The number of individuals diagnosed with colorectal cancer (CRC) has been on an alarming upward trajectory over the past decade. In some countries, this cancer represents one of the most frequently diagnosed types of neoplasia. Therefore, it is an important demand to study the pathology underlying this disease to gain insights into the mechanism of resistance to treatment. Resistance of tumors to chemotherapy and tumor aggressiveness have been associated with a minor population of neoplastic cells, which are considered to be responsible for tumor recurrence. These types of neoplastic cells are known as cancer stem cells, which have been previously reported to serve an important role in pathogenesis of this malignant disease. Slovakia has one of the highest incidence rates of CRC worldwide. In the present study, the aim was to classify the abundance of selected stem cell markers (CD133, CD166 and Lgr5) in CRC tumors using flow cytometry. In addition, the methylation status of selected genomic regions of CRC biomarkers (ADAMTS16, MGMT, PROM1 (CD133), LGR5 and ALCAM) was investigated by pyrosequencing in a cohort of patients from Martin University Hospital, Martin, Slovakia. Samples from both primary tumors and metastatic tumors were tested. Analysis of DNA methylation in the genomic regions of indicated five CRC biomarkers was also performed, which revealed the highest levels of methylation in the A disintegrin and metalloproteinase with thrombospondin motifs 16 and O6-methyguanine-DNA methyl transferase genes, whereas the lowest levels of methylation were found in genes expressing prominin-1, leucine-rich repeat-containing G-protein-coupled receptor 5 and activated leukocyte cell adhesion molecule. Furthermore, tumor tissues from metastases showed significantly higher levels of CD133+ cells compared with that in primary tumors. Higher levels of CD133+ cells correlated with TNM stage and the invasiveness of CRC into the lymphatic system. Although relatively small number of samples was processed, CD133 marker was consider to be important marker in pathology of CRC.

Biomedical Centre Martin Comenius University in Bratislava Jessenius Faculty of Medicine in Martin 03601 Martin Slovakia

Clinic of Surgery and Transplant Center Martin University Hospital Comenius University in Bratislava Jessenius Faculty of Medicine in Martin 03601 Martin Slovakia

Department of Molecular Biology and Genomics Comenius University in Bratislava Jessenius Faculty of Medicine in Martin 03601 Martin Slovakia

Department of Pathological Anatomy Martin University Hospital Comenius University in Bratislava Jessenius Faculty of Medicine in Martin 03601 Martin Slovakia

Department of Pediatric Hematology and Oncology Charles University and University Hospital Motol 15006 Prague Czech Republic

See more in PubMed

Pop MG. Stem cell markers in colon cancer. Basic Princ Pract Surg. 2019

Coppedè F, Lopomo A, Spisni R, Migliore L. Genetic and epigenetic biomarkers for diagnosis, prognosis and treatment of colorectal cancer. World J Gastroenterol. 2014;20:943–956. doi: 10.3748/wjg.v20.i4.943. PubMed DOI PMC

Orjuela S, Menigatti M, Schraml P, Kambakamba P, Robinson MD, Marra G. The DNA hypermethylation phenotype of colorectal cancer liver metastases resembles that of the primary colorectal cancers. BMC Cancer. 2020;20:290. doi: 10.1186/s12885-020-06777-6. PubMed DOI PMC

de Vogel S, Wouters KA, Gottschalk RW, van Schooten FJ, de Goeij AF, de Bruïne AP, Goldbohm RA, van den Brandt PA, Weijenberg MP, van Engeland M. Genetic variants of methyl metabolizing enzymes and epigenetic regulators: Associations with promoter CpG island hypermethylation in colorectal cancer. Cancer Epidemiol Biomarkers Prev. 2009;18:3086–3096. doi: 10.1158/1055-9965.EPI-09-0289. PubMed DOI

Gupta R, Bhatt LK, Johnston TP, Prabhavalkar KS. Colon cancer stem cells: Potential target for the treatment of colorectal cancer. Cancer Biol Ther. 2019;20:1068–1082. doi: 10.1080/15384047.2019.1599660. PubMed DOI PMC

Spelt L, Sasor A, Ansari D, Hilmersson KS, Andersson R. The prognostic role of cancer stem cell markers for long-term outcome after resection of colonic liver metastases. Anticancer Res. 2018;38:313–320. PubMed

