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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 MHC-II+ cells (red) and CD31+ vessels (green). These images were acquired at depths of 70 μm from the tissue surface. (a) Control ear that received HPPH but was not irradiated; (b) PDT-treated site imaged 24 h following irradiation. The field of view in the images is 800μm x 800μm. (c) Mean normalized MHC-II+ cell counts in untreated control and PDT-treated tissue at 24 h following irradiation. MHC-II+ cell population is approximately 1.5 fold higher in PDT-treated vs. control tissue, and the increase is significantly different at the P=0.05 level.
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Figure 6: In vivo confocal images of MHC-II+ cells (red) and CD31+ vessels (green). These images were acquired at depths of 70 μm from the tissue surface. (a) Control ear that received HPPH but was not irradiated; (b) PDT-treated site imaged 24 h following irradiation. The field of view in the images is 800μm x 800μm. (c) Mean normalized MHC-II+ cell counts in untreated control and PDT-treated tissue at 24 h following irradiation. MHC-II+ cell population is approximately 1.5 fold higher in PDT-treated vs. control tissue, and the increase is significantly different at the P=0.05 level.

Mentions: We have recently described imaging of MHC-II+ cells in normal and tumor tissue accomplished using fluorescence labeling in vivo 32. MHC class II proteins are present on the surface of antigen presenting cells (APCs), majority of which are macrophages and dendritic cells. It is well established that these cells internalize antigens and display a fragment of the antigen, bound to a MHC-II molecule which is recognized by T-cells, leading to their activation and initiation of specific immunity. We therefore imaged the MHC-II+ cell population in untreated control and 24 h post-PDT treated tissue. Figures 6(a) and 6(b) illustrate the MHC-II+ cells (red) in control and treated tissue, respectively. The distribution of MHC-II+ observed at a depth of about 70 μm is demonstrated in these representative images with respect to CD31+ vessels (green). In contrast to the 2-3-fold PDT-induced increase in infiltration of Gr1+ cells (figure 3), our analysis shows a modest but significant increase in the population of MHC-II+ cells at the treated site, with cell counts approximately 50% higher relative to control (figure 6(c)). It is possible that infiltrating macrophages and dendritic cells contribute to this modest increase in MHC-II+ cell counts. However, recent studies have also suggested that neutrophils under stimulation can present MHC-II on their surface and thus function as an APC 33, 34. Motivated by these findings, we examined if HHPH-PDT can induce Gr1+ cells to present MHC-II. To test whether the fraction of Gr1+ cells expressing MHC-II changes in response to therapy, we imaged Gr1+ and MHC-II+ cells in the same ear following administration of an antibody cocktail that contained anti-Gr1 and anti-MHC-II conjugated to AlexaFluor488 and Allophycocyanin, respectively. Figures 7(a) and 7(b) illustrate representative in vivo confocal images of anti-Gr1+ cells (green) and anti-MHC-II+ cells (red) pre- and 24 h post-irradiation, respectively. Co-localization analysis of these images indicates that in control tissue roughly 21% of Gr1+ pixels have corresponding MHC-II+ pixels, while in treated sites the fraction increases to approximately 28% and 36% at 5 h and 24 h post-irradiation, respectively (figure 7(c)). This suggests that PDT is inducing Gr1+ cells to express an antigen-presenting phenotype. Similar results have been reported by Sun et al., who observed expression of MHC class II on F4/80-/Gr1+ cells that had infiltrated Photofrin-PDT-treated SCCVII tumors 23. These results are also consistent with the observations of Kousis et al., who examined in detail the role of F4/80-/CD11b+/Gr1+ neutrophils in the stimulation of adaptive immune response 19. The authors reported that PDT-induced inflammation enables neutrophils to access tumor draining lymph nodes and directly play a role in the enhancement of T-cell activation and proliferation. Our results therefore support the contention that neutrophils may play an important role in stimulating T-cell proliferation in an MHC-II dependent manner.


