Inhibition of Specific Cellular Antioxidant Pathways Increases the Sensitivity of Neurons to Meta‐tetrahydroxyphenyl Chlorin‐Mediated Photodynamic Therapy in a 3D Co‐culture Model

The effect of photodynamic therapy (PDT) on neurons is of critical importance when treating cancers within or adjacent to the nervous system. Neurons show reduced sensitivity to meta‐tetrahydroxyphenyl chlorin (mTHPC) mediated PDT, so the aim of this study was to investigate whether neuron sparing is due to endogenous cellular antioxidant activity. Dorsal root ganglion (DRG) neurons and their associated satellite glia were subjected to mTHPC‐PDT in a 3D co‐culture system following incubation with antioxidant inhibitors: diethyl dithiocarbamate (DDC, SOD‐1 inhibitor), 2‐methoxyestradiol (2‐MeOH₂, SOD‐2 inhibitor) and l ‐buthionine sulfoximine (l ‐BSO, glutathione synthase inhibitor). Sensitivity of each cell type was assessed using a combination of live/dead staining and immunofluorescence. Pretreatment with DDC and with l ‐BSO significantly increased the sensitivity of neurons to mTHPC‐PDT and also affected satellite glial cell viability, whereas 2‐MeOE₂ caused only a small increase in neuron sensitivity (not significant). Pretreatment using a combination of DDC and l ‐BSO caused a near total loss of neuron and glial cell viability in treatment and control conditions. These findings suggest that the SOD‐1 and glutathione pathways are likely to be involved in the neuronal sparing associated with mTHPC‐PDT.     

Lysosomal signaling enhances mitochondria-mediated photodynamic therapy in A431 cancer cells: role of iron

In photodynamic therapy (PDT), light activates a photosensitizer added to a tissue, resulting in singlet oxygen formation and cell death. The photosensitizer phthalocyanine 4 (Pc 4) localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. The aim of this study was to determine how lysosomes contribute to PDT-induced cell killing by mitochondria-targeted photosensitizers such as Pc 4. We monitored cell killing of A431 cells after Pc 4-PDT in the presence and absence of bafilomycin, an inhibitor of the vacuolar proton pump of lysosomes and endosomes. Bafilomycin was not toxic by itself, but greatly enhanced Pc 4-PDT-induced cell killing. To investigate whether iron loading of lysosomes affects bafilomycin-induced killing, cells were incubated with ammonium ferric citrate (30 μM) for 30 h prior to PDT. Ammonium ferric citrate enhanced Pc 4 plus bafilomycin-induced cell killing without having toxicity by itself. Iron chelators (desferrioxamine and starch-desferrioxamine) and the inhibitor of the mitochondrial calcium (and ferrous iron) uniporter, Ru360, protected against Pc 4 plus bafilomycin toxicity. These results support the conclusion that chelatable iron stored in the lysosomes enhances the efficacy of bafilomycin-mediated PDT and that lysosomal disruption augments PDT with Pc 4.     

PHOTODYNAMIC THERAPY-INDUCED HYPOXIA IN RAT TUMORS and NORMAL TISSUES

Abstract The administration of misonidazole (MISO) to Fischer x Copenhagen rats whose R3327-H prostate tumors were treated with photodynamic therapy (PDT) produced enhanced tumor growth delays and cures. This potentiation of PDT by MISO was previously observed with R3327-AT tumors and was postulated to result from drug cytotoxicity of naturally-occurring and PDT-induced hypoxic cells. Radioactively-labelled MISO has been developed as a marker for tissue p0₂ at the cellular level and [³H]MISO was administered to R3327-AT and R3327-H tumor-bearing rats before and after standard PDT treatments. The amount of ³H in tissues 24 h after drug administration was a measure of'bound MISO'which reflects average tissue oxygenation. [³H]MISO retained in R3327-AT tumors was ˜4x and in liver tissue ˜2x that retained in muscle, heart, brain and R3327-H tumors (1x). Tumors treated with Photofrin II and lased with 1000 J showed a 6-fold increase in retained [³H]MISO in R3327-H tumors and a 2-fold increase in retained [³H]MISO in R3327-AT tumors. The absolute levels of retained ³H in both tumors after PDT were similar. These data provide direct evidence that PDT induces rapid hypoxia in both tumors. When the gastrocnemius muscle of the rat leg was similarly treated, the amount of [³H]MISO retained was ˜4x greater than that in untreated muscle. This result suggests that PDT-induced hypoxia is not selective to just tumor tissue. These data suggest that the hypoxia-inducing property of PDT might be exploited in combination with hypoxic cell cytotoxins to produce improved tumor responses and cures.     

