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

Preston Campbell J, Mulcrone P, Masood SK, Karolak M, Merkel A, Hebron K, Zijlstra A, Sterling J, Elefteriou F - Sci Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, United States of America [2] Vanderbilt Center for Bone Biology, Vanderbilt University, Nashville, Tennessee, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Alu PCR accurately quantifies tumor cell establishment in the bone.Representative ex vivo imaging of tibiae at different time points after intracardiac injection of MDA-231 tumor cells (a) and quantification of GFP+ bone tumors per mouse (c) (n = 5)with representative GFP images (b). BLI of tibiae injected with known numbers of MDA-231 cells expressing Luciferase (d) and corresponding Alu qPCR from the same bones (e), n = 2.
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f3: Alu PCR accurately quantifies tumor cell establishment in the bone.Representative ex vivo imaging of tibiae at different time points after intracardiac injection of MDA-231 tumor cells (a) and quantification of GFP+ bone tumors per mouse (c) (n = 5)with representative GFP images (b). BLI of tibiae injected with known numbers of MDA-231 cells expressing Luciferase (d) and corresponding Alu qPCR from the same bones (e), n = 2.

Mentions: In the commonly-used preclinical models of bone metastasis, it has been speculated that very few of the metastatic cells survive, and that most die over the course of the first week, resulting in a trough of bioluminescent signal during this first week. Attempts have been made with fluorescence or bioluminescence approaches to quantify the events in the early hours or days of metastatic establishment, but these techniques are limited by low reporter gene expression seen in metabolically quiescent cells12, limited depth of penetration and scattering of signal13, multiple layers of tissue between deep metastatic cells and the detector, and by the relative opacity of dense tissues like bone. Given these limitations, we applied the Alu qPCR technique to reveal the fate of bone metastatic cells in the intracardiac mouse metastasis model, and compared the results with eGFP fluorescence data and eGFP RNA expression from the same samples. According to Alu qPCR, 10–100 cells arrested in any one long bone at 3 hours post intracardiac injection, and cell number increased steadily until mice were sacrificed at 14 days (Fig. 3a). In contrast to the increasing number of cells quantified by Alu qPCR, eGFP qPCR of MDA-MB-231 cells that express eGFP driven by the CMV promoter (Supplementary Fig. 3a) showed a decrease from d1 to d7 that would suggest a reduction in cell number in the tissues. When in vitro eGFP and Alu qPCR signals were compared from MDA-MB-231 cells alone, the slopes were not significantly different (Supplementary Fig. 3a–b) which excludes the possibility of differences in qPCR efficiency between eGFP cDNA and Alu genomic DNA. The discrepancy between in vivo and in vitro Alu DNA vs eGFP RNA signal may be due to metabolic changes in tumor cells, which are known to affect expression of reporter genes, even when under the control of a constitutive promoter14. Using fluorescence imaging, we did not observe an ex vivo signal in bone until 9 days after injection, which corresponded to >1000 cells (Fig. 3b), consistent with previous findings regarding the sensitivity of spectral imaging in vivo15. Furthermore, many large tumor foci were rendered undetectable by ex vivo fluorescence; by simply adjusting the positional angle of the bone during imaging and fluorescence, the resulting data- used to quantify tumor burden- were highly variable (data not shown). Interestingly, the number of bones with detectable metastases (more than 10 Alu + cells) did not increase over two weeks, though detectable metastases by ex vivo fluorescence appeared to increase (Supplementary Fig 2f). This observation further supports the advantage of Alu detection to precisely quantify the number of metastatic tumor cells in the skeleton (and other tissues) at the earliest stage of metastasis, and reinforces the recent observation that circulating tumor cell clusters may have higher metastatic potential than isolated tumor cells16.


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

Preston Campbell J, Mulcrone P, Masood SK, Karolak M, Merkel A, Hebron K, Zijlstra A, Sterling J, Elefteriou F - Sci Rep (2015)

Alu PCR accurately quantifies tumor cell establishment in the bone.Representative ex vivo imaging of tibiae at different time points after intracardiac injection of MDA-231 tumor cells (a) and quantification of GFP+ bone tumors per mouse (c) (n = 5)with representative GFP images (b). BLI of tibiae injected with known numbers of MDA-231 cells expressing Luciferase (d) and corresponding Alu qPCR from the same bones (e), n = 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4536516&req=5

f3: Alu PCR accurately quantifies tumor cell establishment in the bone.Representative ex vivo imaging of tibiae at different time points after intracardiac injection of MDA-231 tumor cells (a) and quantification of GFP+ bone tumors per mouse (c) (n = 5)with representative GFP images (b). BLI of tibiae injected with known numbers of MDA-231 cells expressing Luciferase (d) and corresponding Alu qPCR from the same bones (e), n = 2.
Mentions: In the commonly-used preclinical models of bone metastasis, it has been speculated that very few of the metastatic cells survive, and that most die over the course of the first week, resulting in a trough of bioluminescent signal during this first week. Attempts have been made with fluorescence or bioluminescence approaches to quantify the events in the early hours or days of metastatic establishment, but these techniques are limited by low reporter gene expression seen in metabolically quiescent cells12, limited depth of penetration and scattering of signal13, multiple layers of tissue between deep metastatic cells and the detector, and by the relative opacity of dense tissues like bone. Given these limitations, we applied the Alu qPCR technique to reveal the fate of bone metastatic cells in the intracardiac mouse metastasis model, and compared the results with eGFP fluorescence data and eGFP RNA expression from the same samples. According to Alu qPCR, 10–100 cells arrested in any one long bone at 3 hours post intracardiac injection, and cell number increased steadily until mice were sacrificed at 14 days (Fig. 3a). In contrast to the increasing number of cells quantified by Alu qPCR, eGFP qPCR of MDA-MB-231 cells that express eGFP driven by the CMV promoter (Supplementary Fig. 3a) showed a decrease from d1 to d7 that would suggest a reduction in cell number in the tissues. When in vitro eGFP and Alu qPCR signals were compared from MDA-MB-231 cells alone, the slopes were not significantly different (Supplementary Fig. 3a–b) which excludes the possibility of differences in qPCR efficiency between eGFP cDNA and Alu genomic DNA. The discrepancy between in vivo and in vitro Alu DNA vs eGFP RNA signal may be due to metabolic changes in tumor cells, which are known to affect expression of reporter genes, even when under the control of a constitutive promoter14. Using fluorescence imaging, we did not observe an ex vivo signal in bone until 9 days after injection, which corresponded to >1000 cells (Fig. 3b), consistent with previous findings regarding the sensitivity of spectral imaging in vivo15. Furthermore, many large tumor foci were rendered undetectable by ex vivo fluorescence; by simply adjusting the positional angle of the bone during imaging and fluorescence, the resulting data- used to quantify tumor burden- were highly variable (data not shown). Interestingly, the number of bones with detectable metastases (more than 10 Alu + cells) did not increase over two weeks, though detectable metastases by ex vivo fluorescence appeared to increase (Supplementary Fig 2f). This observation further supports the advantage of Alu detection to precisely quantify the number of metastatic tumor cells in the skeleton (and other tissues) at the earliest stage of metastasis, and reinforces the recent observation that circulating tumor cell clusters may have higher metastatic potential than isolated tumor cells16.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, United States of America [2] Vanderbilt Center for Bone Biology, Vanderbilt University, Nashville, Tennessee, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus