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Recently, it has become clear that a prerequisite requirement for most cancer therapies is controlling the negative impact of the activity of immunosuppressory cell populations. It is therefore of a considerable interest to develop treatments for containing the operation of major myeloid and lymphoid immunoregulatory cell populations. We have reported that acid ceramidase inhibitor LCL521 effectively overrides the activity of immunoregulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) engaged in the context of tumor response to photodynamic therapy (PDT). The present communication dissects and describes in detail the procedure for the use of LCL521 as an adjuvant to PDT for improved cure rates of treated tumors based on restricting the activity of immunoregulatory cell populations.

Our recent investigation uncovered that the acid ceramidase inhibitor LCL521 enhances the direct tumor cell killing effect of photodynamic therapy (PDT) treatment. The present study aimed at elucidating the mechanisms underlying this effect. Exposing mouse squamous cell carcinoma SCCVII cells treated with temoporfin-based PDT to LCL521 (rising ceramide concentration) produced a much greater decrease in cell survival than comparable exposure to the sphingosine kinase-1 inhibitor PF543 (that reduces sphingosine-1-phosphate concentration). This is consistent with recognizing the rising levels of pro-apoptotic sphingolipid ceramide as being more critical in promoting the death of PDT-treated cells than the reduction in the availability of pro-survival acting sphingosine-1 phosphate. This pro-apoptotic impact of LCL521, which was suppressed by the apoptosis inhibitor bongkrekic acid, involves the interaction with the cellular stress signaling network. Hence, inhibiting the key elements of these pathways markedly influenced the adjuvant effect of LCL521 on the PDT response. Particularly effective was the inositol-requiring element-1 (IRE1) kinase inhibitor STF-083010 that dramatically enhanced the killing of cells treated with PDT plus LCL521. An important role in the survival of these cells was exhibited by master transcription factors STAT3 and HIF-1α. The STAT3 inhibitor NSC 74859 was especially effective in further reducing the cell survival rates, suggesting its possible exploitation for therapeutic gain. An additional finding in this study is that LCL521-promoted PDT-mediated cell killing through ceramide-mediated lethal effects is extended to the interaction with other cancer treatment modalities with a rapid cellular stress impact such as photothermal therapy (PTT) and cryoablation therapy (CAT).

The insult delivered by photodynamic therapy (PDT) in treated cells is oxidative stress. The main burden threatening survival of PDT-treated cells is proteotoxic damage that jeopardizes proteostasis in these cells. For dealing with this type of proteostasis impairment, cells have developed protection mechanisms operating by signaling networks. This review will outline various components of signaling networks that can be engaged in stressed cells with highlighting the emerging aspects relevant to response to PDT. It will be also shown how the well known inflammatory/immune response associated with PDT is also based on the activity of these stress signaling networks. Lasers Surg. Med. 50:491-498, 2018. © 2018 Wiley Periodicals, Inc.

Background/aim Combining an anticancer agent fenretinide (HPR) or C6-pyridinium ceramide (LCL29) with Foscan-mediated photodynamic therapy (FoscanPDT) is expected to augment anticancer benefits of each substance. We showed that treatment with FoscanPDT+HPR enhanced accumulation of C16-dihydroceramide, and that fumonisin B1 (FB), an inhibitor of ceramide synthase, counteracted caspase-3 activation and colony-forming ability of head and neck squamous cell carcinoma (HNSCC) cells. Because cancer cells appear to be more susceptible to increased levels of the endoplasmic reticulum (ER) stress than normal cells, herein we tested the hypothesis that FoscanPDT combined with HPR or LCL29 induces FB-sensitive ER stress-associated apoptosis that affects cell survival. Materials and methods Using an HNSCC cell line, we determined: cell survival by clonogenic assay, caspase-3 activity by spectrofluorometry, the expression of the ER markers BiP and CHOP by quantitative real-time polymerase chain reaction and western immunoblotting, and sphingolipid levels by mass spectrometry. Results Similar to HPR+FoscanPDT, LCL29+FoscanPDT induced enhanced loss of clonogenicity and caspase-3 activation, that were both inhibited by FB. Our additional pharmacological evidence showed that the enhanced loss of clonogenicity after the combined treatments was singlet oxygen-, ER stress- and apoptosis-dependent. The combined treatments induced enhanced, FB-sensitive, up-regulation of BiP and CHOP, as well as enhanced accumulation of sphingolipids. Conclusion Our data suggest that enhanced clonogenic cell killing after the combined treatments is dependent on oxidative- and ER-stress, apoptosis, and FB-sensitive sphingolipid production, and should help develop more effective mechanism-based therapeutic strategies.

