Ivana Cecic

Contribution of myeloid and lymphoid host cells to the curative outcome of mouse sarcoma treatment by photodynamic therapy

Selective depletion or inactivation of specific myeloid populations (neutrophils, macrophages) and lymphoid populations (helper T cells, cytolytic T cells) in EMT6 sarcoma-bearing mice was used to determine the contribution of each of these host immune cell types to the curative outcome of Photofrin-based photodynamic therapy (PDT). Immunodepletion of neutrophils and cytolytic T cells initiated immediately after PDT resulted in a marked reduction in PDT-mediated tumor cures. Significant reduction in the cures of EMT6 tumors was also achieved by immunodepletion of helper T cells and inactivation of macrophages by silica treatment. The initial tumor ablation by PDT was not affected by any of the above depletion treatments. These results provide direct evidence that the contribution of neutrophils, macrophages and T lymphocytes is essential for the maintenance of long-term control of PDT-treated tumors.

Mediators of peripheral blood neutrophilia induced by photodynamic therapy of solid tumors

Photodynamic therapy (PDT) of tumors elicits a strong host immune response and one of its manifestations is a pronounced neutrophilia. By blocking their function prior to Photofrin-based PDT of mouse EMT6 tumors, we have identified multiple mediators whose regulated action is responsible for this neutrophilia. In addition to complement fragments (direct mediators) released as a consequence of PDT-induced complement activation, there are at least a dozen secondary mediators that all arise as a result of complement activity. The latter include cytokines IL-1beta, TNF-alpha, IL-6, IL-10, G-CSF and KC, thromboxane, prostaglandins, leukotrienes, histamine, and coagulation factors.

Induction of systemic neutrophil response in mice by photodynamic therapy of solid tumors.

Abstract Photodynamic therapy (PDT) of solid tumors elicits a strong, acute inflammatory response characterized by a rapid and massive infiltration of activated neutrophils into the tumor. The present study investigated the impact of PDT on the systemic and local (treatment site) kinetics of neutrophil trafficking and activity in mouse SCCVII and EMT6 tumor models. Differential leukocyte counts in the peripheral blood of treated mice revealed a pronounced neutrophilia developing rapidly after Photofrin® porfimer sodium (Photofrin)- or tetra(m-tetrahydroxyphenyl)chlorin (mTHPC)-based PDT. Significant neutrophilia was also observed upon PDT treatment of normal dorsal skin but not on the footpad of tumor-free mice. The changes in circulating neutrophil numbers were accompanied by an efflux of these cells from the bone marrow. An increased proportion of cells with high L-selectin (CD62L antigen) expression was found among bone-marrow–residing neutrophils 6–24 h after PDT, and in neutrophils in the peripheral circulation and treated tumors 24 h after therapy. Complement inhibition completely prevented the development of PDT-induced neutrophilia. The results of the present study demonstrate that treatment of solid tumors by PDT induces a strong and protracted increase in systemic neutrophil numbers mediated by complement activation. This reaction reflects rapid and massive mobilization and activation of neutrophils for the destruction of PDT-treated tumor tissue.

Neutrophils as inflammatory and immune effectors in photodynamic therapy-treated mouse SCCVII tumours.

Neutrophils have become recognised as important contributors to the effectiveness of tumour eradication by photodynamic therapy (PDT). In this study, we have used the mouse SCCVII squamous cell carcinoma model to investigate the activity of neutrophils in tumours treated by PDT. Tumour levels of neutrophilic myeloperoxidase (MPO) demonstrated not only a massive and sustained sequestration of these cells in PDT-treated tumours but also revealed their activated state evidenced by the presence of released MPO. Among the adhesion molecules expressed on tumour vascular endothelium, ICAM-1 appears to be of primary importance in the invasion of neutrophils into PDT-treated tumours, because its functional blocking with monoclonal antibodies reduced the tumour cure rate. A marked upregulation of its ligands CD11b/CD18 and CD11c/CD18 found on neutrophils associated with PDT-treated tumours supports this assumption. To evaluate the role of inflammatory cytokines regulating neutrophil activity, neutralising antibodies were given to mice before PDT treatment. The results suggest that IL-1beta activity is critical for the therapeutic outcome, since its neutralisation diminished the cure rates of PDT-treated tumours. No significant effect was observed with anti-IL-6 and anti-TNF-alpha treatment. Further flow cytometry-based examination of neutrophils round in PDT-treated tumours revealed that these cells express MHC class II molecules, which suggests their engagement as antigen-presenting cells and involvement in the development of antitumour immune response.

