Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

Summary

This study presents a protocol of live-3D-cell immunocytochemistry applied to a pediatric diffuse midline glioma cell line, useful to study in real-time the expression of proteins on the plasma membrane during dynamic processes like 3D cell invasion and migration.

Abstract

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using three-dimensional (3D) cell-based invasion and migration assays. In this study, we have optimized these 3D assays for live-cell immunocytochemistry. An Antibody Labeling Reagent was used to detect in real-time the expression of the adhesion molecule CD44, on the plasma membrane of migrating and invading cells of a DMG H3K27M primary patient-derived cell line. CD44 is associated with cancer stem cell phenotype and tumor cell migration and invasion and is involved in the direct interactions with the central nervous system (CNS) extracellular matrix. Neurospheres (NS) from the DMG H3K27M cell line were embedded into the basal membrane matrix (BMM) or placed onto a thin coating layer of BMM, in the presence of an anti-CD44 antibody in conjunction with the antibody labeling reagent (ALR). The live-3D-cell immunocytochemistry image analysis was performed on a live-cell analysis instrument to quantitatively measure the overall CD44 expression, specifically on the migrating and invading cells. The method also allows visualizing in real-time the intermittent expression of CD44 on the plasma membrane of migrating and invading cells. Moreover, the assay also provided new insights into the potential role of CD44 in the mesenchymal to amoeboid transition in DMG H3K27M cells.

Introduction

The ability of tumor cells to evade and disseminate through the surrounding tissue is a hallmark of cancer¹. In particular, tumor cell motility is a characteristic feature of malignant tumors, whether it is a metastatic tumor type such as breast² or colorectal cancer³ or a locally invasive type such as diffuse glioma^4,5.

Imaging has a central role in the investigation of many aspects of tumor cell phenotypes; however, live-cell imaging is definitely to be preferred when studying dynamic cellular processes such a....

Protocol

This protocol follows the guidelines of the institutions' human research ethics committees.

NOTE: This study was performed using Incucyte S3 and/or SX5 Live-Cell Analysis Instrument (referenced as live cell analysis instrument).

1. Generation of reproducibly sized tumor spheroids

NOTE: The protocol (section 1) described by Vinci et al. 2015^7,12, was used as reported below, with some modifications:

1. Collect the DMG H3K27M-mutant neurospheres (NS) and centrifuge at 170 x g for 10 minutes (....

Results

Live-3D-Cell Immunocytochemistry protocol for invasion and migration is summarized in a straightforward and reproducible workflow in Figure 1. By seeding the DMG cells in ULA 96-well round-bottom plates, reproducible sized NS are obtained and used in the steps displayed. When the NS have reached the ideal size of ~300 µm (approximately 4 days post-seeding) the invasion¹² and migration¹⁴ assays are initiated. The addition of the ALR/antibody com.......

Discussion

The live-3D-cell immunocytochemistry we have adopted here for pediatric DMG invasion and migration could be easily adapted also for other highly invasive tumor cell types, including breast and colon cancer cell lines.

Different from previously performed live-2D-cell immunocytochemistry assays¹⁰, when working in 3D, it is suggested to pay attention to some critical steps. In particular, for the invasion assay we describe, it is advised to add the ALR/antibody mix direct.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96 Well TC-Treated Microplates	Corning	3595	size 96 wells, polystyrene plate, flat bottom
Accutase	Euroclone	ECB3056D	solution for neurosphere dissociation
Burker chamber	Mv medical	FFL16034	cell counting chamber
CD-44 (156-3C11)	Cell Signaling Technology	3570	Mouse mAb IgG2a
Corning Matrigel Matrix	Corning	356237	Basement Membrane Matrix (BMM), Phenol Red-free, LDEV-free
Fabfluor-488 Antibody Labeling Dye	Incucyte	4743	Antibody labelling reagent (ALR): Mouse IgG2a 488 antibody for Live-Cell Immunocytochemistry
Incucyte S3 and/or SX5 Live-Cell Analysis Instrument	Sartorius	-	The Incucyte S3 and/or SX5 Instrument is used for real-time cell monitoring and surveillance, cell health and viability, migration and invasion, plus a wide range of phenotypic cell-based assays.
Inverted Microscope		-	any inverted microscope
Opti-Green Background Suppressor Reagent	Incucyte	6500-0045	Backgroung suppressor reagent (BSR)
Tumor stem cell (TSM) medium	-	-	growth cell medium (see reference in the text for details)
Ultra-Low Attachment Multiple Well Plate	Corning Costar	7007	size 96 well, round bottom clear