Abstract
Interrogation and characterization of lung cancer stem cells (CSCs) that are implicated in lung oncogenesis is crucial for our understanding of inter- and intra-tumor heterogeneity and the aggressive nature of the disease, including tumor resistance and relapse in response to conventional therapy. Here, we describe an in vitro surrogate model, namely the “sphere-forming assay,” for the derivation, enrichment, and propagation of lung stem/progenitor cells with CSC properties, including self-renewal, tumor initiation capacity and propagation, and differentiation into cells of the tumor bulk, from a murine Kras-mutant lung adenocarcinoma cell line. Self-renewing cancer stem/progenitor cells, in the form of 3D in vitro spheres, can be phenotypically interrogated using downstream techniques such as gene expression analysis (e.g., whole transcriptome sequencing and quantitative real time PCR), which is the focus of this chapter.
Ansam Sinjab and Reem Daouk Co-contributing first authors.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Reynolds BA, Rietze RL (2005) Neural stem cells and neurospheres--re-evaluating the relationship. Nat Methods 2(5):333–336
Daouk R, Hassane M, Bahmad HF et al (2019) Genome-wide and phenotypic evaluation of stem cell progenitors derived from Gprc5a-deficient murine lung adenocarcinoma with somatic Kras mutations. Front Oncol 9:207
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15):2114–2120
Kim D, Langmead B, Salzberg SL (2015) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12(4):357–360
Pertea M, Pertea GM, Antonescu CM et al (2015) StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol 33(3):290–295
Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12):550
Bahmad HF, Cheaito K, Chalhoub RM et al (2018) Sphere-formation assay: three-dimensional in vitro culturing of prostate cancer stem/progenitor sphere-forming cells. Front Oncol 8:347–347
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Sinjab, A., Daouk, R., Abou-Kheir, W., Kadara, H. (2021). Whole Transcriptome Sequencing Analysis of Cancer Stem/Progenitor Cells Obtained from Mouse Lung Adenocarcinomas. In: Santiago-Cardona, P.G. (eds) Lung Cancer. Methods in Molecular Biology, vol 2279. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1278-1_15
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1278-1_15
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1277-4
Online ISBN: 978-1-0716-1278-1
eBook Packages: Springer Protocols