The effect of EGF culture time on colorectal cancer stem cells enrichment_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

WU Nisha ^1,3 , ZHOU Guojun ^1,2,3 , XU Liming ^1,3 , LUO Bo ^1,3 , DOU Yajun ^1,3 , HUANG Lizheng ^1,3 , SHI Lin ^1,3 , SUN Chengjie ^1,3 ,  LENG Zhengwei ^1,2,3

1. Department of Hepatobiliary Surgery Ⅱ, Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan 637000, P. R. China;
2. Cancer Stem Cells Research Center, Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan 637000, P. R. China;
3. Translational Medicine Research Center, North Sichuan Medical College, Nanchong, Sichuan 637000, P. R. China;

Corresponding author：

LENG Zhengwei, Email: lengzhengwei@163.com

Keywords：

colon cancer; cancer stem cell; serum-free suspension culture; time; epidermal growth factor

DOI：

10.7507/1007-9424.201911124

Video：

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Abstract Full text Figures/Tables Video References Cited by

Objective To investigate the most appropriate culture time with the action of EGF in colon cancer stem cells enrichment by suspension culture.Methods DLD-1 cells were cultured in serum-free medium containing 20 ng/mL EGF to generate spheroid cells. The time gradient was set to 10 d, 20 d, 30 d and 40 d, the cell proportion of CD133⁺, CD44⁺ and CD133⁺CD44⁺ were confirmed by flow cytometery. The ability of self-renewal was detected by the sphere forming assay and the limited dilution assay, and the in vitro tumorigenicity of the cells was detected by the colony formation assay.Results In the 30 d group, the proportion of CD133⁺ and CD133⁺ CD44⁺ cells were significantly higher than those in the other groups (allP<0.05), the CD44⁺ cell was higher than that in the 20 d group (P<0.05), but there was no significant difference with the other two groups (P>0.05). The results of the limited dilution assay and the colony formation assay, the number of spheres in the 30 d or 40 d group was the highest among the 4 groups, and there was no statistical difference between the 30 d group and 40 d group (P>0.05), with statistically significant difference between the 30 d, 10 d and 20 d groups (all P<0.05). The results of the sphere forming assay and the self-renewal ability of 30 d group was significantly higher compared with other groups (all P< 0.05).Conclusion The cancer stem cells could be enriched more efficiently by suspension culture using 20 ng/mL EGF for 30 days.

Citation： WU Nisha, ZHOU Guojun, XU Liming, LUO Bo, DOU Yajun, HUANG Lizheng, SHI Lin, SUN Chengjie, LENG Zhengwei. The effect of EGF culture time on colorectal cancer stem cells enrichment. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2020, 27(8): 959-963. doi: 10.7507/1007-9424.201911124 Copy

