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Confocal fluorescence imaging enables noninvasive quantitative assessment of host cell populations in vivo following photodynamic therapy.

Mitra S, Mironov O, Foster TH - Theranostics (2012)

Bottom Line: The maximum accumulation of Gr1(+) cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point.Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1(+ )cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status.Co-localization analysis reveals an increase in the fraction of Gr1(+) cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Imaging Sciences, University of Rochester Medical Center, Rochester, NY 14642, USA.

ABSTRACT
We report the use of optical imaging strategies to noninvasively examine photosensitizer distribution and physiological and host responses to 2-[1-hexyloxyethyl]-2 devinyl pyropheophorbide-a (HPPH)-mediated photodynamic therapy (PDT) of EMT6 tumors established in the ears of BALB/c mice. 24 h following intravenous (IV) administration of 1 μmol kg(-1) HPPH, wide-field fluorescence imaging reveals tumor selectivity with an approximately 2-3-fold differential between tumor and adjacent normal tissue. Confocal microscopy demonstrates a relatively homogeneous intratumor HPPH distribution. Labeling of host cells using fluorophore-conjugated antibodies allowed the visualization of Gr1(+)/CD11b(+) leukocytes and major histocompatibility complex class II (MHC-II)(+) cells in vivo. Imaging of the treated site at different time-points following irradiation shows significant and rapid increases in Gr1(+) cells in response to therapy. The maximum accumulation of Gr1(+) cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point. Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1(+ )cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status. Dual color confocal imaging experiments demonstrates that about 90% of the anti-Gr1 cell population co-localized with anti-CD11b labeling, thus indicating that majority of the Gr1-labeled cells were neutrophils. At 24 h post-PDT, an approximately 2-fold increase in MHC-II+ cells relative to untreated control is also observed. Co-localization analysis reveals an increase in the fraction of Gr1(+) cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype.

No MeSH data available.


Related in: MedlinePlus

(a) Stereofluorescence image of an EMT6 tumor in the ear of a BALB/c mouse, acquired at 24 h post IV administration of 1 μmol kg-1 HPPH. This representative image is a superposition of a bright field and HPPH fluorescence and highlights the selective uptake of HPPH in the tumor tissue. The HPPH fluorescence intensity in the tumor is approximately 2-3-fold higher than the adjacent normal tissue. (b) In vivo confocal image of intratumor HPPH distribution imaged at a depth of 100 μm into a mouse ear. HPPH fluorescence is in red, and CD31+ vessels labeled by Alexa488-conjugated antibody are in green. (c) HPPH fluorescence as a function of distance from CD31+ vessels, normalized to the fluorescence amplitude at the vessel boundary. The data points are an average of at least four independent measurements, and the error bars represent standard deviations.
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Figure 2: (a) Stereofluorescence image of an EMT6 tumor in the ear of a BALB/c mouse, acquired at 24 h post IV administration of 1 μmol kg-1 HPPH. This representative image is a superposition of a bright field and HPPH fluorescence and highlights the selective uptake of HPPH in the tumor tissue. The HPPH fluorescence intensity in the tumor is approximately 2-3-fold higher than the adjacent normal tissue. (b) In vivo confocal image of intratumor HPPH distribution imaged at a depth of 100 μm into a mouse ear. HPPH fluorescence is in red, and CD31+ vessels labeled by Alexa488-conjugated antibody are in green. (c) HPPH fluorescence as a function of distance from CD31+ vessels, normalized to the fluorescence amplitude at the vessel boundary. The data points are an average of at least four independent measurements, and the error bars represent standard deviations.

Mentions: In vivo, whole-mouse fluorescence imaging using the stereofluorescence microscope showed 2-3-fold tumor-to-normal skin selectivity (Figure 2(a)) at 24 h following IV administration of HPPH. We recently reported on the intratumor distribution of the sensitizer, NPe6, wherein we used the antibody labeling technique to label CD31+ tumor vasculature and imaged the spatial distribution and temporal kinetics of NPe6 with respect to vessels following systemic administration 11. Adopting the same approach, we examined the microscopic pattern of intratumor HPPH distribution using confocal fluorescence imaging.


Confocal fluorescence imaging enables noninvasive quantitative assessment of host cell populations in vivo following photodynamic therapy.

