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TRIzol and Alu qPCR-based quantification of metastatic seeding within the skeleton.

ABSTRACT: Current methods for detecting disseminated tumor cells in the skeleton are limited by expense and technical complexity. We describe a simple and inexpensive method to quantify, with single cell sensitivity, human metastatic cancer in the mouse skeleton, concurrently with host gene expression, using TRIzol-based DNA/RNA extraction and Alu sequence qPCR amplification. This approach enables precise quantification of tumor cells and corresponding host gene expression during metastatic colonization in xenograft models.

SUBMITTER: Preston Campbell J

PROVIDER: S-EPMC4536516 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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TRIzol and Alu qPCR-based quantification of metastatic seeding within the skeleton.

Preston Campbell J J Mulcrone P P Masood S K SK Karolak M M Merkel A A Hebron K K Zijlstra A A Sterling J J Elefteriou F F

Scientific reports 20150814

Current methods for detecting disseminated tumor cells in the skeleton are limited by expense and technical complexity. We describe a simple and inexpensive method to quantify, with single cell sensitivity, human metastatic cancer in the mouse skeleton, concurrently with host gene expression, using TRIzol-based DNA/RNA extraction and Alu sequence qPCR amplification. This approach enables precise quantification of tumor cells and corresponding host gene expression during metastatic colonization i ...[more]

PMID: 26271202

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Project description:Bioluminescence imaging (BLI) is a non-invasive state-of-the-art-method for longitudinal tracking of tumor cells in mice. The technique is commonly used to determine bone metastatic burden in vivo and also suitable ex vivo to detect even smallest bone micro-metastases in spontaneous metastasis xenograft models. However, it is unclear to which extent ex vivo BLI correlates with alternative methods for metastasis quantification. Here, we compared ex vivo BLI, human DNA-based Alu-qPCR, and histology for the quantification of bone vs. lung metastases, which are amongst the most common sites of metastasis in prostate cancer (PCa) patients and spontaneous PCa xenograft models. Data from 93 immunodeficient mice were considered, each of which were subcutaneously injected with luciferase/RGB-labeled human PCa PC-3 cells. The primary tumors were resected at ~ 0.75 cm³ and mice were sacrificed ~ 3 weeks after surgery and immediately examined by ex vivo BLI. Afterwards, the right lungs and hind limbs with the higher BLI signal (BLIHi bone) were processed for histology, whereas the left lung lobes and hind limbs with the lower BLI signal (BLILo bone) were prepared for Alu-qPCR. Our data demonstrate remarkable differences in the correlation coefficients of the different methods for lung metastasis detection (r ~ 0.8) vs. bone metastasis detection (r ~ 0.4). However, the BLI values of the BLIHi and BLILo bones correlated very strongly (r ~ 0.9), indicating that the method per se was reliable under identical limitations; the overall level of metastasis to contralateral bones was astonishingly similar. Instead, the level of lung metastasis only weakly to moderately correlated with the level of bone metastasis formation. Summarized, we observed a considerable discrepancy between ex vivo BLI and histology/Alu-qPCR in the quantification of bone metastases, which was not observed in the case of lung metastases. Future studies using ex vivo BLI for bone metastasis quantification should combine multiple methods to accurately determine metastatic load in bone samples.

Dataset Information

TRIzol and Alu qPCR-based quantification of metastatic seeding within the skeleton.

Publications

TRIzol and Alu qPCR-based quantification of metastatic seeding within the skeleton.

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