Abstract
Abstract Purpose: To develop a robust real-time PCR-based assay with improved accuracy for quantifying the relative fractions of human and mouse DNA in heterogeneous samples such as patient-derived cancer xenografts or cultures. Background: Patient derived mouse xenografts (PDXs), or irradiated mouse fibroblast conditionally reprogrammed cancer cultures (CRCs) provide valuable resources for studying cancers, but as mouse tissue in heterogeneous samples can significantly confound downstream functional and molecular assays (such as next-generation sequencing) it is important to determine the fraction of mouse DNA in mixed samples. Methods: Using an approach different from a previously reported PCR assay (Alcoser SY et al BMC Biotech 2011, 11:124), we established a Taqman real-time PCR assay employing mouse- and human-specific primers that amplified the same chromosomal region of PTGER2 on human chromosome 14q22.1 and mouse chromosome 14 22.68 cM. We also developed a method to enrich human tumor cells from PDXs using enzymatic digestion with Liberase (Roche), followed by differential centrifugation and adherence. Results: Utilizing serial dilutions of heterogeneous DNA mixtures of mouse spleen DNA and normal human white blood cell DNA at varied DNA ratios we demonstrated the assay to be linear (Pearson r = 0.9984, human DNA; r = 0.9998, mouse DNA; p<0.0001) and sensitive (>6.4 pg DNA/reaction). The assay was evaluated in 66 samples of human tumors (lung, ovary, neuroblastoma, colon, pancreas, stomach and melanoma) including 25 PDXs before and after tumor cell enrichment, 12 non-enriched PDXs, 2 CRC-derived xenografts and 2 CRCs. Of the PDXs 28 were subcutaneous tumors and 9 were malignant ascites. A wide range of human:mouse DNA ratios were found from zero to 99% among all samples, with two PDXs and 2 CRC-derived xenografts lacking detectable human DNA. The highest human cell fractions were in the malignant ascites samples. Of the enriched samples, 18 of 25 (72%) showed tumor cell enrichment of 2 to 71-fold as compared with the value of matching non-enriched PDX. Conclusions: We developed a robust real-time PCR assay for measuring the relative fraction of species-specific DNA in human:mouse heterogenous samples. The human:mouse DNA ratios in PDX samples varied widely and the human tumor cell populations in PDXs can be effectively enriched in vitro. Assessing human tumor cell content with this assay will facilitate molecular studies with PDXs and mouse-human cocultivated cells. Citation Format: Yu-An Zhang, Victor Stastny, Mahboubeh Papari-Zareei, Heather Davidson, Boning Gao, Brenda Timmons, Jingsheng Yan, C Patrick Reynolds, John D. Minna, Adi F. Gazdar. Assessing and enriching human tumor cell content in patient-derived cancer xenografts and co-cultures. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 225. doi:10.1158/1538-7445.AM2015-225
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