Quantitation of Murine Stroma and Selective Purification of the Human Tumor Component of Patient-Derived Xenografts for Genomic Analysis

• Published in: PloS one Vol. 11; no. 9; p. e0160587
• Main Authors: Schneeberger, Valentina E, Allaj, Viola, Gardner, Eric E, Poirier, J T, Rudin, Charles M
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 09-09-2016; Public Library of Science (PLoS)
• Subjects: Animal models; Animals; Bioinformatics; Biology and Life Sciences; Cancer; Cloning; Computational Biology - methods; Deoxyribonucleic acid; Disease Models, Animal; DNA; DNA sequencing; Epigenetics; Flow cytometry; Fluorescence; Gene expression; Gene sequencing; Genes; Genetic aspects; Genetic engineering; Genomes; Genomic analysis; Genomics; Genomics - methods; Health aspects; Heterografts; High-Throughput Nucleotide Sequencing; Histology; Humans; Lung cancer; Lung diseases; Medical research; Medicine; Medicine and Health Sciences; Mice; Mutation; Neoplasms - genetics; Neoplasms - pathology; Pharmacology; Polymerase chain reaction; Positive selection; Purification; Quantitation; Research and Analysis Methods; Rodents; Stroma; Stromal Cells - metabolism; Stromal Cells - pathology; Tumors; Virology; Xenografts
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0160587

Patient-derived xenograft (PDX) mouse models are increasingly used for preclinical therapeutic testing of human cancer. A limitation in molecular and genetic characterization of PDX tumors is the presence of integral murine stroma. This is particularly problematic for genomic sequencing of PDX models. Rapid and dependable approaches for quantitating stromal content and purifying the malignant human component of these tumors are needed. We used a recently developed technique exploiting species-specific polymerase chain reaction (PCR) amplicon length (ssPAL) differences to define the fractional composition of murine and human DNA, which was proportional to the fractional composition of cells in a series of lung cancer PDX lines. We compared four methods of human cancer cell isolation: fluorescence-activated cell sorting (FACS), an immunomagnetic mouse cell depletion (MCD) approach, and two distinct EpCAM-based immunomagnetic positive selection methods. We further analyzed DNA extracted from the resulting enriched human cancer cells by targeted sequencing using a clinically validated multi-gene panel. Stromal content varied widely among tumors of similar histology, but appeared stable over multiple serial tumor passages of an individual model. FACS and MCD were superior to either positive selection approach, especially in cases of high stromal content, and consistently allowed high quality human-specific genomic profiling. ssPAL is a dependable approach to quantitation of murine stromal content, and MCD is a simple, efficient, and high yield approach to human cancer cell isolation for genomic analysis of PDX tumors.