Enhanced tumor cures after Foscan photodynamic therapy combined with the ceramide analog LCL29. Evidence from mouse squamous cell carcinomas for sphingolipids as biomarkers of treatment response.

To improve anticancer therapeutic success of photodynamic therapy (PDT), combination treatments represent a viable strategy. Sphingolipid analogs combined with anticancer drugs can enhance tumor response. We have shown that LCL29, a C6-pyridinium ceramide, promotes therapeutic efficacy of Photofrin-PDT in mouse SCCVII squamous cell carcinoma tumors. The long-term effect of the combination PDT + LCL29 is unknown. In this study we used the same model to test the long-term curative potential of Foscan-PDT + LCL29. We show that treatment of SCCVII tumors with the combination led to enhanced long-term tumor cure compared to PDT alone. LCL29 itself did not prevent tumor growth. All treatments triggered early increases in tumor-associated C16-ceramide, C18-ceramide, dihydrosphingosine, and global levels of dihydroceramides. PDT-evoked increases in tumor-associated sphingosine-1-phosphate and dihydrosphingosine-1-phosphate remained elevated or were attenuated after the combination, respectively; in contrast, LCL29 had no effect on these two sphingolipids. Our data demonstrate that adjuvant LCL29 improves PDT long-term therapeutic efficacy, implying translational potential of the combination. Furthermore, our findings indicate that changes in the sphingolipid profile might serve as predictive biomarkers of tumor response to treatments.

Increased tumour dihydroceramide production after Photofrin-PDT alone and improved tumour response after the combination with the ceramide analogue LCL29. Evidence from mouse squamous cell carcinomas.

Photodynamic therapy (PDT) has been proven effective for treatment of several types of cancer. Photodynamic therapy alone, however, attains limited cures with some tumours and there is need for its improved efficacy in such cases. Sphingolipid (SL) analogues can promote tumour response in combination with anticancer drugs. In this study, we used mouse SCCVII squamous cell carcinoma tumours to determine the impact of Photofrin-PDT on the in vivo SL profile and the effect of LCL29, a C6-pyridinium ceramide, on PDT tumour response. Following PDT, the levels of dihydroceramides (DHceramides), in particular C20-DHceramide, were elevated in tumours. Similarly, increases in DHceramides, in addition to C20:1-ceramide, were found in PDT-treated SCCVII cells. These findings indicate the importance of the de novo ceramide pathway in Photofrin-PDT response not only in cells but also in vivo. Notably, co-exposure of SCCVII tumours to Photofrin-PDT and LCL29 led to enhanced tumour response compared with PDT alone. Thus, we show for the first time that Photofrin-PDT has a distinct signature effect on the SL profile in vitro and in vivo, and that the combined treatment advances PDT therapeutic gain, implying translational significance of the combination.

Photodynamic therapy-generated vaccines: relevance of tumour cell death expression.

Recent investigations have established that tumour cells treated in vitro by photodynamic therapy (PDT) can be used for generating potent vaccines against cancers of the same origin. In the present study, cancer vaccines were prepared by treating mouse SCCVII squamous cell carcinoma cells with photosensitiser chlorin e6-based PDT and used against poorly immunogenic SCCVII tumours growing in syngeneic immunocompetent mice. The vaccine potency increased when cells were post-incubated in culture after PDT treatment for 16 h before they were injected into tumour-bearing mice. Interfering with surface expression of phosphatidylserine (annexin V treatment) and apoptosis (caspase inhibitor treatment) demonstrated that this post-incubation effect is affiliated with the expression of changes associated with vaccine cell death. The cured mice acquired resistance to re-challenge with the same tumour, while the engagement of cytotoxic T lymphocytes was demonstrated by detection of high numbers of degranulating CD8+ cells in vaccinated tumours. The vaccines prepared from ex vivo PDT-treated SCCVII tumour tissue were also highly effective, implying that surgically removed tumour tissue can be directly used for PDT vaccines. This opens attractive prospects for employing PDT vaccines tailored for individual patients targeting specific antigens of the patient's tumour.

Potentiation of photodynamic therapy of cancer by complement: the effect of gamma-inulin.

