Jinghai Sun

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.

Enhancement of Laser Cancer Treatment by a Chitosan-derived Immunoadjuvant¶

Abstract A chitosan derivative, glycated chitosan (GC), has been used as an immunostimulant for cancer treatment in laser immunotherapy. The function of GC is to enhance the host immune response after direct cancer cell destruction by a selective laser photothermal interaction. To further test its effects, laser immunotherapy was extended to include several different adjuvants for immunological stimulation and to include photodynamic therapy (PDT) as a different tumor-destruction mechanism. Complete Freund (CF) adjuvant, incomplete Freund (IF) adjuvant and Corynebacterium parvum (CP) were selected for treatment of metastatic mammary tumors in rats, in combination with a selective photothermal interaction. The solution of the immunoadjuvants admixed with indocyanine green (ICG), a light-absorbing dye, was injected directly into the tumors, followed by noninvasive irradiation of an 805 nm laser. Combined with PDT, in the treatment of tumors in mice, GC was administered peritumorally immediately after laser irradiation. The survivals of treated animals were compared with untreated control animals. In the treatment of rat tumors, CF, IF and CP raised the cure rates from 0% to 18%, 7% and 9%, respectively. In comparison, GC resulted in a 29% long-term survival. In the treatment of EMT6 mammary sarcoma in mice, GC of 0.5% and 1.5% concentrations increased the cure rates of Photofrin-based PDT treatment from 38% to 63% and 75%, respectively. In the treatment of Line 1 lung adenocarcinoma in mice, a 1.67% GC solution enabled a noncurative meso-substituted tetra(meta-hydroxy-phenyl)chlorin–based PDT to cure 37% of the tumor-bearing mice. The experimental results of this study confirmed our previous studies, showing that immunoadjuvants played an active role in laser-related cancer treatment and that GC significantly enhanced the efficacy of laser cancer treatment.

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.

Cancer treatment by photodynamic therapy combined with adoptive immunotherapy using genetically altered natural killer cell line

Adoptive immunotherapy mediated by human natural killer (NK) cell line genetically altered to produce interleukin‐2 (NK92MI) was evaluated as adjuvant to photodynamic therapy (PDT) of subcutaneous tumors. The combined effect of these two modalities was first examined with SiHa tumors (human cervical squamous cell carcinoma) growing in NOD‐scid mice. The most effective protocol for NK92MI cell transfer in conjunction with PDT mediated by photosensitizer mTHPC was the injection of 5 × 107 cells (peritumoral or intravenous) given immediately after PDT, which produced a marked improvement in the therapeutic outcome compared with the effect of PDT alone. The same protocol was tested with HT‐29 tumor model (human colorectal adenocarcinoma) xenografted in NOD‐scid mice. The results demonstrate that the adoptive immunotherapy with NK92MI cells (which when used alone were not effective in controlling tumor growth) significantly improved the cures of PDT‐treated HT‐29 tumors, whereas such benefit was not observed with the parental cell line NK92 (not producing interleukin‐2). Flow cytometry‐based analysis revealed a higher percentage of p.t. injected NK92MI cells in PDT‐treated than in non‐treated HT‐29 tumors. Further investigation showed that the NK92MI cell‐based adoptive immunotherapy is also a highly effective adjuvant for PDT treatment of murine EMT6 tumors growing in immunocompetent syngeneic BALB/c mice. This result diminishes the concern that adoptively transferred NK92MI cells may be rendered ineffective by an allogenic reaction of the host. The findings of this study suggest that advanced protocols of NK cell‐based adoptive immunotherapy can be developed as efficient adjuvants to PDT used for the treatment of solid malignant tumors.

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.

Interaction Between Photodynamic Therapy and BCG Immunotherapy Responsible for the Reduced Recurrence of Treated Mouse Tumors

Subcutaneous mouse EMT6 tumors were treated by individual or combined regimens of a single Bacillus Calmette–Guérin (BCG) vaccine administration and photodynamic therapy (PDT). Six clinically relevant photosensitizers characterized by different action mechanisms were used: Photofrin, benzoporphyrin derivative, tetra(m‐hydroxyphenyl)chlorin (foscan), mono‐l‐aspartyl‐chlorin e6, lutetium texaphyrin or zinc phthalocyanine. Irrespective of the type of photosensitizer used, the optimized BCG protocols improved the cure rate of PDT‐treated tumors. This indicates that the interaction does not take place during the early phase of tumor ablation but at later events involved in preventing tumor recurrence. Beneficial effects on tumor cure were observed even when the BCG injection was delayed to 7 days after PDT. The accumulation of activated myeloid cells that markedly increases in tumors treated by Photofrin‐based PDT was not additionally affected by BCG treatment. However, the incidence of immune memory T cells in tumor‐draining lymph nodes that almost doubled at 6 days after Photofrin‐PDT further increased close to three‐fold with adjuvant BCG. This suggests that BCG immunotherapy amplifies the T‐lymphocyte–mediated immune response against PDT‐treated tumors. Since both these modalities are established for the treatment of superficial bladder carcinomas, use of their combination for this condition should be clinically tested.

