Expression profiling of stem cell signaling alters with spheroid formation in CD133high/CD44high prostate cancer stem cells

Cancer stem cells (CSC) isolated from multiple tumor types differentiate in vivo and in vitro when cultured in serum; however, the factors responsible for their differentiation have not yet been identified. The first aim of the present study was to identify CD133high/CD44high DU145 prostate CSCs and compare their profiles with non-CSCs as bulk counterparts of the population. Subsequently, the two populations continued to be three-dimensional multicellular spheroids. Differentiation was then investigated with stem cell-related genomic characteristics. Polymerase chain reaction array analyses of cell cycle regulation, embryonic and mesenchymal cell lineage-related markers, and telomerase reverse transcriptase (TERT) and Notch signaling were performed. Immunohistochemistry of CD117, Notch1, Jagged1, Delta1, Sox2, c-Myc, Oct4, KLF4, CD90 and SSEA1 were determined in CSC and non-CSC monolayer and spheroid subcultures. Significant gene alterations were observed in the CD133high/CD44high population when cultured as a monolayer and continued as spheroid. In this group, marked gene upregulation was determined in collagen type 9 α1, Islet1 and cyclin D2. Jagged1, Delta-like 3 and Notch1 were respectively upregulated genes in the Notch signaling pathway. According to immunoreactivity, the staining density of Jagged1, Sox2, Oct4 and Klf-4 increased significantly in CSC spheroids. Isolated CSCs alter their cellular characterization over the course of time and exhibit a differentiation profile while maintaining their former surface antigens at a level of transcription or translation. The current study suggested that this differentiation process may be a mechanism responsible for the malignant process and tumor growth.


Introduction
It has been widely accepted that tumor growth is sustained by a rare subpopulation of putative cancer stem cells (CSCs)/progenitor-like cells that share specific characteristics with normal stem cells, namely self-renewal, clonogenicity and multipotency (1)(2)(3). Previous investigations have shown that a number of tumors may actually arise from the transformation of progenitor cells rather than stem cells (4,5). Normal stem cells and CSCs share significant properties, such as heterogeneity and plasticity. Maturation and differentiation are important in cancer cell heterogeneity, and tumor cell heterogeneity may result from clonal evolution driven by genetic instability of stem-like cells, frequently called CSCs or tumor-initiating cells (6). Cells in this heterogeneous population exist in various stages throughout their lifetime. During early tumor development or in unperturbed tumor conditions, CSCs mainly undergo one-way maturation by developing into tumor progenitor cells and even differentiated tumor cells (7). It is possible to assume that these differentiated cells may arise from CSCs, which have self-renewal capacity and/or phenotypically differentiated tumor cells that functionally possess low or no tumor-regenerating capacity (non-CSCs/bulk population). CSCs are the cell subpopulation that are most likely responsible for treatment failure and cancer recurrence, while the bulk population of tumor cells exhibit low self-renewal capacity and a higher probability of terminal differentiation (i.e. transit-amplifying cancer progenitor cells) (8).
CSCs have been previously isolated and identified using common cell surface markers in the majority of cancer types, including brain (9,10), kidney (11), liver (12,13), colon (14), Expression profiling of stem cell signaling alters with spheroid formation in CD133 high /CD44 high prostate cancer stem cells pancreas (15) and prostate (16). CD133, also known as prominin-1 or AC133 (a glycoprotein comprising of five transmembrane domains), has been described as a marker of stem cells in several organs and appears to be the CSC marker for a number of tumor types (17). However, there have been accumulating results demonstrating that CD133 + and CD133subpopulations are tumorigenic in metastatic glioblastoma and colon cancer (18)(19)(20). CD44 is a member of the cell adhesion protein family and the expression of several CD44 proteins has been found to correlate with aggressive stages of various types of human cancer (21). An evident function of the CD44 family members is their alternative splicing. Previously, Ponta et al demonstrated that CD44 family members differ in the extracellular domain by the insertion of variable regions through alternative splicing (22). A small subset of CD44 + cells in prostate cell cultures and xenograft tumors are more tumorigenic, proliferative, clonogenic and metastatic as compared with the CD44subpopulation. This CD44 + subset expresses higher mRNA levels of several genes that are characteristic of embryonic stem cells (23). In addition, Collins et al have shown that prostate cancer tumorigenic cells have a CD44 + /1α2β1 high /CD133 + phenotype (24).
A challenge has been encountered with regard to the enrichment of CSCs from the established cell lines of a variety of solid tumors that develop as three-dimensional (3D) cell cultures. The 3D spheroid model is a new technique for the propagation of cells in vitro using serum-free medium and cultured under low-adherence conditions (25). An additional usage of spheroids constitutes the liquid overlay technique, namely multicellular tumor spheroids (26) The 3D spheroid model presents a convenient model to investigate cancer cells and has been increasingly used for this purpose. It reproduces in vitro results in accordance with in vivo results and generates significant in vitro characteristics not observed in monolayers or suspension cultures.
The present study hypothesized that the structure of CSCs may show differentiation when compared with non-CSCs, and differentiation of stem cell markers may aid therapeutic strategies of cancer. Therefore, the current study describes approaches to present and analyze the differentiation properties of human prostate CSCs within 3D spheroids, which may serve as the basis for defining the gene and protein trace of CSCs.