Host response elicited by photodynamic therapy (PDT) of cancerous lesions is a critical contributor to the clinical outcome, and complement system has emerged as its important element. Amplification of complement action was shown to improve tumour PDT response. In search of a clinically relevant complement activator for use as a PDT adjuvant, this study focused on gamma-inulin and examined its effects on PDT response of mouse tumours. Intralesional gamma-inulin (0.1 mg mouse(-1)) delivered immediately after PDT rivaled zymosan (potent classical complement activator) in delaying the recurrence of B16BL6 melanomas. This effect of gamma-inulin was further enhanced by IFN-gamma pretreatment. Tumour C3 protein levels, already elevated after individual PDT or gamma-inulin treatments, increased much higher after their combination. With fibrosarcomas MCA205 and FsaR, adjuvant gamma-inulin proved highly effective in reducing recurrence rates following PDT using four different photosensitisers (BPD, ce6, Photofrin, and mTHPC). At 3 days after PDT plus gamma-inulin treatment, over 50% of cells found at the tumour site were CTLs engaged in killing specific targets via perforin-granzyme pathway. This study demonstrates that gamma-inulin is highly effective PDT adjuvant and suggests that by amplifying the activation of complement system, this agent potentiates the development of CTL-mediated immunity against PDT-treated tumours.

Ischaemia-reperfusion injury in photodynamic therapy-treated mouse tumours.

Prompted by the observation of ischaemia development during the treatment of tumours by photodynamic therapy (PDT) that is typically followed by a restoration of tumour blood flow and by the indications of secondary superoxide generation after PDT, we aimed in this study to obtain evidence of the induction of ischaemia-reperfusion (I/R) injury in PDT-treated tumours. Using subcutaneous mouse FsaR fibrosarcoma model and Photofrin-based PDT treatment, we have examined the activity of xanthine oxidase (XO, a key enzyme in the I/R injury development) in tumours before and after the therapy. Compared to the levels in nontreated tumours, there was a five-fold increase in the activity of this enzyme in tumours excised immediately after PDT. This burst of elevated XO activity declined rapidly, returning to the pretreatment levels within the next 30 min. Visible reflectance spectroscopy confirmed the occurrence of a PDT-induced strong but temporary reduction in tumour oxygenation. The administration of XO inhibitor oxypurinol prevented this PDT-induced rise in XO activity. The oxypurinol treatment also decreased the extent of neutrophil accumulation in PDT-treated tumours and reduced the level of PDT-mediated cures. These results demonstrate the induction of I/R injury in PDT-treated tumours, and show that it can contribute to the therapy outcome. Since I/R injury is a well-recognised proinflammatory insult, we suggest that its induction in PDT-treated tumours promotes the development of inflammatory response that has become established as a key element of the antitumour effect of PDT.

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

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.

Oral aminolevulinic acid induces protoporphyrin IX fluorescence in psoriatic plaques and peripheral blood cells.

Photodynamic therapy (PDT) with topical aminolevulinic acid (ALA) has been shown in previous studies to improve psoriasis. However, topical ALA-PDT may not be practical for the treatment of extensive disease. In order to overcome this limitation we have explored the potential use of oral ALA administration in psoriatic patients. Twelve patients with plaque psoriasis received a single oral ALA dose of 10, 20 or 30 mg/kg followed by measurement of protoporphyrin IX (PpIX) fluorescence in the skin and circulating blood cells. Skin PpIX levels were determined over time after ALA administration by the quantification of the 635 nm PpIX emission peak with in vivo fluorescence spectroscopy under 442 nm laser excitation. Administration of ALA at 20 and 30 mg/kg induced preferential accumulation of PpIX in psoriatic as opposed to adjacent normal skin. Peak fluorescence intensity in psoriatic and normal skin occurred between 3 and 5 h after the administration of 20 and 30 mg/kg, respectively. Ratios of up to 10 for PpIX fluorescence between psoriatic versus normal skin were obtained at the 30 mg/kg dose of ALA. Visible PpIX fluorescence was also observed on normal facial skin, and nonspecific skin photosensitivity occurred only in patients who received the 20 or 30 mg/kg doses. PpIX fluorescence intensity was measured in circulating blood cells by flow cytometry. PpIX fluorescence was higher in monocytes and neutrophils as compared to CD4+ and CD8+ T lymphocytes. PpIX levels in these cells were higher in patients who received higher ALA doses and peaked between 4 and 8 h after administration of ALA. There was only a modest increase in PpIX levels in circulating CD4+ and CD8+ T lymphocytes. In conclusion oral administration of ALA induced preferential accumulation of PpIX in psoriatic plaques as compared to adjacent normal skin suggesting that PDT with oral ALA should be further explored for the treatment of psoriasis.