It is becoming apparent that to obtain robust and prolonged antitumor responses in cancer immunotherapy, appropriate adjunct agents promoting both tumor antigen delivery and immune rejection enhancement are critically required. The semisynthetic biopolymer N-dihydrogalactochitosan (GC) is emerging as a promising such candidate. In the present study, the effects of GC were investigated when combined with cancer vaccines generated by photodynamic therapy (PDT) using mouse tumor model SCCVII (squamous cell carcinoma). The adjunct GC treatment was found to enhance therapeutic benefit obtained with PDT vaccine, while reducing the numbers of myeloid-derived suppressor cells. Another important property of GC is promoting directly the death of SCCVII cells sustaining injury from PDT mediated by various photosensitizers. This effect is extended to cells treated by cryoablation therapy (CAT) performed by exposure to −80 °C. A capacity of GC for preferential binding to PDT treated cells was demonstrated using fluorescence microscopy. In vitro testing with specific caspase-1 inhibitor revealed a pro-survival role of this enzyme in membrane lipid repair mechanisms following combined PDT plus GC treatment. In conclusion, GC represents a uniquely promising adjunct for various PDT protocols, photothermal and similar rapid tumor-ablating therapies.

Photothermal therapy (PTT) is recently clinically established cancer therapy that uses near-infrared light for thermal ablation of solid tumors. The biopolymer N-dihydrogalactochitosan (GC) was shown in multiple reports to act as a very effective adjunct to tumor PTT. In the present study, mouse tumor model SCCVII (squamous cell carcinoma) was used with two protocols, in situ tumor PTT and therapeutic PTT vaccine for tumors, for investigating the effects of GC. The results reveal that GC can potentiate tumoricidal action of PTT through both direct and indirect mechanisms. In addition to previously known capacity of GC for activating immune effector cells, the indirect means is shown to include reducing the populations of immunoregulatory T cells (Tregs) in PTT-treated tumors. Testing the effects of GC on PTT-treated SCCVII tumor cells in vitro uncovered the existence of a direct mechanism evident by reduced colony survival of these cells. Fluorescence microscopy demonstrated increased binding of fluorescein-labeled GC to PTT-treated compared to untreated SCCVII cells that can be blocked by pre-exposure to annexin V. The results of additional in vitro testing with specific inhibitors demonstrate that these direct mechanisms do not involve the engagement of death surface receptors that trigger extrinsic apoptosis pathway signaling but may be linked to pro-survival activity of caspase-1. Based on the latter, it can be suggested that GC-promoted killing of PTT-treated cells stems from interference of GC bound to damaged membrane components with the repair of these structures that consequently hinders cell survival.

Ablation therapies have emerged as an effective tool for destroying cancerous tissue, but for advanced and disseminated tumors their application remains mainly a palliative measure. However, it is becoming increasingly clear that this limitation can be redressed by the use of intratumoral immune stimulating agents for amplifying potential antitumor immune responses that are induced by ablation therapies. A novel immune stimulating drug IP-001, a specific variant of the N-dihydrogalactochitosan (GC) family of molecules, has shown to be effective against metastatic tumors, when combined with different forms tumor ablation. It acts as a multi-function immune stimulant both by directly inhibiting cell membrane repair and recycling of ablation-damaged tumor cells, and indirectly by sequestering ablation-released tumor antigens, as well as recruiting and stimulating antigen presenting cells to induce a potent Th1 type T cell response against the cancer. In this review, we briefly discuss the current applications of local ablation for cancer treatment and the effects of GC in combination with other ablation therapies, a therapeutic approach that is pioneering the field of Interventional Immuno-Oncology (IIO).

Acid ceramidase has been identified as a promising target for cancer therapy. One of its most effective inhibitors, LCL521, was examined as adjuvant to photodynamic therapy (PDT) using mouse squamous cell carcinoma SCCVII model of head and neck cancer. Lethal effects of PDT, assessed by colony forming ability of in vitro treated SCCVII cells, were greatly enhanced when combined with 10 µM LCL521 treatment particularly when preceding PDT. When PDT-treated SCCVII cells are used to vaccinate SCCVII tumor-bearing mice (PDT vaccine protocol), adjuvant LCL521 treatment (75 mg/kg) resulted in a marked retardation of tumor growth. This effect can be attributed to the capacity of LCL521 to effectively restrict the activity of two main immunoregulatory cell populations (Tregs and myeloid-derived suppressor cells, MDSCs) that are known to hinder the efficacy of PDT vaccines. The therapeutic benefit with adjuvant LCL521 was also achieved with SCCVII tumors treated with standard PDT when using immunocompetent mice but not with immunodeficient hosts. The interaction of LCL521 with PDT-based antitumor mechanisms is dominated by immune system contribution that includes overriding the effects of immunoregulatory cells, but could also include a tacit contribution from boosting direct tumor cell kill.

Photodynamic therapy (PDT)-generated cancer vaccine represents an attractive potential application of PDT, therapeutic modality destroying targeted lesions by localized photooxidative stress. Since immunoregulatory cell activity has become recognized as a major obstacle to effective cancer immunotherapy, the present study examined their participation in the therapeutic effect of PDT cancer vaccine. Following protocols from previous studies, mouse with squamous cell carcinoma SCCVII tumors were vaccinated by SCCVII cells treated by PDT and response monitored by tumor size measurement. The effects of low-dose cyclophosphamide (50 mg/kg) and all-trans retinoic acid (ATRA) on the numbers of Tregs and myeloid-derived suppressor cells (MDSCs) were determined by antibody staining followed by flow cytometry, while their impact on PDT vaccine therapy was evaluated by monitoring changes in tumor responses. Cyclophosphamide effectively reduced the numbers of Tregs, which became elevated following PDT vaccine treatment, and this resulted in an increase in the vaccine’s effectiveness. A similar benefit for the therapy outcome with PDT vaccine was attained by ATRA treatment. The activities of Tregs and MDSCs thus have a critical impact on therapy outcome with PDT vaccine and reducing their numbers substantially improves the vaccine’s effectiveness.