Constitution of spheroids and sphere formation assay.
For spheroid cultures, the tumor cells grown as monolayer were resuspended with trypsin and the clonogenic potential of various phenotypic populations was analyzed in a 3D non-adherent culture condition (plates coated with 3% Noble agar; Difco Laboaratories Inc., BD Diagnostic Systems, Detroit, MI, USA). The cells were counted, resuspended and plated with 1x10 3 cells per well in a six-well plate. Two weeks following initiation, the plates were inspected for colony (sphere) growth. The number of colonies within each well was counted under the microscope (Olympus BX-51, Olympus, Hamburg, Germany) and images of representative fields were captured. First passage floating spheres were removed and gently disaggregated with a new 3% Noble agar-coated well.
Immunohistochemical analysis. Immunohistochemistry was adapted and modified from our previous protocols (27). Briefly, monolayer cells were maintained in 24-well plates and fixed with paraformaldehyde. The spheroids were processed in routine histological processing for embedding in paraffin wax. Cells were incubated with primary antibodies overnight at 40˚C in a humidity chamber. The modified Streptavidin-Peroxidase technique was then used. Following incubation with 3,3'-diaminobenzidine (Invitrogen Life Technologies), sections were counterstained with Mayer's hematoxylin (Sigma-Aldrich). Immunoreactivity of molecules was assessed by light microscopy using Olympus BX-51 and C-5050 digital cameras. Staining was graded independently by two observers, blinded to the groups, who evaluated semi-quantitatively using the following scale: Mild, +; moderate, ++; and strong, +++.

Results
CD133 high /CD44 high CSC and non-CSC subpopulation purity and sorting rates. Prior to performing the microarray, the purity of CSC and non-CSC samples was tested with CD133  In order to confirm the flow cytometry analysis, cells were re-evaluated following sorting, and this analysis was repeated after one passage. Results showed that the purity of the cells was 85% and immunofluorescence staining yielded a cell purity of >85% in all samples.

Analysis of TERT and cell cycle regulation gene products.
Following cell separation with FACS ( Fig. 1), the differentially expressed genes of the DU145 human prostate cell line were analyzed in CD133 high /CD44 high (CSCs) and their bulk counterpart (non-CSCs) cultured as monolayer cells or 3D spheroids. In general, notable differences were observed between the CSCs spheroid (S + ) and monolayer (M + ) groups. These two groups constituted of CSCs and TERT expression was increased significantly in the S + group when compared with the M + group ( Fig. 2A). An additional large population of genes related to cell cycle regulation were analyzed, including CCND1, CCND2, CCNE1, CDK1, CDC42, EP300 and MYC. These genes were upregulated in the S + group versus the M + group. CCND2 expression increased in the non-CSC counterpart of the Sgroup compared with the S + group. On the other hand, CCND2 was significantly reduced in the M + group compared with the Mgroup ( Fig. 2A).   The Notch signaling pathway is important in cell-to-cell communications that regulate multiple cell differentiation processes during embryonic and adult life (28). In the present study, the expression of NOTCH1, NOTCH2, JAG1, DLL1, DLL3, DTX1, DTX2, DVL1, KAT2A, HDAC2 and NUMB was investigated. The expression of these genes significantly increased in the S + versus the M + populations compared with the other groups. The Notch signaling genes, JAG1, DLL3, NOTCH1, DTX1 and DLL1, were of higher levels when compared with other upregulated genes (Fig. 2B).