Nosrati A, Naghshvar F, Maleki I, Salehi F. Cancer stem cells CD133 and CD24 in colorectal cancers in Northern Iran. Gastroenterol Hepatol Bed Bench. 2016;9:132–139. PubMed PMC

Kazama S, Kishikawa J, Kiyomatsu T, Kawai K, Nozawa H, Ishihara S, Watanabe T. Expression of the stem cell marker CD133 is related to tumor development in colorectal carcinogenesis. Asian J Surg. 2018;41:274–278. doi: 10.1016/j.asjsur.2016.12.002. PubMed DOI

Schmohl JU, Vallera DA. CD133, selectively targeting the root of cancer. Toxins (Basel) 2016;8:165. doi: 10.3390/toxins8060165. PubMed DOI PMC

Barzegar Behrooz A, Syahir A, Ahmad S. CD133: Beyond a cancer stem cell biomarker. J Drug Target. 2019;27:257–269. doi: 10.1080/1061186X.2018.1479756. PubMed DOI

Sahlberg SH, Spiegelberg D, Glimelius B, Stenerlöw B, Nestor M. Evaluation of cancer stem cell markers CD133, CD44, CD24: Association with AKT isoforms and radiation resistance in colon cancer cells. PLoS One. 2014;9:e94621. doi: 10.1371/journal.pone.0094621. PubMed DOI PMC

Yamamoto S, Tanaka K, Takeda K, Akiyama H, Ichikawa Y, Nagashima Y, Endo I. Patients with CD133-negative colorectal liver metastasis have a poor prognosis after hepatectomy. Ann Surg Oncol. 2014;21:1853–1861. doi: 10.1245/s10434-014-3549-1. PubMed DOI

Narita M, Oussoultzoglou E, Chenard MP, Fuchshuber P, Yamamoto T, Addeo P, Jaeck D, Bachellier P. Predicting early intrahepatic recurrence after curative resection of colorectal liver metastases with molecular markers. World J Surg. 2015;39:1167–1176. doi: 10.1007/s00268-014-2916-1. PubMed DOI

Chen S, Song X, Chen Z, Li X, Li M, Liu H, Li J. CD133 expression and the prognosis of colorectal cancer: A systematic review and meta-analysis. PLoS One. 2013;8:e56380. doi: 10.1371/journal.pone.0056380. PubMed DOI PMC

Wang K, Xu J, Zhang J, Huang J. Prognostic role of CD133 expression in colorectal cancer: A meta-analysis. BMC Cancer. 2012;12:573. doi: 10.1186/1471-2407-12-573. PubMed DOI PMC

Geddert H, Braun A, Kayser C, Dimmler A, Faller G, Agaimy A, Haller F, Moskalev EA. Epigenetic regulation of CD133 in gastrointestinal stromal tumors. Am J Clin Pathol. 2017;147:515–524. doi: 10.1093/ajcp/aqx028. PubMed DOI

Fanali C, Lucchetti D, Farina M, Corbi M, Cufino V, Cittadini A, Sgambato A. Cancer stem cells in colorectal cancer from pathogenesis to therapy: Controversies and perspectives. World J Gastroenterol. 2014;20:923–942. doi: 10.3748/wjg.v20.i4.923. PubMed DOI PMC

Yi JM, Tsai HC, Glöckner SC, Lin S, Ohm JE, Easwaran H, James CD, Costello JF, Riggins G, Eberhart CG, et al. Abnormal DNA methylation of CD133 in colorectal and glioblastoma tumors. Cancer Res. 2008;68:8094–8103. doi: 10.1158/0008-5472.CAN-07-6208. PubMed DOI PMC

Dana H, Marmari V, Mahmoodi G, Mahmoodzadeh H, Ebrahimi M, Mehmandoost N. CD166 as a stem cell marker? A potential target for therapy colorectal cancer? J Stem Cell Res Ther. 2016;1:226–229.

