Molecular determinants of the interaction between glioblastoma CD133⁺ cancer stem cells and the extracellular matrix

Abstract

Glioblastoma multiforme (GBM) is the most common primary tumor of the human brain. It is characterized by invasive growth and strong resistance to treatment, and the median survival time of patients is 15 months. The invasive growth of this tumor type is associated with tumor cells with an aggressive phenotype, while its treatment resistance is attributed to cancer stem cells (CSCs). It remains unclear if CSCs have a more invasive nature than differentiated glioblastoma cells (DGCs), and what contribution CSCs make to the aggressive phenotype of GBM. Interaction with the extracellular matrix (ECM) is a key factor in the development of invasion. The aim of the present study was to compare the expression levels of signaling pathway proteins involved in interaction of receptors with the ECM in CSCs and DGCs. The U-87MG GBM cell line was used in the present study CSCs were extracted from gliomaspheres through magnetic-activated cell sorting based on the expression of cluster of differentiation 133 (CD133); CD133-negative DCGs were used as a control. HPLC and mass spectrometry were also used, and biological and molecular functions, signaling pathways and protein-protein interactions were analyzed using publicly available databases. Increased expression levels of the following 10 proteins involved in interaction with the ECM were identified in CSCs, compared with expression levels in DGCs: COL6A1, COL6A3, FN1, ITGA2, ITGA5, ITGAV, ITGB1, ITGB3, LAMB1 and LAMC1. The proteome of CSCs was observed to have > 2-fold higher expression of these key proteins, when compared with the DGC proteome. Increased expression levels of four proteins (FERMT2, LOXL2, HDAC2 and FBN1) involved in activating signaling in response to receptor interaction with the ECM was also observed, indicating that CSCs may have highly invasive nature. LOXL2 expression level was > 9-fold higher in CSCs compared to DGCs, suggesting that this protein may have potential as an marker for CSCs and as a target for this cell type in GBM.

Introduction

Glioblastoma multiforme (GBM) is one of the most aggressive types of human brain cancer. It is characterized by rapid invasive growth, significant brain infiltration and strong resistance to treatment. Despite advances in surgery, radical elimination of the tumor without causing severe and irreversible neurological damage to the patient remains a challenge (Costa, Lawson, Lelotte, et al., 2019). Therefore, the primary treatment type is high-dose radiation with multiple rounds of chemotherapy (Lukas, Wainwright, Ladomersky, et al., 2019). Prognosis for patients with this type of tumor is unfavorable, as the survival rate at 2 years following diagnosis is 27–43%, and the median survival time of patients with GBM is 15 months (Stupp & Ram, 2018). Due to their invasive ability, cancer cells that have deeply penetrated the brain tissue from the original lesion inevitably cause relapses, requiring new approaches and treatment protocols for patients with this disease.

The ability to penetrate the brain matter (Perrin, Samuel, Koszyca, et al., 2019) is due to the mesenchymal-like aggressive phenotype of cancer cells, and is influenced by coordinated intercellular interactions with the local microenvironment (Roos, Ding, Loftus, et al., 2017). The key properties of this phenotype include switching from adhesive E-cadherins to migratory N-cadherins, extending the range of integrin receptors on the cellular surface, developing the ability to produce extracellular matrix (ECM) components, synthesizing matrix metalloproteases (Bryukhovetskiy & Shevchenko, 2016).

Focal adhesion proteins are critical to underpinning the invasive potential of cancer cells with an aggressive phenotype (Bolteus, Berens, & Pilkington, 2001). GBM cells penetrate the brain tissue, adhere to ECM proteins and create cellular-matrix connections with structural and signaling functions that interact with the microenvironment (Huttenlocher & Horwitz, 2011). These connections between the ECM and the actin cytoskeleton of the cells also generate tension that is required for migration beyond the primary lesion. Inhibition of the interaction between cancer cells and the ECM is one of the aims of GBM treatment.

GBM relapse and progression is also attributed to cancer stem cells (CSCs) that have unique signaling and morphological properties (Singh, Clarke, Terasaki, et al., 2003), including an ability to initiate and support rapid tumor growth. CSCs occupy the dominant position in the GBM cellular hierarchy, and are detected at the anterior border of the tumor (Gimple, Bhargava, Dixit, et al., 2019), indicating that they are involved in invasive processes. However, it remains unclear if CSCs are more invasive than differentiated tumor cells, and if they contribute to the development of GBM with an aggressive phenotype.

The CD133 antigen is the most reliable marker for CSCs (Singh, Hawkins, Clarke, et al., 2004). This marker is introduced into GBM tissue by the symmetrical division of CSCs and by reprogramming of differentiated CD133-negative (CD133 −) tumor cells resulting in phenotypic plasticity (Li, Zhou, Xu, & Xiao, 2013) in the tumor cell population. The ability of to recruit normal neural CD133-positive (CD133 +) stem cells further complicates our understanding of its heterogeneity (Okawa, Gagrica, & Blin, 2017). The aim of the present study was, therefore, to compare the expression profiles of proteins involved in the interaction with the ECM, and signaling pathways in CD133 + CSCs and differentiated glioblastoma cells (DGCs). The present study may lead to the discovery of new molecular targets for regulating the invasive activity of CSCs.