Abstract 1947: Plasticity of CD44+ colorectal cancer stem cells depends on TGF-beta-induced epithelial mesenchymal transition(EMT): Evidences from an ex vivo culture

Abstract

Abstract Background EpCAMhigh CD44+ colorectal cancer (CRC) cells are thought to be cancer stem cells. Recently CD44- CRC cells are also suggested to gain a character of cancer stem cells, but the molecular mechanisms have not been clarified. Epithelial-mesenchymal transition (EMT), which is observed in several caner cell lines, could be a possible process of cancer cells to obtain stemness. However, whether primary human CRC cells underwent EMT has not been proven. Patients and Methods We obtained surgical specimens of 30 cases of advanced CRC, and cultured isolated primary cancer cells in matrigel-based medium containing several growth factors. For induction of EMT, TGF-beta was added into the culture of CD44+ and CD44- cells. Immunocytochemistry staining and flow cytometry of the spheroid cells and single cell PCR analysis of the cultured cells using 48-gene set that included EMT-related genes (BioMark HD System:Fluidigm Corporation) were performed. Results Cancer cells grew spherically (50 μm> diameter) in 20 out of 30 samples after a week culture of whole isolated cancer cells. The sphere mostly consisted of CD44+ cells, and non-sphere forming single cells were CD44-negative. Isolated CD44+ cells from primary samples gave rise to CD44+ and CD44- cells in a week culture. Interestingly, 10% of CD44- cell from primary samples also expressed CD44. Similar to the previous findings of cell lines that TGF-beta-induced EMT could convert CD44- cells into CD44+ cancer initiating cells, increased number of CD44+ cells appeared from primary CD44- cells cultured with TGF-beta. In this culture, N-cadherin+ cells increased time-dependent manner and most of this N-cadherin expressing cells co-expressed CD44. In single cell PCR analysis, increased expression of EMT-related genes, vimentin and TWIST1, were preferentially detected in CD44+N-Cadherin+ cells. Conclusions To our knowledge, this is the first report that CD44- cells from primary samples undergo EMT and convert into CD44+ cells by TGF-β treatment. Investigation whether these CD44+ converted cells have cancer stem cell properties such as tumorigenic potential in immunodeficient mouse, self renewing ability and drug resistance is underway. Citation Format: Michitaka Nakano, Hiroshi Ariyama, Shingo Tamura, Taichi Isobe, Kohta Miyawaki, Yuta Okumura, Hitoshi Kusaba, Eishi Baba, Koichi Akashi. Plasticity of CD44+ colorectal cancer stem cells depends on TGF-beta-induced epithelial mesenchymal transition(EMT): Evidences from an ex vivo culture. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1947. doi:10.1158/1538-7445.AM2014-1947