Photodynamic therapy (PDT) shows great potential as a therapeutic modality in cancer. Although most studies in this field have employed porphyrins as tumor-localizing photosensitizers, the phthalo-cyanine )Pc) class of compounds has emerged as an effective alternative. Efforts to optimize PcPDT protocols have been complicated by the fact that little is known about the in vivo mechanisms by which these agents cause tumor destruction or about the contribution of immunological and inflammatory mediators in the response. The objectives of this proposal will be to delineate these mechanisms in mice, utilizing the RIF-1 tumor system as a model. Among the mechanic issues that we will address are the direct vs indirect effects of Pc PDT on tumors, Pc PDT effects on the tumor vasculature, and the type of leukocytes that infiltrate tumors. Direct vs. indirect effects on tumor will be evaluated by removing tumors immediately after Pc PDT has been completed -- before the indirect effects have taken place -- and determining whether there is enhanced survival of tumor cells in clonogenicity assays. Another approach will be to determine the hypoxic tumor cell fraction following Pc PDT. A large hypoxic tumor cell fraction in the face of extensive tumor destruction would suggest that indirect effects predominate. Additional mechanistic studies will include fluorescein angiography to characterize Pc PDT effects on the tumor vasculature, and immunohistology to identify immunological and inflammatory cell populations that infiltrate tumors prior to and after Pc PDT. Once these experiments have been completed, detailed studies will be performed to evaluate whether leukocytes that infiltrate tumors participate in the anti-tumor efficacy of Pc PDT. This will be accomplished by subjecting RIF-1 tumor-bearing mice that have been depleted of specific leukocyte subpopulations in vivo to our Pc PDT protocol and determining whether such procedures alter the anti-tumor response. Our preliminary studies have shown that Pcs and light are potent stimuli for cytokine production. Further evaluation in the RIF-1 tumor system will be performed and in situ hybridization studies will be conducted on Pc PDT-treated tumors to determine sites of cytokine mRNA expression. The role of cytokines in Pc PDT mediated tumor regression will also be assessed by treating animals with neutralizing anti-cytokine antibodies in vivo and determining their effect on tumor destruction and regrowth. In vitro and in vivo experiments will be conducted to define the profile of eicosanoids released following Pc PDT. We will then attempt to evaluate their participation in tumor regression by treating animals with inhibitors of the cyclooxygenase and lipoxygenase pathways prior to Pc PDT treatment and examining whether they alter the response. Finally, using the TNF-alpha gene as a model, we will examine the transcriptional and post-transcriptional effects of Pc PDT. Knowledge obtained from these studies may help to identify safe and effective Pcs for PDT protocols and may enhance our understanding of the mechanisms that underlie the anti-tumor effects of this exciting class of compounds.
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