Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

Abstract

Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133⁺ cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

Introduction

Worldwide, approximately 700,000 people develop head and neck carcinomas each year, especially squamous cell carcinoma of the oral cavity, pharynx, and larynx [1]. Head and neck cancer is listed in sixth place among the most common malignant tumors worldwide and is responsible for ∼350,000 cancer deaths per year [2]. Unfortunately, the incidence of oral tumors is increasing around the world [3], [4].

The current treatment strategy for carcinomas attempts to attack homogenous cancerous tissues. Recently, data shows that tumors are organized as hierarchical tissues containing various differentiated cells with a small subpopulation of undifferentiated cells, so-called cancer stem cells (CSCs) that are the focus of a new strategy of treatment [5], [6], [7]. In models of CSCs, these cells exclusively have the capability for tumor formation and growth, and are possibly slow-cycling cells resistant to therapies targeting fast proliferating cells [8]. This tumor-initiating cells hypothesis has recently been tested for a number of diseases including squamous cell carcinoma [9]. The isolation of CSCs makes the discovery of more efficient cancer treatment strategies possible.

Although the existence of CSCs has been demonstrated for many tumors, the isolation of these tumor-initiating cells is still difficult because a definitely unique CSC-marker is not available. One possible marker of CSCs is the surface marker CD133 [10], [11]. This surface marker, also known as AC133 and Prominin-1, is expressed in glioblastoma cells [12]. A method for the enrichment of oral squamous cell carcinoma CSCs (OSCCSCs) was the cultivation of oral squamous cell carcinoma lines in serum-free cell culture medium [13]. Here, cells formed spheres and expressed pluripotent stem cell markers such as Oct-4 and the CSC-marker CD133. Interestingly, in comparison to their parental OSCC cell line, these OSCCSCs demonstrated increased cell viability after treatment with known anticancer treatments such as Paclitaxel or radiation therapy under in vivo and in vitro conditions [13], [10]. CSC-like cells, which have some properties of multipotent stem cells, can also be enriched from established cancer cell lines after treatment with anticancer drugs such as cisplatin [14], [15]. Interestingly, the proliferation of a myriad of CSCs depends on the activation of the WNT pathway, an important target of novel CSC drugs [14], [10], [9].

Harper et al. identified CSCs in head and neck squamous cell carcinoma (HNSCC) cell lines [16], [17]. This study obtained colonies with variable morphologies from single cell suspension of HNSCC cells, which defines the degree of cell differentiation. Colonies with holoclone morphology contain undifferentiated, stem cell-like cells. The formation of paraclones or meroclones reveals cells with a higher degree of differentiation that are less likely that to contain CSCs [16], [17]. Moreover, Locke et al. have demonstrated that the pattern of stem cells and differentiated cells is robust and persists even in cancer cell lines [16].

We investigated OSCC cell lines from tissues with variable degrees of differentiation for CSCs. Cell lines were evaluated for their expression of stem cell markers such as CD133, their ability to form colonies in standard medium and their potential for cultivation and enrichment of CSC-like cells under serum-free cell culture conditions. We reveal that cell lines with CSC-like qualities could be obtained from holoclone colonies of established OSCC cell lines. Cells obtained from these holoclone colonies had differentially expressed the stem cell marker CD133. In contrast to parental cell lines, WNT signaling stimulates cell proliferation of these holoclone-derived cells.