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 x 10(7) 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.

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-aspartylchlorin 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.

Nitric oxide production by tumour tissue: impact on the response to photodynamic therapy.

The role of nitric oxide (NO) in the response to Photofrin-based photodynamic therapy (PDT) was investigated using mouse tumour models characterized by either relatively high or low endogenous NO production (RIF and SCCVII vs EMT6 and FsaR, respectively). The NO synthase inhibitors Nomega-nitro-L-arginine (L-NNA) or Nomega-nitro-L-arginine methyl ester (L-NAME), administered to mice immediately after PDT light treatment of subcutaneously growing tumours, markedly enhanced the cure rate of RIF and SCCVII models, but produced no obvious benefit with the EMT6 and FsaR models. Laser Doppler flowmetry measurement revealed that both L-NNA and L-NAME strongly inhibit blood flow in RIF and SCCVII tumours, but not in EMT6 and FsaR tumours. When injected intravenously immediately after PDT light treatment, L-NAME dramatically augmented the decrease in blood flow in SCCVII tumours induced by PDT. The pattern of blood flow alterations in tumours following PDT indicates that, even with curative doses, regular circulation may be restored in some vessels after episodes of partial or complete obstruction. Such conditions are conducive to the induction of ischaemia-reperfusion injury, which is instigated by the formation of superoxide radical. The administration of superoxide dismutase immediately after PDT resulted in a decrease in tumour cure rates, thus confirming the involvement of superoxide in the anti-tumour effect. The results of this study demonstrate that NO participates in the events associated with PDT-mediated tumour destruction, particularly in the vascular response that is of critical importance for the curative outcome of this therapy. The level of endogenous production of NO in tumours appears to be one of the determinants of sensitivity to PDT.

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.

Photodynamic therapy-mediated immune response against subcutaneous mouse tumors.

The curative ability of photodynamic therapy (PDT) is severely compromised if treated tumors are growing in immunodeficient hosts. Reconstitution of severe combined immunodeficient (scid) mice with splenocytes from naive immunologically intact BALB/c mice did not improve the response to Photofrin-based PDT of EMT6 tumors growing in these animals. In contrast, adoptive transfer of BALB/c splenocytes containing EMT6 tumor-sensitized immune cells had a dramatic effect on tumor regrowth after PDT. For instance, full restoration of the curative effect of PDT was achieved with scid mice that received splenocytes from BALB/c donors that were cured of EMT6 tumors by PDT 5 weeks before adoptive transfer. Splenocytes obtained from donors cured of EMT6 tumors using X-rays were much less effective. Selective in vitro depletion of specific T-cell populations from engrafting splenocytes indicated that CTLs are the main immune effector cells responsible for conferring the curative outcome to PDT in this experimental model, whereas helper T lymphocytes play a supportive role. The immune specificity of these T-cell populations was demonstrated by the absence of cross-reactivity between the EMT6 and Meth-A tumor models (mismatch between tumors growing in splenocyte donors and recipients). The immunocompetent BALB/c mice that received adoptively transferred splenocytes containing PDT-generated, tumor-sensitized immune cells also benefited from the improved outcome of PDT of tumors they were bearing. This was demonstrated not only with the fairly immunogenic EMT6 tumor model but also with weakly immunogenic Line 1 carcinomas. The results of this study indicate that PDT is a highly effective means of generating tumor-sensitized immune cells that can be recovered from lymphoid sites distant to the treated tumor at protracted time intervals after PDT, which asserts their immune memory character. It is also shown that the treatment of tumors by PDT creates the conditions necessary for converting the inactive adoptively transferred pre-effector, tumor-sensitized immune cells into fully functional antitumor effector cells. An additional finding of this study is the evidence of NK cell activation in PDT-treated Meth-A sarcomas.

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.

Photodynamic therapy.

