Quantification of B16 melanoma cells in lungs using triplex Q-PCR--a new approach to evaluate melanoma cell metastasis and tumor control.

Skin cancer is the most common type of all cancers. However, it comprises several different types of cancers, one of which is malignant melanoma. Even though melanomas only make up about 5% of skin cancers, they are responsible for the majority of skin cancer deaths due to the poor chance of surviva...

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Autores principales:	Maria R Sorensen, Sara R Pedersen, Annika Lindkvist, Jan P Christensen, Allan R Thomsen
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f71a5c390951464e918fa57e1338854a
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spelling	oai:doaj.org-article:f71a5c390951464e918fa57e1338854a2021-11-18T08:34:26ZQuantification of B16 melanoma cells in lungs using triplex Q-PCR--a new approach to evaluate melanoma cell metastasis and tumor control.1932-620310.1371/journal.pone.0087831https://doaj.org/article/f71a5c390951464e918fa57e1338854a2014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24498205/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Skin cancer is the most common type of all cancers. However, it comprises several different types of cancers, one of which is malignant melanoma. Even though melanomas only make up about 5% of skin cancers, they are responsible for the majority of skin cancer deaths due to the poor chance of survival once the tumor has metastasized. In the present study, we have developed a new assay for quantitative analysis of B16 melanoma metastasis in the lungs. We have used a triplex Q-PCR to determine the expression of the melanoma genes GP100/Pmel and tyrosinase-related protein 2 (TRP-2), and found that B16.F10gp cells were detectable in the lungs as early as 2 hours after intravenous challenge with ≥ 10(4) tumor cells. When investigating the gene expression as a function of time, we observed a gradual decrease from 2-24 hours post tumor challenge followed by an increase of approximately 2 log10 on day 11. The early decrease was accelerated in the presence of activated NK cells. To further evaluate our assay, we also investigated the level of metastasis in the context of vaccination with replication defective adenoviral vectors, Ad-Ii-GP and Ad-GP, previously found to significantly delay the outgrowth of subcutaneous melanomas. Results obtained using Q-PCR were compared to conventional counting of metastatic foci under a dissection microscope. A marked reduction in gene expression was observed in the lungs after vaccination with both vectors; however, Ad-Ii-GP showed the highest protection, and matching results were obtained by enumeration of visible tumor nodules on the lung surfaces. Finally, we could show that inhibition of tumor metastasis required antigen-specific CD8 T cells and IFNγ, but not perforin. In conclusion, the presented results validate triplex Q-PCR as a fast, objective, and quantitative method for analysis of melanoma metastasis in the lungs.Maria R SorensenSara R PedersenAnnika LindkvistJan P ChristensenAllan R ThomsenPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 1, p e87831 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Maria R Sorensen Sara R Pedersen Annika Lindkvist Jan P Christensen Allan R Thomsen Quantification of B16 melanoma cells in lungs using triplex Q-PCR--a new approach to evaluate melanoma cell metastasis and tumor control.
description	Skin cancer is the most common type of all cancers. However, it comprises several different types of cancers, one of which is malignant melanoma. Even though melanomas only make up about 5% of skin cancers, they are responsible for the majority of skin cancer deaths due to the poor chance of survival once the tumor has metastasized. In the present study, we have developed a new assay for quantitative analysis of B16 melanoma metastasis in the lungs. We have used a triplex Q-PCR to determine the expression of the melanoma genes GP100/Pmel and tyrosinase-related protein 2 (TRP-2), and found that B16.F10gp cells were detectable in the lungs as early as 2 hours after intravenous challenge with ≥ 10(4) tumor cells. When investigating the gene expression as a function of time, we observed a gradual decrease from 2-24 hours post tumor challenge followed by an increase of approximately 2 log10 on day 11. The early decrease was accelerated in the presence of activated NK cells. To further evaluate our assay, we also investigated the level of metastasis in the context of vaccination with replication defective adenoviral vectors, Ad-Ii-GP and Ad-GP, previously found to significantly delay the outgrowth of subcutaneous melanomas. Results obtained using Q-PCR were compared to conventional counting of metastatic foci under a dissection microscope. A marked reduction in gene expression was observed in the lungs after vaccination with both vectors; however, Ad-Ii-GP showed the highest protection, and matching results were obtained by enumeration of visible tumor nodules on the lung surfaces. Finally, we could show that inhibition of tumor metastasis required antigen-specific CD8 T cells and IFNγ, but not perforin. In conclusion, the presented results validate triplex Q-PCR as a fast, objective, and quantitative method for analysis of melanoma metastasis in the lungs.
format	article
author	Maria R Sorensen Sara R Pedersen Annika Lindkvist Jan P Christensen Allan R Thomsen
author_facet	Maria R Sorensen Sara R Pedersen Annika Lindkvist Jan P Christensen Allan R Thomsen
author_sort	Maria R Sorensen
title	Quantification of B16 melanoma cells in lungs using triplex Q-PCR--a new approach to evaluate melanoma cell metastasis and tumor control.
title_short	Quantification of B16 melanoma cells in lungs using triplex Q-PCR--a new approach to evaluate melanoma cell metastasis and tumor control.
title_full	Quantification of B16 melanoma cells in lungs using triplex Q-PCR--a new approach to evaluate melanoma cell metastasis and tumor control.
title_fullStr	Quantification of B16 melanoma cells in lungs using triplex Q-PCR--a new approach to evaluate melanoma cell metastasis and tumor control.
title_full_unstemmed	Quantification of B16 melanoma cells in lungs using triplex Q-PCR--a new approach to evaluate melanoma cell metastasis and tumor control.
title_sort	quantification of b16 melanoma cells in lungs using triplex q-pcr--a new approach to evaluate melanoma cell metastasis and tumor control.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/f71a5c390951464e918fa57e1338854a
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Quantification of B16 melanoma cells in lungs using triplex Q-PCR--a new approach to evaluate melanoma cell metastasis and tumor control.

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