CD44 as a molecular marker to screen cancer stem cells in hypopharyngeal cancer

Abstract

Conclusions: The CD44⁺ cells have a stronger proliferative capacity and higher tumorigenic potential than the CD44^– cells, which suggests that the cancer stem cells of hypopharyngeal cancer may exist in the CD44⁺ tumor cell population. Therefore, we propose that CD44 is an important biological marker to screen cancer stem cells of hypopharyngeal cancer. Objectives: To study the significance of CD44 as a molecular marker for screening cancer stem cells in hypopharyngeal cancer. Methods: The CD44 expression levels in the hypopharyngeal cancer cell line FaDu were analyzed using flow cytometry. To investigate the biological significance of the CD44⁺ population, we sorted the CD44⁺ and CD44^– cell populations by using magnetic-associated cell sorting (MACS) technology. After the separation, the purity of the CD44⁺ cells was determined using flow cytometry. The MTT method was used to detect the different proliferation capabilities of the CD44⁺ and CD44^– cells in vitro. The tumorigenicity of the CD44⁺ and CD44^– cells was determined by injecting CD44⁺ or CD44^– cells (1 × 10⁶ and 1 × 10⁵) into the body of NOD/SCID mice. Results: Some (21.1 ± 1.56)% of the hypopharyngeal cancer cell line FaDu cells expressed CD44. The CD44⁺ population was efficiently sorted by MACS, and after separation, the purity of the CD44⁺ cells was (99.4 ± 0.29)%. The MTT assay indicated that the sorted CD44⁺ cells had a stronger proliferative capacity than the CD44^– cells. The tumorigenicity study showed that all the mice injected with 1 × 10⁶ CD44⁺ cells developed tumors (8/8), half the mice injected with 1 × 10⁶ CD44^– cells developed tumors (4/8), 1 of the 8 mice injected with 1 × 10⁵ CD44⁺ cells developed tumors (12.5%), but none of the mice injected with 1 × 10⁵ CD44^– cells developed any tumors (0/8). At the same concentration, the difference in tumorigenic rates between the CD44⁺ and CD44^– groups was statistically significant (Fisher's exact test, p < 0.05). Furthermore, the CD44⁺ group had a shorter incubation period than the CD44^– group. In addition, the average tumor volume of the CD44⁺ group was (2017.81 ± 538.50) mm³; however, the average tumor volume of the CD44^– group was (1153.25 ± 503.18) mm³. The difference was statistically significant (t = 2.67, p < 0.05).

Keywords::

• Tumor stem cell
• cell surface marker
• MACS
• NOD/SCID
• biological marker
• hypopharyngeal carcinoma

Acknowledgments

Mingliang Xiang was the director of this research and in charge of funds. Chenling Shen contributed to laboratory experiments and wrote the manuscript. The study was supported by the National Natural Science Foundation of China, no. 81271088; and the Natural Science Foundation of Shanghai, no. 11ZR1423600.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.