Cancer-initiating cells derived from established cervical cell lines exhibit stem-cell markers and increased radioresistance

Abstract

Background: Cancer-initiating cells (CICs) are proposed to be responsible for the generation of metastasis and resistance to therapy. Accumulating evidences indicates CICs are found among different human cancers and cell lines derived from them. Few studies address the characteristics of CICs in cervical cancer. We identify biological features of CICs from four of the best-know human cell lines from uterine cervix tumors. (HeLa, SiHa, Ca Ski, C-4 I).

Methods: Cells were cultured as spheres under stem-cell conditions. Flow cytometry was used to detect expression of CD34, CD49f and CD133 antigens and Hoechst 33342 staining to identify side population (SP). Magnetic and fluorescence-activated cell sorting was applied to enrich and purify populations used to evaluate tumorigenicity in nude mice. cDNA microarray analysis and in vitro radioresistance assay were carried out under standard conditions.

Results: CICs, enriched as spheroids, were capable to generate reproducible tumor phenotypes in nu-nu mice and serial propagation. Injection of 1 × 10³ dissociated spheroid cells induced tumors in the majority of animals, whereas injection of 1 × 10⁵ monolayer cells remained nontumorigenic. Sphere-derived CICs expressed CD49f surface marker. Gene profiling analysis of HeLa and SiHa spheroid cells showed up-regulation of CICs markers characteristic of the female reproductive system. Importantly, epithelial to mesenchymal (EMT) transition-associated markers were found highly expressed in spheroid cells. More importantly, gene expression analysis indicated that genes required for radioresistance were also up-regulated, including components of the double-strand break (DSB) DNA repair machinery and the metabolism of reactive oxygen species (ROS). Dose-dependent radiation assay indicated indeed that CICs-enriched populations exhibit an increased resistance to ionizing radiation (IR).

Conclusions: We characterized a self-renewing subpopulation of CICs found among four well known human cancer-derived cell lines (HeLa, SiHa, Ca Ski and C-4 I) and found that they express characteristic markers of stem cell, EMT and radioresistance. The fact that CICs demonstrated a higher degree of resistance to radiation than differentiated cells suggests that specific detection and targeting of CICs could be highly valuable for the therapy of tumors from the uterine cervix.

Figure 1

Spheroids of self-renewing cells from established human cell lines of squamous carcinomas and adenocarcinomas of the cervix. A, Morphology of cells cultured in anchorage-independent conditions as non-adherent clusters one week after plating HeLa (a), SiHa (b), Ca Ski (c), and C-4 I (d) cells. Magnification is 10X, size bar = 100 μm. B, Serial photographs during culture demonstrated progressive increase diameter at 4 (a), 8 (b), 12 (c) and 16 days (d). Magnification is 10X, size bar = 100 μm. C, Sphere-forming efficiency (SFE) of spheroid cells on serial passage from first to fifth generation (G1-G5). Cells were plated at a density of 100 cells/well in 200-μl of serum-free medium (SFM), spheres were counted weekly. Bar graph represents the SFE mean calculated by counting the number of spheres formed in a given well and dividing by the total number of cells seeded in the well, represented as a percentage. Error bars represent standard deviation (SD) (n = 3); * p < 0.05 and ** p < 0.005

Figure 2

Immune phenotype analysis of cervical tumor sphere-forming cells. Sphere-forming cells and monolayer cells were stained with CD34, CD49f, and CD133 conjugated primary antibodies and subjected to flow cytometry. Red line corresponds to sphere-forming cells, green line to monolayer cells, and black line to isotype control

Figure 3

A side population (SP) is present in HeLa and SiHa cell lines. Flow cytometry trace from cervical cell preparation incubated for 90 min at 37°C with Hoechst dye as previously described by Goodell et al. (1996). Delimited red region is the (SP) fraction with and without Verapamil

Figure 4

Cervical cancer-initiating cells (CICs) differentially express characteristic genes of CICs and epithelial to mesenchymal transition (EMT). A, Expression of CICs markers in HeLa and SiHa spheroid cells. B, Expression of EMT markers in HeLa and SiHa spheroid cells. Bar graph represents mean fold change (FC) values in spheroid cells compared with those in monolayer, data shown are representative of two independent experiments

Figure 5

Cervical cancer-initiating cells (CCICs) are more resistant to X-ray treatment. A, DNA repair double-strand breaks (DSB) genes differentially expressed in HeLa CCICs. B, as in A but SiHa CICs. C, Reactive oxygen species (ROS) metabolism genes overrepresented in HeLa and SiHa CCICs-enriched spheroids compared with monolayer counterparts. D, Cell survival curves for CCICs-enriched spheroid and monolayer cells. For x-ray treatment, cells were dissociated and irradiated in suspension with 0, 2, 4, 6, 8, and 10 Gy with a linear accelerator Clinac 2100C operating at 200 cGy/min and 6MV. Cells were then seeded in six-well dishes at an appropriate number with DMEM and noble agar (0.3%) for clonogenic survival assay. Twenty one days after irradiation colonies were fixed and stained. Colonies comprising >50 cells were counted for each dose point. To determine surviving fractions (SFs) counts were normalized using the plating efficiency (PE) of the unirradiated corresponding control. Error bars represent standard deviation (SD). Data shown are representative of three independent experiments; S: CCICs-enriched spheroid cells