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14.	Gatti M, Pattarozzi A, Bajetto A, et al. Inhibition of CXCL12/CXCR4 autocrine/paracrine loop reduces viability of human glioblastoma stem-like cells affecting self-renewal activity. Toxicology, 2013, 314(2-3): 209-220.
15.	Roudi R, Madjd Z, Ebrahimi M, et al. CD44 and CD24 cannot act as cancer stem cell markers in human lung adenocarcinoma cell line A549. Cell Mol Biol Lett, 2014, 19(1): 23-36.
16.	Markert S, Musmann C, Hülsmann P, et al. Automated and enhanced clone screening using a fully automated microtiter plate-based system for suspension cell culture. Biotechnol Prog, 2019, 35(2): e2760.
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19.	Shimokawa M, Ohta Y, Nishikori S, et al. Visualization and targeting of LGR5+ human colon cancer stem cells. Nature, 2017, 545(7653): 187-192.
20.	Yang L, Shi P, Zhao G, et al. Targeting cancer stem cell pathways for cancer therapy. Signal Transduct Target Ther, 2020, 5: 8.
21.	Ponti D, Costa A, Zaffaroni N, et al. Isolation and in vitro propagation of tumorigenic breast cancer cells withstem/progenitor cell properties. Cancer Res, 2005, 65(13): 5506-5511.
22.	Hemmati HD, Nakano I, Lazareff JA, et al. Cancerous stem cells can arise from pediatric brain tumors. Proc Natl Acad Sci U S A, 2003, 100(25): 15178-15183.
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24.	Ricci-Vitiani L, Lombardi DG, Pilozzi E, et al. Identification and expansion of human colon-cancer-initiating cells. Nature, 2007, 445(7123): 111-115.
25.	Rybak AP, He L, Kapoor A, et al. Characterization of sphere-propagating cells with stem-like properties from DU145 prostate cancer cells. Biochim Biophys Acta, 2011, 1813(5): 683-694.
26.	Louis SA, Rietze RL, Deleyrolle L, et al. Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay. Stem Cells, 2008, 26(4): 988-996.
27.	Manupati K, Dhoke NR, Debnath T, et al. Inhibiting epidermal growth factor receptor signalling potentiatesmesenchymal-epithelial transition of breast cancer stem cells and theirres-ponsiveness to anticancer drugs. FEBS J, 2017, 284(12): 1830-1854.
28.	De Angelis ML, Bruselles A, Francescangeli F, et al. Colorectal cancer spheroid biobanks: multi-level approaches to drug sensitivity studies. Cell Biol Toxicol, 2018, 34(6): 459-469.
29.	Xu Q, Zhang Q, Ishida Y, et al. EGF induces epithelial-mesenchymal transition and cancer stem-like cell properties in human oral cancer cells via promoting Warburg effect. Oncotarget, 2017, 8(6): 9557-9571.
30.	Ding C, Zou Q, Wu Y, et al. EGF released from human placental mesenchymal stem cells improves prematureovarian insufficiency via NRF2/HO-1 activation. Aging (Albany NY), 2020, 12(3): 2992-3009.
31.	Lee K, Sheung P, Chou C, et al. Elevation of CD109 promotes metastasis and drug resistance in lung cancer viaactivation of EGFR-AKT-mTOR signaling. Cancer Sci, 2020 Mar 4. [Epub ahead of print].

