live-3d-cell-immunocytochemistry-assays-pediatric-diffuse-midline

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

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...

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The intratumor heterogeneity represents one of the most difficult challenges for the development of effective therapies to treat pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG). These brain tumors are composed of heterogeneous cell subpopulations that coexist and cooperate to build a functional network responsible for their aggressive phenotype. Understanding the cellular and molecular mechanisms sustaining such network will be crucial for the identification of new therapeutic strategies. To study more in-depth these mechanisms, we sought to apply the Multifluorescent Marking Technology. We generated multifluorescent pGBM and DIPG bulk cell lines randomly expressing six different fluorescent proteins and from which we derived stable optical barcoded single cell-derived clones. In this study, we focused on the application of the Multifluorescent Marking Technology in 2D and 3D in vitro/ex vivo culture systems. We discuss how we integrated different multimodal fluorescence analysis platforms, identifying their strengths and limitations, to establish the tools that will enable further studies on the intratumor heterogeneity and interclonal interactions in pGBM and DIPG.

Integration of Multiple Platforms for the Analysis of Multifluorescent Marking Technology Applied to Pediatric GBM and DIPG.

3D models are increasingly used to study mechanisms driving tumor progression and mimicking  processes such as invasion and migration. However, there is a need to establish more protocols based on 3D culture systems that allow for downstream molecular biology investigations.  Here we present a method for optimal RNA extraction from highly aggressive primary glioma cells invading into Matrigel. The method has been established by comparing previously reported protocols, available commercial kits and optimizing specific steps for matrix dissociation, RNA separation and purification.  The protocol allows RNA extraction from cells embedded into Matrigel, with optimal yield, purity and integrity suitable for subsequent sequencing analysis of both high and low molecular weight RNA.

Article	Year
Integration of Multiple Platforms for the Analysis of Multifluorescent Marking Technology Applied to Pediatric GBM and DIPG. International journal of molecular sciences\| PubMed ID: 32942636	2020
Tumor cell invasion into Matrigel: optimized protocol for RNA extraction. BioTechniques\| PubMed ID: 33969693	2021

Roberta Ferretti

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