Photodynamic therapy involves administration of a tumor-localizing photosensitizing agent, which may require metabolic synthesis (i.e., a prodrug), followed by activation of the agent by light of a specific wavelength. This therapy results in a sequence of photochemical and photobiologic processes that cause irreversible photodamage to tumor tissues. Results from preclinical and clinical studies conducted worldwide over a 25-year period have established photodynamic therapy as a useful treatment approach for some cancers. Since 1993, regulatory approval for photodynamic therapy involving use of a partially purified, commercially available hematoporphyrin derivative compound (Photofrin) in patients with early and advanced stage cancer of the lung, digestive tract, and genitourinary tract has been obtained in Canada, The Netherlands, France, Germany, Japan, and the United States. We have attempted to conduct and present a comprehensive review of this rapidly expanding field. Mechanisms of subcellular and tumor localization of photosensitizing agents, as well as of molecular, cellular, and tumor responses associated with photodynamic therapy, are discussed. Technical issues regarding light dosimetry are also considered.

The value of serum alpha-N-acetylgalactosaminidase measurement for the assessment of tumour response to radio- and photodynamic therapy.

Serum activity of alpha-N-acetylgalactosaminidase (NaGalase), the extracellular matrix-degrading enzyme that appears to be produced exclusively by cancer cells, was measured in mice bearing SCCVII tumours (squamous cell carcinoma). The NaGalase levels in these mice increased with time of tumour growth and were directly proportional to tumour burden. After exposure of SCCVII tumours to a single X-ray dose of 20 Gy, the serum NaGalase levels gradually decreased during the first 10 days after treatment (to approximately one-third of the initial value) and then began to increase. The decrease in serum NaGalase activity was more rapid after the treatment of SCCVII and EMT6 tumours by photodynamic therapy (PDT) and was dependent on the PDT dose. The treatments (based on photosensitizers Photofrin or mTHPC) that were fully curative resulted in the reduction of NaGalase activity to background levels within 2 or 3 days after PDT. A slower decrease in NaGalase activity was found after PDT treatments that attain an initial tumour ablation but are not fully curative. The regrowth of PDT-treated SCCVII tumours was preceded by an increase in serum NaGalase levels, which was detected as early as 8 days before the visible tumour reappearance. These findings ascertain the validity of serum NaGalase measurement for the assessment of tumour response to different treatments and support the concept that the NaGalase measurement could serve as a diagnostic and prognostic index that might allow oncologists to design the dosage or nature of treatment.

Macrophage-directed immunotherapy as adjuvant to photodynamic therapy of cancer.

The effect of Photofrin-based photodynamic therapy (PDT) and adjuvant treatment with serum vitamin D3-binding protein-derived macrophage-activating factor (DBPMAF) was examined using a mouse SCCVII tumour model (squamous cell carcinoma). The results show that DBPMAF can markedly enhance the curative effect of PDT. The most effective DBPMAF therapy consisted of a combination of intraperitoneal and peritumoral injections (50 and 0.5 ng kg-1 respectively) administered on days 0, 4, 8 and 12 after PDT. Used with a PDT treatment curative to 25% of the treated tumours, this DBPMAF regimen boosted the cures to 100%. The DBPMAF therapy alone showed no notable effect on the growth of SCCVII tumour. The PDT-induced immunosuppression, assessed by the evaluation of delayed-type contact hypersensitivity response in treated mice, was greatly reduced with the combined DBPMAF treatment. These observations suggest that the activation of macrophages in PDT-treated mice by adjuvant immunotherapy has a synergistic effect on tumour cures. As PDT not only reduces tumour burden but also induces inflammation, it is proposed that recruitment of the activated macrophages to the inflamed tumour lesions is the major factor for the complete eradication of tumours.

The role of host lymphoid populations in the response of mouse EMT6 tumor to photodynamic therapy.

Photodynamic therapy (PDT) treatment of murine EMT6 mammary sarcoma using Photofrin (10 mg/kg) and light (110 J/cm2) cured all these lesions growing in syngeneic BALB/c mice. In contrast, the same treatment produced initial ablation but no long-term cures of EMT6 tumors growing in either scid or nude mice, the immunodeficient strains sharing the same genetic background with BALB/c mice. No difference was detected in either the level of Photofrin accumulated per g of tumor tissue or the extent of tumor cell killing during the first 24 h after PDT of EMT6 tumors growing in BALB/c or scid mice. The assumption that the difference in tumor cures could be ascribed to the absence of functional lymphoid cells in scid and nude mice was supported by the results of experiments involving the adoptive T-cell transfer into scid mice or bone marrow transfer between BALB/c and scid mice. The adoptive transfer of splenic virgin T lymphocytes from BALB/c mice into scid mice performed 9 days before PDT of EMT6 tumors growing in the recipients was successful in delaying the recurrence of treated tumors. Adoptive transfer done immediately after PDT or 7 days after PDT had no obvious benefit. Even better improvement and a high cure rate of PDT-treated tumors was obtained with scid mice reconstituted with BALB/c bone marrow. In contrast, a marked drop in tumor cure rate was observed with BALB/c mice reconstituted with scid bone marrow. These results suggest that the activity of host lymphoid populations was essential for preventing the recurrence of EMT6 tumors following the PDT treatment used in this study. The contribution of PDT-induced immune reaction may, therefore, be of critical importance for the cure with at least some tumors.

