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Detection of tumor cell-specific mRNA and protein in exosome-like microvesicles from blood and saliva.

Yang J, Wei F, Schafer C, Wong DT - PLoS ONE (2014)

Bottom Line: RNA content was analyzed in cell free-saliva and ELM-enriched fractions of saliva.Altogether, our results demonstrate that ELMs carry tumor cell-specific mRNA and protein from blood to saliva in a xenografted mouse model of human lung cancer.These results therefore strengthen the link between distal tumor progression and the biomarker discovery of saliva through the ELMs.

View Article: PubMed Central - PubMed

Affiliation: School of Dentistry, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
The discovery of disease-specific biomarkers in oral fluids has revealed a new dimension in molecular diagnostics. Recent studies have reported the mechanistic involvement of tumor cells derived mediators, such as exosomes, in the development of saliva-based mRNA biomarkers. To further our understanding of the origins of disease-induced salivary biomarkers, we here evaluated the hypothesis that tumor-shed secretory lipidic vesicles called exosome-like microvesicles (ELMs) that serve as protective carriers of tissue-specific information, mRNAs, and proteins, throughout the vasculature and bodily fluids. RNA content was analyzed in cell free-saliva and ELM-enriched fractions of saliva. Our data confirmed that the majority of extracellular RNAs (exRNAs) in saliva were encapsulated within ELMs. Nude mice implanted with human lung cancer H460 cells expressing hCD63-GFP were used to follow the circulation of tumor cell specific protein and mRNA in the form of ELMs in vivo. We were able to identify human GAPDH mRNA in ELMs of blood and saliva of tumor bearing mice using nested RT-qPCR. ELMs positive for hCD63-GFP were detected in the saliva and blood of tumor bearing mice as well as using electric field-induced release and measurement (EFIRM). Altogether, our results demonstrate that ELMs carry tumor cell-specific mRNA and protein from blood to saliva in a xenografted mouse model of human lung cancer. These results therefore strengthen the link between distal tumor progression and the biomarker discovery of saliva through the ELMs.

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Salivary RNAs reside within salivary ELMs.(a) Representative electron microscopy images of salivary ELMs, and anti-CD63 immunogold-labeled ELMs in human saliva. (b) Western blot analysis of salivary ELMs using antibodies against human CD63 and GAPDH. Lane 1 to 7 are protein extracts of the salivary ELM pellets obtained from seven donors using Exoquick precipitation. (c) Ribogreen RNA quantitation from seven donors. Error bars represent means ± SD. (d) Representiave electron microscopy images of salivary ELMs and salivary ELMs treated with Tx. (e) RT-qPCR results of three reference genes from the following samples: cell-free saliva; salivary ELMs; salivary ELMs treated with RNase; salivary ELMs treated with Tx and RNase and ELM-depleted saliva. Data are expressed as means ± SD. *P<0.05, statistically significant difference from the saliva group.
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pone-0110641-g001: Salivary RNAs reside within salivary ELMs.(a) Representative electron microscopy images of salivary ELMs, and anti-CD63 immunogold-labeled ELMs in human saliva. (b) Western blot analysis of salivary ELMs using antibodies against human CD63 and GAPDH. Lane 1 to 7 are protein extracts of the salivary ELM pellets obtained from seven donors using Exoquick precipitation. (c) Ribogreen RNA quantitation from seven donors. Error bars represent means ± SD. (d) Representiave electron microscopy images of salivary ELMs and salivary ELMs treated with Tx. (e) RT-qPCR results of three reference genes from the following samples: cell-free saliva; salivary ELMs; salivary ELMs treated with RNase; salivary ELMs treated with Tx and RNase and ELM-depleted saliva. Data are expressed as means ± SD. *P<0.05, statistically significant difference from the saliva group.

Mentions: Previous studies have demonstrated that cell-free saliva supernatant contains more than 3000 different mRNA species [1], [2], [3], and the exRNAs remain stable unless they are exposed to detergents [46]. Lipidic vesicles, exosomes, protected the breakdown of exRNAs in saliva [42]. To evaluate whether all exRNAs in saliva are subject to this type of vesicular protection, we compared exRNAs in cell-free saliva and salivary ELMs. We first isolated salivary ELMs from the cell-free saliva of seven healthy subjects, which were characterized by the expression of exosomal surface marker CD63 (Fig. 1a, b). exRNAs in salivary ELMs and cell-free salivary were quantified and found to range from from 74 to 300 ng/ml and 87 to 350 ng/ml, respectively, with no statistically significant difference (Fig. 1c). Consistent with previous observation [42], the exRNAs in salivary ELMs is protected from RNase degradation. Quantitative PCR analysis of extracted RNAs revealed no notable differences in Ct values of the reference genes GAPDH, ß-actin, or RPS9 between cell-free saliva, salivary ELMs, and salivary ELMs with RNase treatment (Fig. 1e). 1% Triton X-100 (Tx) solution compromised structural integrity of salivary ELMs (Fig. 1d). ELMs with Triton X-100 plus RNase treatment or ELMs-depleted saliva exhibited substantially higher Ct values of reference genes GAPDH, ß-actin, or RPS9 (Fig. 1e) These results indicate that exRNAs in cell-free saliva are largely encased within salivary ELMs.