Effect and Mechanism of a New Photodynamic Therapy with Glycoconjugated Fullerene

When irradiated, fullerene efficiently generates reactive oxygen species (ROS) and is an attractive photosensitizer for photodynamic therapy (PDT). Ideally, photosensitizers for PDT should be water-soluble and tumor-specific. Because cancer cells endocytose glucose more effectively than normal cells, the characteristics of fullerene as a photosensitizer were improved by combining it with glucose. The cytotoxicity of PDT was studied in several cancer cell lines cultured with C₆₀-(Glc)1 (d -glucose residue pendant fullerene) and C₆₀-(6Glc)1 (a maltohexaose residue pendant fullerene) subsequently irradiated with UVA₁. PDT alone induced significant cytotoxicity. In contrast, PDT with the glycoconjugated fullerene exhibited no significant cytotoxicity against normal fibroblasts, indicating that PDT with these compounds targeted cancer cells. To investigate whether the effects of PDT with glycoconjugated fullerene were because of the generation of singlet oxygen (¹O₂), NaN₃ was added to cancer cells during irradiation. NaN₃ extensively blocked PDT-induced apoptosis, suggesting that PDT-induced cell death was a result of the generation of ¹O₂. Finally, to investigate the effect of PDT in vivo, melanoma-bearing mice were injected intratumorally with C₆₀-(Glc)1 and irradiated with UVA₁. PDT with C₆₀-(Glc)1 suppressed tumor growth. These findings indicate that PDT with glycoconjugated fullerene exhibits tumor-specific cytotoxicity both in vivo and in vitro via the induction of ¹O₂.     

NUCLEAR FACTOR κB BINDING ACTIVITY IN MOUSE L1210 CELLS FOLLOWING PHOTOFRIN II-MEDIATED PHOTOSENSITIZATION

Clinical photodynamic therapy (PDT) uses the photosensitizer photofnn II to produce singlet molecular oxygen and other reactive oxygen intermediates for localized tumor tissue cytotoxicity. In this report, we show that PDT enhances the DNA binding activity of nuclear factor kappa B (NFkB), a transacti vator of cytokine gene expression. Photosensitization following a 16 h incubation of photofrin II induced NFkB binding activity in mouse leukemia L1210 cells 10-fold above that observed in exponentially growing cultures. Serum starvation, as well as drug-alone and light-alone controls, elevated basal NF k B binding activity two- to three-fold. Upstream stimulatory factor binding activity was not modulated by any of the cell treatments and was used to standardize gel mobility shift data. This study identifies porphynn-mediated PDT as an inducer of NF k B binding activity, extending recent findings that NF k B activation is a general response to oxidative stress.     

Reactive Oxygen Species Explicit Dosimetry for Photofrin-mediated Pleural Photodynamic Therapy

Explicit dosimetry of treatment light fluence and implicit dosimetry of photosensitizer photobleaching are commonly used methods to guide dose delivery during clinical PDT. Tissue oxygen, however, is not routinely monitored intraoperatively even though it is one of the three major components of treatment. Quantitative information about in vivo tissue oxygenation during PDT is desirable, because it enables reactive oxygen species explicit dosimetry (ROSED) for prediction of treatment outcome based on PDT-induced changes in tumor oxygen level. Here, we demonstrate ROSED in a clinical setting, Photofrin-mediated pleural photodynamic therapy, by utilizing tumor blood flow information measured by diffuse correlation spectroscopy (DCS). A DCS contact probe was sutured to the pleural cavity wall after surgical resection of pleural mesothelioma tumor to monitor tissue blood flow (blood flow index) during intraoperative PDT treatment. Isotropic detectors were used to measure treatment light fluence and photosensitizer concentration. Blood-flow-derived tumor oxygen concentration, estimated by applying a preclinically determined conversion factor of 1.5 × 10⁹ μMs cm⁻² to the blood flow index, was used in the ROSED model to calculate the total reacted reactive oxygen species [ROS]rx. Seven patients and 12 different pleural sites were assessed and large inter- and intrapatient heterogeneities in [ROS]rx were observed although an identical light dose of 60 J cm⁻² was prescribed to all patients.     

