Rapid Isolation of Gastric Adenocarcinoma Cancer Stem Cells as a Target for Autologous Dendritic Cell-Based Immunotherapy

Document Type : Research Paper

Authors

1 Cellular and Molecular Research Center, Birjand University of Medical Sciences, Birjand, Iran

2 Immunology Department, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.

3 Immunology Department, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

4 Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad, Iran

5 Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

Abstract

Background: Gastric cancer (GC) is a malignancy cause associated with a high death rate in the world. Cancer stem cells 
(CSCs) are a rare immortal subpopulation of cells within tumors with characteristics of the ability to self-renew, initiate 
tumor, and differentiate into defined progenies as well as and high resistance to conventional therapies. 
Objectives: Despite the use of surgery and chemotherapy for GC therapy, there are no efficient therapeutic protocols for 
it to date. Therefore, rapid isolation of CSCs in order to therapeutic targets, especially immunotherapy is very important.
Materials and Methods: Cancerous cell suspension isolated from patients with GC was cultured in the serum-free 
medium containing EGF, bFGF, LIF, and heparin under non-adherent culture conditions to generate spheres. Expression 
of mRNA level stemness transcription factors (OCT4, SOX2, SALL4, and Cripto-1), CD44 variable isoforms (CD44s, 
CD44v3, CD44v6, CD44V8-10) of spheroid-forming single cells compared with gastric normal tissue cells using real 
time PCR and molecules of CD44, CD54, and EpCAM as gastric CSC markers, and stemness factor Oct4 using flow 
cytometry, as well as tumorgenicity using subcutaneous injection of sphere-forming cells to nude mice were investigated.
Results: Few cancerous cells isolated from patients with GC were able to generate three-dimensional spheroid colonies in 
the serum-free medium containing EGF, bFGF, LIF, and heparin under non-adherent culture conditions, and form xenograft 
tumors in immunodeficient nude mice after subcutaneous injection. Spheroid-forming single cells upregulated stemness 
transcription factors OCT4, SOX2, SALL4, and Cripto-1 that are associated with pluripotency and self-renewal and CD44 
isoforms (CD44s, CD44v3, CD44v6, CD44V8-10) compared with gastric normal tissue cells. Finally, molecules of CD44, 
CD54, and EpCAM as gastric CSC markers and stemness factor Oct4 were expressed in sphere-forming cells.
Conclusion: We suggested that the sphere formation and tumorigenicity assays are two procedures, leading to the rapid 
isolation of cancer cells with certain stem-like properties in order to target CSCs using autologous dendritic cell therapy, 
especially in patients with advanced disease.