Induction of tumor immunity by photodynamic therapy.

The mechanism of tumor destruction by photodynamic therapy (PDT) incorporates a variety of events leading to inactivation of tumor cells. The unique feature of PDT is the mobilization of the host to participate in the eradication of treated cancer. A critical element is the induced inflammation at the treated site associated with massive invasion of activated myeloid cells. In addition to further destruction of cancer cells, conditions are created for the presentation of tumor antigens with subsequent activation of lymphoid cells, leading to tumor-specific immunity. This inflammation-primed immune development process results in generation of tumor-specific immune memory cells that appear to be elicited against both strongly and poorly immunogenic PDT-treated cancers. Once generated by PDT, it is conceivable that these immune cells (especially if further expanded and activated by adjuvant immunotherapy) can be engaged in additional eradication of disseminated and/or metastatic lesions of the same cancer. A number of immunotherapy regimens have already been proven effective in enhancing the curative effect of PDT with various animal tumor models. Inflamed cancerous tissue at the PDT-treated site appears to exert powerful attracting signals for immune cells activated by different immunotherapy regimens.

Potentiation of photodynamic therapy-elicited antitumor response by localized treatment with granulocyte-macrophage colony-stimulating factor.

Murine squamous cell carcinoma (SCCVII) cells were genetically engineered to produce marine granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF immunotherapy, based on the peritumoral injection of lethally irradiated GM-CSF-producing SCCVII cells, was examined as adjuvant to photodynamic therapy (PDT) treatment of this tumor. The GM-CSF immunotherapy administered three times in 48-h intervals, starting 2 days before the light treatment, substantially improved the curative effect of Photofrin-mediated PDT. A comparable effect of GM-CSF immunotherapy was observed in the combination with benzoporphyrin derivative-mediated PDT. The tumor-localized GM-CSF immunotherapy alone had no obvious effect on the growth of parental SCCVII tumors. This treatment did not significantly alter the differential peripheral WBC count and appeared not to affect tumor leukocyte infiltration. However, GM-CSF treatment did increase the cytotoxic activity of tumor-associated macrophages against SCCVII tumor cells. It appears, therefore, that tumor-localized immune stimulation by GM-CSF amplifies a PDT-induced antitumor immune reaction, which has a potentiating effect on tumor control.

Photofrin accumulation in malignant and host cell populations of various tumours.

Photofrin accumulation in malignant and host cell populations of various tumours was studied by flow cytometry analysis of cells dissociated from the tumour tissue. The transplantable mouse tumour models included in this analysis were sarcomas EMT6, RIF, KHT and FsaN, Lewis lung carcinoma, SCCVII squamous cell carcinoma (SCC) and slowly growing moderately differentiated AT17 SCC. An example of spontaneous mouse adenocarcinoma was also examined. Staining with specific monoclonal antibodies was used to identify the various cell populations present in these tumours. The main characteristic of Photofrin cellular accumulation was a very high photosensitiser content found exclusively in a subpopulation of tumour-associated macrophages (TAMs). Photosensitiser levels similar to or lower than in malignant cells were observed in the remaining TAMs and other tumour-infiltrating host cells. Photofrin accumulation in malignant cells was not equal in all tumour models, but may have been affected by tumour blood perfusion/vascularisation. Results consistent with the above findings were obtained with SCC of buccal mucosa induced by 9,10-dimethyl-1,2-benzanthracene in Syrian hamsters. The TAM subpopulation that accumulates by far the highest cellular Photofrin levels in tumours is suggested to be responsible for the tumour-localised photosensitiser fluorescence.