Isolation of Stem-Like Cancer Cells in Primary Endometrial Cancer Using Cell Surface Markers CD133 and CXCR4 Yi Sun, Toshiko Yoshida, Motonori Okabe,

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Isolation of Stem-Like Cancer Cells in Primary Endometrial Cancer Using Cell Surface Markers CD133 and CXCR4 Yi Sun, Toshiko Yoshida, Motonori Okabe, Kaixuan Zhou, Fang Wang, Chika Soko, Sigeru Saito, Toshio Nikaido Translational Oncology Volume 10, Issue 6, Pages 976-987 (December 2017) DOI: 10.1016/j.tranon.2017.07.007 Copyright © 2017 The Authors Terms and Conditions

Figure 1 The expression of stemness genes and surface markers in primary endometrial cancer cells. (A) RT-PCR showed both two patients expressed stemness related genes including c-Myc, Sox-2, Nanog, Oct4A, ABCG2, BMI-1, CK-18, Nestin and β-actin, β-actin is the negative control. (B-1 and B-2) The expression levels of CD24, CD133, CD47, CD29, CD44, CXCR4, SSEA3, and SSEA4 by flow cytometry. (C) The mRNA expression of comparison and analysis between CD24, CD133, and CXCR4 positive and negative subpopulation in the two patients by RT-PCR. (D) The double CD133+CXCR4+ cells ration is 7.2% and 9.3%, respectively. Translational Oncology 2017 10, 976-987DOI: (10.1016/j.tranon.2017.07.007) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Reverse transcriptase PCR analysis between double positive and double negative EC cell. (A) The cells from patient's tissue expressed higher stemness genes such as c-Myc, Oct4A, Sox2, Nanog, ABCG2, and Nestin in CD133+CXCR4+ cells than CD133−CXCR4− cells. β-actin was used as parameter. (B) The other patient showed the similar result, include BMI-1. (C) The immunocytochemistry stain of the each subgroups. Nuclei were stain with DAPI. Translational Oncology 2017 10, 976-987DOI: (10.1016/j.tranon.2017.07.007) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Characteristics of unsorted cells, positive and negative cells. (A) The growth curve of EC cells that counted day 2 to day 12 after sorting cell. (B) The photograph of spheres after sorting, cultured for 7 days. (C) The number and size of spheres. Spheres were observed in all subpopulations, but whether the numbers or the size of the spheres, the CD133+CXCR4+ cells were the most and largest (* P<.05). (D) Colony-forming assay demonstrated the difference in the each subgroups (* P<0.05, ** P<0.01). Translational Oncology 2017 10, 976-987DOI: (10.1016/j.tranon.2017.07.007) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Tumorigenicity ability of sorted cells in vivo. (A) Tumor formation in nude mice after subcutaneous injection (1.0×104). (B) Tumor formation groups were compared between the double positive and negative cells. Translational Oncology 2017 10, 976-987DOI: (10.1016/j.tranon.2017.07.007) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Drug resistance of the each subpopulation. (A and B) Cisplatin and Paclitaxel were added into the sorted cells with different concentration. Then evaluated the cell viability by CCK-8 kit (*P<.05). Translational Oncology 2017 10, 976-987DOI: (10.1016/j.tranon.2017.07.007) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Immunohistochemistry of CD133, CXCR4 in tumor tissue which from the endometrial cancer patient. The expression of CD133 in the patients (A 1-3) and the tissue of xenograft tumor (A 4). The expression of CXCR4 in the patients (B 1-3) and the tissue of xenograft tumor (B 4). Translational Oncology 2017 10, 976-987DOI: (10.1016/j.tranon.2017.07.007) Copyright © 2017 The Authors Terms and Conditions