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An optimized triple modality reporter for quantitative in vivo tumor imaging and therapy evaluation.

Levin RA, Felsen CN, Yang J, Lin JY, Whitney MA, Nguyen QT, Tsien RY - PLoS ONE (2014)

Bottom Line: We present an optimized triple modality reporter construct combining a far-red fluorescent protein (E2-Crimson), enhanced firefly luciferase enzyme (Luc2), and truncated wild type herpes simplex virus I thymidine kinase (wttk) that allows for sensitive, long-term tracking of tumor growth in vivo by fluorescence, bioluminescence, and positron emission tomography.Two human cancer cell lines (MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer) were successfully transduced to express this triple modality reporter.This is the first reported use of both fluorescence and bioluminescence signals from a multi-modality reporter construct to measure drug efficacy in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, UCSD School of Medicine, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
We present an optimized triple modality reporter construct combining a far-red fluorescent protein (E2-Crimson), enhanced firefly luciferase enzyme (Luc2), and truncated wild type herpes simplex virus I thymidine kinase (wttk) that allows for sensitive, long-term tracking of tumor growth in vivo by fluorescence, bioluminescence, and positron emission tomography. Two human cancer cell lines (MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer) were successfully transduced to express this triple modality reporter. Fluorescence and bioluminescence imaging of the triple modality reporter were used to accurately quantify the therapeutic responses of MDA-MB-231 tumors to the chemotherapeutic agent monomethyl auristatin E in vivo in athymic nude mice. Positive correlation was observed between the fluorescence and bioluminescence signals, and these signals were also positively correlated with the ex vivo tumor weights. This is the first reported use of both fluorescence and bioluminescence signals from a multi-modality reporter construct to measure drug efficacy in vivo.

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Comparison of four far-red and infrared fluorescent proteins.(A) The mean quartile (mean 25%) fluorescence intensities (FLI) of E2-Crimson (C), infrared fluorescent protein (I), mNeptune (N), and mPlum (P) in HT-1080 cells measured by fluorescent-activated cell sorting (FACS) were compared (100 mW laser with ex 568 nm and em 650–670 nm for C, N, and P; ex 690 nm and em 710–900 nm for I). (B) The top 5% brightest HT-1080 cells from each fluorescent protein cell type were injected into athymic nude mice (1×106 cells/injection; exposure time: 500 msec; ex 590/23 nm and em 645LP for C, N, and P; ex 640/48 nm and em 700LP for I).
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pone-0097415-g001: Comparison of four far-red and infrared fluorescent proteins.(A) The mean quartile (mean 25%) fluorescence intensities (FLI) of E2-Crimson (C), infrared fluorescent protein (I), mNeptune (N), and mPlum (P) in HT-1080 cells measured by fluorescent-activated cell sorting (FACS) were compared (100 mW laser with ex 568 nm and em 650–670 nm for C, N, and P; ex 690 nm and em 710–900 nm for I). (B) The top 5% brightest HT-1080 cells from each fluorescent protein cell type were injected into athymic nude mice (1×106 cells/injection; exposure time: 500 msec; ex 590/23 nm and em 645LP for C, N, and P; ex 640/48 nm and em 700LP for I).

Mentions: Fluorescent proteins used in previously published triple reporters have been excited below 600 nm, overlapping with heme absorbance, which contributes to depth attenuation. Because E2-Crimson [13] (excitation maximum 611 nm), infrared fluorescent protein [14] (IFP, excitation maximum 684 nm), mNeptune [15] (excitation maximum 600 nm), and mPlum [16] (excitation maximum 590 nm) have all been published separately, the expression of these far-red to infrared FPs in stably transfected cells has not been compared head-to-head in vivo. Stable, consistent, and high expression of these proteins is necessary for in vivo tumor imaging where fluorescent signal is monitored for weeks to months of tumors growth. Comparing human fibrosarcoma HT-1080 cells stably expressing E2-Crimson, IFP, mNeptune, or mPlum, HT-1080 E2-Crimson cells had the highest mean quartile (mean 25% of cells) fluorescence according to fluorescent-activated cell sorting (FACS) analysis, even though the far-red settings were optimized for mPlum (ex 568 nm and em 650-670 nm; IFP was assessed at ex 690 nm and em 710–900 nm) (Figure 1A). The same was true for HT-1080 tumors in vivo, in which HT-1080 E2-Crimson tumors had the highest in vivo fluorescence at the far-red imaging settings (ex 590/23 nm and em 645LP for E2-Crimson, mNeptune, and mPlum; ex 640/48 nm and em 700LP for IFP) (Figure 1B). E2-Crimson may be brighter in vitro and in vivo from the combination of its high quantum yield, tetrameric structure, and rapid folding stability. As a result, E2-Crimson was incorporated into our optimized triple modality reporter design.


An optimized triple modality reporter for quantitative in vivo tumor imaging and therapy evaluation.