Because photodynamic therapy (PDT) alone is not always effective as an anticancer treatment, PDT is combined with other anticancer agents for improved efficacy. The clinically-relevant fenretinide [N-(4-hydroxyphenyl) retinamide; 4HPR], was combined with the silicon phthalocyanine photosensitizer Pc4-mediated PDT to test for their potential to enhance killing of SCC17B cells, a clinically-relevant model of human head and neck squamous cell carcinoma. Because each of these treatments induces apoptosis and regulates the de novo sphingolipid (SL) biosynthesis pathway, the role of ceramide synthase, the pathway-associated enzyme, in PDT+4HPR-induced apoptotic cell death was determined using the ceramide synthase inhibitor fumonisin B1 (FB). PDT+4HPR enhanced loss of clonogenicity. zVAD-fmk, a pan-caspase inhibitor, and FB, protected cells from death post-PDT+4HPR. In contrast, the anti-apoptotic protein Bcl2 inhibitor ABT199 enhanced cell killing after PDT+4HPR. Combining PDT with 4HPR led to FB-sensitive, enhanced Bax associated with mitochondria and cytochrome c redistribution. Mass spectrometry data showed that the accumulation of C16-dihydroceramide, a precursor of ceramide in the de novo SL biosynthesis pathway, was enhanced after PDT+4HPR. Using quantitative confocal microscopy, we found that PDT+4HPR enhanced dihydroceramide/ceramide accumulation in the ER, which was inhibited by FB. The results suggest that SCC17B cells are sensitized to PDT by 4HPR via the de novo SL biosynthesis pathway and apoptosis, and imply potential clinical relevance of the combination for cancer treatment.

The sphingolipid ceramide modulates stress-induced cell death and apoptosis. We have shown that ceramide generated via de novo sphingolipid biosynthesis is required to initiate apoptosis after photodynamic therapy (PDT). The objective of this study was to define the role of ceramide synthase (CERS) in PDT-induced cell death and apoptosis using fumonisin B1 (FB), a CERS inhibitor. We used the silicon phthalocyanine Pc4 for PDT, and SCC17B cells, as a clinically-relevant model of human head and neck squamous carcinoma. zVAD-fmk, a pan-caspase inhibitor, as well as FB, protected cells from death after PDT. In contrast, ABT199, an inhibitor of the anti-apoptotic protein Bcl2, enhanced cell killing after PDT. PDT-induced accumulation of ceramide in the endoplasmic reticulum and mitochondria was inhibited by FB. PDT-induced Bax translocation to the mitochondria and cytochrome c release were also inhibited by FB. These novel data suggest that PDT-induced cell death via apoptosis is CERS/ceramide-dependent.

Photodynamic therapy (PDT) is not always effective as an anticancer treatment, therefore, PDT is combined with other anticancer agents for improved efficacy. The combination of dasatinib and PDT with the silicone phthalocyanine photosensitizer Pc 4 was assessed for increased killing of SCCVII mouse squamous cell carcinoma cells, a preclinical model of head and neck squamous cell carcinoma, using apoptotic markers and colony formation as experimental end-points. Because each of these treatments regulates the metabolism of the sphingolipid ceramide, their effects on mRNA levels of ceramide synthase, a ceramide-producing enzyme, and the sphingolipid profile were determined. PDT + dasatinib induced an additive loss of clonogenicity. Unlike PDT alone or PDT + dasatinib, dasatinib induced zVAD-fmk-dependent cell killing. PDT or dasatinib-induced caspase-3 activation was potentiated after the combination. PDT alone induced mitochondrial depolarization, and the effect was inhibited after the combination. Annexin V+ and propidium iodide+ cells remained at control levels after treatments. In contrast to PDT alone, dasatinib induced upregulation of ceramide synthase 1 mRNA, and the effect was enhanced after the combination. Dasatinib induced a modest increase in C20:1- and C22-ceramide but had no effect on total ceramide levels. PDT increased the levels of 12 individual ceramides and total ceramides, and the addition of dasatinib did not affect these increases. PDT alone decreased substantially sphingosine levels and inhibited the activity of acid ceramidase, an enzyme that converts ceramide to sphingosine. The data suggest that PDT-induced increases in ceramide levels do not correlate with ceramide synthase mRNA levels but rather with inhibition of ceramidase. Cell killing was zVAD-fmk-sensitive after dasatinib but not after either PDT or the combination and enhanced cell killing after the combination correlated with potentiated caspase-3 activation and upregulation of ceramide synthase 1 mRNA but not the production of ceramide. The data imply potential significance of the combination for cancer treatment.