Keywords

Main Subjects


  1. References

    1. Herrero R, Parsonnet J, Greenberg ER. Prevention of gastric cancer. JAMA. 2014;312(12):1197-1198. doi: 10.1001/jama.2014.10498.
    2. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87-108. doi: 10.3322/caac.21262.
    3. Bray F, Ren JS, Masuyer E, Ferlay J. Global estimates of cancer prevalence for 27 sites in the adult population in 2008. Int J Cancer. 2013;132(5):1133-1145. doi: : 10.1002/ijc.27711.
    4. Newton AD, Datta J, Loaiza-Bonilla A, Karakousis GC, Roses RE. Neoadjuvant therapy for gastric cancer: current evidence and future directions. J Gastrointest Oncol. 2015;6(5):534-543. doi: 10.3978/j.issn.2078-6891.2015.047.
    5. Parizi PK, Yarahmadi F, Tabar HM, Hosseini Z, Sarli A, Kia N, et al. MicroRNAs and target molecules in bladder cancer. Medical Oncology. 2020;37(12):1-33. doi: 10.1007/s12032-020-01435-0.
    6. Abbaszadegan MR, Bagheri V, Razavi MS, Momtazi AA, Sahebkar A, Gholamin M. Isolation, identification, and characterization of cancer stem cells: A review. J Cell Physiol. 2017;232(8):2008-2018. doi: 10.1002/jcp.25759.
    7. Eaves CJ. Cancer stem cells: Here, there, everywhere? Nature. 2008;456(7222):581-582. doi: 10.1038/456581a.
    8. Zhang X, Yashiro M, Qiu H, Nishii T, Matsuzaki T, Hirakawa K. Establishment and characterization of multidrug-resistant gastric cancer cell lines. Anticancer Res. 2010;30(3):915-921. doi: 10.7314/apjcp.2014.15.16.6849.
    9. Radmanesh F, Mahmoudi M, Yazdanpanah E, Keyvani V, Kia N, Nikpoor AR, et al. The immunomodulatory effects of mesenchymal stromal cell-based therapy in human and animal models of systemic lupus erythematosus. IUBMB life. 2020;72(11):2366-2381. doi: 10.1002/iub.2387.
    10. Tan BT, Park CY, Ailles LE, Weissman IL. The cancer stem cell hypothesis: a work in progress. Lab Invest. 2006;86(12):1203-1207. doi: 10.1038/labinvest.3700488.
    11. Visvader JE. Cells of origin in cancer. Nature. 2011;469(7330):314-322. doi: 10.1038/nature09781.
    12. Prasetyanti PR, Medema JP. Intra-tumor heterogeneity from a cancer stem cell perspective. Mol Cancer. 2017;16(1):41. doi: 10.1186/s12943-017-0600-4.
    13. Aguilar-Gallardo C, Simon C. Cells, stem cells, and cancer stem cells. Semin Reprod Med. 2013;31(1):5-13. doi: 10.1055/s-0032-1331792.
    14. Keyvani V, Farshchian M, Esmaeili S-A, Yari H, Moghbeli M, Nezhad S-RK, et al. Ovarian cancer stem cells and targeted therapy. J Ovarian Res. 2019;12(1):1-11. doi: 10.1186/s13048-019-0588-z.
    15. Esmaeili S-A, Sahranavard S, Salehi A, Bagheri V. Selectively targeting cancer stem cells: Current and novel therapeutic strategies and approaches in the effective eradication of cancer. IUBMB Life. 2021;73(8):1045-1059. doi: 10.1002/iub.2524.
    16. Bagheri V, Abbaszadegan MR, Memar B, Motie MR, Asadi M, Mahmoudian RA, et al. Induction of T cell-mediated immune response by dendritic cells pulsed with mRNA of sphere-forming cells isolated from patients with gastric cancer. Life Sci. 2019;219:136-143. doi: 10.1016/j.lfs.2019.01.016.
    17. Vik-Mo EO, Nyakas M, Mikkelsen BV, Moe MC, Due-Tonnesen P, Suso EM, et al. Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma. Cancer Immunol Immunother. 2013;62(9):1499-1509. doi: 10.1007/s00262-013-1453-3.
    18. Dashti A, Ebrahimi M, Hadjati J, Memarnejadian A, Moazzeni SM. Dendritic cell based immunotherapy using tumor stem cells mediates potent antitumor immune responses. Cancer Lett. 2016;374(1):175-185. doi: 10.1016/j.canlet.2016.01.021.
    19. Yin T, Shi P, Gou S, Shen Q, Wang C. Dendritic cells loaded with pancreatic Cancer Stem Cells (CSCs) lysates induce antitumor immune killing effect in vitro. PLoS One. 2014;9(12):e114581. doi: 10.1371/journal.pone.0114581.
    20. Pastrana E, Silva-Vargas V, Doetsch F. Eyes wide open: a critical review of sphere-formation as an assay for stem cells. Cell Stem Cell. 2011;8(5):486-498. doi:10.1016/j.stem. 2011.04.007.
    21. Bagheri V, Memar B, Behzadi R, Aliakbarian M, Jangjoo A, Bahar MM, et al. Isolation and identification of chemotherapy-enriched sphere-forming cells from a patient with gastric cancer. J Cell Physiol. 2018;233(10):7036-7046. doi: 10.1002/jcp.26627.
    22. Takaishi S, Okumura T, Tu S, Wang SS, Shibata W, Vigneshwaran R, et al. Identification of gastric cancer stem cells using the cell surface marker CD44. Stem Cells. 2009;27(5):1006-1020. doi: 10.1002/stem.30.
