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In vivo fluorescence lifetime imaging monitors binding of specific probes to cancer biomarkers.

Ardeshirpour Y, Chernomordik V, Zielinski R, Capala J, Griffiths G, Vasalatiy O, Smirnov AV, Knutson JR, Lyakhov I, Achilefu S, Gandjbakhche A, Hassan M - PLoS ONE (2012)

Bottom Line: One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells.Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention.Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the "image and treat" concept, especially for early evaluation of the efficacy of the therapy.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the "image and treat" concept, especially for early evaluation of the efficacy of the therapy.

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In vivo fluorescence imaging of xenograft mouse with high HER2 expressing human tumor model (BT-474) after injection of HER2-specific Affibody® (His6-ZHER2:GS-Cys) conjugated to Dylight750.(A) Fluorescence intensity map at the tumor region. (B)Fluorescence intensity map at the contralateral site (C) The difference of fluorescence intensity at the tumor region and the contralateral site, mapped on the tumor region. (D) Pharmacokinetics of the fluorescence intensity at the tumor region and contralateral site after the injection over time. The fluorescent intensity was averaged over 16 pixels at the contralateral site. The data in Figs. (D) and (H) are the average data of four mice. Markers show the average and bars show the standard deviation. (E) Fluorescence lifetime map at the tumor region. (F) Fluorescence lifetime map at the contralateral site. (G) The difference of fluorescence lifetime at the tumor and the contralateral site mapped on the tumor region. (E)Pharmacokinetics of the fluorescent lifetime at the tumor region and contralateral site after injection over time. All lifetime and fluorescence intensity maps in figures 2,4–7 are from measurements at 1 hour after the injection of Affibody probe. In all measurements, the photons were counted over two seconds integration time, t0. To exclude saturation effect of the 16 bit camera, in the brightest pixels, where the corresponding limit of 65536 counts was reached before time t0, we have renormalized the data by multiplying photon counts, measured for lower integration time t, by factor t0/t.
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pone-0031881-g002: In vivo fluorescence imaging of xenograft mouse with high HER2 expressing human tumor model (BT-474) after injection of HER2-specific Affibody® (His6-ZHER2:GS-Cys) conjugated to Dylight750.(A) Fluorescence intensity map at the tumor region. (B)Fluorescence intensity map at the contralateral site (C) The difference of fluorescence intensity at the tumor region and the contralateral site, mapped on the tumor region. (D) Pharmacokinetics of the fluorescence intensity at the tumor region and contralateral site after the injection over time. The fluorescent intensity was averaged over 16 pixels at the contralateral site. The data in Figs. (D) and (H) are the average data of four mice. Markers show the average and bars show the standard deviation. (E) Fluorescence lifetime map at the tumor region. (F) Fluorescence lifetime map at the contralateral site. (G) The difference of fluorescence lifetime at the tumor and the contralateral site mapped on the tumor region. (E)Pharmacokinetics of the fluorescent lifetime at the tumor region and contralateral site after injection over time. All lifetime and fluorescence intensity maps in figures 2,4–7 are from measurements at 1 hour after the injection of Affibody probe. In all measurements, the photons were counted over two seconds integration time, t0. To exclude saturation effect of the 16 bit camera, in the brightest pixels, where the corresponding limit of 65536 counts was reached before time t0, we have renormalized the data by multiplying photon counts, measured for lower integration time t, by factor t0/t.

Mentions: In the first experiment, HER2-specific Affibody was injected into four mice with a high level (+3) HER2-expressing human tumor carcinoma, BT-474. Fig. 2 shows the results of in vivo measurements of fluorescence intensity and lifetime at the tumor area and the contralateral site. Fig. 2C shows the difference between the fluorescence intensity at the tumor (Fig. 2A) and the contralateral sites (Fig. 2B) 1 hour after injection, mapped on the tumor area. Fig. 2D shows the dynamics of the maximum fluorescence intensity at the tumor region and contralateral site for 6 hours after the injection. The pixel with maximum intensity was used to characterize the tumor. The fluorescence mapping at the contralateral site was averaged over 16 pixels since there was no specific point to target at the contralateral site. Averaging over 16 pixels has been used for noise reduction. To demonstrate the variations of the lifetime and fluorescence intensity throughout the scanned area we presented maps of fluorescence intensity and lifetime at the contralateral and tumor regions. The data shown in the Fig. 2D and Fig. 2H are the average values of the measurements over 4 mice (Markers show the average and bars show the standard deviation). Fig. 2G shows the difference between the fluorescence lifetime at the tumor (Fig. 2E) and contralateral sites (Fig. 2F) 1 hour after injection, mapped on the tumor region. The fluorescence lifetime in the tumor area and contralateral site for over 6 hours after injection is shown in Fig. 2H. In Figs. 2H, the data, corresponding to the pixel with maximum intensity were used for lifetime calculations since it had the highest signal to noise ratio.


