We demonstrate the use of an enzyme-activatable fluorogenic probe, Neutrophil Elastase 680 FAST (NE680), for in vivo imaging of neutrophil elastase (NE) activity in tumors subjected to photodynamic therapy (PDT). NE protease activity was assayed in SCC VII and EMT6 tumors established in C3H and BALB/c mice, respectively. Four nanomoles of NE680 was injected intravenously immediately following PDT irradiation. 5 h following administration of NE680, whole-mouse fluorescence imaging was performed. At this time point, levels of NE680 fluorescence were at least threefold greater in irradiated versus unirradiated SCC VII and EMT6 tumors sensitized with Photofrin. To compare possible photosensitizer-specific differences in therapy-induced elastase activity, EMT6 tumors were also subjected to 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH)-PDT. NE levels measured in HPPH-PDT-treated tumors were twofold higher than in unirradiated controls. Ex vivo labeling of host cells using fluorophore-conjugated antibodies and confocal imaging were used to visualize Gr1 + cells in Photofrin-PDT-treated EMT6 tumors. These data were compared with recently reported analysis of Gr1 + cell accumulation in EMT6 tumors subjected to HPPH-PDT. The population density of infiltrating Gr1 + cells in treated versus unirradiated drug-only control tumors suggests that the differential in NE680 fold enhancement observed in Photofrin versus HPPH treatment may be attributed to the significantly increased inflammatory response induced by Photofrin-PDT. The in vivo imaging of NE680, which is a fluorescent reporter of NE extracellular release caused by neutrophil activation, demonstrates that PDT results in increased NE levels in treated tumors, and the accumulation of the cleaved probe tracks qualitatively with the intratumor Gr1 + cell population.
We measured the optical properties of freshly excised kidneys with renal parenchymal tumors to assess the feasibility of photodynamic therapy (PDT) in these patients. Kidneys were collected from 16 patients during surgical nephrectomies. Spatially resolved, white light, steady-state diffuse reflectance measurements were performed on normal and neoplastic tissue identified by a pathologist. Reflectance data were fit using a radiative transport model to obtain absorption (μ a ) and transport scattering coefficients (μ ′ s ), which define a characteristic light propagation distance, δ . Monte Carlo (MC) simulations of light propagation from cylindrical diffusing fibers were run using the optical properties extracted from each of the kidneys. Interpretable spectra were obtained from 14 kidneys. Optical properties of human renal cancers exhibit significant inter-lesion heterogeneity. For all diagnoses, however, there is a trend toward increased light penetration at longer wavelengths. For renal cell carcinomas (RCC), mean values of δ increase from 1.28 to 2.78 mm as the PDT treatment wavelength is increased from 630 to 780 nm. MC simulations of light propagation from interstitial optical fibers show that fluence distribution in tumors is significantly improved at 780 versus 630 nm. Our results support the feasibility of PDT in selected renal cancer patients, especially with photosensitizers activated at longer wavelengths.
Using integrated Raman and angular scattering microscopy (IRAM), we follow the response of EMT6 cancer cells
to photodynamic therapy (PDT) treatment. The study combines two non-labelling light scattering techniques
to extract chemical information and organelle sizes from single cells. Each cell is measured repeatedly over
several hours to follow changes in these parameters as the cell responds to the PDT treatment. An automated
algorithm identifies which parameters are changing in time. Size parameters extracted from angular scattering
measurements show a decrease in the size of 1-micron-diameter scatterers in treated cells. Treated cells also
exhibit trends in several Raman peaks, denoting changes in chemical concentrations of proteins, nucleic acids,
and lipids. Each of these parameters - acquired from both measurement modalities - can be monitored on a
cell-by-cell basis. The ability to track these chemical and structural changes over time allows access to greater
knowledge of biological processes.
Candida albicans is an opportunistic human fungal pathogen that requires an intact host immune response to prevent disease. Thus, studying host-pathogen interactions is critical to understanding and preventing this disease. We report a new model infection system in which ongoing C. albicans infections can be imaged at high spatial resolution in the ears of living mice. Intradermal inoculation into mouse ears with a C. albicans strain expressing green fluorescent protein results in systemic C. albicans infection that can be imaged in vivo using confocal microscopy. We observed filamentous growth of the organism in vivo as well as formation of microabscesses. This model system will allow us to gain significant new information about C. albicans pathogenesis through studies of host-C. albicans interactions in the native environment.
Motivated by recent successes in growing intradermal tumors in the ears of mice and establishing the
feasibility of in vivo confocal imaging of anatomic vessels in these tumors using fluorophore-conjugated
antibodies to CD31, we are exploring a number of applications of optical fluorescence imaging in
superficial murine tumor models in vivo. Immune responses induced by photodynamic therapy (PDT) are
dynamic processes that occur in a spatially and temporally specific manner. To visualize these processes
noninvasively, we have made progress in developing optical molecular imaging strategies that take
advantage of intradermal injection of fluorophore-conjugated-antibodies against surface antigens on
immune cells. This enables confocal imaging of the fluorescently labeled host cells to depths of at least
100 microns, and using this technique we have achieved in vivo imaging of granulocyte (GR-1)- and major
histocompatibility complex class II (MHC-II)-positive cell trafficking in tumors in response to PDT. The
latter include macrophages and dendritic cells. Data from tumors that were subjected to PDT with the
photosensitizer, HPPH, reveals a significantly enhanced level of
GR-1+ cell infiltration compared to
untreated control tumor. The temporal kinetics of GR-1+ and
MHC-II+ cells at different time intervals
post-PDT are being examined. The ability to image host responses in vivo without excising or perturbing the
tissue has opened up opportunities to explore means of optimizing them to therapeutic advantage.
We report an optical molecular imaging technique that exploits local administration of fluorophore-conjugated antibodies and confocal fluorescence microscopy to achieve high-contrast imaging of host cell populations in normal and tumor tissue in living mice. The method achieves micron-scale spatial resolution to depths greater than 100 µm. We illustrate the capabilities of this approach by imaging two dendritic cell populations in the skin and normal and tumor vasculature in vivo.
Motivated by recent successes in fluorescence imaging of whole mount tissue preparations and by rapid progress
in the fields of molecular imaging and molecular biology, we are exploring a number of applications of optical
fluorescence imaging in superficial murine tumor models in vivo. Imaging the PDT-induced expression of the
heat shock protein 70 (HSP70) in cells and in vivo is accomplished using stably transfected EMT6 cells in which
the gene for GFP is under the control of the HSP70 promoter. These cells readily form solid tumors in BALB/c
mice, enabling the direct imaging of the extent and time course of the activation of this promoter, with each
mouse serving as its own control. Imaging of similarly transfected EMT6 cells with a HIF-1&agr;/GFP fusion
protein vector enables visualization of HIF-1&agr; translocation to the nucleus. Recently, we have accomplished
fluorescent labeling of surface antigens in vivo using intratumor and intravenous injection of fluorophore-conjugated
antibodies. Injection of deep-red fluorophore-conjugated-anti-CD31 enables confocal fluorescence
imaging of the tumor vasculature to depths of at least 100 microns. With the vessels rendered fluorescent in this
way, a number of interesting studies become possible in the living mouse, including the direct visualization of
photosensitizer distribution from perfused vessels. Using the appropriate fluorophore-conjugated antibodies, we
have also been able to image infiltrating granulocytes in EMT6 tumors in response to PDT in vivo.
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