Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Summary

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Abstract

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Introduction

Gliomas are primary solid tumors of the central nervous system classified by their phenotypic and genotypic characteristics according to the World Health Organization¹. This classification incorporates the presence or absence of driver mutations, which represent an attractive source of biomarkers². Biomarkers are biological characteristics that can be measured and evaluated to indicate normal and pathological processes, as well as pharmacological responses to a therapeutic intervention³. Biomarkers can be detected in tumor tissue and cells derived from glioma, enabling its biological characterizat....

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Michigan.

1. Generation of Brain Tumor Neurospheres Derived from a Mouse Model

1. Before the dissection procedure, prepare neural stem cell media (NSC Media: DMEM/F12 with 1x B27 supplement, 1x N2 supplement, 1x normocin, and 1x antibiotic/antimycotic supplemented with human recombinant EGF and basic-FGF at concentrations of 20 ng/mL each). Fill a 1.5 mL tube with 300 µL of NSC media and reserve for later.
2. Select a mouse with a large tumor.
   NOTE: The size of a tumor can be monitored by ....

Results

To monitor tumor development and evaluate if the tumor has reached the size necessary to generate NS, we analyzed the luminescence emitted by tumors using bioluminescence. This allows the study of transduction efficiency in the pups and tumor progression by the luminescence signal of luciferase (Figure 1A and 1B). When the tumor size reaches a signal of 1 x 10⁷ photons/s/cm2/sr (Figure 1

Discussion

In this article, we detail a versatile and reproducible method to perform immunohistochemistry on paraffin-embedded glioma NS, which maintain the characteristic 3D structure of tumor cells growing in the brain in situ. This technique possesses the following advantages over using cells plated onto glass or plastic surfaces: i) preservation of the 3D structure of NS; ii) avoiding of stress of cell dissociation before analysis of protein expression; iii) quenching of any endogenous fluorophore expression from genet.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Coated microtome blade HP35	Thermo Scientific	3150734
Microtome RM 2135	Leica	MR2135
Formalin solution, neutral buffered, 10%	Sigma-aldrich	HT501128-4L
Rabbit polyclonal anti-ATRX,	Santa Cruz Biotechnology	sc-15408	IHC, 1:250 dilution
Rabbit polyclonal anti-Ki-67	Abcam	Ab15580	IHC, 1:1000 dilution
Rabbit polyclonal anti-OLIG2	Millipore	AB9610	IHC, 1:500 dilution
Goat polyclonal anti-rabbit biotin-conjugated	Dako	E0432	IHC, 1:1000 dilution
Vectastain Elite ABC HRP kit	Vector Laboratories Inc	PK-6100
BETAZOID DAB CHROMOGEN KIT	Biocare medical	BDB2004 L/price till 12/18
N-2 Supplement	ThermoFisher	17502048
N-27 Supplement	ThermoFisher	A3582801
Accutase® Cell Detachment Solution	Biolegend	423201
AGAROSE LE	GoldBio	A-201-1000
Genesee Sc. Corporation	Olympus 15 ml	21-103
Genesee Sc. Corporation	TC-75 treated Flask	25-209
Genesee Sc. Corporation	TC-25 treated Flask	25-207
DMEM/F12	Gibco	11330-057	NS media
HBSS	GibcoTM	14175-103	balanced salt solution
C57BL/6	Taconic	B6-f	C57BL/6 mouse