Development of New Cancer Therapies that Target the Cancer Stem Cell

Kawakami, Miyuki

Development of New Cancer Therapies that Target the Cancer Stem Cell

Miyuki Kawakami DDS, PhD, Depertment of Oral and Maxillofacial Surgery, The Nippon Dental University School of Life Dentistry at Niigata, Niigata, Japan

Hideaki Sato DDS, PhD, Oral and Maxillofacial Surgery, The Nippon Dental University Niigata Hospital, Niigata, Japan

Kaname Sakuma DDS, PhD, Oral and Maxillofacial Surgery, The Nippon Dental University Niigata Hospital, Niigata, Japan

Akira Tanaka DDS, PhD, Department of Oral and Maxillofacial Surgery, The Nippon Dental University School of Life Dentistry at Niigata, Niigata, Japan

Hiroshi Ishikawa MD, DMS, Department of NDU Life Sciences, School of Life Dentistry, The Nippon Dental University, Tokyo, Japan

Haruka Takahashi DDS, Department of NDU Life Sciences, School of Life Dentistry, The Nippon Dental University, Tokyo, Japan

Akihiro Ohyama PhD, Department of NDU life sciences, School of Life Dentistry, The Nippon Dental University, Tokyo, Japan

Minako Suzuki , Department of Oral and Maxillofacial Surgery, School of Life Dentistry, The Nippon Dental University at Niigata, Japan., Niigata-shi, Japan

[Purpose]

Recent research suggest that cancer stem cells (CSCs)¹⁾ contribute to tumorigenesis, metastasis, recurrence, chemoresistance and radioresistance. In this study, we report an overview of the cell-killing effect of peripheral blood-derived NK cells to CSC cell lines. The CSC cell line, derived from metastatic foci of lymph node of squamous cell carcinoma of the tongue, was established in our group.

[Methods]

The metastatic lymphnode of squamous cell carcinoma was cut into small pieces by razor blades within 60-mm dish. The fragments were dissociated with trypsin-EDTA solution at 37℃ for 30 min. After centrifugation, sediment was resuspended with growth medium (DMEM/F12, 15% FBS) and cultured in CO₂ incubator. The CSC cell line was established by colonial cloning of pilling up colony composed of small round cells in the long-term primary cultures of metastatic foci of lymph nodes. The identification of cells was performed with gene expression analysis of RT-PCR and observation of cell morphology by electron microscope. To analyze drug resistance of the cells, anti-cancer drug sensitivity test using an oxygen electrode apparatus was carried out. NK cells were isolated from human peripheral blood using a RossetteStep kit. The effect of NK cells²⁾to CSC cell line was examined by co-culturing CSC cell line with NK cells. Apoptotic cells stained with Tunnel method were calculated using an image analyzer. The amount of secreted HLA-G²⁾was determined by ELISA.

[Results]

The characteristic of CSCs is was small sphere cell, and their cell organelles were poorly developed. The gene expression analysis confirmed expression of stem cell markers (Nanog, Oct3/4, Sox2 and aldehyde dehydrogenase). In the anti-cancer drug sensitivity testing of CSCs, showed resistance to TXT and CDDP. The CSCs did not secrete little HLA-G. Furthermore, as a result of co-cultured with NK cells and CSCs, showed significant cell-killing effect against CSCs.

[Conclusion]

The CSC, showing the anti-cancer agent resistance, co-cultured with NK cells showed a significant cytotoxic effect. Possibility of the development of new NK cell-based immunotherapy for treatment-resistant oral squamous cell carcinoma was suggested.

References

1) Biological characteristics of a cell subpopulation in tongue squamous cell carcinoma. Sun Y, Han J, Lu Y, Yang X, Fan M. Oral Dis 2012; 18(2): 169-77.

2) Surface expression of HLA-G is involved in mediating immunomodulatory effects of placenta-derived multipotent cells (PDMCs) towards natural killer lymphocytes. Liu KJ, Wang CJ, Chang CJ, et al. Cell Transplant. 2011; 20: 1721-30.