A 3D Spheroid Model for Glioblastoma

Summary

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Abstract

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Introduction

In most studies using primary or commercially available cell lines, assays are performed on cells grown on plastic surfaces as monolayer cultures. Managing cell culture in 2D represents disadvantages, as it does not mimic an in vivo 3D cell environment. In 2D cultures, the entire cell surface is directly in contact with the medium, altering cell growth and modifying drug availability. Furthermore, the nonphysiological plastic surface triggers cell differentiation¹. Three-dimensional culture models have been developed to overcome these difficulties. They have the advantage of mimicking the multicellular architecture and heterogeneity of tumors

Protocol

Informed written consent was obtained from all patients (from the Haukeland Hospital, Bergen, Norway according to local ethics committee regulations). Our protocol follows the guidelines of our institution’s human research ethics committee. 

1. Generation of uniform size tumor spheroids

NOTE: Stem-like cells are cultured in neurobasal medium complemented with B27 supplement, heparin, FGF-2, penicillin, and streptomycin, as described in previous articles¹². These cells spontaneously form spheroids in culture.

1. Wash the tumor cells with 5 mL phosphate-buffered saline (P....

Results

Spheroids were prepared as described in the protocols section and observations were made regarding migration, invasion, proliferation, and microscopy. To measure hypoxia in distinct areas of the spherical structure, carboxic anhydrase IX staining was used for determining hypoxic activity (Figure 1A-C). More CAIX-positive cells were observed in the spheroid center (Figure 1A-C). Hypoxic cells loca.......

Discussion

Tumor spheroid assays are well adapted to study tumor characteristics including proliferation, invasion, and migration, as well as cell death and drug response. Cancer cells invade the 3D matrix forming an invasive microtumor, as seen in Figure 4B,C. During the invasive process, matrix metalloproteinases (MMP) digest matrices surrounding tumor cells¹³, and MMP inhibitors (e.g., GM6001 or Rebimastat) may impair cell invasion but not migration

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96 well round-bottom plate	Falcon	08-772-212
Accutase	Gibco	A11105-01	Stored at 4 °C, sphere dissociation enzyme
B27	Gibco	12587	Stored at -20 °C, defrost before use
Basic Fibroblast Growth Factor	Peprotech	100-18B	Stored at -20 °C, defrost before use
Countess Cell Counting Chamber Slides	Invitrogen	C10283
DPBS 10X	Pan Biotech	P04-53-500	Stored at 4 °C
Fiji software	ImageJ		Used to analyze pictures
Flask 75 cm2	Falcon	10497302
Matrigel	Corning	354230	Stored at -20 °C, diluted to a final concentration of 0.2 mg/mL in cold NBM
Methylcellulose	Sigma	M0512	Diluted in NBM for a 2% final concentration
NBM	Gibco	21103-049	Stored at 4 °C
Neurobasal medium	Gibco	21103049	Stored at 4 °C
Penicillin - Streptomycin	Gibco	15140-122	Stored at 4 °C
Trypan blue 0.4%	ThermoFisher	T10282	Used to cell counting
Type I Collagen	Corning	354236	Stored at 4 °C