Quantitation of Murine Stroma and Selective Purification of the Human Tumor Component of Patient-Derived Xenografts for Genomic Analysis.
Quantitation of Murine Stroma and Selective Purification of the Human Tumor Component of Patient-Derived Xenografts for Genomic Analysis.
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DOI：
10.1371/journal.pone.0160587
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Rudin CM
中科院分区：
文献类型：
--
作者：
Schneeberger VE;Allaj V;Gardner EE;Poirier JT;Rudin CM
患者来源的异种移植（PDX）小鼠模型越来越多地用于人类癌症的临床前治疗测试。PDX肿瘤的分子和遗传表征的限制是存在完整的鼠基质。这对于PDX模型的基因组测序尤其成问题。需要快速可靠的方法来定量基质含量和纯化这些肿瘤的恶性人类成分。我们使用了最近开发的技术，利用物种特异性聚合酶链反应（PCR）扩增子长度（ssPAL）的差异，以确定小鼠和人的DNA，这是成比例的细胞在一系列的肺癌PDX线的分数组成的分数组成。我们比较了四种人类癌细胞分离方法：荧光激活细胞分选（FACS），免疫磁性小鼠细胞耗竭（MCD）方法，以及两种不同的基于EpCAM的免疫磁性阳性选择方法。我们使用临床验证的多基因组通过靶向测序进一步分析了从所得富集的人癌细胞中提取的DNA。基质含量在相似组织学的肿瘤之间变化很大，但在单个模型的多个连续肿瘤传代中表现稳定。FACS和MCD上级任何一种阳性选择方法，特别是在高基质含量的情况下，并始终允许高质量的人类特异性基因组分析。ssPAL是一种可靠的定量鼠间质含量的方法，MCD是一种简单、有效、高产的人癌细胞分离方法，用于PDX肿瘤的基因组分析。
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.
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DOI：
10.1158/1541-7786.mcr-12-0307
发表时间：
2012-11
期刊：
Molecular cancer research : MCR
影响因子：
--
作者：
Karagiannis GS;Poutahidis T;Erdman SE;Kirsch R;Riddell RH;Diamandis EP
通讯作者：
Diamandis EP
影响因子：
82.9
作者：
通讯作者：
--
影响因子：
3.8
作者：
Gorges TM;Tinhofer I;Drosch M;Röse L;Zollner TM;Krahn T;von Ahsen O
通讯作者：
von Ahsen O
影响因子：
8.8
作者：
Liu, Jie;Kibiki, Gibson;Houpt, Eric
通讯作者：
Houpt, Eric
影响因子：
6.4
作者：
LEFRANCOIS, D;OLSCHWANG, S;DUTRILLAUX, B
通讯作者：
DUTRILLAUX, B