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Tumour photodynamic therapy-induced changes of complement gene expression Stott, Don Brandon

Abstract

Photodynamic therapy (PDT), a clinically established modality for treatment of tumours and other lesions, involves the administration of a light-activated drug that is activated at the targeted site by exposure to light. Light energy transforms the drug into a chemically active state and, in the presence of oxygen, leads to the formation of toxic oxygen derivatives. This oxidative, PDT-inflicted damage elicits a response from the host that plays an important role in the outcome of therapy. A major element of this host response is the activation of the complement system. Complement proteins are produced in the liver and it was presumed until recently that all components were synthesized there and released into the circulation. Recently, it has become clear that other cells besides hepatocytes are capable of synthesizing some or all of the cascade components. The purpose of this study was to examine the origin of complement production by the semi-quantitative RT-PCR analysis of key complement component genes C3, C5 and C9. Specifically we wanted to determine whether upregulation of these genes occurred following PDT and if so, was it happening in the liver or locally at the tumour. Our hypothesis was: PDT results in an increase of the tumourlocalized expression of key complement component genes. An in vivo time course experiment using Lewis Lung Carcinoma (LLC) growing in C57BL/6J mice examined the local and hepatic expression of C3, C5 and C9 following PDT. The results indicated no hepatic up-regulation of these genes but local expression in the tumour significantly increased (3.5 fold for C3, 3.2 fold for C5 and 1.7 fold for C9) at 24 hours following PDT light administration. These levels declined but remained elevated until five days post-treatment. Because this peak time point coincides with immune cell infiltration in vivo we wanted to examine the ability of macrophages in vitro to respond to PDT-treated LLC cells. This was done by co-incubating macrophages with PDT-treated LLC cells. Gene expression analysis revealed that macrophages significantly up-regulate the expression of complement genes C3, C5 and C9 by 8 hours following treatment and that this increase of gene expression results in a significant increase of complement protein levels in these cells by 16 hours co-incubation as compared to the control (no PDT) as determined by FACS. It was also discovered that malignant LLC cells themselves produce basal levels of complement proteins, but do not respond to PDT by increasing complement gene expression. Further experiments revealed that PDT-induced complement gene up-regulation was less pronounced in tumours growing in Toll-like receptor 4 (TLR-4) knockout mice compared to tumours in WT hosts. Using a series of specific inhibitors or blocking antibodies in the in vitro system with macrophages co-incubated with PDT treated tumour cells, it was confirmed that the TLR signaling pathway leading to NF - KB activation has a major role in this phenomenon. The results of this thesis shed light on complement engagement during the local host response which carries implications for the effective treatment of tumours by this therapy. These components may be harnessed and targeted to improve therapy by means of better controlling the complement response to PDT and therefore controlling a large component of the critical immune response which is necessary for a positive outcome after therapy.

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