Levin RA, Felsen CN, Yang J, Lin JY, Whitney MA, Nguyen QT, Tsien RY - PLoS ONE (2014)

Comparison of four far-red and infrared fluorescent proteins.(A) The mean quartile (mean 25%) fluorescence intensities (FLI) of E2-Crimson (C), infrared fluorescent protein (I), mNeptune (N), and mPlum (P) in HT-1080 cells measured by fluorescent-activated cell sorting (FACS) were compared (100 mW laser with ex 568 nm and em 650–670 nm for C, N, and P; ex 690 nm and em 710–900 nm for I). (B) The top 5% brightest HT-1080 cells from each fluorescent protein cell type were injected into athymic nude mice (1×106 cells/injection; exposure time: 500 msec; ex 590/23 nm and em 645LP for C, N, and P; ex 640/48 nm and em 700LP for I).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4016317&req=5

pone-0097415-g001: Comparison of four far-red and infrared fluorescent proteins.(A) The mean quartile (mean 25%) fluorescence intensities (FLI) of E2-Crimson (C), infrared fluorescent protein (I), mNeptune (N), and mPlum (P) in HT-1080 cells measured by fluorescent-activated cell sorting (FACS) were compared (100 mW laser with ex 568 nm and em 650–670 nm for C, N, and P; ex 690 nm and em 710–900 nm for I). (B) The top 5% brightest HT-1080 cells from each fluorescent protein cell type were injected into athymic nude mice (1×106 cells/injection; exposure time: 500 msec; ex 590/23 nm and em 645LP for C, N, and P; ex 640/48 nm and em 700LP for I).
Mentions: Fluorescent proteins used in previously published triple reporters have been excited below 600 nm, overlapping with heme absorbance, which contributes to depth attenuation. Because E2-Crimson [13] (excitation maximum 611 nm), infrared fluorescent protein [14] (IFP, excitation maximum 684 nm), mNeptune [15] (excitation maximum 600 nm), and mPlum [16] (excitation maximum 590 nm) have all been published separately, the expression of these far-red to infrared FPs in stably transfected cells has not been compared head-to-head in vivo. Stable, consistent, and high expression of these proteins is necessary for in vivo tumor imaging where fluorescent signal is monitored for weeks to months of tumors growth. Comparing human fibrosarcoma HT-1080 cells stably expressing E2-Crimson, IFP, mNeptune, or mPlum, HT-1080 E2-Crimson cells had the highest mean quartile (mean 25% of cells) fluorescence according to fluorescent-activated cell sorting (FACS) analysis, even though the far-red settings were optimized for mPlum (ex 568 nm and em 650-670 nm; IFP was assessed at ex 690 nm and em 710–900 nm) (Figure 1A). The same was true for HT-1080 tumors in vivo, in which HT-1080 E2-Crimson tumors had the highest in vivo fluorescence at the far-red imaging settings (ex 590/23 nm and em 645LP for E2-Crimson, mNeptune, and mPlum; ex 640/48 nm and em 700LP for IFP) (Figure 1B). E2-Crimson may be brighter in vitro and in vivo from the combination of its high quantum yield, tetrameric structure, and rapid folding stability. As a result, E2-Crimson was incorporated into our optimized triple modality reporter design.

Bottom Line: We present an optimized triple modality reporter construct combining a far-red fluorescent protein (E2-Crimson), enhanced firefly luciferase enzyme (Luc2), and truncated wild type herpes simplex virus I thymidine kinase (wttk) that allows for sensitive, long-term tracking of tumor growth in vivo by fluorescence, bioluminescence, and positron emission tomography.Two human cancer cell lines (MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer) were successfully transduced to express this triple modality reporter.This is the first reported use of both fluorescence and bioluminescence signals from a multi-modality reporter construct to measure drug efficacy in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, UCSD School of Medicine, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
We present an optimized triple modality reporter construct combining a far-red fluorescent protein (E2-Crimson), enhanced firefly luciferase enzyme (Luc2), and truncated wild type herpes simplex virus I thymidine kinase (wttk) that allows for sensitive, long-term tracking of tumor growth in vivo by fluorescence, bioluminescence, and positron emission tomography. Two human cancer cell lines (MDA-MB-231 breast cancer and HT-1080 fibrosarcoma cancer) were successfully transduced to express this triple modality reporter. Fluorescence and bioluminescence imaging of the triple modality reporter were used to accurately quantify the therapeutic responses of MDA-MB-231 tumors to the chemotherapeutic agent monomethyl auristatin E in vivo in athymic nude mice. Positive correlation was observed between the fluorescence and bioluminescence signals, and these signals were also positively correlated with the ex vivo tumor weights. This is the first reported use of both fluorescence and bioluminescence signals from a multi-modality reporter construct to measure drug efficacy in vivo.

Show MeSH
Related in: MedlinePlus