Supplementary MaterialsS1 Fig: Clonogenic potential of RCC cell lines under different serum concentration and normoxic and hypoxic condition. shown that tumors are highly heterogeneous, and multiple phenotypically different cell populations DC_AC50 are found in a single tumor. Cancer development and tumor growth are driven by specific types of cellsstem cell-like cancer cells (SCLCCs)which are also responsible for metastatic spread and drug resistance. This research was designed to verify the presence of SCLCCs in renal cell cancer cell lines. Subsequently, we aimed to characterize phenotype and cell biology of CD105+ cells, defined previously as renal cell carcinoma tumor-initiating cells. The main goal of the project was to describe the gene-expression profile of stem cell-like cancer cells of primary tumor and metastatic origin. Materials and Methods Real-time PCR analysis of stemness genes (Oct-4, Nanog and Ncam) and soft agar colony formation assay were conducted to check the stemness properties of renal cell carcinoma (RCC) cell lines. FACS analysis of CD105+ and CD133+ cells was performed on RCC cells. Isolated CD105+ cells were verified for expression of mesenchymal markersCD24, CD146, CD90, CD73, CD44, CD11b, CD19, CD34, CD45, HLA-DR and alkaline phosphatase. Hanging drop assay was used to investigate CD105+ cell-cell cohesion. Analysis of free-floating 3D spheres formed by isolated CD105+ was verified, as spheres have been hypothesized to contain undifferentiated multipotent progenitor cells. Finally, CD105+ cells were sorted from primary (Caki-2) and metastatic (ACHN) renal cell cancer cell lines. Gene-expression profiling of sorted CD105+ cells was performed with Agilents human GE 4x44K v2 microarrays. Differentially expressed genes were further categorized into canonical pathways. Network analysis and downstream analysis were performed with Ingenuity Pathway Analysis. Results Metastatic RCC cell lines (ACHN and Caki-1) demonstrated higher colony-forming ability in comparison to primary RCC DC_AC50 cell lines. Metastatic RCC cell lines harbor numerous CD105+ cell subpopulations and have higher expression of stemness genes (Oct-4 and Nanog). CD105+ cells adopt 3D grape-like floating structures under handing drop conditions. Sorted CD105+ cells are positive for human mesenchymal stem cell (MSC) markers CD90, CD73, CD44, CD146, and alkaline phosphatase activity, but not for CD24 and hematopoietic lineage markers CD34, CD11b, CD19, CD45, and HLA-DR. 1411 genes are commonly differentially DC_AC50 expressed in CD105+ cells (both from primary [Caki-2] and metastatic RCC [ACHN] cells) in comparison to a healthy kidney epithelial cell line (ASE-5063). TGF-, Wnt/-catenine, epithelial-mesenchymal transition (EMT), Rap1 signaling, PI3K-Akt signaling, and Hippo signaling pathway are deregulated in CD105+ cells. TGFB1, ERBB2, and TNF are the most significant transcriptional regulators activated in these cells. Conclusions All together, RCC-CD105+ cells present stemlike properties. These stem cell-like cancer cells may represent a novel target for therapy. A unique gene-expression profile of CD105+ cells could be used as initial data for subsequent functional studies and drug design. Introduction Renal cell carcinoma (RCC) is the most common type of kidney cancer and accounts for 3% of all cancer cases worldwide. The incidence of RCC has been steadily rising over the last 30 years [1]. The prognosis for patients with RCC is poor; it is believed that approximately 30%C40% of primary localized RCC patients will develop metastatic disease if it is not detected early [2]. Late detection and rapid metastasis of RCC spread has a negative impact on a patients survival. Metastatic RCC is resistant to conventional therapies, including chemotherapy and radiotherapy. Over the past ten years, targeted therapies have been developed and have shown a significant objective Rabbit polyclonal to GMCSFR alpha response rate, long progression-free survival (PFS), and overall survival (OS) in phase III clinical trials [3C5]. Resistance may have developed in the course of treatment [6]. At the same time, treatment may result in development of diverse adverse effects [7]. It was recently hypothesized that drug resistance, disease progression, and recurrence are mediated by stem cell-like cancer cells (SCLCCs) also referred to as cancer stem cells/tumor-initiating cells (CSCs/TICs) [8, 9]. This remains in accordance with recent progress in cancer research that has.