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.

Ceramide and sphingosine-1-phosphate act as photodynamic therapy-elicited damage-associated molecular patterns: cell surface exposure.

Molecules that appear on the surface of tumor cells after their therapy treatment may have important roles either as damage-associated molecular patterns (DAMPs) or signals for phagocytes influencing the disposal of these cells. Treatment of SCCVII and CAL27 cells, models of mouse and human squamous cell carcinoma respectively, by photodynamic therapy (PDT) resulted in the presentation of ceramide and sphingosine-1-phosphate (S1P) on the cell surface. This was documented by anti-ceramide and anti-S1P antibody staining followed by flow cytometry. The exposure of these key sphingolipid molecules on PDT-treated tumor cells was PDT dose-dependent and it varied in intensity with different photosensitizers used for PDT. The above results, together with the finding that both ceramide and S1P can activate NFκB signaling in macrophages co-incubated with PDT-treated tumor cells, establish that these two sphingolipids can act as DAMPs stimulating inflammatory/immune reactions critical for tumor therapy response.

Dying cells program their expedient disposal: serum amyloid P component upregulation in vivo and in vitro induced by photodynamic therapy of cancer

Serum amyloid P component (SAP) is known as a prototypic acute phase reactant in the mouse and the protein that binds to dying cells securing their swift disposal by phagocytes. Treatment of solid tumors by photodynamic therapy (PDT) triggers SAP production in the liver of host mice, its release in the circulation and accumulation in PDT-targeted lesions. In the present study, mouse Lewis lung carcinoma (LLC) cells treated in vitro by PDT are shown to upregulate their gene encoding SAP. This effect was manifested following PDT treatment mediated by various types of photosensitizers (Photofrin, BPD, mTHPC, ALA). Generated SAP protein was not detected in tissue supernatants but remained localized to producing PDT-treated cells. The upregulation of SAP gene was observed also in untreated IC-21 macrophages after they were co-incubated for 4 h with PDT-treated LLC cells. Based on these findings, SAP that accumulates in PDT-treated tumors may originate from both systemic sources (released from the liver as acute phase reactant) and local sources; the latter could include tumor cells directly sustaining PDT injury and macrophages invading the tumor that become stimulated by signals from these affected tumor cells. Since SAP gene upregulation in LLC cells increased with the lethality of PDT dose used for their treatment, we propose that cells sensing they are inflicted with mortal injury can turn on molecular programs insuring not only that they die an innocuous form of death (apoptosis) but also that once they are dead their elimination is (facilitated by SAP) swift and efficient.

Activation of Poly(adenosine diphosphate-ribose) Polymerase in Mouse Tumors Treated by Photodynamic Therapy {

Poly(adenosine diphosphate–ribose) polymerase (PARP) has recently been characterized as a key regulator of cell death–survival transcriptional programs associated with stress and inflammation. Possible participation of this enzyme in the response of tumors to photodynamic therapy (PDT) was investigated in this study. Immunohistochemical analysis of mouse FsaR tumors treated by PDT based on photosensitizers Photofrin or 5,10,15,20‐tetra‐(m‐hydroxyphenyl)chlorine (mTHPC) revealed a strong positive staining for PARP product poly(ADP‐ribose) at 30 min and 1 h after PDT, respectively, and even more intense positivity at 2 h after PDT with both photosensitizers. Flow cytometry–based examination showed the induction of poly‐ADP‐ribosylation in FsaR tumors at 30 min after PDT, with a trend for a further increase in the intensity by 2 h after PDT in both cancer cells and tumor‐associated leukocytes. In FsaR cells treated in vitro by mTHPC‐based PDT, flow cytometric analysis indicated that the activation of PARP concentrated in cells undergoing apoptosis and reached a maximum by 30 min after PDT. The administration of PARP inhibitors, 3‐aminobenzamide or 1,5‐isoquinolinediol, to FsaR tumor–bearing mice before PDT light treatment increased the resistance of these tumors to PDT. PARP appears to control the balance between apoptotic and necrotic cell death in PDT‐treated tumors and regulate the progression of PDT‐induced inflammatory or innate immune response.