    23. Cao L, Zhou Y, Zhai B, Liao J, Xu W, Zhang R, et al. Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines. BMC Gastroenterol. 2011;11:71. doi: 10.1186/1471-230x-11-71.
    24. Yue D, Zhang Z, Li J, Chen X, Ping Y, Liu S, et al. Transforming growth factor-beta1 promotes the migration and invasion of sphere-forming stem-like cell subpopulations in esophageal cancer. Exp Cell Res. 2015;336(1):141-149. doi: 10.1016/j.yexcr.2015.06.007.
    25. Bahmad HF, Cheaito K, Chalhoub RM, Hadadeh O, Monzer A, Ballout F, et al. Sphere-Formation Assay: Three-Dimensional in vitro Culturing of Prostate Cancer Stem/Progenitor Sphere-Forming Cells. Front Oncol. 2018;8:347. doi: 10.3389/fonc.2018.00347.
    26. Michelson S, Slate D. Emergence of the drug-resistant phenotype in tumor subpopulations: a hybrid model. J Natl Cancer 1989;81(18):1392-1401. doi: 10.1093/jnci/81.18.1392.
    27. Perryman SV, Sylvester KG. Repair and regeneration: opportunities for carcinogenesis from tissue stem cells. J Cell Mol Med. 2006;10(2):292-308. doi: 10.1111/j.1582-4934.2006.tb00400.x.
    28. Brungs D, Aghmesheh M, Vine KL, Becker TM, Carolan MG, Ranson M. Gastric cancer stem cells: evidence, potential markers, and clinical implications. J Gastroenterol. 2016;51(4):313-326. doi: 10.1007/s00535-015-1125-5.
    29. Zhang C, Li C, He F, Cai Y, Yang H. Identification of CD44+CD24+ gastric cancer stem cells. J Cancer Res Clin Oncol. 2011;137(11):1679-1686. doi: 10.1007/s00432-011-1038-5.
    30. Han ME, Jeon TY, Hwang SH, Lee YS, Kim HJ, Shim HE, et al. Cancer spheres from gastric cancer patients provide an ideal model system for cancer stem cell research. Cell Mol Life Sci. 2011;68(21):3589-3605. doi: 10.1007/s00018-011-0672-z.
    31. Chen T, Yang K, Yu J, Meng W, Yuan D, Bi F, et al. Identification and expansion of cancer stem cells in tumor tissues and peripheral blood derived from gastric adenocarcinoma patients. Cell Res. 2012;22(1):248-258. doi: 10.1038/cr.2011.109.
    32. Matsuoka J, Yashiro M, Sakurai K, Kubo N, Tanaka H, Muguruma K, et al. Role of the stemness factors sox2, oct3/4, and nanog in gastric carcinoma. J Surg Res. 2012;174(1):130-135. doi: 10.1016/j.jss.2010.11.903.
    33. Tian T, Zhang Y, Wang S, Zhou J, Xu S. Sox2 enhances the tumorigenicity and chemoresistance of cancer stem-like cells derived from gastric cancer. J Biomed Res. 2012;26(5):336-345. doi: 10.7555/jbr.26.20120045.
    34. Yuan X, Zhang X, Zhang W, Liang W, Zhang P, Shi H, et al. SALL4 promotes gastric cancer progression through activating CD44 expression. Oncogenesis. 2016;5(11):e268. doi: 10.1038/oncsis.2016.69.
    35. Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ. Prospective identification of tumorigenic prostate cancer stem cells. Cancer research. 2005;65(23):10946-10951. doi: 10.1158/0008-5472.can-05-2018.
    36. Flanagan S. ‘Nude’, a new hairless gene with pleiotropic effects in the mouse. Genetics Research. 1966;8(3):295-309. doi: 10.1017/s0016672300010168.
    37. Hu Y, Smyth GK. ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol methods. 2009;347(1-2):70-78. doi: 10.1016/j.jim.2009.06.008.
    38. Lau WM, Teng E, Chong HS, Lopez KA, Tay AY, Salto-Tellez M, et al. CD44v8-10 is a cancer-specific marker for gastric cancer stem cells. Cancer Res. 2014;74(9):2630-2641. doi: 10.1158/0008-5472.can-13-2309.
    39. Osta WA, Chen Y, Mikhitarian K, Mitas M, Salem M, Hannun YA, et al. EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy. Cancer Res. 2004;64(16):5818-5824. doi: 10.1158/0008-5472.can-04-0754.
    40. Castella EM, Ariza A, Pellicer I, Fernandez-Vasalo A, Ojanguren I. Differential expression of CD44v6 in metastases of intestinal and diffuse types of gastric carcinoma. J Clin Pathol. 1998;51(2):134-137. doi: 10.1136/jcp.51.2.134.
    41. Heider KH, Dammrich J, Skroch-Angel P, Muller-Hermelink HK, Vollmers HP, Herrlich P, et al. Differential expression of CD44 splice variants in intestinal- and diffuse-type human gastric carcinomas and normal gastric mucosa. Cancer Res. 1993;53(18):4197-4203.
    42. Kyte JA, Mu L, Aamdal S, Kvalheim G, Dueland S, Hauser M, et al. Phase I/II trial of melanoma therapy with dendritic cells transfected with autologous tumor-mRNA. Cancer Gene Ther. 2006;13(10):905-918. doi: 10.1038/sj.cgt.7700961.