Lugli A, Iezzi G, Hostettler I, Muraro MG, Mele V, Tornillo L, Carafa V, Spagnoli G, Terracciano L, Zlobec I. Prognostic impact of the expression of putative cancer stem cell markers CD133, CD166, CD44s, EpCAM, and ALDH1 in colorectal cancer. Br J Cancer. 2010;103:382–390. doi: 10.1038/sj.bjc.6605762. PubMed DOI PMC

Weichert W, Knösel T, Bellach J, Dietel M, Kristiansen G. ALCAM/CD166 is overexpressed in colorectal carcinoma and correlates with shortened patient survival. J Clin Pathol. 2004;57:1160–1164. doi: 10.1136/jcp.2004.016238. PubMed DOI PMC

Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, Cho RW, Hoey T, Gurney A, Huang EH, Simeone DM, et al. Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci USA. 2007;104:10158–10163. doi: 10.1073/pnas.0703478104. PubMed DOI PMC

Darvishi B, Boroumandieh S, Majidzadeh-A K, Salehi M, Jafari F, Farahmand L. The role of activated leukocyte cell adhesion molecule (ALCAM) in cancer progression, invasion, metastasis and recurrence: A novel cancer stem cell marker and tumor-specific prognostic marker. Exp Mol Pathol. 2020;115:104443. doi: 10.1016/j.yexmp.2020.104443. PubMed DOI

Chen C, Zhao S, Karnad A, Freeman JW. The biology and role of CD44 in cancer progression: Therapeutic implications. J Hematol Oncol. 2018;11:64. doi: 10.1186/s13045-018-0605-5. PubMed DOI PMC

Vincent A, Ouelkdite-Oumouchal A, Souidi M, Leclerc J, Neve B, Van Seuningen I. Colon cancer stemness as a reversible epigenetic state: Implications for anticancer therapies. World J Stem Cells. 2019;11:920–936. doi: 10.4252/wjsc.v11.i11.920. PubMed DOI PMC

Tachezy M, Zander H, Gebauer F, Marx A, Kaifi JT, Izbicki JR, Bockhorn M. Activated leukocyte cell adhesion molecule (CD166)-its prognostic power for colorectal cancer patients. J Surg Res. 2012;177:e15–e20. doi: 10.1016/j.jss.2012.02.013. PubMed DOI

Levin TG, Powell AE, Davies PS, Silk AD, Dismuke AD, Anderson EC, Swain JR, Wong MH. Characterization of the intestinal cancer stem cell marker CD166 in the human and mouse gastrointestinal tract. Gastroenterology. 2010;139:2072–2082.e5. doi: 10.1053/j.gastro.2010.08.053. PubMed DOI PMC

Han S, Yang W, Zong S, Li H, Liu S, Li W, Shi Q, Hou F. Clinicopathological, prognostic and predictive value of CD166 expression in colorectal cancer: A meta-analysis. Oncotarget. 2017;8:64373–64384. doi: 10.18632/oncotarget.17442. PubMed DOI PMC

Beumer J, Clevers H. Regulation and plasticity of intestinal stem cells during homeostasis and regeneration. Development. 2016;143:3639–3649. doi: 10.1242/dev.133132. PubMed DOI

Wu XS, Xi HQ, Chen L. Lgr5 is a potential marker of colorectal carcinoma stem cells that correlates with patient survival. World J Surg Oncol. 2012;10:244. doi: 10.1186/1477-7819-10-244. PubMed DOI PMC

Zheng Z, Yu H, Huang Q, Wu H, Fu Y, Shi J, Wang T, Fan X. Heterogeneous expression of Lgr5 as a risk factor for focal invasion and distant metastasis of colorectal carcinoma. Oncotarget. 2018;9:30025–30033. doi: 10.18632/oncotarget.23144. PubMed DOI PMC

Hsu HC, Liu YS, Tseng KC, Hsu CL, Liang Y, Yang TS, Chen JS, Tang RP, Chen SJ, Chen HC. Overexpression of Lgr5 correlates with resistance to 5-FU-based chemotherapy in colorectal cancer. Int J Colorectal Dis. 2013;28:1535–1546. doi: 10.1007/s00384-013-1721-x. PubMed DOI

Wahab SMR, Islam F, Gopalan V, Lam AK. The identifications and clinical implications of cancer stem cells in colorectal cancer. Clin Colorectal Cancer. 2017;16:93–102. doi: 10.1016/j.clcc.2017.01.011. PubMed DOI

Wu W, Cao J, Ji Z, Wang J, Jiang T, Ding H. Co-expression of Lgr5 and CXCR4 characterizes cancer stem-like cells of colorectal cancer. Oncotarget. 2016;7:81144–81155. doi: 10.18632/oncotarget.13214. PubMed DOI PMC