Treatment of mouse tumors by photodynamic therapy (PDT) was reported to trigger the production of serum amyloid P component (SAP), a prototypic acute phase reactant in the mouse, that occurs in the targeted tumor as well as distant sites dominated by host's liver. It was also shown that the SAP gene becomes upregulated and protein produced in mouse tumor cells treated by PDT in vitro. Present study revealed that, in addition to SAP, increased expression of genes encoding related pentraxin and complement proteins, including PTX3, C1q and ficolin B, can be found in mouse LLC tumor cells treated by PDT. Since in humans C-reactive protein (CRP) is more important acute phase reactant than SAP, the expression of gene encoding this pentraxin protein was examined in human lung tumor A549 cells treated by PDT. The results demonstrated a PDT dose-dependent upregulation of CRP gene, as well as of PTX3 and ficolin 1 genes in these cells. Investigation into the signal transduction process underlying PDT-induced human CRP gene upregulation using specific inhibitors of critical signaling elements revealed critical role played by PI3K/Akt pathway. Downstream DNA transcription factor largely responsible for this increased CRP gene expression is AP-1 with possible cooperation of HIF-1. It was suggested that cells sensing to have sustained a mortal injury from PDT can turn on molecular programs ensuring that the disposal of their corpses (facilitated by CRP and related pentraxin and complement components) is swift and efficient.

Myeloid regulatory cells (Mregs) are, together with regulatory T cells (Tregs), a dominant effector population responsible for restriction of the duration and strength of antitumor immune response. Photodynamic therapy (PDT) and cancer vaccines generated by PDT are modalities whose effectiveness in tumor destruction is closely dependent on the associated antitumor immune response. The present study investigated whether the immunodepletion of granulocytic Mregs in host mice by anti-GR1 antibody would improve the response of tumors to PDT or PDT vaccines in these animals. Anti-GR1 administration immediately after Temoporfin-PDT of mouse SCCVII tumors abrogated curative effect of PDT. The opposite effect, increasing PDT-mediated tumor cure-rates was attained by delaying anti-GR1 treatment to 1 h post PDT. With PDT vaccines, multiple anti-GR1 administrations (days 0, 4, and 8 post vaccination) improved the therapy response with SCCVII tumors. The results with PDT suggest that neutrophils (boosting antitumor effect of this therapy) that are engaged immediately after photodynamic light treatment are within one hour replaced with a different myeloid population, presumably Mregs that hampers the therapy-mediated antitumor effect. Anti-GR1 antibody, when used with optimal timing, can improve the efficacy of both PDT of tumors in situ and PDT-generated cancer vaccines.

We and others have shown that stresses, including photodynamic therapy (PDT)1, can disrupt the de novo sphingolipid biosynthesis pathway, leading to changes in the levels of sphingolipids, and subsequently, modulation of cell death. The de novo sphingolipid biosynthesis pathway includes a ceramide synthase-dependent reaction, giving rise to dihydroceramide, which is then converted in a desaturase-dependent reaction to ceramide. In this study we tested the hypothesis that combining Foscan-mediated PDT with desaturase inhibitor fenretinide (HPR) enhances cancer cell killing. We discovered that by subjecting SCC19 cells, a human head and neck squamous cell carcinoma cell line, to PDT+HPR resulted in enhanced accumulation of C16-dihydroceramide, not ceramide. Concomitantly, mitochondrial depolarization was enhanced by the combined treatment. Enhanced activation of caspase-3 after PDT +HPR was inhibited by FB. Enhanced clonogenic cell death after the combination was sensitive to FB, as well as Bcl2- and caspase inhibitors. Treatment of mouse SCCVII squamous cell carcinoma tumors with PDT+HPR resulted in improved long-term tumor cures. Overall, our data showed that combining PDT with HPR enhanced apoptotic cancer cell killing and antitumor efficacy of PDT. The data suggest the involvement of the de novo sphingolipid biosynthesis pathway in enhanced apoptotic cell killing after PDT+HPR, identify PDT+HPR as a more effective combination than either treatment alone, and that the combination has potential for cancer treatment.

Acid ceramidase has been identified as a promising target for cancer therapy. One of its most effective inhibitors, LCL521, was examined as adjuvant to photodynamic therapy (PDT) using mouse squamous cell carcinoma SCCVII model of head and neck cancer. Lethal effects of PDT, assessed by colony forming ability of in vitro treated SCCVII cells, were greatly enhanced when combined with 10 µM LCL521 treatment particularly when preceding PDT. When PDT-treated SCCVII cells are used to vaccinate SCCVII tumor-bearing mice (PDT vaccine protocol), adjuvant LCL521 treatment (75 mg/kg) resulted in a marked retardation of tumor growth. This effect can be attributed to the capacity of LCL521 to effectively restrict the activity of two main immunoregulatory cell populations (Tregs and myeloid-derived suppressor cells, MDSCs) that are known to hinder the efficacy of PDT vaccines. The therapeutic benefit with adjuvant LCL521 was also achieved with SCCVII tumors treated with standard PDT when using immunocompetent mice but not with immunodeficient hosts. The interaction of LCL521 with PDT-based antitumor mechanisms is dominated by immune system contribution that includes overriding the effects of immunoregulatory cells, but could also include a tacit contribution from boosting direct tumor cell kill.