1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin, 2020, 70(1): 7-30.
2. 游金辉, 黄霓. 结直肠癌 PET/CT 显像的应用与研究进展. 川北医学院学报, 2019, 34(1): 155-159.
3. Zhou Y, Xia L, Wang H, et al. Cancer stem cells in progression of colorectal cancer. Oncotarget, 2018, 9(70): 33403-33415.
4. Hirata A, Hatano Y, Niwa M, et al. Heterogeneity of colon cancer stem cells. Adv Exp Med Biol, 2019, 1139: 115-126.
5. Shen Y, Cao D. Hepatocellular carcinoma stem cells: origins and roles in hepatocarcinogenesis and disease progression. Front Biosci (Elite Ed), 2012, 4: 1157-1169.
6. Pradhan T, Padmanabhan K, Prasad M, et al. Augmented CD133 expression in distal margin correlates with poor prognosis incolorectal cancer. J Cell Mol Med, 2019, 23(6): 3984-3994.
7. Reya T, Morrison SJ, Clarke MF, et al. Stem cells, cancer, and cancer stem cells. Nature, 2001, 414(6859): 105-111.
8. Efremov YR, Proskurina AS, Potter EA, et al. Cancer stem cells: emergent nature of tumor emergency. Front Genet, 2018, 9: 544.
9. Wang D, Kong X, Li Y, et al. Curcumin inhibits bladder cancer stem cells by suppressing Sonic Hedgehogpathway. Biochem Biophys Res Commun, 2017, 493(1): 521-527.
10. Li Y, Wu S, Lu S, et al. (-)-Epigallocatechin-3-gallate inhibits nasopharyngeal cancer stem cellself-renewal and migration and reverses the epithelial-mesenchymal transition via NF-kappaB p65 inactivation. Tumour Biol, 2015, 36(4): 2747-2761.
11. Xu S, Yue Y, Zhang S, et al. STON2 negatively modulates stem-like properties in ovarian cancer cells viaDNMT1/MUC1 pathway. J Exp Clin Cancer Res, 2018, 37(1): 305.
12. Xiong B, Ma L, Hu X, et al. Characterization of side population cells isolated from the colon cancer cellline SW480. Int J Oncol, 2014, 45(3): 1175-1183.
13. Wang L, Huang X, Zheng X, et al. 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, 2013, 9(5): 472-479.
14. Gatti M, Pattarozzi A, Bajetto A, et al. Inhibition of CXCL12/CXCR4 autocrine/paracrine loop reduces viability of human glioblastoma stem-like cells affecting self-renewal activity. Toxicology, 2013, 314(2-3): 209-220.
15. Roudi R, Madjd Z, Ebrahimi M, et al. CD44 and CD24 cannot act as cancer stem cell markers in human lung adenocarcinoma cell line A549. Cell Mol Biol Lett, 2014, 19(1): 23-36.
16. Markert S, Musmann C, Hülsmann P, et al. Automated and enhanced clone screening using a fully automated microtiter plate-based system for suspension cell culture. Biotechnol Prog, 2019, 35(2): e2760.
17. Kumar SV, Er PX, Lawlor KT, et al. Kidney micro-organoids in suspension culture as a scalable source of human pluripotent stem cell-derived kidney cells. Development, 2019, 146(5): dev172361.
18. Munro MJ, Wickremesekera SK, Peng L, et al. Cancer stem cells in colorectal cancer: a review. J Clin Pathol, 2018, 71(2): 110-116.
19. Shimokawa M, Ohta Y, Nishikori S, et al. Visualization and targeting of LGR5+ human colon cancer stem cells. Nature, 2017, 545(7653): 187-192.
20. Yang L, Shi P, Zhao G, et al. Targeting cancer stem cell pathways for cancer therapy. Signal Transduct Target Ther, 2020, 5: 8.
21. Ponti D, Costa A, Zaffaroni N, et al. Isolation and in vitro propagation of tumorigenic breast cancer cells withstem/progenitor cell properties. Cancer Res, 2005, 65(13): 5506-5511.
22. Hemmati HD, Nakano I, Lazareff JA, et al. Cancerous stem cells can arise from pediatric brain tumors. Proc Natl Acad Sci U S A, 2003, 100(25): 15178-15183.
23. Singh SK, Clarke ID, Terasaki M, et al. Identification of a cancer stem cell in human brain tumors. Cancer Res, 2003, 63(18): 5821-5828.
24. Ricci-Vitiani L, Lombardi DG, Pilozzi E, et al. Identification and expansion of human colon-cancer-initiating cells. Nature, 2007, 445(7123): 111-115.
25. Rybak AP, He L, Kapoor A, et al. Characterization of sphere-propagating cells with stem-like properties from DU145 prostate cancer cells. Biochim Biophys Acta, 2011, 1813(5): 683-694.
26. Louis SA, Rietze RL, Deleyrolle L, et al. Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay. Stem Cells, 2008, 26(4): 988-996.
27. Manupati K, Dhoke NR, Debnath T, et al. Inhibiting epidermal growth factor receptor signalling potentiatesmesenchymal-epithelial transition of breast cancer stem cells and theirres-ponsiveness to anticancer drugs. FEBS J, 2017, 284(12): 1830-1854.
28. De Angelis ML, Bruselles A, Francescangeli F, et al. Colorectal cancer spheroid biobanks: multi-level approaches to drug sensitivity studies. Cell Biol Toxicol, 2018, 34(6): 459-469.
29. Xu Q, Zhang Q, Ishida Y, et al. EGF induces epithelial-mesenchymal transition and cancer stem-like cell properties in human oral cancer cells via promoting Warburg effect. Oncotarget, 2017, 8(6): 9557-9571.
30. Ding C, Zou Q, Wu Y, et al. EGF released from human placental mesenchymal stem cells improves prematureovarian insufficiency via NRF2/HO-1 activation. Aging (Albany NY), 2020, 12(3): 2992-3009.
31. Lee K, Sheung P, Chou C, et al. Elevation of CD109 promotes metastasis and drug resistance in lung cancer viaactivation of EGFR-AKT-mTOR signaling. Cancer Sci, 2020 Mar 4. [Epub ahead of print].

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

The effect of EGF culture time on colorectal cancer stem cells enrichment

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