Zhou Y, Xia L, Wang H, Oyang L, Su M, Liu Q, Lin J, Tan S, Tian Y, Liao Q, Cao D. Cancer stem cells in progression of colorectal cancer. Oncotarget. 2017;9:33403–33415. doi: 10.18632/oncotarget.23607. PubMed DOI PMC

Jang BG, Kim HS, Chang WY, Bae JM, Kim WH, Kang GH. Expression profile of LGR5 and its prognostic significance in colorectal cancer progression. Am J Pathol. 2018;188:2236–2250. doi: 10.1016/j.ajpath.2018.06.012. PubMed DOI

Alizadeh Naini M, Kavousipour S, Hasanzarini M, Nasrollah A, Monabati A, Mokarram P. O6-methyguanine-DNA Methyl transferase (MGMT) promoter methylation in serum DNA of Iranian patients with colorectal cancer. Asian Pac J Cancer Prev. 2018;19:1223–1227. PubMed PMC

Inno A, Fanetti G, Di Bartolomeo M, Gori S, Maggi C, Cirillo M, Iacovelli R, Nichetti F, Martinetti A, de Braud F, et al. Role of MGMT as biomarker in colorectal cancer. World J Clin Cases. 2014;2:835–839. doi: 10.12998/wjcc.v2.i12.835. PubMed DOI PMC

Cervena K, Siskova A, Buchler T, Vodicka P, Vymetalkova V. Methylation-based therapies for colorectal cancer. Cells. 2020;9:1540. doi: 10.3390/cells9061540. PubMed DOI PMC

Kordowski F, Kolarova J, Schafmayer C, Buch S, Goldmann T, Marwitz S, Kugler C, Scheufele S, Gassling V, Németh CG, et al. Aberrant DNA methylation of ADAMTS16 in colorectal and other epithelial cancers. BMC Cancer. 2018;18:796. doi: 10.1186/s12885-018-4701-2. PubMed DOI PMC

Strnadel J, Woo SM, Choi S, Wang H, Grendar M, Fujimura K. 3D culture protocol for testing gene knockdown efficiency and cell line derivation. Bio Protoc. 2018;8:e2874. doi: 10.21769/BioProtoc.2874. PubMed DOI PMC

Meršaková S, Holubeková V, Grendár M, Višňovský J, Ňachajová M, Kalman M, Kúdela E, Žúbor P, Bielik T, Lasabová Z, Danko J. Methylation of CADM1 and MAL together with HPV status in cytological cervical specimens serves an important role in the progression of cervical intraepithelial neoplasia. Oncol Lett. 2018;16:7166–7174. PubMed PMC

R Core Team (2021). R, corp-author. R Foundation for Statistical Computing; Vienna, Austria: A language and environment for statistical computing.

World Cancer Research Fund International, corp-author. Colorectal cancer statistics. https://www.wcrf.org/cancer-trends/colorectal-cancer-statistics/ [ April 26; 2022 ];

Horst D, Kriegl L, Engel J, Kirchner T, Jung A. Prognostic significance of the cancer stem cell markers CD133, CD44, and CD166 in colorectal cancer. Cancer Invest. 2009;27:844–850. doi: 10.1080/07357900902744502. PubMed DOI

Fang C, Fan C, Wang C, Huang Q, Meng W, Yu Y, Yang L, Hu J, Li Y, Mo X, Zhou Z. Prognostic value of CD133+ CD54+ CD44+ circulating tumor cells in colorectal cancer with liver metastasis. Cancer Med. 2017;6:2850–2857. doi: 10.1002/cam4.1241. PubMed DOI PMC

Morgan RG, Mortensson E, Williams AC. Targeting LGR5 in colorectal cancer: Therapeutic gold or too plastic? Br J Cancer. 2018;118:1410–1418. doi: 10.1038/s41416-018-0118-6. PubMed DOI PMC

Miller TJ, McCoy MJ, Hemmings C, Bulsara MK, Iacopetta B, Platell CF. The prognostic value of cancer stem-like cell markers SOX2 and CD133 in stage III colon cancer is modified by expression of the immune-related markers FoxP3, PD-L1 and CD3. Pathology. 2017;49:721–730. doi: 10.1016/j.pathol.2017.08.007. PubMed DOI