Acute phase response induction by cancer treatment with photodynamic therapy

Inflammation and immunity development are well recognized as responses to tumor treatment by photodynamic therapy (PDT). To demonstrate that another major host response effector process, acute phase response, may be also induced by this cancer treatment modality, the expression of serum amyloid P component (SAP) acknowledged as a hallmark acute phase reactant in the mouse was investigated following PDT of murine FsaR fibrosarcomas. The results reveal almost 150‐fold increase in the expression of SAP gene in the liver of mice bearing tumors treated by Photofrin‐mediated PDT, while serum SAP levels increased around 50‐fold at the peak interval about 24 hr post PDT. The same tumor treatment induced also the liver gene upregulation and serum levels elevation of another established acute phase reactant, mannose‐binding lectin A (MBL‐A). Both SAP and MBL‐A were found to accumulate in PDT‐treated tumors, but this includes local production because their genes in these tumor tissues were upregulated as well. Gene encoding C‐reactive protein (CRP) was also upregulated almost 7‐fold in the same tumor tissues, suggesting a rare example of CRP participation in host response of the mouse. Interleukin‐6 and glucocorticoid hormones were identified as major mediators promoting tumor PDT‐induced upregulation of liver SAP gene. Moreover, glucocorticoids were found to act as critical inducers of SAP gene upregulation in PDT‐treated tumors. The study definitely proves the occurrence of a strong acute phase response following tumor PDT, and reveals that glucocorticoid hormones released during this development impact the expression of host response‐relevant genes in PDT‐treated tumors.

Enhancement of tumour response to photodynamic therapy by adjuvant mycobacterium cell-wall treatment

Mycobacterium cell-wall extract (MCWE) is a potent non-specific immunostimulant that elicits a local inflammatory response associated with antitumour activity. Tumour-localized administration of MCWE has been examined as an adjuvant to photodynamic therapy (PDT) mediated by the photosensitizers Photofrin, benzoporphyrin derivative monoacid (BPD), metatetrahydroxyphenylchlorin (mTHPC), or zinc (II)-phthalocyanine (ZnPc). A single MCWE treatment, given immediately after light treatment of murine EMT6 tumours, potentiates the curative effect of PDT. A similar enhancement of tumour response to Photofrin-based PDT is obtained with the live Bacillus Calmette-Guérin (BCG) vaccine. Despite differences in the kinetics/intensity of damage induction to tumour microvasculature and other characteristics underlying the mechanism of antitumour activity of Photofrin, BPD, mTHPC and ZnPc, there appear to be no marked differences in the therapeutic benefit of adjuvant MCWE therapy combined with the PDT mediated by these various photosensitizers. This may be related to the fact that MCWE elicits a wide range of immunomodulatory effects that could amplify and sustain the inflammatory/immune responses triggered by PDT. The enhancement of inflammatory effector cell activity is indicated by the increased infiltration of neutrophils and other myeloid cells at the expense of malignant cells found in the MCWE plus mTHPC-based PDT treatment group compared to the PDT-only group.

Role of Complement Anaphylatoxin C3a in Photodynamic Therapy‐elicited Engagement of Host Neutrophils and Other Immune Cells

Abstract Tumor treatment by photodynamic therapy (PDT) provokes a host-protective inflammatory and acute-phase response and an immune reaction. Neutrophilia manifested in this context is driven by multiple mediators of neutrophil chemotaxis orchestrated by an activated complement system. Mouse FsaR fibrosarcoma was used in this study to further investigate neutrophilia induced by Photofrin-based PDT. The complement anaphylatoxin C3a was identified as a major chemoattractant in the advanced phase of PDT-induced neutrophilia, because injecting mice with antibodies blocking its receptor C3aR significantly inhibited the increase in neutrophil levels 8 h after PDT. At the same time point, an increased C3aR expression was detected in neutrophils, monocytes and B lymphocytes in the blood of host mice. Peritoneal macrophages and mast cells harvested from treatment-naive mice exhibited elevated C3aR expression after coincubation in vitro for 8 h with PDT-treated FsaR cells. Thus, C3a emerges as one of the key effector molecules engaged in PDT-induced host response.