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 MHC-II+ cells (red) and CD31+ vessels (green). These images were acquired at depths of 70 μm from the tissue surface. (a) Control ear that received HPPH but was not irradiated; (b) PDT-treated site imaged 24 h following irradiation. The field of view in the images is 800μm x 800μm. (c) Mean normalized MHC-II+ cell counts in untreated control and PDT-treated tissue at 24 h following irradiation. MHC-II+ cell population is approximately 1.5 fold higher in PDT-treated vs. control tissue, and the increase is significantly different at the P=0.05 level.
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Related In: Results  -  Collection

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Figure 6: In vivo confocal images of MHC-II+ cells (red) and CD31+ vessels (green). These images were acquired at depths of 70 μm from the tissue surface. (a) Control ear that received HPPH but was not irradiated; (b) PDT-treated site imaged 24 h following irradiation. The field of view in the images is 800μm x 800μm. (c) Mean normalized MHC-II+ cell counts in untreated control and PDT-treated tissue at 24 h following irradiation. MHC-II+ cell population is approximately 1.5 fold higher in PDT-treated vs. control tissue, and the increase is significantly different at the P=0.05 level.
Mentions: We have recently described imaging of MHC-II+ cells in normal and tumor tissue accomplished using fluorescence labeling in vivo 32. MHC class II proteins are present on the surface of antigen presenting cells (APCs), majority of which are macrophages and dendritic cells. It is well established that these cells internalize antigens and display a fragment of the antigen, bound to a MHC-II molecule which is recognized by T-cells, leading to their activation and initiation of specific immunity. We therefore imaged the MHC-II+ cell population in untreated control and 24 h post-PDT treated tissue. Figures 6(a) and 6(b) illustrate the MHC-II+ cells (red) in control and treated tissue, respectively. The distribution of MHC-II+ observed at a depth of about 70 μm is demonstrated in these representative images with respect to CD31+ vessels (green). In contrast to the 2-3-fold PDT-induced increase in infiltration of Gr1+ cells (figure 3), our analysis shows a modest but significant increase in the population of MHC-II+ cells at the treated site, with cell counts approximately 50% higher relative to control (figure 6(c)). It is possible that infiltrating macrophages and dendritic cells contribute to this modest increase in MHC-II+ cell counts. However, recent studies have also suggested that neutrophils under stimulation can present MHC-II on their surface and thus function as an APC 33, 34. Motivated by these findings, we examined if HHPH-PDT can induce Gr1+ cells to present MHC-II. To test whether the fraction of Gr1+ cells expressing MHC-II changes in response to therapy, we imaged Gr1+ and MHC-II+ cells in the same ear following administration of an antibody cocktail that contained anti-Gr1 and anti-MHC-II conjugated to AlexaFluor488 and Allophycocyanin, respectively. Figures 7(a) and 7(b) illustrate representative in vivo confocal images of anti-Gr1+ cells (green) and anti-MHC-II+ cells (red) pre- and 24 h post-irradiation, respectively. Co-localization analysis of these images indicates that in control tissue roughly 21% of Gr1+ pixels have corresponding MHC-II+ pixels, while in treated sites the fraction increases to approximately 28% and 36% at 5 h and 24 h post-irradiation, respectively (figure 7(c)). This suggests that PDT is inducing Gr1+ cells to express an antigen-presenting phenotype. Similar results have been reported by Sun et al., who observed expression of MHC class II on F4/80-/Gr1+ cells that had infiltrated Photofrin-PDT-treated SCCVII tumors 23. These results are also consistent with the observations of Kousis et al., who examined in detail the role of F4/80-/CD11b+/Gr1+ neutrophils in the stimulation of adaptive immune response 19. The authors reported that PDT-induced inflammation enables neutrophils to access tumor draining lymph nodes and directly play a role in the enhancement of T-cell activation and proliferation. Our results therefore support the contention that neutrophils may play an important role in stimulating T-cell proliferation in an MHC-II dependent manner.

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