Elsevier

Human Pathology

Volume 41, Issue 11, November 2010, Pages 1516-1529
Human Pathology

Original contribution
Prognostic impact of CD133 expression as a tumor-initiating cell marker in endometrial cancer

https://doi.org/10.1016/j.humpath.2010.05.006Get rights and content

Summary

Tumor-initiating cells are known to be the major source of tumor propagation and might be an attractive therapeutic target. The present study dissected the roles of CD133 as a tumor-initiating cell marker in endometrial cancer and investigated the prognostic impact of this marker expression. Flow cytometry using 6 endometrial cancer cell lines revealed that the frequency of CD133+ cells varied widely among the cell types and that Ishikawa and MFE280 cells contained significantly higher ratio (10%-20%) of such cells; therefore, these were subjected to the subsequent analyses. Sorted CD133+ cells showed more aggressive proliferative potential in vitro and more increased tumorigenicity in nude or NOD/SCID mice than CD133 cells and generated both CD133+ and CD133 cells. Furthermore, they showed apparent resistance to cisplatin- or paclitaxel-induced cytotoxicity compared with CD133 cells. CD133+ cells had a greater S-phase fraction than CD133 cells, and the serum starvation that induced G0/G1 accumulation decreased the population of CD133+ cells. Finally, we immunohistochemically analyzed the CD133 expression in endometrial cancer specimens from 62 patients. CD133 expression was not significantly associated with any of the clinicopathologic characteristic of tumors. However, the Kaplan-Meier analysis revealed that tumors with high CD133 expression showed worse overall survival (P = .023, log-rank test) than those with low CD133 expression; and the Cox regression hazard model found that high CD133 expression was an independent prognostic factor (P = .045). Thus, the present study demonstrates that CD133 is not only a tumor-initiating cell marker but also a critical prognostic marker in endometrial cancer.

Introduction

Endometrial cancer is the third most common gynecologic malignant tumor in Japan, and its incidence in Japan has increased dramatically over the last decades [1]. Although surgery, radiation, and chemotherapy protocols were established for the treatment of endometrial cancer, alternative therapies are urgently required, especially for recurrent diseases that have acquired radio- or chemoresistance. Attempts to better understand the carcinogenesis of endometrial cancer have focused on genetic alternations and molecular pathways that might be targeted by molecular-based therapies. However, most of these have not been specially designed to overcome radio- or chemoresistance.

Recently, it has been reported that a small subpopulation of cancer cells has a great advantage of cell proliferation, proposing the concept of cancer stem cells (CSCs) in malignant tumors. Demonstration of CSC existence is accomplished through an experimental strategy that combines the sorting of tumor cell subpopulations, identified on the basis of differing expressions of surface markers, with functional transplantation into appropriate animal models [2]. These studies have shown that the tumor clone is heterogeneous with respect to proliferation and differentiation and that CSCs are responsible for tumor formation and progression. Interestingly, these CSCs share with other stem cell types the key feature of self-renewal.

The existence of CSCs was first reported in acute myeloid leukemia (AML) [3], [4]. A subfraction of cells in AML resembled normal hematopoietic stem cells based upon morphologic and immunohistochemical characteristics. This subset of cells, but not the rest of the tumor cells, could form AML when xenotransplanted into immunodeficient mice. The corresponding secondary AML in mice possessed histopathologic characteristics similar to the primary tumor. Subsequently, further evidence of CSCs in breast cancer has been published [5], in which the CD44+/CD24 expression pattern associated with normal ductal stem cells was identified as a CSC marker. When small numbers of CSCs were injected into immunodeficient mice, tumors were formed at very high frequency, whereas the stem cell–negative fraction did not form tumors. The secondary tumors formed by CSCs were histologically similar to the primary tumors and also contained a subpopulation of CD44+/CD24 cells capable of forming tumors in other mice. Thereafter, emerging evidence of the existence of cells with CSC-like properties has been demonstrated as tumor-initiating cells (TICs) in a variety of solid tumors including brain tumors [6], [7], prostate cancer [8], [9], lung cancer [10], pancreatic cancer [11], colorectal cancers [12], [13], and melanoma [14], in which CD133, a 5-transmembrane glycoprotein with a molecular weight of 117 kd, has widely been used to isolate TICs. CD133 is now considered to be a potential marker of TICs in a variety of tumor types. However, little information is available regarding TICs in endometrial cancer, prompting us to investigate the significance of CD133 expression in endometrial cancer.

The present study dissected the in vitro and in vivo functional characteristics of CD133+ cells sorted from endometrial cancer cell lines. We also characterized CD133 with regard to chemosensitivity as well as cell cycle DNA profile. More importantly, we evaluated the prognostic impact of CD133 expression in endometrial cancer. Our data provide novel findings that CD133 is a TIC marker closely associated with cell proliferation and is an independent prognostic marker of endometrial cancer.

Section snippets

Cell culture

The human endometrial cancer cell lines Ishikawa, HEC1A, AN3CA, RL95-2, MFE280, and MFE296 cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), streptomycin (100 μg/mL), and penicillin (100 IU/mL) in the presence of 5% CO2.

Flow cytometry and cell sorting

Cells were incubated in phosphate-buffered saline containing 0.5% bovine serum albumin and 2 mmol/L EDTA with phycoerythrin-conjugated anti-human CD133/1 (clone AC133) or CD133/2 (clone 293C3) antibodies

Frequency of CD133-expressing cells in endometrial cancer cells

To clarify the mechanistic role of CD133 as a candidate CSC marker in endometrial cancer, we first sought to examine the frequency of CD133+ cells in 6 endometrial cancer cell lines (Ishikawa, HEC1A, AN3CA, RL95-2, MFE280, and MFE296 cells). Flow cytometric analyses using anti-human CD133/1 antibody (AC133) revealed that the ratio of the CD133+ population varied among cell lines and that Ishikawa and MFE280 cells exhibited higher ratio than others, with approximately 15.5% and 9.3% frequency,

Discussion

Among several cell surface markers identified as CSC markers, CD133 is a representative one in solid tumors. Singh et al [6] reported that a CD133+ cell subpopulation from human brain tumors exhibited stem cell properties; only 100 CD133+ cells produced a tumor that could be serially transplanted in NOD/SCID mice while keeping the phenotype of the patient's original tumor, whereas injection of 105 CD133 cells did not form a tumor. O'Brien et al [12] also identified CD133+ as a maker of TICs in

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  • Cited by (0)

    This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and the Megumi Medical Foundation of Kanazawa University (Kanazawa, Japan).

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