Detection of tumor cell-specific mRNA and protein in exosome-like microvesicles from blood and saliva.

Yang J, Wei F, Schafer C, Wong DT - PLoS ONE (2014)

Salivary RNAs reside within salivary ELMs.(a) Representative electron microscopy images of salivary ELMs, and anti-CD63 immunogold-labeled ELMs in human saliva. (b) Western blot analysis of salivary ELMs using antibodies against human CD63 and GAPDH. Lane 1 to 7 are protein extracts of the salivary ELM pellets obtained from seven donors using Exoquick precipitation. (c) Ribogreen RNA quantitation from seven donors. Error bars represent means ± SD. (d) Representiave electron microscopy images of salivary ELMs and salivary ELMs treated with Tx. (e) RT-qPCR results of three reference genes from the following samples: cell-free saliva; salivary ELMs; salivary ELMs treated with RNase; salivary ELMs treated with Tx and RNase and ELM-depleted saliva. Data are expressed as means ± SD. *P<0.05, statistically significant difference from the saliva group.
© Copyright Policy
Related In: Results  -  Collection

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pone-0110641-g001: Salivary RNAs reside within salivary ELMs.(a) Representative electron microscopy images of salivary ELMs, and anti-CD63 immunogold-labeled ELMs in human saliva. (b) Western blot analysis of salivary ELMs using antibodies against human CD63 and GAPDH. Lane 1 to 7 are protein extracts of the salivary ELM pellets obtained from seven donors using Exoquick precipitation. (c) Ribogreen RNA quantitation from seven donors. Error bars represent means ± SD. (d) Representiave electron microscopy images of salivary ELMs and salivary ELMs treated with Tx. (e) RT-qPCR results of three reference genes from the following samples: cell-free saliva; salivary ELMs; salivary ELMs treated with RNase; salivary ELMs treated with Tx and RNase and ELM-depleted saliva. Data are expressed as means ± SD. *P<0.05, statistically significant difference from the saliva group.
Mentions: Previous studies have demonstrated that cell-free saliva supernatant contains more than 3000 different mRNA species [1], [2], [3], and the exRNAs remain stable unless they are exposed to detergents [46]. Lipidic vesicles, exosomes, protected the breakdown of exRNAs in saliva [42]. To evaluate whether all exRNAs in saliva are subject to this type of vesicular protection, we compared exRNAs in cell-free saliva and salivary ELMs. We first isolated salivary ELMs from the cell-free saliva of seven healthy subjects, which were characterized by the expression of exosomal surface marker CD63 (Fig. 1a, b). exRNAs in salivary ELMs and cell-free salivary were quantified and found to range from from 74 to 300 ng/ml and 87 to 350 ng/ml, respectively, with no statistically significant difference (Fig. 1c). Consistent with previous observation [42], the exRNAs in salivary ELMs is protected from RNase degradation. Quantitative PCR analysis of extracted RNAs revealed no notable differences in Ct values of the reference genes GAPDH, ß-actin, or RPS9 between cell-free saliva, salivary ELMs, and salivary ELMs with RNase treatment (Fig. 1e). 1% Triton X-100 (Tx) solution compromised structural integrity of salivary ELMs (Fig. 1d). ELMs with Triton X-100 plus RNase treatment or ELMs-depleted saliva exhibited substantially higher Ct values of reference genes GAPDH, ß-actin, or RPS9 (Fig. 1e) These results indicate that exRNAs in cell-free saliva are largely encased within salivary ELMs.

Bottom Line: RNA content was analyzed in cell free-saliva and ELM-enriched fractions of saliva.Altogether, our results demonstrate that ELMs carry tumor cell-specific mRNA and protein from blood to saliva in a xenografted mouse model of human lung cancer.These results therefore strengthen the link between distal tumor progression and the biomarker discovery of saliva through the ELMs.

View Article: PubMed Central - PubMed

Affiliation: School of Dentistry, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
The discovery of disease-specific biomarkers in oral fluids has revealed a new dimension in molecular diagnostics. Recent studies have reported the mechanistic involvement of tumor cells derived mediators, such as exosomes, in the development of saliva-based mRNA biomarkers. To further our understanding of the origins of disease-induced salivary biomarkers, we here evaluated the hypothesis that tumor-shed secretory lipidic vesicles called exosome-like microvesicles (ELMs) that serve as protective carriers of tissue-specific information, mRNAs, and proteins, throughout the vasculature and bodily fluids. RNA content was analyzed in cell free-saliva and ELM-enriched fractions of saliva. Our data confirmed that the majority of extracellular RNAs (exRNAs) in saliva were encapsulated within ELMs. Nude mice implanted with human lung cancer H460 cells expressing hCD63-GFP were used to follow the circulation of tumor cell specific protein and mRNA in the form of ELMs in vivo. We were able to identify human GAPDH mRNA in ELMs of blood and saliva of tumor bearing mice using nested RT-qPCR. ELMs positive for hCD63-GFP were detected in the saliva and blood of tumor bearing mice as well as using electric field-induced release and measurement (EFIRM). Altogether, our results demonstrate that ELMs carry tumor cell-specific mRNA and protein from blood to saliva in a xenografted mouse model of human lung cancer. These results therefore strengthen the link between distal tumor progression and the biomarker discovery of saliva through the ELMs.

Show MeSH
Related in: MedlinePlus