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

Abstract

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.

Dual IGF1R/IR inhibitors in combination with GD2-CAR T-cells display a potent anti-tumor activity in diffuse midline glioma H3K27M-mutant

“…Primary patient-derived cell lines were derived as previously described. 21 Cell authenticity and mycoplasma test were performed. Experimental details are outlined in the Supplementary Material .…”

“…Whole brain organotypic slices (WBOS) were prepared and cocultured with DIPG NS as previously described. 21 Experimental details including treatment, staining, and image analysis are reported in the Supplementary Material .…”

Tumor Cell Invasion into Matrigel: Optimized Protocol for RNA Extraction

“…All the cell lines were tested for their authenticity by DNA fingerprinting (Eurofins Genomics Germany GmbH, Ebersberg, Germany) (Table 1) and verified mycoplasma free. Cells were cultured in 'Tumor Stem Media (TSM)' as previously described [24,25] and expanded as neurospheres (NSs). Once the NSs reached the diameter of 100-400 μm, they were collected and used for the invasion assay.…”

“…In fact, we modified this assay to be performed in a 24-well plate format in order to increase the number of NSs by well, which would ensure a higher yield of RNA. Moreover, having more and differently sized NSs per well would also allow better capture of intratumoral heterogeneity, characteristic of many tumor types and a crucial feature of pediatric high-grade gliomas (pHGGs) [20,24,[28][29][30][31][32][33]. Compared with 6-or 12-well plates, using the 24-well plates would require less reagents for the RNA extraction and thus would lower the cost of the experiments.…”

Multi-Modal and Molecular Imaging of Cellular Microenvironment and Tissue Development

“…With the aim of dissecting cell population heterogeneity in pediatric brain tumors Pericoli et al [14] developed a method to generate fluorescent barcoded cell lines derived from primary pediatric glioblastoma and pontine glioma. The barcoding was achieved by random expression of six different fluorescent fusion proteins, an approach dubbed Multifluorescent Marking Technology.…”