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Astrocytic trans-differentiation completes a multicellular paracrine feedback loop required for medulloblastoma tumor growth

ABSTRACT: We used RNA-sequencing technology for high-throughput profiling of tumor-derived astrocytes. Laser capture microdissection of individual cells was performed on an ArcturusXT LCM system (Applied Biosystems) with an infrared laser using CapSure HS LCM caps after optimization of laser power and duration. For each sample, 250 cells were microdissected per LCM cap and split into five 50-cell technical replicates (as a control, one replicate did not undergo reverse transcription) after elution from the cap. Reamplified cDNA (~500 bp 3’ ends) was purified away from primer concatemers by two rounds of purification with 0.7x volume of AMPure XP beads (Beckman) according to the manufacturer’s recommendations, and purified cDNA was quantified by Qubit assay (Life Technologies). For each sample, 1 ng was tagmented with the Nextera XT kit (Illumina) and sequenced as 75 bp paired-end reads on a NextSeq instrument using v2 reagents (Illumina). 14–22 million reads per sample were filtered for signal to noise (chastity filtered), assessed for overall quality with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and then mapped with STAR (Dobin et al., 2013) against the mouse genome build mm10.

ORGANISM(S): Mus musculus

PROVIDER: GSE111734 | GEO | 2020/01/08

REPOSITORIES: GEO

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Project description:Lung cancers are a heterogeneous group of diseases with respect to biology and clinical behavior. Currently, diagnosis and classification are based on histological morphology and immunohistological methods for discrimination between two main histologic groups: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) which account for 20% and 80% of lung carcinomas, respectively. NSCLCs, which are divided into the three major subtypes adenocarcinoma, squamous cell carcinoma and dedifferentiated large cell carcinoma, show different characteristics such as the expression of certain keratins or production of mucin and lack of neuroedocrine differentiation. The molecular pathogenesis of lung cancer involves the accumulation of genetic und epigenetic alterations including the activation of proto-oncogenes and inactivation of tumor suppressor genes which are different for lung cancer subgroups. The development of microarray technologies opened up the possibility to quantify the expression of a large number of genes simultaneously in a given sample. There are several recent reports on expression profiling on lung cancers but the analysis interpretation of the results might be difficult because of the heterogeneity of cellular components. The methods used for sample selection and processing can have a strong influence on the expression values obtained through microarray profiling. Laser capture microdissection (LCM) provides higher specificity in the selection of target cells compared to traditional bulk tissue selection methods, but at an increased processing cost. Here we describe the use of an expression microarray study on NSCLC samples and surrounding tissue, comparing macroscopic lung tumor and tissue samples (“grind and bind”), versus tumor and alveolar compartment cells laser capture microdissected (LCM) from the same macroscopic lung samples. In this study, an initial set of 30 pairs of macroscopic tumor and non-tumor samples (10 pairs squamous-cell carcinoma, 18 pairs adenocarcinoma, 2 pairs adeno-squamous carcinoma) and 2 unpaired samples was selected for bulk/macro sampling. Of these 30 pairs and 2 unpaired samples, 17 pairs and 9 unpaired samples were reanalyzed using laser capture microdissection (LCM) for sampling the cells (7 pairs squamous and 1 unpaired sample, 9 pairs adenocarcinomas and 7 unpaired samples, 1 pair adeno-squamous carcinoma and 1 unpaired sample). For macroscopic samples, 50 to 80 µg of tissue was used to isolate total RNA. Gene expression profile was determined using Affymetrix Human Genome Gene 1.0 ST genechip. For the LCM samples, from representative slides histologically confirmed and mapped by a pathologist, approximately 1000 cells/sample were collected by LCM. cDNA was amplified using Nugen WT-Ovation One-Direct amplification system. In order to validate Nugen amplification bias of WT-Ovation One-Direct amplification system, the total RNA samples of 10 pairs of macroscopic tumor and non-tumor samples were amplified with this amplification system, and their cDNA was used to microarray.

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Astrocytic trans-differentiation completes a multicellular paracrine feedback loop required for medulloblastoma tumor growth

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