Mărgaritescu C, Pirici D, Cherciu I, Bărbălan A, Cârtână T, Săftoiu A. CD133/CD166/Ki-67 triple immunofluorescence assessment for putative cancer stem cells in colon carcinoma. J Gastrointest Liver Dis. 2014;23:161–170. doi: 10.15403/jgld.2014.1121.232.cm1. PubMed DOI

Pietrantonio F, Perrone F, de Braud F, Castano A, Maggi C, Bossi I, Gevorgyan A, Biondani P, Pacifici M, Busico A, et al. Activity of temozolomide in patients with advanced chemorefractory colorectal cancer and MGMT promoter methylation. Ann Oncol. 2014;25:404–408. doi: 10.1093/annonc/mdt547. PubMed DOI

Atlasy N, Amidi F, Mortezaee K, Fazeli MS, Mowla SJ, Malek F. Expression patterns for TETs, LGR5 and BMI1 in cancer stem-like cells isolated from human colon cancer. Avicenna J Med Biotechnol. 2019;11:156–161. PubMed PMC

Wang BB, Li ZJ, Zhang FF, Hou HT, Yu JK, Li F. Clinical significance of stem cell marker CD133 expression in colorectal cancer. Histol Histopathol. 2016;31:299–306. PubMed

Park YY, An CH, Oh ST, Chang ED, Lee J. Expression of CD133 is associated with poor prognosis in stage II colorectal carcinoma. Medicine (Baltimore) 2019;98:e16709. doi: 10.1097/MD.0000000000016709. PubMed DOI PMC

Pellacani D, Packer RJ, Frame FM, Oldridge EE, Berry PA, Labarthe MC, Stower MJ, Simms MS, Collins AT, Maitland NJ. Regulation of the stem cell marker CD133 is independent of promoter hypermethylation in human epithelial differentiation and cancer. Mol Cancer. 2011;10:94. doi: 10.1186/1476-4598-10-94. PubMed DOI PMC

Dame MK, Attili D, McClintock SD, Dedhia PH, Ouillette P, Hardt O, Chin AM, Xue X, Laliberte J, Katz EL, et al. Identification, isolation and characterization of human LGR5-positive colon adenoma cells. Development. 2018;145:dev153049. doi: 10.1242/dev.153049. PubMed DOI PMC

Leng Z, Xia Q, Chen J, Li Y, Xu J, Zhao E, Zheng H, Ai W, Dong J. Lgr5+CD44+EpCAM+ strictly defines cancer stem cells in human colorectal cancer. Cell Physiol Biochem. 2018;46:860–872. doi: 10.1159/000488743. PubMed DOI

Costello JF, Futscher BW, Tano K, Graunke DM, Pieper RO. Graded methylation in the promoter and body of the O6-methylguanine DNA methyltransferase (MGMT) gene correlates with MGMT expression in human glioma cells. J Biol Chem. 1994;269:17228–17237. doi: 10.1016/S0021-9258(17)32544-9. PubMed DOI

Qian X, von Wronski MA, Brent TP. Localization of methylation sites in the human O6-methylguanine-DNA methyltransferase promoter: Correlation with gene suppression. Carcinogenesis. 1995;16:1385–1390. doi: 10.1093/carcin/16.6.1385. PubMed DOI

Shen L, Kondo Y, Rosner GL, Xiao L, Hernandez NS, Vilaythong J, Houlihan PS, Krouse RS, Prasad AR, Einspahr JG, et al. MGMT promoter methylation and field defect in sporadic colorectal cancer. J Natl Cancer Inst. 2005;97:1330–1338. doi: 10.1093/jnci/dji275. PubMed DOI

Fornaro L, Vivaldi C, Caparello C, Musettini G, Baldini E, Masi G, Falcone A. Pharmacoepigenetics in gastrointestinal tumors: MGMT methylation and beyond. Front Biosci (Elite Ed) 2016;8:170–180. doi: 10.2741/e758. PubMed DOI

Margison GP, Povey AC, Kaina B, Santibáñez Koref MF. Variability and regulation of O6-alkylguanine-DNA alkyltransferase. Carcinogenesis. 2003;24:625–635. doi: 10.1093/carcin/bgg005. PubMed DOI