Establishment and Characterization of Brain Cancer Primary Cell Cultures From Patients to Enable Phenotypic Screening for New Drugs.

Aim: Desmoplastic infantile ganglioglioma (DIG), is a rare tumor arising mainly during the first 2 years of life. Molecular characterization of these benign yet rapidly proliferating tumors has been limited to evaluating a few mutations in few genes. Our aim was to establish a live cell culture to enable the understanding of the cellular processes driving the non-malignant growth of these tumors. Methods: Tumor tissue from a rare non-infantile 8-year-old female DIG patient was dissociated and digested using collagenase to establish live cultures. Both 2D monolayer and 3D neurospheres were successfully cultured and characterized for proliferative potential, intrinsic plasticity, presence of cancer stem cells and the expression of stem cell markers. Cells cultured as 3D were embedded as tissue blocks. Immunohistochemistry was performed in both tissue and 3D sections for markers including synaptophysin, vimentin, neurofilament and MIB-1. Mutation analysis by NGS was performed using a-100 gene panel. Results: Using immunohistochemistry, the 3D cultures were shown to express markers as in the original DIG tumor tissue indicating that the spheroid cultures were able to maintain the heterogeneity found in the original tumor. Cells continued proliferating past passage 10 indicative of immortalization. Enrichment of cancer stem cells was observed in neurospheres by FACS using CD133 antibody and RT-PCR. Mutation analysis indicated the presence of germline mutations in three genes and somatic mutations in two other genes. Conclusion: A spontaneous cell line-like cell culture with high percentage of stem cells has been established from a DIG tumor for the first time.

Urokinase-type plasminogen activator induces proliferation in breast cancer cells.

Urokinase-type plasminogen activator (uPA) is implicated in various pathophysiological processes, including extracellular matrix turnover, cell migration and invasion. Our study aimed to determine the role of uPA in both proliferation and mitogen-activated protein kinase (MAPK) pathway. Hence, we analyzed the effects induced by exogeneous addition of domain-specific uPA antibodies and uPA-interacting molecules on proliferation of uPA-suppressed MDA-MB-231 breast cancer cells. uPA expression was reduced to 53% by stable transfection with an antisense/vector construct and to 65% by siRNA transfection. Immunocytochemical Ki67 staining and flow cytometry (S-phase) analysis indicated a strong decrease of cellular proliferation activity (35% and 38%, respectively). Exogenous addition of high molecular weight-uPA (HMW-uPA) or incubation with the amino terminal fragment (ATF), which lacks the enzymatic activity of uPA, lead to increased cell proliferation. A strong increase of proliferation was absent when the monoclonal anti-uPAR antibody IIIF10 (blocking uPA binding site), soluble uPAR (scavenger effect) and phosphatidyl-inositol-specific phospholipase C (PI-PLC, degrading uPAR) was added prior to the addition of HMW-uPA. In conclusion, HMW-uPA and ATF induce proliferation of breast cancer cells by binding to uPAR. Thereby, integrins situated adjacent to uPAR carry the signals into the cell, thus stimulating proliferation that is mediated via the MAPK pathway.

In vitro suppression of urokinase plasminogen activator in breast cancer cells--a comparison of two antisense strategies.

High level expression of urokinase plasminogen activator (uPA) has been well documented in a variety of tumors. In breast cancer, expression of uPA is essential for tumor cell invasion, metastasis and proliferation. By contrast, the primary objective of tumor therapy is to reduce the uPA expression level within the tumor, which results in abrogation of proliferation, invasion and metastasizing of the tumor cells. We investigated the effects of uPA on the MDA-MB-231 cell line. MDA-MB-231 cells are highly invasive and express high levels of uPA. In our study, uPA inhibition was achieved by two methodologies: a) stable transfection with an antisense uPA vector, b) transfection with siRNA molecules (small interfering RNA). A plasmid vector was constructed by cloning a uPA-specific cDNA (612 bp) fragment into pBK-CMV plasmid in antisense orientation. In contrast, a double-stranded 21-mer siRNA was designed for targeting uPA. The antisense-transfected cells revealed decreased uPA mRNA and protein as detected by real-time PCR, immunocytochemistry, ELISA, and Western blotting. Moreover, the transfected cells exhibited a significantly reduced proliferation activity as determined by a fluorometric proliferation assay. As a conclusion of our study siRNA-technique is the superior method also regarding time saving for clone selection and instant availability of the transfected cells. Moreover, even if both strategies lead to uPA suppression, a stronger inhibitory effect could be obtained by application of the siRNA-based technique.

Identification and characterization of estrogen receptor-related receptor alpha and gamma in human glioma and astrocytoma cells.

The purpose of this study was to examine expression and function of estrogen receptor-related receptors (ERRs) in human glioma and astrocytoma cell lines. These estrogen receptor-negative cell lines expressed ERRalpha and ERRgamma proteins to varying degree in a cell context dependent manner, with U87MG glioma cells expressing both orphan nuclear receptors. Cell proliferation assays were performed in the presence of ERR isoform-specific agonists and antagonists, and the calculated EC(50) and IC(50) values were consistent with previous reported values determined in other types of cancer cell lines. Induction of luciferase expression under the control of ERR isoform-specific promoters was also observed in these cells. These results indicate that ERRalpha and ERRgamma are differentially expressed in these tumor cell lines and likely contribute to agonist-dependent ERR transcriptional activity.

The urokinase-system--role of cell proliferation and apoptosis.

The serine protease urokinase-type plasminogen activator (uPA) and its receptor (uPAR) are involved in the control of extracellular matrix turnover, cell migration, invasion and cell signalling leading to a variety of different responses, under both physiological and pathological conditions. The urokinase receptor, binding to the growth factor-like domain of uPA, directs membrane-associated extracellular proteolysis and signals through transmembrane proteins, thus regulating tissue regeneration, angiogenesis, cancer growth and metastasis. Since these physiological and patho-physiological processes of the uPA-system are known, less informations concerning uPA-induced cell proliferation and anti-apoptotic effects of the uPA-system are available. Recent studies show a close relationship of the uPA-system and cell proliferation/ apoptosis. uPA is responsible for the activation and release of different growth factors and modulates the cell proliferation/apoptosis ratio through the dynamic control of cell-matrix interactions. This article focuses on the important role of the uPA/uPAR-system for cell proliferation and apoptosis.