Adjuvant treatment for complement activation increases the effectiveness of photodynamic therapy of solid tumors

Phototoxic lesions generated in tumor tissue by photodynamic therapy (PDT) are recognized by the host as a threat to the integrity and homeostasis at the affected site. Among the canonical pathways invoked by the host for dealing with this type of challenge is the activation of the complement system, integrating proteins that serve as molecular sensors of danger signals produced by PDT and those initiating signalling cascades coupled into the network of inflammatory and immune responses. Since the activated complement system is a salient participant of the antitumor response produced by PDT, it is worth exploring whether its manipulation can be exploited for the therapeutic benefit. Using mouse tumor models, the present study examined the potential of representative complement-activating agents to act as effective adjuvants to PDT. Tumor-localized treatment with zymosan, an alternative complement pathway activator, reduced the recurrence-rate of PDT-treated tumors, markedly increasing the percentage of permanent cures. In contrast, a similar treatment with heat aggregated gamma globulin (complement activator via the classical pathway) was of no significant benefit as a PDT adjuvant. Systemic complement activation with streptokinase treatment had no detectable effect on complement deposition at the tumor site without PDT, but it augmented the extent of complement activity in PDT-treated tumors. This finding based on immunohistochemistry analysis explains the results of tumor therapy experiments, which showed that systemic treatment with streptokinase or a similar agent, urokinase, enhances the PDT-mediated tumor response. Zymosan and streptokinase administrations produced no beneficial results with PDT of tumors growing in complement-deficient mice. This study, therefore, establishes the potential of complement-activating agents to serve as effective adjuvants to PDT for cancer treatment.

Complement activation cascade and its regulation: relevance for the response of solid tumors to photodynamic therapy.

The complement system has emerged as a prominent participant in host response elicited following treatment of solid tumors by photodynamic therapy (PDT). Activity of the complement system is tightly controlled by a series of endogenous regulatory proteins. The expression of decay-accelerating factor (DAF), complement-receptor-1-related protein y (Crry), and protectin, which are the three major mouse membrane-bound complement regulatory proteins (mCRPs), was examined following treatment of murine squamous cell carcinomas SCCVII by PDT mediated by the photosensitizer Photofrin. A marked decrease was detected in the expression of all three mCRPs on cancer cells from tumors following PDT, indicating that these cells were made more vulnerable to complement attack. In order to amplify this effect, following PDT mice were injected with antibodies neutralizing either Crry, protectin, or DAF. With anti-Crry and anti-protectin this resulted in increased tumor cure rate compared to PDT alone, while the contrary was observed with PDT plus anti-DAF combination (presumably owing to additional role of DAF in T cell signaling). Further examination including other complement regulatory proteins showed that combining antitumor PDT with antibody-mediated neutralization of factor H (soluble negative complement regulator) or injection of properdin (positive complement regulator) increased the cure rates of PDT-treated tumors. The use of various agents promoting complement activity appears promising for employment as adjuvants to PDT.

The impact of complement activation on tumor oxygenation during photodynamic therapy.

The response to photodynamic therapy (PDT) mediated by photosensitizer Photofrin was examined with Lewis lung carcinomas growing in either complement‐proficient C57BL/6 (B6) or complement‐deficient complement C3 knockout (C3KO) mice. The results reveal that Photofrin‐PDT was more effective in attaining cures of tumors in C3KO than in B6 hosts. Colony‐forming ability of cells from tumors excised immediately after Photofrin‐PDT confirmed that the direct cell killing effect was more pronounced in C3KO than in B6 hosts. In contrast, PDT mediated by photosensitizer benzoporphyrin derivative (BPD) produced higher cure rates of tumors in B6 hosts than those in C3KO hosts. Determination of tumor C3 levels by ELISA showed that Photofrin‐PDT induced markedly more pronounced complement activation than BPD‐PDT. Measurements of tumor oxygen tension immediately after PDT by Eppendorf pO2 histograph showed that Photofrin‐PDT induced a marked decline in the oxygenation of tumors growing in B6 mice that was much less pronounced in C3KO hosts. With BPD‐PDT the oxygen tensions in tumors in B6 and C3KO hosts decreased to a similar extent. This study indicates that complement activation in PDT‐treated tumors that varies with different photosensitizers is an important determinant of tumor oxygen limitation effects directly associated with photodynamic action.