Mitra S, Mironov O, Foster TH - Theranostics (2012)

(a) Stereofluorescence image of an EMT6 tumor in the ear of a BALB/c mouse, acquired at 24 h post IV administration of 1 μmol kg-1 HPPH. This representative image is a superposition of a bright field and HPPH fluorescence and highlights the selective uptake of HPPH in the tumor tissue. The HPPH fluorescence intensity in the tumor is approximately 2-3-fold higher than the adjacent normal tissue. (b) In vivo confocal image of intratumor HPPH distribution imaged at a depth of 100 μm into a mouse ear. HPPH fluorescence is in red, and CD31+ vessels labeled by Alexa488-conjugated antibody are in green. (c) HPPH fluorescence as a function of distance from CD31+ vessels, normalized to the fluorescence amplitude at the vessel boundary. The data points are an average of at least four independent measurements, and the error bars represent standard deviations.
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Related In: Results  -  Collection

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Figure 2: (a) Stereofluorescence image of an EMT6 tumor in the ear of a BALB/c mouse, acquired at 24 h post IV administration of 1 μmol kg-1 HPPH. This representative image is a superposition of a bright field and HPPH fluorescence and highlights the selective uptake of HPPH in the tumor tissue. The HPPH fluorescence intensity in the tumor is approximately 2-3-fold higher than the adjacent normal tissue. (b) In vivo confocal image of intratumor HPPH distribution imaged at a depth of 100 μm into a mouse ear. HPPH fluorescence is in red, and CD31+ vessels labeled by Alexa488-conjugated antibody are in green. (c) HPPH fluorescence as a function of distance from CD31+ vessels, normalized to the fluorescence amplitude at the vessel boundary. The data points are an average of at least four independent measurements, and the error bars represent standard deviations.
Mentions: In vivo, whole-mouse fluorescence imaging using the stereofluorescence microscope showed 2-3-fold tumor-to-normal skin selectivity (Figure 2(a)) at 24 h following IV administration of HPPH. We recently reported on the intratumor distribution of the sensitizer, NPe6, wherein we used the antibody labeling technique to label CD31+ tumor vasculature and imaged the spatial distribution and temporal kinetics of NPe6 with respect to vessels following systemic administration 11. Adopting the same approach, we examined the microscopic pattern of intratumor HPPH distribution using confocal fluorescence imaging.

Bottom Line: The maximum accumulation of Gr1(+) cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point.Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1(+ )cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status.Co-localization analysis reveals an increase in the fraction of Gr1(+) cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Imaging Sciences, University of Rochester Medical Center, Rochester, NY 14642, USA.

ABSTRACT
We report the use of optical imaging strategies to noninvasively examine photosensitizer distribution and physiological and host responses to 2-[1-hexyloxyethyl]-2 devinyl pyropheophorbide-a (HPPH)-mediated photodynamic therapy (PDT) of EMT6 tumors established in the ears of BALB/c mice. 24 h following intravenous (IV) administration of 1 μmol kg(-1) HPPH, wide-field fluorescence imaging reveals tumor selectivity with an approximately 2-3-fold differential between tumor and adjacent normal tissue. Confocal microscopy demonstrates a relatively homogeneous intratumor HPPH distribution. Labeling of host cells using fluorophore-conjugated antibodies allowed the visualization of Gr1(+)/CD11b(+) leukocytes and major histocompatibility complex class II (MHC-II)(+) cells in vivo. Imaging of the treated site at different time-points following irradiation shows significant and rapid increases in Gr1(+) cells in response to therapy. The maximum accumulation of Gr1(+) cells is found at 24 h post-irradiation, followed by a decrease at the 48 h time-point. Using IV-injected FITC-conjugated dextran as a fluorescent perfusion marker, we imaged tissue perfusion at different times post-irradiation and found that the reduced Gr1(+ )cell density at 48 h correlated strongly with functional damage to the vasculature as reported via decreased perfusion status. Dual color confocal imaging experiments demonstrates that about 90% of the anti-Gr1 cell population co-localized with anti-CD11b labeling, thus indicating that majority of the Gr1-labeled cells were neutrophils. At 24 h post-PDT, an approximately 2-fold increase in MHC-II+ cells relative to untreated control is also observed. Co-localization analysis reveals an increase in the fraction of Gr1(+) cells expressing MHC-II, suggesting that HPPH-PDT is stimulating neutrophils to express an antigen-presenting phenotype.

No MeSH data available.


Related in: MedlinePlus