholar : PubMed/NCBI | |
|
Kahn M: Wnt signaling in stem cells and cancer stem cells: A tale of two coactivators. Prog Mol Biol Transl Sci. 153:209–244. 2018. View Article : Google Scholar : PubMed/NCBI | |
|
Najafi M, Mortezaee K and Majidpoor J: Cancer stem cell (CSC) resistance drivers. Life Sci. 234:1167812019. View Article : Google Scholar : PubMed/NCBI | |
|
Saltanatpour Z, Johari B, Alizadeh A, Lotfinia M, Majidzadeh-A K, Nikbin B and Kadivar M: Enrichment of cancer stem-like cells by the induction of epithelial-mesenchymal transition using lentiviral vector carrying E-cadherin shRNA in HT29 cell line. J Cell Physiol. 234:22935–22946. 2019. View Article : Google Scholar : PubMed/NCBI |
