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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

In vivo confocal images of Alexa488-conjugated anti-Gr1+ cells (green) and Alexa647-conjugated anti-CD31 vessels (red) in the tumor-bearing ears of BALB/c mice.. (a) Control ear that received HPPH but was not irradiated, and (b) ear that was PDT-treated. The FOV in these images is 800μm x 800μm. The image in (c) is an expanded view of an anti-Gr1 imaged field to illustrate the high contrast staining of host cells. (d) Mean normalized neutrophil counts in control and PDT-treated sites at time-points of 5, 24 and 48 hours following irradiation. 24 h post-PDT, the treatment induces approximately 2.5-fold higher neutrophil accumulation in the irradiated tissue. The neutrophil counts at 24 h are significantly different from those at control, 5 h and 48 h time-points (P < 0.05).
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Figure 3: In vivo confocal images of Alexa488-conjugated anti-Gr1+ cells (green) and Alexa647-conjugated anti-CD31 vessels (red) in the tumor-bearing ears of BALB/c mice.. (a) Control ear that received HPPH but was not irradiated, and (b) ear that was PDT-treated. The FOV in these images is 800μm x 800μm. The image in (c) is an expanded view of an anti-Gr1 imaged field to illustrate the high contrast staining of host cells. (d) Mean normalized neutrophil counts in control and PDT-treated sites at time-points of 5, 24 and 48 hours following irradiation. 24 h post-PDT, the treatment induces approximately 2.5-fold higher neutrophil accumulation in the irradiated tissue. The neutrophil counts at 24 h are significantly different from those at control, 5 h and 48 h time-points (P < 0.05).

Mentions: PDT induces a local inflammatory response that is characterized by leukocyte infiltration, with a significant fraction of these infiltrating cells being neutrophils 23. To examine the extent of this response to HPPH-PDT using treatment parameters that resulted in 90% tumor cures, we imaged the influx of Gr1+ cells in vivo into the treated site at 5, 24 and 48 h post-irradiation. Figures 3(a) and (b) illustrate the fluorophore-labeled infiltrating Gr1+ cells imaged in an untreated control and PDT-treated ear, respectively. The image shown in figure 3(c) is a magnified view of an imaged field and demonstrates that confocal imaging can resolve the antibody-labeled Gr1+ cells at an individual cell level. The data summarized in figure 3(d) from multiple independent measurements show an approximately 2.5-fold enhanced Gr1+ cell infiltration in the treated site 24 h post-irradiation compared to untreated control. We also observe a smaller but significant increase in Gr1+ cell accumulation as early as 5 h following treatment. At 48 h post-irradiation, the Gr1+ cell counts decrease and are not significantly different from those at the 5 h time-point. This trend in Gr1+ cell accumulation is remarkably similar to that reported by Gollnick et al. in an elegant study that used identical HPPH-PDT treatment conditions to investigate various mediators of the inflammatory response 18. Using flow cytometry, the authors found that HPPH-PDT induces an influx of nearly 3-fold higher neutrophils into tumors compared to untreated control at 24 h post-irradiation. The comparable finding between our study and that reported by Gollnick et al. serves as a measure of validation for the in vivo imaging strategy as an assay for host cell populations.


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 images of Alexa488-conjugated anti-Gr1+ cells (green) and Alexa647-conjugated anti-CD31 vessels (red) in the tumor-bearing ears of BALB/c mice.. (a) Control ear that received HPPH but was not irradiated, and (b) ear that was PDT-treated. The FOV in these images is 800μm x 800μm. The image in (c) is an expanded view of an anti-Gr1 imaged field to illustrate the high contrast staining of host cells. (d) Mean normalized neutrophil counts in control and PDT-treated sites at time-points of 5, 24 and 48 hours following irradiation. 24 h post-PDT, the treatment induces approximately 2.5-fold higher neutrophil accumulation in the irradiated tissue. The neutrophil counts at 24 h are significantly different from those at control, 5 h and 48 h time-points (P < 0.05).
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Related In: Results  -  Collection

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Figure 3: In vivo confocal images of Alexa488-conjugated anti-Gr1+ cells (green) and Alexa647-conjugated anti-CD31 vessels (red) in the tumor-bearing ears of BALB/c mice.. (a) Control ear that received HPPH but was not irradiated, and (b) ear that was PDT-treated. The FOV in these images is 800μm x 800μm. The image in (c) is an expanded view of an anti-Gr1 imaged field to illustrate the high contrast staining of host cells. (d) Mean normalized neutrophil counts in control and PDT-treated sites at time-points of 5, 24 and 48 hours following irradiation. 24 h post-PDT, the treatment induces approximately 2.5-fold higher neutrophil accumulation in the irradiated tissue. The neutrophil counts at 24 h are significantly different from those at control, 5 h and 48 h time-points (P < 0.05).
Mentions: PDT induces a local inflammatory response that is characterized by leukocyte infiltration, with a significant fraction of these infiltrating cells being neutrophils 23. To examine the extent of this response to HPPH-PDT using treatment parameters that resulted in 90% tumor cures, we imaged the influx of Gr1+ cells in vivo into the treated site at 5, 24 and 48 h post-irradiation. Figures 3(a) and (b) illustrate the fluorophore-labeled infiltrating Gr1+ cells imaged in an untreated control and PDT-treated ear, respectively. The image shown in figure 3(c) is a magnified view of an imaged field and demonstrates that confocal imaging can resolve the antibody-labeled Gr1+ cells at an individual cell level. The data summarized in figure 3(d) from multiple independent measurements show an approximately 2.5-fold enhanced Gr1+ cell infiltration in the treated site 24 h post-irradiation compared to untreated control. We also observe a smaller but significant increase in Gr1+ cell accumulation as early as 5 h following treatment. At 48 h post-irradiation, the Gr1+ cell counts decrease and are not significantly different from those at the 5 h time-point. This trend in Gr1+ cell accumulation is remarkably similar to that reported by Gollnick et al. in an elegant study that used identical HPPH-PDT treatment conditions to investigate various mediators of the inflammatory response 18. Using flow cytometry, the authors found that HPPH-PDT induces an influx of nearly 3-fold higher neutrophils into tumors compared to untreated control at 24 h post-irradiation. The comparable finding between our study and that reported by Gollnick et al. serves as a measure of validation for the in vivo imaging strategy as an assay for host cell populations.

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