The role of immune cells in photodynamic therapy
Creators: name: Krosl, Gorazd
Publisher: name: University of British Columbia
Year: 2009

description: The mechanisms of tumor destruction by photodynamic therapy (PDT) have proven to be very complex. The evidence from published work suggests that host factors may have an important role in both the uptake of photosensitizers and the effect of PDT. Focusing on clinically established photosensitizer Photofrin®, the role of tumor associated macrophages (TAMs) in accumulation of the drug using an experimental murine tumor mode! was examined in this thesis. To provide additional information about the inflammation processes that develop in the tumor after PDT treatment, the host cell infiltration into the treated tumor during and after the photodynamic light delivery was also investigated. The hypothesis that enhancing the host immune response can increase the curative potential of PDT was tested with two PDT-immunotherapy protocols. Photosensitizer accumulation in tumors is an important factor in the destruction of malignant lesions by PDT. The role of tumor associated macrophages in the accumulation of Photofrin in mouse SCCVII squamous cell carcinoma was studied by two different approaches. First, in vitro uptake and retention of Photofrin in mouse peritoneal macrophages were compared to that in SCCVII cells. Second, the accumulation of Photofrin in SCCVII tumor cells and tumor associated macrophages in vivo was studied using flow cytometry analysis. In all experiments, it was found that macrophages have a superior capacity for Photofrin uptake in comparison to that observed in SCCVII tumor cells. The in vivo accumulation of Photofrin showed consistently higher levels of the drug in TAMs compared to SCCVII malignant cells throughout the observation period (up to 96 hours past Photofrin administration). At 24 hours after Photofrin administration, the drug content was 74±15 (SD) ng/ug cell protein for TAMs compared to 15+2 ng/ug cell protein for SCCVII tumor cells. In addition, factors that modify macrophage phagocytic activity also influenced the ability of these cells to accumulate Photofrin. The infiltration of inflammatory cells is a central event in an acute inflammatory reaction. The major cellular populations contained in SGCVII tumors were analyzed at various time points after PDT. The cell populations were identified by staining with monoclonal antibodies against leukocyte membrane markers using indirect immunoperoxidase technique or by flow cytometry. Photofrin-based PDT induced profound changes in the levels of different cell populations comprising the treated tumor. The most immediate change was a dramatic increase in the content of neutrophils, which occurred within 5 min. after the onset of light treatment. This was followed by an increase in mast cell numbers, while monocyte/macrophage infiltration followed between 0 and 2 h after PDT. The examination of in vitro cytotoxicity of macrophages against SCCVII tumor cells revealed a pronounced increase in cytolysis mediated by TAMs harvested from the tumor at 2 h after PDT compared to that seen with the TAMs recovered from an untreated tumor. Such increased cytotoxicity of TAMs suggests that PDT induces an acute inflammatory infiltration of myeloid cells into the treated tumor, that is associated with functional activation of immune cells. This inflammatory reaction may provide an appropriate environment for activation of immune cells, and therapy with immunostimulators may effectively enhance its contribution to tumor destruction by, PDT. Two different PDT-immunotherapy protocols were tested for their ability to improve the curative potential of PDT. In the first case, treatment of SCCVII tumor bearing mice with glucan SPG which raised the relative content of Mac-1 positive cells infiltrating the tumor was examined. The tumor cure rate following Photofrin-based PDT (25 mg/kg Photofrin; 60J/cm2 light) increased approximately 3 times if PDT was preceded by the SPG therapy. In vitro colony formation assay revealed a greater killing of tumor cells in groups pre-treated with SPG, and this enhancement was particularly pronounced when the tumor excision was delayed to 8 h after PDT. In another part of the study, the SCCVII tumor cells were genetically engineered to produce the cytokine granulocyte-macrophage colonystimulating factor. (GM-CSF). 1x107 lethally irradiated GM-CSF producing cells were injected under the parental SCCVII tumor, followed by PDT (25 mg/kg Photofrin; 60 J/cm2 light) 48 h later. This therapy resulted in a dramatic increase in tumor cures (62% cure compared to 0% for PDT alone). Similarly, the POT with 10 mg/kg Photofrin and 150 J/cm2 light (that resulted in 6% tumor cure) was enhanced to 75% cure by the GM-CSF immunotherapy administered 3 times in 2 day intervals starting at 2 days before the light treatment. A comparable effect of GM-CSF immunotherapy was observed in the combination with benzoporphyrin derivative mediated PDT. The immunotherapy alone did not have a visible effect on tumor growth, and did not significantly affect peripheral white blood cell counts. However, a marked increase in TAM cytotoxicity against syngeneic tumor cells was detected.