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The role of neutrophils in the response of solid cancers to photodynamic therapy Cecić, Ivana F. K.


Photodynamic Therapy (PDT) has been employed as a treatment for the eradication of solid neoplastic lesions over the last decade. With the FDA approval of Photofrin for use in PDT, this therapy has been clinically established for the treatment of several cancers in the Netherlands, Japan, France, the USA, and Canada. PDT involves the administration of a photosensitive drug, excited by light of appropriate wavelength, causing localized tumor cell death in the presence of oxygen. The mechanism of cell death by PDT is complex, yet is increasingly understood, with accumulating evidence suggesting that the host response to PDT plays a major role in the success of this treatment. A marked feature of PDT is the induction of strong, acute inflammation characterized by edema formation, and a wave of infiltrating inflammatory cells, first of which is the neutrophil. It has been documented that activated neutrophils sequester in tumors during and following PDT, and their role is indispensable for the effectiveness of this treatment modality. For instance, as shown in this thesis, in the absence of circulating neutrophils (achieved by the administration of 5 mg/kg of the monoclonal anti-GRL antibody) long-term tumor control by PDT is diminished for the murine squamous cell carcinoma SCCVII grown in syngeneic C3H/HeN mice. Using this tumor model and also the murine EMT6 mammary sarcoma, we further examined the systemic response of neutrophils to PDT. Based on Wright stain analysis, it was determined that PDT induced a rise in relative circulating neutrophil content up to 2.3 times normal levels, from approximately 25% to 60% of nucleated cells in blood. Consequently, total neutrophil numbers in circulation rose from 2.95 ± 1.1 x 10⁶ to 18.3 ± 5 x 10⁶ per ml of blood at the peak interval 3 hours following PDT light treatment of subcutaneous back tumors. These increases were significantly higher than the effect on neutrophil levels by any stress-related reaction the animals experienced in handling during treatment (tail bleeding for blood collection, i.v. injection, immobilization in lead holders, PDT light only). No changes were observed in the total numbers of other white blood cell types, such as lymphocytes and monocytes. Identifying neutrophils by their high level of GR1 expression, flow cytometry analysis was used to compare the neutrophil cell content in tumors, blood, lungs, and bone marrow of tumor-bearing mice over a 24-hour interval following PDT. A temporal rise in the levels of these cells in tumors, blood, and lungs, corresponded with a 50% drop in the granulocyte cell content of bone marrow. It therefore appears that a release of neutrophils from bone marrow occurred in response to PDT. L-selectin (CD62L) expression was analyzed in these neutrophil populations, as an indicator of age and activation state. Flow cytometry analysis detected a significant increase in L-selectin expression in the neutrophil populations of bone marrow and tumors from 2 and 8%, to 33 and 53%, respectively. In circulation, the majority of neutrophils expressed medium concentrations of L-selectin, with a very small fraction expressing high levels. There was an increase in L - selectin medlum with a corresponding decrease in L-selectin low populations 10 hours after PDT treatment, while a marked rise in L-selectin high neutrophils was detected 24 hours post PDT. No significant changes were observed in the lungs. Collectively, these results suggest that localized PDT induced a systemic response characterized by the release of younger, less mature, L-selectin high neutrophils from bone marrow. Hence, tumor-infiltrating neutrophil populations may consist of both mature and young neutrophils (present due to their accelerated release from bone marrow) which together contribute to tumor eradication by PDT. Therefore, PDT-induced inflammation partly characterized by the continuous sequestration of neutrophils into PDT treated mouse tumors during the first day following PDT treatment, is causing a systemic response dominated by neutrophilia and an increased activation status of these cells. This condition reflects a massive mobilization of neutrophils from their storage pools and myeloid precursors, as they are recruited to participate in the destruction of PDT treated tumor tissue.

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