Analysis of embryonic cell lineage and
Analysis of mesenchymal cell linage gene products. The ACAN, ALPI, BGLAP, COL1A1, COL2A1, COL9A1 and PPARG genes were evaluated. Significantly, the COL9A1 gene was upregulated in the CSC spheroid as compared with the CSC monolayer. The expression of COL2A1 and COL9A1 genes was reduced in the non-CSC spheroids when compared with the monolayers (Svs. M -) (Fig. 2C).
Immunohistochemical analysis of stem cell markers. Results of the immunohistochemical analyses revealed that embry-onic stem cell markers increased following the differentiation of CSCs when the cells constituted a spheroid formation. Immunohistochemistry of CD117, Notch1, Jagged1, Delta1, Sox2, c-Myc, Oct4, KLF4, CD90 and SSEA1 was determined in the various groups. Positive immunoreactivity was observed in CSCs and non-CSCs whether the cells were maintained in monolayer culture or as spheroid. The monolayer CSCs showed low (+) immunoreactivity scores (Fig. 3), while the monolayer non-CSCs (Fig. 4) showed moderate (++) immunoreactivity. Increased nucleus/cytoplasm ratios, decreased cell diameter and enhanced immunoreactivity were observed in the CD133 high /CD44 high population. The staining density of Jagged1, Sox2, Oct4 and Klf-4 increased significantly in this monolayer CSCs population. On the other hand, strong (+++) immunoreactivity was observed in the CSC spheroids (Fig. 5) when compared with the non-CSC spheroids (Fig. 6). Among these spheroids, a moderate (++) immunoreactivity score was observed for Notch1, Jagged1 and Delta1 in the non-CSCs spheroids while strong (+++) immunoreactivity was observed in the other groups. Moreover, the highest immunoreactivity was observed in the CSC spheroid group when compared with the monolayer CSCs, monolayer non-CSCs or spheroid non-CSCs group.

Discussion
Despite limited data in the previous literature, the differentiation of CSCs may be investigated. Cancer cells capable of undergoing proliferation have the ability to self-renew and their differentiation properties are unique to CSCs. In the present study, this differentiation hypothesis was examined by using an in vitro 3D-tumor differentiation spheroid model. The cells were found to alter their gene expression profiles during this process. Our hypothesis was supported by the observation that significant gene alterations were observed in the CD133 high /CD44 high population when the monolayer cells were allowed to grow as spheroid. In this group, a marked upregulation was determined in COL9A1 and ISL1 compared with other genes. Type IX collagen is covalently bound to the surface of type II collagen fibrils within the cartilage extracellular matrix (29). Collagen IX is required for the integrity of collagen II fibrils and the regulation of vascular plexus formation (30 (32). The present study reported, for the first time, that ISL1 is an additional significantly upregulated gene in prostate spheroid CSCs. ISL1 + multipotent precursors have the potential of self-renewal and differentiation into endothelial, cardiomyocyte and smooth muscle lineages. These features highlight postnatal angiogenesis and vasculogenesis by improving the angiogenic properties of endothelial cells and mesenchymal stem cells (33). Angiogenesis is critical for tumor growth, and the VEGF pathway and Notch signaling are perhaps two of the most important mechanisms in the regulation of embryonic vascular development and tumor angiogenesis (34). According to our recent study, Notch signaling affects ovarian carcinomas and Notch1 expression correlates with metastasis, while Jagged1 expression correlates with tumor grade (27). However, it was demonstrated that in spheroids, all genes in Notch signaling are significantly upregulated, particularly Jagged1, DLL3 and Notch1. High Jagged1 expression has been demonstrated to predict a worse outcome in breast cancer (35,36), renal cell carcinoma (37) and colon adenocarcinoma (38). It has also been reported that high Jagged1 expression is associated with prostate cancer recurrence (39). Furthermore, Jagged1 signaling regulates hemangioma stem cell-to-pericyte/vascular smooth muscle cell differentiation (40). The abovementioned observations indicate that cellular organizations in CSCs accompany vascular development or extracellular structuring with the possible tendency of epithelial mesenchymal transition. The most upregulated cyclin was CCND2, which is implicated in cell differentiation and malignant transformation and is inactivated by promoter hypermethylation in several types of human cancer. High DNA methylation levels of CCND2 cause deregulation of the G1/S checkpoint and correlate with clinicopathological features of tumor aggressiveness in breast and types of prostate cancer (41,42).
In conclusion, isolated CSCs in human tumors may alter their cellular characterization with time and exhibit differentiation by maintaining their former surface antigens at the level of transcription or translation. This differentiation may be a principal mechanism that is responsible for the malignant process and tumor growth. As demonstrated in the current study, upregulated genes of angiogenesis and mesenchymal transition or cellular tendency to the vascular development appear to be due to malignancy and tumor progression. Overall, these determinations indicated the differentiation of CSCs, but must be further validated with a series of patient samples derived from primary and/or metastatic lesions of prostate cancer.