Eradication of tumours by photodynamic therapy (PDT) is accompanied by marked changes in local sphingolipid (SL) engagement. Because of the heterogeneity of cellular composition, analysis of tumour tissue homogenates to quantify SL species is inadequate for evaluating their levels in parenchymal cancer cell population. By staining tumour-derived single cell suspensions with antibodies specific to ceramide and sphingosine 1-phosphate (S1P) followed by flow cytometry, we were able to document changes in the levels of these two key SLs in cancer cells and tumour-associated macrophages (TAMs) of mouse SCCVII tumours following PDT. The results confirm previously obtained indications that tumour treatment by PDT induces a marked rise in ceramide levels in cancer cells within these lesions. Cancer cells from PDT-treated SCCVII tumours undergoing apoptosis were found to have much higher ceramide levels and substantially lower S1P levels than their viable counterparts. Compared to cancer cells, considerably higher ceramide and S1P levels were consistently found in TAMs. Treatment of SCCVII tumour-bearing mice with ceramide analog LCL29 induced a rise in ceramide levels in TAMs but not in cancer cells. When combined with PDT, LCL29 treatment produced a further increase in ceramide levels in TAMs while having no evident impact on ceramide content in cancer cells within same tumours. The results highlight SLs as important participants in tumour response to PDT and potential adjuvant therapeutic targets to PDT.

Background and Objectives C6-ceramide analog LCL29 was recently shown to improve the cure rates of mouse tumors treated by photodynamic therapy (PDT), but the mechanism underlying the therapeutic gain remains unclear. Since LCL29 is a pro-apoptotic agent, the main objective of the present study was to determine how LCL29 affects cancer cell apoptosis in PDT-treated tumors. Study Design/Materials and Methods Mice bearing SCCVII tumors (syngeneic squamous cell carcinomas) were treated by PDT with photosensitizer Foscan. Adjuvant LCL29 treatment (40 mg/kg) was 24 hours before PDT. The tumors were excised 3 hours after PDT, disaggregated into single cell suspensions that were stained for flow cytometry to detect apoptosis-related events in cancer cells (caspase activation, loss of mitochondrial transmembrane potential, and intracellular calcium). In addition, phagocytic activity in tumor-associated macrophages was assessed by phalloidin staining. Results While 5–10% apoptotic cancer cells were found in tumors treated by PDT or LCL29 alone, close to 40% of such cells were found in tumors treated by LCL29 combined with PDT. Mitochondrial depolarization was detected in PDT, LCL29, and LCL29 + PDT groups, reaching similar values in all groups. Intracellular calcium release triggered by PDT was more pronounced when PDT was combined with LCL29. Macrophage phagocytic activity, negatively affected by PDT, was stimulated by adjuvant LCL29. Conclusions Combining LCL29 treatment with PDT can enhance intracellular calcium release in cancer cells and strongly amplify their apoptosis. Lasers Surg. Med. 43:614–620, 2011. © 2011 Wiley-Liss, Inc.

The development of photodynamic therapy (PDT)-generated cancer vaccines is potentially one of the most significant achievements in the field of PDT. Employing vaccine protocols optimizes the capacity of PDT of inducing a strong immune response against treated tumor due to the establishment of highly favourable conditions for maximizing the avidity of the immune reaction while sustaining and prolonging its tumor-destroying attack. While the introduction of PDT vaccines into the clinics and testing on patients is still in a very early phase, much work can still be done on further improvement of the potency of PDT vaccines. Considerable advances can be expected by identifying the most effective adjuvants to be used with PDT vaccines, which will most likely be different with different types of cancerous lesions.

We have developed a micro-Raman spectrometer system for use to differentiate tumor lesions from normal skin using an in vivo animal model. A study of 494 Raman spectra from 24 mice revealed different spectral patterns at different depths and between normal and tumor-bearing skin sites. A peak at 899 cm−1 (possibly from proline or fatty acids) and one with higher intensity in the 1325–1330 cm−1 range (assigned to nucleic acids) were correlated with the presence of tumors, which can potentially be used as biomarkers for skin cancer detection. Spectral diagnosis performed on the murine tumor model achieved a diagnostic sensitivity of 95.8% and specificity of 93.8%. These results encourage us to develop further the use of confocal Raman spectroscopy as a clinical tool for noninvasive human skin biochemical analysis, particularly in relation to skin cancer. Copyright © 2010 John Wiley & Sons, Ltd.

Treatment of solid tumors by photodynamic therapy (PDT) was recently shown to trigger a strong acute phase response. Using the mouse Lewis lung carcinoma (LLC) model, the present study examined complement and pentraxin proteins as PDT-induced acute phase reactants. The results show a distinct pattern of changes in the expression of genes encoding these proteins in the tumor, as well as host liver and spleen, following PDT mediated by photosensitizer Photofrin™. These changes were influenced by glucocorticoid hormones, as evidenced by transcriptional activation of glucocorticoid receptor and the upregulation of gene encoding this receptor. The expression of gene for glucocorticoid-induced zipper (GILZ) protein, whose activity is particularly susceptible to glucocorticoid regulation, was also changed in PDT-treated tumors. A direct demonstration that tumor PDT induces glucocorticoid hormone upregulation is provided by documenting elevated levels of serum corticosterone in mice bearing PDT-treated LLC tumors. Tumor response to PDT was negatively affected by blocking glucocorticoid receptor activity, which suggests that glucocorticoid hormones have a positive impact on the therapeutic outcome with this therapy.