In vitro fungicidal photodynamic effect of hypericin on Candida species

Hypericin is a natural photosensitizer considered for the new generation of photodynamic therapy (PDT) drugs. The aim of this study was to evaluate the in vitro fungicidal effect of hypericin PDT on various Candida spp., assessing its photocytotoxicity to keratinocytes (HaCaT) and dermal fibroblasts (hNDF) to determine possible side effects. A 3 log fungicidal effect was observed at 0.5 McFarland for two Candida albicans strains, Candida parapsilosis and Candida krusei with hypericin concentrations of 0.625, 1.25, 2.5 and 40 μm, respectively, at a fluence of 18 J cm(-2) (LED lamp emitting at 602 ± 10 nm). To obtain a 6 log reduction, significantly higher hypericin concentrations and light doses were needed (C. albicans 5 μM, C. parapsilosis 320 μM and C. krusei 320 μM; light dose 37 J cm(-2)). Keratinocytes and fibroblasts can be preserved by keeping the hypericin concentration below 1 μm and the light dose below 37 J cm(-2). C. albicans appears to be suitable for treatment with hypericin PDT without significant damage to cutaneous cells.     

Modeling of a type II photofrin-mediated photodynamic therapy process in a heterogeneous tissue phantom

We present a quantitative framework to model a Type II photodynamic therapy (PDT) process in the time domain in which a set of rate equations are solved to describe molecular reactions. Calculation of steady-state light distributions using a Monte Carlo method in a heterogeneous tissue phantom model demonstrates that the photon density differs significantly in a superficial tumor of only 3 mm thickness. The time dependences of the photosensitizer, oxygen and intracellular unoxidized receptor concentrations were obtained and monotonic decreases in the concentrations of the ground-state photosensitizer and receptor were observed. By defining respective decay times, we quantitatively studied the effects of photon density, drug dose and oxygen concentration on photobleaching and cytotoxicity of a photofrin-mediated PDT process. Comparison of the dependences of the receptor decay time on photon density and drug dose at different concentrations of oxygen clearly shows an oxygen threshold under which the receptor concentration remains constant or PDT exhibits no cytotoxicity. Furthermore, the dependence of the photosensitizer and receptor decay times on the drug dose and photon density suggests the possibility of PDT improvement by maximizing cytotoxicity in a tumor with optimized light and drug doses. We also discuss the utility of this model toward the understanding of clinical PDT treatment of chest wall recurrence of breast carcinoma.     

Kazinol Q from Broussonetia kazinoki enhances cell death induced by Cu(II) through increased reactive oxygen species

The ability of the flavan kazinol Q (KQ) to induce DNA breakage in the presence of Cu(II) was examined by agarose gel electrophoresis using supercoiled plasmid DNA. In KQ-mediated DNA breakage reaction, the involvement of reactive oxygen species (ROS), H(2)O(2) and O(2)- was established by the inhibition of DNA breakage by catalase and revealed DNA breakage by superoxide dismutase (SOD). The cell viability of gastric carcinoma SCM-1 cells treated with various concentrations of KQ was significantly decreased by cotreatment with Cu(II). Treatment of SCM-1 cells with 300 μM Cu(II) enhanced the necrosis induced by 100 μM KQ. Treatment of SCM-1 cells with 100 mM KQ in the presence of 300 mM Cu(II) increased the generation of H(2)O(2). Taken together, the above finding suggested that KQ cotreatment with Cu(II) produced increased amounts of H(2)O(2), thus enhancing subsequent cell death due to necrosis.     

Photodynamic treatment with fractionated light decreases production of reactive oxygen species and cytotoxicity in vitro via regeneration of glutathione

Photodynamic therapy removes unwanted or harmful cells by overproduction of reactive oxygen species (ROS). Fractionated light delivery in photodynamic therapy may enhance the photodynamic effect in tumor areas with insufficient blood supply by enabling the reoxygenation of the treated area. This study addresses the outcome of fractionated irradiation in an in vitro photodynamic treatment (PDT) system, where deoxygenation can be neglected. Our results show that fractionated irradiation with light/dark intervals of 45/60 s decreases ROS production and cytotoxicity of PDT. This effect can be reversed by addition of 1,3-bis-(2-chlorethyl)-1-nitrosurea (BCNU), an inhibitor of the glutathione reductase. We suggest that the dark intervals during irradiation allow the glutathione reductase to regenerate reduced glutathione (GSH), thereby rendering cells less susceptible to ROS produced by PDT compared with continuous irradiation. Our results could be of particular clinical importance for photodynamic therapy applied to well-oxygenated tumors.     