In vivo fluorescence lifetime imaging monitors binding of specific probes to cancer biomarkers.

Ardeshirpour Y, Chernomordik V, Zielinski R, Capala J, Griffiths G, Vasalatiy O, Smirnov AV, Knutson JR, Lyakhov I, Achilefu S, Gandjbakhche A, Hassan M - PLoS ONE (2012)

In vivo fluorescence imaging of xenograft mouse with high HER2 expressing human tumor model (BT-474) after injection of HER2-specific Affibody® (His6-ZHER2:GS-Cys) conjugated to Dylight750.(A) Fluorescence intensity map at the tumor region. (B)Fluorescence intensity map at the contralateral site (C) The difference of fluorescence intensity at the tumor region and the contralateral site, mapped on the tumor region. (D) Pharmacokinetics of the fluorescence intensity at the tumor region and contralateral site after the injection over time. The fluorescent intensity was averaged over 16 pixels at the contralateral site. The data in Figs. (D) and (H) are the average data of four mice. Markers show the average and bars show the standard deviation. (E) Fluorescence lifetime map at the tumor region. (F) Fluorescence lifetime map at the contralateral site. (G) The difference of fluorescence lifetime at the tumor and the contralateral site mapped on the tumor region. (E)Pharmacokinetics of the fluorescent lifetime at the tumor region and contralateral site after injection over time. All lifetime and fluorescence intensity maps in figures 2,4–7 are from measurements at 1 hour after the injection of Affibody probe. In all measurements, the photons were counted over two seconds integration time, t0. To exclude saturation effect of the 16 bit camera, in the brightest pixels, where the corresponding limit of 65536 counts was reached before time t0, we have renormalized the data by multiplying photon counts, measured for lower integration time t, by factor t0/t.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285647&req=5

pone-0031881-g002: In vivo fluorescence imaging of xenograft mouse with high HER2 expressing human tumor model (BT-474) after injection of HER2-specific Affibody® (His6-ZHER2:GS-Cys) conjugated to Dylight750.(A) Fluorescence intensity map at the tumor region. (B)Fluorescence intensity map at the contralateral site (C) The difference of fluorescence intensity at the tumor region and the contralateral site, mapped on the tumor region. (D) Pharmacokinetics of the fluorescence intensity at the tumor region and contralateral site after the injection over time. The fluorescent intensity was averaged over 16 pixels at the contralateral site. The data in Figs. (D) and (H) are the average data of four mice. Markers show the average and bars show the standard deviation. (E) Fluorescence lifetime map at the tumor region. (F) Fluorescence lifetime map at the contralateral site. (G) The difference of fluorescence lifetime at the tumor and the contralateral site mapped on the tumor region. (E)Pharmacokinetics of the fluorescent lifetime at the tumor region and contralateral site after injection over time. All lifetime and fluorescence intensity maps in figures 2,4–7 are from measurements at 1 hour after the injection of Affibody probe. In all measurements, the photons were counted over two seconds integration time, t0. To exclude saturation effect of the 16 bit camera, in the brightest pixels, where the corresponding limit of 65536 counts was reached before time t0, we have renormalized the data by multiplying photon counts, measured for lower integration time t, by factor t0/t.
Mentions: In the first experiment, HER2-specific Affibody was injected into four mice with a high level (+3) HER2-expressing human tumor carcinoma, BT-474. Fig. 2 shows the results of in vivo measurements of fluorescence intensity and lifetime at the tumor area and the contralateral site. Fig. 2C shows the difference between the fluorescence intensity at the tumor (Fig. 2A) and the contralateral sites (Fig. 2B) 1 hour after injection, mapped on the tumor area. Fig. 2D shows the dynamics of the maximum fluorescence intensity at the tumor region and contralateral site for 6 hours after the injection. The pixel with maximum intensity was used to characterize the tumor. The fluorescence mapping at the contralateral site was averaged over 16 pixels since there was no specific point to target at the contralateral site. Averaging over 16 pixels has been used for noise reduction. To demonstrate the variations of the lifetime and fluorescence intensity throughout the scanned area we presented maps of fluorescence intensity and lifetime at the contralateral and tumor regions. The data shown in the Fig. 2D and Fig. 2H are the average values of the measurements over 4 mice (Markers show the average and bars show the standard deviation). Fig. 2G shows the difference between the fluorescence lifetime at the tumor (Fig. 2E) and contralateral sites (Fig. 2F) 1 hour after injection, mapped on the tumor region. The fluorescence lifetime in the tumor area and contralateral site for over 6 hours after injection is shown in Fig. 2H. In Figs. 2H, the data, corresponding to the pixel with maximum intensity were used for lifetime calculations since it had the highest signal to noise ratio.

Bottom Line: One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells.Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention.Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the "image and treat" concept, especially for early evaluation of the efficacy of the therapy.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the "image and treat" concept, especially for early evaluation of the efficacy of the therapy.

Show MeSH
Related in: MedlinePlus