A Protocol for Explant Cultures of IDH1-mutant Diffuse Low-grade Gliomas

Summary

IDH1-mutant diffuse low-grade gliomas are rare tumors that are difficult to culture. We introduce a simple, non-enzymatic explant method that preserves IDH1-mutant protein expression and sustains cultures for months. Importantly, this technique works with cryopreserved tumors, offering a valuable resource for glioma research.

Abstract

Diffuse gliomas, the most prevalent primary brain tumors, are categorized into low and high grades based on histopathological criteria and genetic mutations. Diffuse low-grade gliomas constitute a subset, predominantly afflicting young adults and predisposing to high-grade gliomas. There are two main types of diffuse low-grade gliomas: astrocytomas and oligodendrogliomas, each defined by distinct genetic and histological features. These tumors characteristically exhibit a mutated variant of the metabolic enzyme IDH1, disrupting cellular differentiation. They are heterogeneous, comprising both proliferative stem cells and more differentiated, quiescent glial cells.

Patient-specific, in vitro tumor models hold significant potential for unraveling oncogenic mechanisms and tailoring drug selection for personalized therapy. However, the absence of reliable in vitro models specifically for IDH1-mutant low-grade gliomas has hindered fundamental and translational research. Here, we present a simple method for culturing IDH1-mutant tumors as explants in defined media without needing enzymatic dissociation. These explants can be sustained for extended periods, maintaining IDH1-mutant protein expression as well as markers for oligodendrocyte progenitor cells such as ASCL1 (MASH1), OLIG1, and OLIG2 and for astrocytes (GFAP, SOX9). They can also be established from frozen tumors in DMSO. These cultures can serve as a valuable platform for drug screening, videomicroscopy, and gene studies, thus facilitating advancements in understanding and treating these challenging tumors. They also offer the potential to derive IDH1-mutant cell lines.

Introduction

Diffuse gliomas are the most frequent primary brain tumors. These tumors are currently incurable despite very intense treatment (surgery, radio, and chemotherapy)¹. Increasing evidence supports the hypothesis that gliomagenesis originates from adult stem or glial progenitor cells resident in the brain². Although glioblastomas represent the most aggressive type, diffuse low-grade gliomas (DLGG, grade 2 tumors) are also prevalent (15% of diffuse gliomas)^1,3. DLGGs grow slowly and migrate along white matter tracts, a hallmark of this 'diffuse' neoplasm<....

Protocol

The tumor fragments utilized in this protocol were obtained from a well-characterized and ethically approved collection, with patient consent. This research was conducted under the project titled Gliomacult: Characterization and Primary Cultures of Low-Grade Gliomas (Project No. RECH/P722/1-5) and was approved by the tumor biobank's scientific council on August 28, 2023.

1. Preparation of culture plates

NOTE: Perform these steps under a sterile tissue culture laminar flow hood.

1. At least 1 h before culture, prepare poly-D-lysine (PDL) and laminin-coated 24 well plates.
   ....

Results

After a few days, some tumor fragments will adhere to the well surfaces, initiating the outgrowth of cells from the explants. The time between the initial plating and the appearance of tumoral growing cells is different from sample to sample. The initial cultures often contain substantial cellular debris, making it difficult to assess the presence of viable cells. Consequently, the culture should be maintained for a minimum of 4 weeks. Once the cells start to grow, elongated cells with a bipolar or multipolar appearance .......

Discussion

Developing in vitro and preclinical models for IDH1-mutant low-grade gliomas is particularly challenging due to the slow growth of these tumors and the complexity of replicating the extensive genomic and epigenomic alterations caused by the IDH mutation. Moreover, these infiltrative tumor cells actively interact with surrounding cells, including immune, endothelial, glial, and neuronal cells, potentially relying on them for survival. This complex interplay is difficult to replicate in a laboratory setting.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-ASCL1	BD Pharmingen™	556604	Dilution 1:500
Anti-ATRX	Sigma-Aldrich	HPA001906	Dilution 1:100
Anti-CD68	Agilent	M081401-2	Dilution 1:500
Anti-GFAP	Aves Labs	AB_2313547	Dilution 1:500
Anti-IBA1	Fujifilm Wako Antibodies	019-19741	Dilituion 1:1000
Anti-IDH1 (R132H) mouse Monoclonal Antibody	Dianova	DIA-H09
Anti-Ki67	BD Pharmingen™	556003	Dilituion 1:1000
Anti-OLIG1	R&D Systems®	AF2417	Dulituion 1:300
Anti-OLIG2	Sigma-Aldrich	MABN50	Dilution 1:250
Anti-SOX9	Abcam®	EPR14335-78	Dilution 1:100
B-27 Supplement (50x), minus vitamin A	Thermo Fisher Scientific	12587010
Bovine Serum Albumin (BSA)	CiteAb	A-421-100
Ciprofloxacin HCl	Sigma-Aldrich	PHR1044-1G
CoolCell	Corning®	432000	Cell Freezing Container, for 12 x 1 mL or 2 mL Cryogenic Vials
Cover slip Ø, 13 mm	Karl Hecht™ Assistent™	328017
Dimethyl Sulfoxide (DMSO)	Sigma-Aldrich	D8418
DMEM/F-12, no glutamine	Thermo Fisher Scientific	21331020
DNAse I	Sigma-Aldrich	10104159001
Dulbecco′s Phosphate Buffered Saline (PBS) 1x	Sigma-Aldrich	D8537
Falcon 100 mm TC-treated Cell Culture Dish	Corning®	353003
Falcon 24-well Clear Flat Bottom	Corning®	353047	TC-treated Multiwell Cell Culture Plate, with Lid, Sterile
Fungin	InvivoGen	ant-fn-1
Gentamicin	Thermo Fisher Scientific	15750-037
Heparin	Sigma-Aldrich	H3149-100KU
HEPES	Sigma-Aldrich	H-3375
Human EGF	PeproTech	AF-100-15-1MG
Human FGF-basic	PeproTech	100-18B-1MG
Laminin	Sigma-Aldrich	L2020
L-Glutamine	Thermo Fisher Scientific	25030-024
N-2 Supplement (100x )	Thermo Fisher Scientific	17502-048
PDL	Sigma-Aldrich	P7886
Serum acrodisc 37 mm syringe filter with GF/0.2 µm Supor	PALL Corporation	4525
Trypsin Inhibitor	Thermo Fisher Scientific	17075-029
Trypsin-EDTA (0.05%), phenol red	Thermo Fisher Scientific	25300054
Zeiss Z1 Apotome 3 microscope	Zeiss		straight widefield epifluorescence microscope