Collaboration of 3D context and extracellular matrix in the development of glioma stemness in a 3D model

Abstract

A hierarchy of cellular stemness exists in certain cancers, and any successful strategy to treat such cancers would have to eliminate the self-renewing tumor-initiating cells at the apex of the hierarchy. The cellular microenvironment, in particular the extracellular matrix (ECM), is believed to have a role in regulating stemness. In this work, U251 glioblastoma cells are cultured on electrospun polystyrene (ESPS) scaffolds coated with an array of 7 laminin isoforms to provide a 3D model for stem cell-related genes and proteins expression studies. We observed collaboration between 3D context and laminins in promoting glioma stemness. Depending on the laminin isoform presented, U251 cells cultured on ESPS scaffolds (3D) exhibited increased expression of stemness markers compared to those cultured on tissue culture polystyrene (2D). Our results indicate the influence of 3D (versus 2D) context on integrin expression, specifically, the upregulation of the laminin-binding integrins alpha 6 and beta 4. By a colony forming assay, we showed enhanced clonogenicity of cells grown on ESPS scaffolds in collaboration with laminins 411, 421, 511 and 521. Evaluation of patient glioma databases demonstrated significant enrichment of integrin and ECM pathway networks in tumors of worse prognosis, consistent with our observations. The present results demonstrate how 3D versus 2D context profoundly affects ECM signaling, leading to stemness.

Introduction

The stem cell niche is comprised of factors that help the stem cell to maintain its characteristics, factors which include interactions with other cells, adhesion molecules, soluble factors and the extracellular matrix (ECM) [1]. While the ECM has been documented to play a role in several stem cell niches [2], [3], [4], [5] and successful in vitro culture of stem cells on specific naturally-derived [6] and engineered substrates [7] offers some leads, the exact mechanisms by which the ECM acts in promoting stemness remain unclear and warrant further investigation. An enhanced understanding on the role of the ECM in regulating stemness would not only advance regenerative medicine, it would also open up new avenues in the fight against cancer. Here, certain cancers can be regarded as a heterogenous population of cells with different levels of stemness, where stem-like cells critical for tumor initiation and growth occupy the apex of a stemness hierarchy [8]. Seen in this way, transitions of cancer cells up or down this hierarchy could foreseeably determine the self-renewal, growth and drug resistant characteristics of the cancer.

Such a view may be applicable to glioblastoma (GBM), which is the most common primary malignant brain tumor in adults [9]. The growth and persistence of GBM has been shown to depend on cancer stem cells, and involve a cancer stem cell transcription network [10], [11], [12]. In a manner analogous to the reprogramming of somatic cells to pluripotent stem cells, Suva et al. showed that the introduction of four transcription factors (POU3F2, SOX2, SALL2 and OLIG2) could reprogram glioblastoma cells to acquire characteristics of glioblastoma cancer stem cells [12]. In our present work, we attempted to probe the role of the microenvironment in the regulation of glioma stemness. In order to do so, we looked at the expression of a panel of 21 related markers, which included the abovementioned transcription factors, general stemness markers, drug transporters and integrins.

In recent years, it has been increasingly recognized that cells behave differently when cultured on 2D tissue culture polystyrene (TCP) plates as compared to 3D culture systems, and certainly different when compared to cells in vivo [13], [14], [15]. The effect of the microenvironment on the cell is seen to be a product of context (3D or 2D) and the biochemical components that surround it. In this paper, the term context is used to describe the dimensional (i.e. 3D or 2D) context.

To tease apart the putative synergistic effects of the dimensional context and ECM, we have established a 3D glioma model based on a scaffold of electrospun polystyrene (ESPS) fibers that can be further coated with specific ECM components. In this way, we could investigate how various ECM types collaborate with context in affecting the development of glioma stemness, by performing gene and protein expression profiling for a panel of stemness markers under both 3D (ESPS) and 2D (TCP) conditions. For this study, we focused on a series of laminin isoforms, a class of ECM proteins that play an essential role in providing biochemical cues and structural support in almost every tissue of an organism [16]. To correlate gene and protein expression profiling findings to a functional outcome, we investigated the clonogenicity of U251 cells cultured on laminin-coated TCP and ESPS scaffolds.