br Estimated enriched pathway analysis
Estimated enriched pathway analysis revealed the diﬀerences be-tween features of genes with high expression levels in S clone Ruxolitinib and
LL clone cells. Molecules involved in tissue development pathways (such as embryo, skeletal, sensory organ and mesoderm development) are enriched in S clone cells, and S clone cells have a tendency to have stem cell features. On the other hand, pathways related to cell pro-liferation are enriched in LL clone cells. Second-messenger signaling, which is related to cell proliferation and diﬀerentiation is enriched in LL clone cells. Recently, a strategy for cancer therapy targeting calcium signaling which is second-messenger signaling has been reported (Cui et al., 2017). MAPK signaling has been reported to be related to tumor progression and it can be a target of cancer therapy (Cheng et al., 2013). Genes related to the MAPK pathway are enriched in LL clone cells. These pathways might have a role in the higher tumorigenicity of LL clone cells.
CSCs/CICs have been reported to have high levels of tumorigenicity and chemo-resistance (Hirohashi et al., 2010; Murase et al., 2009; Park et al., 2009; Visvader and Lindeman, 2008). However, we could not explain these features of CSCs/CICs by single gene or one clone cells, even by analysis of the sample from the patient. We think that the degree of malignancy and the prognosis for a patient are determined not by one gene or one clone cells but by multiple genes or multiple phenotypical CSCs/CICs. In this study, the phenotypes as carcinoma were shared by multiple clones, including LL clone cells as tumor-in-itiating clones and S clones as chemo-resistant cells (Fig. 5B), and the gene expression profiles were quite diﬀerent among the clones. Therefore, analysis of a sample from one patient at clonal level, might
be nucessary to elucidate the complexity of CSC/CIC phenotypes.
In summary, we established several clone cells from a primary en-dometrial adenocarcinoma sample and analyzed their functions, and we found that heterogenous CSC/CIC includes tumorigenic clone cells and chemo-resistant clone cells.
The authors have no conflict of interest.
Author contribution statement
YT, YH, TS and TTorigoe wrote the main manuscript. YT, SH, TM, TA, KI, TKuroda, AM, SU and NK performed experiments. YT, YH, SH, KI, TKubo, MN, TKanaseki, TTsukahara, TS and TTorigoe analyzed data. All authors reviewed the manuscript.
This work was supported by KAKENHI (15H04722 and 17H01540) (to Y.H. and T.T.), the project for Development of Innovative Research on Cancer Therapeutics from the Japan Agency for Medical Research and Development, AMED, Ono Cancer Research Fund (to Y.H. and T.T.) and Grants-in-Aid for Regional R&D Proposal-Based Program from Northern Advancement Center for Science & Technology of Hokkaido Japan (to Y.H. and T.T.).
Al-Hajj, M., et al., 2003. Prospective identification of tumorigenic breast cancer cells.
Asano, T., et al., 2016. Brother of the regulator of the imprinted site (BORIS) variant subfamily 6 is involved in cervical cancer stemness and can be a target of im-munotherapy. Oncotarget 7, 11223–11237. Brasseur, K., et al., 2016. Chemoresistance and targeted therapies in ovarian and en-dometrial cancers. Oncotarget 8, 4008–4042. Chen, Z., et al., 2015. Stem cell protein Piwil1 endowed endometrial cancer cells with stem-like properties via inducing epithelial-mesenchymal transition. BMC Cancer 15, 811.
Y. Tabuchi et al.
Ginestier, C., et al., 2007. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1, 555–567.