Oxidative stress in photodynamic therapy (PDT)-treated tumor cells is known to instigate a strong upregulation of the expression of heat shock proteins. However, the treatment of mouse Lewis lung carcinoma (LLC) cells with Photofrin™ PDT resulted in the upregulation of heat shock protein 70 (Hsp70) gene not only in these cells but also in co-incubated untreated Hepa 1-6 cells. To investigate whether this phenomenon extends in vivo, LLC tumors growing in C57BL/6 mice were treated with Photofrin™ PDT. The tumors and the livers from the mice were collected at 4, 8, or 24 h after therapy for quantitative reverse transcriptase polymerase chain reaction-based analysis of Hsp70 gene expression. Increased Hsp70 gene expression was detected in both the tumor and liver tissues and was most pronounced at 4 h after PDT. This effect was inhibited by treatment of host mice with glucocorticoid synthesis inhibitor metyrapone. Hsp70 protein levels in the livers of mice bearing PDT-treated tumors gradually decreased after therapy while serum levels increased at 4 h after therapy and then continually decreased. The exposure of in vitro PDT-treated LLC cells to Hsp70 and subsequent flow cytometry analysis revealed binding of this protein to cells that was dependent on PDT dose and more pronounced with dying than viable cells. Thus, following the induction of tumor injury by PDT, Hsp70 can be produced in the liver and spleen as acute phase reactant and released into circulation, from where it can be rapidly sequestered to damaged tumor tissue to facilitate the disposal of dying cells.

A diaminophenyl derivative of hypocrellin B (SL052) has been developed as a photosensitizer for use in photodynamic therapy (PDT) of solid tumors. Testing SL052-PDT on mouse carcinoma and fibrosarcoma models revealed a typical response seen with clinically established photosensitizers featuring initial rapid tumor ablation with ensuing recurrence at rates dependent on photosensitizer/light doses. Elevated numbers of immune cells were found in lymph nodes draining SCCVII mouse squamous cell carcinomas treated by SL052-PDT (in particular T cells), and the accumulation of degranulating cytotoxic T cells was detected at the tumor-treated site. This indicates that a significant contribution to tumor cures is elicited by an antitumor adaptive immune response. Two different immunotherapy agents, gamma-interferon and antibody blocking inhibitory FcgammaRIIB receptor, were both found to be highly effective in potentiating the curative effect of SL052-PDT with SCCVII tumors. Combining SL052-PDT with FcgammaRIIB-blocking antibody treatment caused a further increase in the number of cells in tumor-draining lymph nodes and in degranulating CD8+ cells, suggesting the amplification of the immune response induced by PDT. Vaccines consisting of SCCVII cells treated with SL052-PDT in vitro were effective in reducing growth of established subcutaneous SCCVII tumors. In conclusion, PDT mediated by SL052 is suitable to be integrated with various immunotherapy protocols.

Recent investigations have established that complement activation is induced following treatment of solid tumors by photodynamic therapy (PDT). The present study using mouse SCCVII tumor model affirms the proficiency of tumor-associated macrophages (TAMs) to produce complement proteins upon receiving signals such as Hsp70 released from PDT-treated cancer cells. Such communication is shown to trigger signaling involving Toll-like receptors (TLR) 2 and 4 and the activation of transcription factor NFkappaB leading to complement protein production. This example of complement system regulation by TLRs adds to a growing awareness of cross-talk within these two key components of innate immunity.

We have measured the micro-Raman spectra of mouse tissues invaded by Lewis lung carcinoma (LLC). We have also carried out categorical principal component analysis (CATPCA) on the acquired spectra. The results indicate that the tumor tissues can be well discriminated from normal tissues by the first two principal components extracted from the spectra. Furthermore, we have found that the concentrations of nucleic acids and lipids/fatty acids in the tumor are considerably higher than those in the normal tissue, whereas the collagen concentration is lower. These differences can be detected and characterized by Raman images using the 788 cm−1 DNA/RNA band and the 1301 cm−1 lipid/fatty acid band. Copyright © 2009 John Wiley & Sons, Ltd.

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.

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.

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.

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.

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.

In current clinical practice, photodynamic therapy (PDT) is carried out with prescribed drug doses and light doses as well as fixed drug-light intervals and illumination fluence rates. This approach can result in undesirable treatment outcomes of either overtreatment or undertreatment because of biological variations between different lesions and patients. In this study, we explore the possibility of improving PDT dosimetry by monitoring drug photobleaching and photoproduct formation. The study involved 60 mice receiving the same drug dose of a novel verteporfin-like photosensitizer, QLT0074, at 0.3 mg/kg body weight, followed by different light doses of 5, 10, 20, 30, 40 or 50 J/cm2 at 686 nm and a fluence rate of 70 mW/cm2. Photobleaching and photoproduct formation were measured simultaneously, using fluorescence spectroscopy. A ratio technique for data processing was introduced to reliably detect the photoproduct formed by PDT on mouse skin in vivo. The study showed that the QLT0074 photoproduct is stable and can be reliably quantified. Three new parameters, photoproduct score (PPS), photobleaching score (PBS) and percentage photobleaching score (PBS%), were introduced and tested together with the conventional dosimetry parameter, light dose, for performance on predicting PDT-induced outcome, skin necrosis. The statistical analysis of experimental results was performed with an ordinal logistic regression model. We demonstrated that both PPS and PBS improved the prediction of skin necrosis dramatically compared to light dose. PPS was identified as the best single parameter for predicting the PDT outcome.

