Abstract 1520: Plasticity of CD44+ colorectal cancer stem cells depends on TGF-beta-induced epithelial mesenchymal transition (EMT): evidences from ex vivo culture system
Authors: Nakano Michitaka , Ariyama Hioshi , Tamura Shingo , Isobe Taichi , Miyawaki Kohta , Okumura Yuta , Kusaba Hitoshi , Ueki Takashi , Baba Eishi , Akashi Koichi 
Published: August 2015

Abstract
Background
EpCAMhigh CD44+ colorectal cancer (CRC) cells are thought to be cancer stem cells. Recently CD44- CRC cells are also suggested to acquire a character of cancer stem cells, but the molecular mechanisms have not been clarified. Epithelial-mesenchymal transition (EMT) is a possible process of cancer cells to acquire stemness. However, it is unclear whether EMT can be induced in primary human CRC.
Patients and Methods
We obtained surgical specimens from 51 CRC patients, and cultured isolated cancer cells on matrigel-coated dish with medium containing several growth factors. For induction of EMT, TGF-beta was added into the culture medium. Immunocytochemistry staining, flow cytometric analysis of the spheroid cells and single cell PCR analysis using 48-gene set containing and Embryonic stem cell (ES)-related genes EMT-related genes were performed.
Results
Cancer cells proliferated and formed sphere (&gt;50 μm in diameter) in 15 out of 51 samples after one week culture. The sphere-forming ability was related with clinical stage (Stage1 and 2:16.7% Stage 3 and 4:41.3%). Sorted single CD44+ cell had higher sphere-forming ability, compared to CD44- cell. The higher expression of ES- and EMT-related genes was observed in sphere-forming cells than in non-sphere forming cells. To confirm the heterogeneity of sphere forming cells derived from sorted single cell, we performed single cell PCR using C1 single cell auto prep system. Cells consisted of a sphere were classified into 2 different population on the basis of primary component analysis. In addition, flow cytometric analysis revealed that sphere forming CD44+ cells gave rise to CD44- cells. These results suggest that CD44+ cells have an ability to reconstruct the heterogeneous population. Next we isolated sphere-forming cells and cultured on the same condition. Isolated sphere-forming CD44+ cells could form sphere with high efficiency. This result suggests that CD44+ cell have a self-renewal ability. Previously, it has been reported that EMT was induced after TGF-beta treatment in cancer cell lines and these cells acquired cancer stem cell properties. In our culture system, we could detect very few CD44+ cells after 8-days culture of CD44- cells, and TGF-beta treatment also increased the number of CD44+ cells. These CD44 expressing cells expressed N-cadherin. Furthermore N-cadherin expressing cells expressed ES-and EMT-related genes. At last, TGF-beta enhanced sphere-forming ability in CD44- cells.
Conclusions
To our knowledge, this is the first report that CD44- cells from primary CRC samples undergo EMT and induced CD44+ cells by TGF-beta treatment. Investigation whether these induced CD44+ cells have cancer stem cell like properties such as tumorigenic potential in immunosuppressive mouse is underway. Also we are going to identify cancer stem cell specific gene expression in our culture system using DNA microarray.
Citation Format: Michitaka Nakano, Hioshi Ariyama, Shingo Tamura, Taichi Isobe, Kohta Miyawaki, Yuta Okumura, Hitoshi Kusaba, Takashi Ueki, Eishi Baba, Koichi Akashi. Plasticity of CD44+ colorectal cancer stem cells depends on TGF-beta-induced epithelial mesenchymal transition (EMT): evidences from ex vivo culture system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1520. doi:10.1158/1538-7445.AM2015-1520