The Kaplan-Meier curves of Figure demonstrate the tumor response to a fluence of 100 J cm-2 delivered at an irradiance of 75 mW cm-2. Among the 10 mice treated with this PDT dose, 9 (90%) were cured as defined by no evidence of tumor 90 days after irradiation. Untreated controls ((−) light, (−) drug) displayed a median tumor doubling time of 6 days (n = 4).
Kaplan-Meier curves of tumor responses to HPPH-PDT. Laser irradiation at 667 nm was performed at an irradiance of 75 mW cm-2 for a fluence of 100 J cm-2 at 24 h after intravenous administration of 1 μmol kg-1 HPPH.
In vivo, whole-mouse fluorescence imaging using the stereofluorescence microscope showed 2-3-fold tumor-to-normal skin selectivity (Figure (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.
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 (more ...)
Figure (b) shows a fluorescence image of HPPH distribution (red) with respect to anti-CD31 labeled vasculature (green) at a depth in tissue of 100 μm. The image was acquired 24 h following IV injection of 1 μmol kg-1 HPPH. As illustrated, at this time point HPPH has partitioned from the vasculature into the adjacent parenchyma relatively uniformly. Figure (c) shows analysis of HPPH distribution from fields of view from four independent experiments. The mean HPPH fluorescence amplitude is plotted as a function of distance from a vessel wall. Even at a distance of 100 μm the HPPH fluorescence does not drop below 30% of the intensity at the vessel boundary.
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 (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 (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 (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.
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. (more ...)
Perfused tumor blood vessels have important functions as the source of local tumor oxygenation and as a route for host cell trafficking 24
. With this motivation, bulk tumor perfusion in vivo was imaged with the stereofluorescence microscope, and microscopic imaging of small numbers of individual vessels was performed using the confocal microscope. Perfusion in live mice was visualized at pre-, 24 h, and 48 h post-irradiation using IV-injected FITC-conjugated high molecular weight dextran as an optical perfusion marker. Figures (a-c) are representative stereofluorescence images of perfusion status in an EMT6 ear tumor in the same mouse, followed up to 48 h post-PDT. As illustrated in figures (a) and 4(b), there was no detectable difference in perfusion status between the control and treated tumor at 24 h post-treatment. However, at the 48 h time-point a severe perfusion deficit is observed (figure (c)). Figures (d-f) are confocal images of microscopic patterns of perfusion level (green) in CD31+
labeled vessels (red). Consistent with the stereofluorescence images, we find that relative to the highly perfused vessels observed in control tissue, most of the CD31-positive vasculature in the treated region at 48 h post-irradiation exhibits an absence or low levels of the perfusion marker. This observation of perfusion loss at the 48 h correlates positively with the reduced Gr1+
population density at the same time-point. It is well established that vascular damage may be mediated by neutrophil secretion products such as chemokines, heparin-binding protein and arachidionic acid 25, 26
. Therefore, the reduced perfusion status at 48 h post-PDT is likely triggered by the large accumulation of neutrophils in the tumor tissue. This mode of vascular response is in contrast to the vascular shutdown induced by Visudyne-PDT, where direct damage to ECs leads to rapid loss of vascular barrier function 27
. Further, recent evidence has shown that neutrophils release proteolytic enzymes like MMP-9 upon activation 28
. Therefore, degradation of the extracellular matrix and damage to blood vessels following HPPH-PDT may also be mediated in part by the release of MMPs from the accumulated neutrophil population.
Figure 4 (a-c) Series of stereofluorescence images illustrating vessel perfusion status up to 48 h in an individual ear tumor treated with 100 J cm-2. (a) Untreated control, (b) 24 h post HPPH-PDT and (c) 48 h post-irradiation. These images were acquired from (more ...)
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 (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 (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.
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 (more ...)
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 (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 ), 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 (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+
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 (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 (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-
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-
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.
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 (more ...)
Figure 7 In vivo confocal images of Alexa488-conjugated anti-Gr1+ cells (green) and APC-conjugated anti-MHC-II+ cells (red) in the same field of view (a) pre- and (b) 24 h post-irradiation. These images were analyzed to evaluate the extent of host cells that co-expressed (more ...)
In conclusion, this study takes advantage of recently established optical imaging strategies to perform an in vivo examination of determinants, ranging from drug distribution to inflammatory response, that are associated with HPPH-PDT efficacy. We present evidence that PDT conditions that yield long term tumor control elicit a strong inflammatory response characterized by influx of Gr1+ cells. The findings suggest a role of these Gr1+ cells as mediators of HPPH-PDT-induced vascular damage and as effectors of adaptive immune response through their release of several secretion products and the expression of an antigen-presenting phenotype, respectively. Our work also highlights certain limitations of optical imaging, at least as implemented here. Flow cytometry offers distinct advantages in identifying cell types using multiple markers because of its ability to label intracellular proteins, its access to angle-resolved scatter, and because contemporary flow systems utilize spectral compensation algorithms and multivariate analysis techniques that are more sophisticated than those available in our optical imaging system and others with which we are familiar.