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.

Glycated chitosan (GC), a water soluble galactose-conjugated natural polysaccharide, has proven to be an effective immunoadjuvant for treatment of tumors based on laser thermal therapy. It was also shown to act as adjuvant for tumor therapy with high-intensity ultrasound and in situ photodynamic therapy (PDT). In the present study, GC was examined as potential adjuvant to PDT-generated cancer vaccine. Two other agents, pure calreticulin protein and acid ceramidase inhibitor LCL521, were also tested as prospective adjuvants for use in conjunction with PDT vaccines. Single treatment with GC, included with PDT vaccine cells suspension, improved the therapeutic efficacy when compared to vaccine alone. This attractive prospect of GC application remains to be carefully optimized and mechanistically elucidated. Both calreticulin and LCL521 proved also effective adjuvants when combined with PDT vaccine tumor treatment.

Calreticulin is recognized as one of pivotal damage-associated molecular pattern (DAMP) molecules alerting the host of the presence of distressed cells. In this role, calreticulin becomes exposed on the surface of tumor cells treated by several types of cancer therapy including photodynamic therapy (PDT). The goal of the present study was to examine the potential of externally added calreticulin for augmenting antitumor effect mediated by PDT. Recombinant calreticulin was found to bind to mouse SCCVII tumor cells treated by PDT. Compared to the outcome with PDT alone, cure-rates of SCCVII tumors grown in immunocompetent C3H/HeN mice were elevated when calreticulin (0.4 mg/mouse) was injected peritumorally immediately after PDT. Such therapeutic gain with PDT plus calreticulin combination was not obtained with SCCVII tumors growing in immunodeficient NOD-scid mice. In PDT vaccine protocol, where PDT-treated SCCVII cells are used for vaccination of SCCVII tumor-bearing mice, adding recombinant calreticulin to cells before their injection produced improved therapeutic effect. The expression of calreticulin gene was reduced in PDT-treated cells, while no changes were observed with the expression of this gene in tumor, liver, and spleen tissues in PDT vaccine-treated mice. These findings reveal that externally added recombinant calreticulin can boost antitumor responses elicited by PDT or PDT-generated vaccines, and can thus serve as an effective adjuvant for cancer treatment with PDT and probably other cancer cell stress-inducing modalities.

Macrophages are one of the principal host cell populations in solid tumors. They are capable, due to their plasticity, of acquiring phenotypes that either combat (M1 type) or promote (M2 type) neoplastic growth. These cells, known as tumor-associated macrophages (TAMs), play complex but pivotal roles in the outcome of photodynamic therapy (PDT) of malignant lesions. Among the various parenchymal and stromal cell populations found in tumors, TAMs have been shown to have the greatest capacity for the uptake of systemically administered photosensitizers. Both the tumor-localizing property of photosensitizers and their tumor-localized fluorescence could be partly attributed to the activity of TAMs. Since resident TAMs with accumulated high photosensitizer content will sustain high degrees of PDT damage, this population (predominantly M2 in most tumors) is selectively destroyed, and during the ensuing inflammatory reaction is replaced with newly invading macrophages of M1 phenotype. These macrophages are sentinels responding to DAMP signals from PDT-treated tumor cells and in turn are mobilized to generate a variety of inflammatory/immune mediators and opsonins. They have a critical role in contributing to the therapeutic effect of PDT by mediating disposal of killed cancer cells and by processing/presenting tumor antigens to T lymphocytes. However, TAMs accumulating in the later post-PDT phase can acquire the M2 (healing) phenotype, and could have a role in tumor recurrence by releasing factors that promote angiogenesis and the survival/proliferation of remaining cancer cells. Various therapeutic strategies modulating TAM activity in the PDT response have potential for clinical use for improving PDT-mediated tumor control.

Combining photodynamic therapy (PDT)1 with another anticancer treatment modality is an important strategy for improved efficacy. PDT with Pc4, a silicon phthalocyanine photosensitizer, was combined with C6-pyridinium ceramide (LCL29) to determine their potential to promote death of SCC17B human head and neck squamous cell carcinoma cells. PDT+LCL29-induced enhanced cell death was inhibited by zVAD-fmk, a pan-caspase inhibitor, and fumonisin B1 (FB), a ceramide synthase inhibitor. Quantitative confocal microscopy showed that combining PDT with LCL29 enhanced FB-sensitive ceramide accumulation in the mitochondria. Furthermore, PDT+LCL29 induced enhanced FB-sensitive redistribution of cytochrome c and caspase-3 activation. Overall, the data indicate that PDT+LCL29 enhanced cell death via FB-sensitive, mitochondrial ceramide accumulation and apoptosis.

