Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Andrea Comba; Patrick  J. Dunn; Phillip  E. Kish; Padma Kadiyala; Alon Kahana; Maria  G. Castro; Pedro  R. Lowenstein

doi:10.3791/60939

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Method Article

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

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DOI:

10.3791/60939

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April 12th, 2020

Andrea Comba¹^,²^,⁴ , Patrick J. Dunn¹^,²^,⁴ , Phillip E. Kish¹^,³ , Padma Kadiyala¹^,²^,⁴ , Alon Kahana³ , Maria G. Castro¹^,²^,⁴ , Pedro R. Lowenstein¹^,²^,⁴

¹Dept. of Neurosurgery, University of Michigan Medical School, ²Dept. of Cell and Developmental Biology, University of Michigan Medical School, ³Dept. of Ophthalmology & Visual Science, University of Michigan Medical School, ⁴Rogel Cancer Center, University of Michigan Medical School

Corresponding Authors: Pedro R. Lowenstein <pedrol@umich.edu>

Summary

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

Abstract

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma's growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Introduction

Gliomas are the most aggressive primary tumors of the central nervous system. They are highly invasive and heterogeneous¹. Analysis of cellular and molecular components of the tumor will reveal novel therapeutic targets.

Among different methods currently available, laser capture microdissection (LMD) of frozen brain tumor tissue is a cost-effective, reliable technique that allows the isolation of discrete anatomical areas or specific cell populations from tumor tissues to study their molecular profile²^,³. LMD allows the analysis of mRNA gene expression profil....