Quantitation of Murine Stroma and Selective Purification of the Human Tumor Component of Patient-Derived Xenografts for Genomic Analysis.
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.
Molecular Pharmacology & Chemistry Program and Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.
Valentina E
VE
Schneeberger
Molecular Pharmacology & Chemistry Program and Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.
Viola
V
Allaj
Pharmacology Graduate Training Program, Johns Hopkins University, Baltimore, MD, United States of America.
Eric E
EE
Gardner
Molecular Pharmacology & Chemistry Program and Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.|Department of Medicine, Weill Cornell Medical College, New York, NY, United States of America.
J T
http://orcid.org/0000-0001-9795-5644
JT
Poirier
Molecular Pharmacology & Chemistry Program and Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.|Department of Medicine, Weill Cornell Medical College, New York, NY, United States of America.
Charles M
CM
Rudin
D000818:Animals; D019295:Computational Biology / Q000379:methods; D004195:Disease Models, Animal; D023281:Genomics / Q000379:methods; D064593:Heterografts; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D051379:Mice; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology*; D017154:Stromal Cells / Q000378:metabolism* / Q000473:pathology