Photodynamic therapy (PDT) inflicts tumor tissue injury that is experienced by the host as a local trauma. This provokes a strong host response with pronounced neutrophilia as one of its manifestations. Mouse FsaR fibrosarcoma model was used for investigating photodynamic therapy (PDT)-induced neutrophilia and its link to the acute phase response. Compared to normal mice, the extent of neutrophilia induced following Photofrin-based tumor PDT in adrenalectomized host mice was less pronounced revealing the elicited engagement of the adrenal-pituitary axis, which is one of the principal characteristics of the acute phase response. Neutrophilia was demonstrated after tumor-localized PDT even in the host mice previously depleted of circulating neutrophils. The rise in serum levels of complement C3 protein, which is an acute phase reactant and a principal mediator of tumor PDT-induced neutrophilia, occurred at the post PDT time period when the neutrophilia was largely resolved. However, the activation of complement system (assessed by the standard erythrocyte hemolysis assay) peaked already at 6 h after PDT and correlated with the time kinetics of PDT-induced neutrophilia. The findings of this study uncover the link between tumor PDT-induced neutrophilia and key acute phase response manifestations, the activation of adrenal-pituitary axis and the expression of a complement C3 protein (major acute phase reactant).

Background and Objectives: The outcome of the treatment of solid tumors by photodynamic therapy (PDT) is critically dependent on the contribution from the host. This host response is provoked by the rapidly induced massive tumor tissue injury delivered by PDT that is experienced as a local trauma threatening the integrity and homeostasis at the affected site. Study Design/Materials and Methods: Mouse tumor models were extensively employed in pre-clinical studies investigating various aspects of host-tumor interaction following PDT, but important input was also derived from clinical data. Results: The recognition of this PDT-inflicted insult by innate immune sensors detecting danger signals from the distressed/altered tumor tissue, triggers host-protecting responses dominantly manifested as acute inflammation that are elicited and orchestrated by the innate immune system. To secure the affected PDT-targeted site, the inflammatory reaction attacks tumor vasculature and then neutralizes the focal source of danger signals by eliminating the injured tumor cells. Conclusion: The provoked highly intensified phagocytosis of dead tumor cells occurring in the context of a vigorous innate immune reaction emerges as a key factor responsible for the development of tumor antigen-specific adaptive immune response that contributes to the eradication of PDT-treated cancers. Lasers Surg. Med. 38:500–508, 2006. 2006 Wiley-Liss, Inc.

A target tumor-derived whole cancer cell therapeutic vaccine was developed based on an in vitro pre-treatment by photodynamic therapy (PDT) and was investigated using a poorly immunogenic tumor model. The vaccine was produced by incubating in vitro expanded mouse squamous cell carcinoma SCCVII cells for 1 h with photosensitizer benzoporphyrin derivative (BPD), then exposing to light (690 nm, 1 J/cm2) and finally to a lethal X-ray dose. Treatment of established subcutaneous SCCVII tumors growing in syngeneic C3H/HeN mice with 2 x 10(7) PDT-vaccine cells per mouse by a peritumoral injection produced a significant therapeutic effect, including growth retardation, regression and cures. Tumor specificity of this PDT-generated vaccine was demonstrated by its ineffectiveness when prepared from a mismatched tumor cell line. Vaccine cells retrieved from the treatment site at 1 h postinjection were intermixed with dendritic cells (DC), exhibited heat shock protein 70 on their surface, and were opsonized by complement C3. Tumor-draining lymph nodes treated by the PDT-vaccine contained dramatically increased numbers of DC as well as B and T lymphocytes (with enlarged memory phenotype fraction in the latter), while high levels of surface-bound C3 were detectable on DC and to a lesser extent on B cells. The PDT-vaccine produced no therapeutic benefit against tumors growing in C3-deficient hosts. It is suggested that surface expression of heat shock proteins and complement opsonization are the two unique features of PDT-treated cells securing avid immune recognition of vaccinated tumor and the development of a strong and effective antitumor adaptive immune response.

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.

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-phposphate (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.

A recent finding showed that ceramide and sphingosine-1-phosphate (S1P) become exposed on the surface of cells treated by photodynamic therapy (PDT) and acquire the capacity to act as danger-associated molecular patterns (DAMPs). To explore this further, the present study examined whether ceramide and S1P can be released from PDTtreated cells and investigated changes in the levels of these sphingolipids in tumor-associated macrophages (TAMs) left in contact with PDT-treated tumor cells. Mass spectroscopy-based analysis detected increased levels of C16ceramide and dihydroC16-ceramide in media supernatants from SCCVII cells collected three hours after they were treated by PDT, compared to untreated cell supernatants. While no release of S1P was detected, elevated levels of its precursor sphingosine were found in the supernatants of PDT-treated cells. The co-incubation of TAMs-containing primary cultures derived from mouse SCCVII tumors with PDT-treated SCCVII cells was followed by ceramide and S1P analysis in these cells based on staining with specific antibodies and flow cytometry. Levels of both ceramide and S1P as well as inflammasome protein NLRP3 were found to rise in TAMs when they were co-cultured with PDT-treated SCCVII cells, while no significant change was seen with cancer cells. Such changes were induced also in TAMs incubated with supernatants from PDT-treated cells. The findings of the present study affirm the potential of sphingolipids including ceramide, S1P, and sphingosine to act, either exposed on cell surface or released in the microenvironment, as DAMPs in the response of tumors to PDT.