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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.

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In vivo confocal fluorescence images of (a) CD11b+ and (b) Gr1+ cells in the same field of view 24 h post-PDT. (c) Merged image showing the extent of overlap between cells expressing CD11b and Gr1 on the surface. The FOV in the images is 800μm x 800μm.
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Figure 5: In vivo confocal fluorescence images of (a) CD11b+ and (b) Gr1+ cells in the same field of view 24 h post-PDT. (c) Merged image showing the extent of overlap between cells expressing CD11b and Gr1 on the surface. The FOV in the images is 800μm x 800μm.

Mentions: Gr1 is expressed on neutrophils, macrophages, and plasmacytoid dendritic cells 29, 30. Therefore, the results reported above could potentially reflect cell populations other than neutrophils. In flow cytometry, identification of neutrophils includes positive staining for Gr1 and CD11b and negative staining for F4/80 19. We attempted to label in vivo with antibody against F4/80 but observed that commercially available fluorophore-conjugated anti-F4/80 antibodies resulted in unacceptably poor staining. Other investigators have reported similar issues with anti-F4/80 for ex-vivo tissue staining protocols 31. In the absence of a reliable F4/80 marker and in an effort to further scrutinize the Gr1+ cells in the images, we therefore performed two-color imaging experiments in several control and treated ears using an antibody cocktail that contained anti-Gr1 and anti-CD11b conjugated to AlexaFluor647 and AlexaFluor488, respectively. Figures 5(a) and 5(b) are representative in vivo images from identical fields of view 24 h post-PDT that are comprised exclusively of anti-CD11b or anti-Gr1 fluorescence when excited by 488 or 639 nm light, respectively. Figure 5(c) is a merged image of the two channels and displays strong overlap between the two channels in pixels exhibiting yellow / orange color. The high degree of overlap between pixels that display anti-CD11b and anti-Gr1 labeling is confirmed by co-localization analysis, which yields Manders coefficients of approximately 90%. These coefficients in the treated tissue at 24 h and 48 h post-HPPH-PDT were not different from those measured in untreated controls.


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

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

In vivo confocal fluorescence images of (a) CD11b+ and (b) Gr1+ cells in the same field of view 24 h post-PDT. (c) Merged image showing the extent of overlap between cells expressing CD11b and Gr1 on the surface. The FOV in the images is 800μm x 800μm.
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Related In: Results  -  Collection

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Figure 5: In vivo confocal fluorescence images of (a) CD11b+ and (b) Gr1+ cells in the same field of view 24 h post-PDT. (c) Merged image showing the extent of overlap between cells expressing CD11b and Gr1 on the surface. The FOV in the images is 800μm x 800μm.
Mentions: Gr1 is expressed on neutrophils, macrophages, and plasmacytoid dendritic cells 29, 30. Therefore, the results reported above could potentially reflect cell populations other than neutrophils. In flow cytometry, identification of neutrophils includes positive staining for Gr1 and CD11b and negative staining for F4/80 19. We attempted to label in vivo with antibody against F4/80 but observed that commercially available fluorophore-conjugated anti-F4/80 antibodies resulted in unacceptably poor staining. Other investigators have reported similar issues with anti-F4/80 for ex-vivo tissue staining protocols 31. In the absence of a reliable F4/80 marker and in an effort to further scrutinize the Gr1+ cells in the images, we therefore performed two-color imaging experiments in several control and treated ears using an antibody cocktail that contained anti-Gr1 and anti-CD11b conjugated to AlexaFluor647 and AlexaFluor488, respectively. Figures 5(a) and 5(b) are representative in vivo images from identical fields of view 24 h post-PDT that are comprised exclusively of anti-CD11b or anti-Gr1 fluorescence when excited by 488 or 639 nm light, respectively. Figure 5(c) is a merged image of the two channels and displays strong overlap between the two channels in pixels exhibiting yellow / orange color. The high degree of overlap between pixels that display anti-CD11b and anti-Gr1 labeling is confirmed by co-localization analysis, which yields Manders coefficients of approximately 90%. These coefficients in the treated tissue at 24 h and 48 h post-HPPH-PDT were not different from those measured in untreated controls.

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