Hypoxia Induced by Upconversion‐Based Photodynamic Therapy: Towards Highly Effective Synergistic Bioreductive Therapy in Tumors

Local hypoxia in tumors is an undesirable consequence of photodynamic therapy (PDT), which will lead to greatly reduced effectiveness of this therapy. Bioreductive pro‐drugs that can be activated at low‐oxygen conditions will be highly cytotoxic under hypoxia in tumors. Based on this principle, double silica‐shelled upconversion nanoparticles (UCNPs) nanostructure capable of co‐delivering photosensitizer (PS) molecules and a bioreductive pro‐drug (tirapazamine, TPZ) were designed (TPZ‐UC/PS), with which a synergetic tumor therapeutic effect has been achieved first by UC‐based (UC‐) PDT under normal oxygen environment, immediately followed by the induced cytotoxicity of activated TPZ when oxygen is depleted by UC‐PDT. Treatment with TPZ‐UC/PS plus NIR laser resulted in a remarkably suppressed tumor growth as compared to UC‐PDT alone, implying that the delivered TPZ has a profound effect on treatment outcomes for the much‐enhanced cytotoxicity of TPZ under PDT‐induced hypoxia.     

Optimization of photodynamic therapy response by survivin gene knockdown in human metastatic breast cancer T47D cells

Photodynamic therapy (PDT) leads to the generation of cytotoxic oxygen species that appears to stimulate several different signaling pathways, some of which lead to cell death, whereas others mediate cell survival. In this context, we observed that PDT mediated by methyl-5-aminolevulinic acid as the photosensitizer resulted in over-expression of survivin, a member of the inhibitor of apoptosis (IAP) family that correlates inversely with patient prognosis. The role of survivin in resistance to anti-cancer therapies has become an area of intensive investigation. In this study, we demonstrate a specific role for survivin in modulating PDT-mediated apoptotic response. In our experimental system, we use a DNA vector-based siRNA, which targets exon-1 of the human survivin mRNA (pSil_1) to silence survivin expression. Metastatic T47D cells treated with both pSil_1 and PDT exhibited increased apoptotic indexes and cytotoxicity when compared to single-agent treated cells. The treatment resulted in increased PARP and caspase-3 cleavage, a decrease in the Bcl-2/Bak ratio and no participation of heat shock proteins. In contrast, the overexpression of survivin by a survivin-expressed vector increased cell viability and reduced cell death in breast cancer cells treated with PDT. Therefore, our data suggest that combining PDT with a survivin inhibitor may attribute to a more favorable clinical outcome than the use of single-modality PDT.     

Enhanced Photodynamic Therapy by Reduced Levels of Intracellular Glutathione Obtained By Employing a Nano‐MOF with CuII as the Active Center

In photodynamic therapy (PDT), the level of reactive oxygen species (ROS) produced in the cell directly determines the therapeutic effect. Improvement in ROS concentration can be realized by reducing the glutathione (GSH) level or increasing the amount of photosensitizer. However, excessive amounts photosensitizer may cause side effects. Therefore, the development of photosensitizers that reduce GSH levels through synergistically improving ROS concentration in order to strengthen the efficacy of PDT for tumor is important. We report a nano‐metal–organic framework (Cu^II‐metalated nano‐MOF {CuL‐[AlOH]₂}_n (MOF‐2, H₆L=mesotetrakis(4‐carboxylphenyl)porphyrin)) based on Cu^II as the active center for PDT. This MOF‐2 is readily taken up by breast cancer cells, and high levels of ROS are generated under light irradiation. Meanwhile, intracellular GSH is considerably decreased owing to absorption on MOF‐2; this synergistically increases ROS concentration and accelerates apoptosis, thereby enhancing the effect of PDT. Notably, based on the direct adsorption of GSH, MOF‐2 showed a comparable effect with the commercial antitumor drug camptothecin in a mouse breast cancer model. This work provides strong evidence for MOF‐2 as a promising new PDT candidate and anticancer drug.     

Hybrid TiO2–Ruthenium Nano‐photosensitizer Synergistically Produces Reactive Oxygen Species in both Hypoxic and Normoxic Conditions

Photodynamic therapy (PDT) is widely used to treat diverse diseases, but its dependence on oxygen to produce cytotoxic reactive oxygen species (ROS) diminishes the therapeutic effect in a hypoxic environment, such as solid tumors. Herein, we developed a ROS‐producing hybrid nanoparticle‐based photosensitizer capable of maintaining high levels of ROS under both normoxic and hypoxic conditions. Conjugation of a ruthenium complex (N3) to a TiO₂ nanoparticle afforded TiO₂‐N3. Upon exposure of TiO₂‐N3 to light, the N3 injected electrons into TiO₂ to produce three‐ and four‐fold more hydroxyl radicals and hydrogen peroxide, respectively, than TiO₂ at 160 mmHg. TiO₂‐N3 maintained three‐fold higher hydroxyl radicals than TiO₂ under hypoxic conditions via N3‐facilitated electron–hole reduction of adsorbed water molecules. The incorporation of N3 transformed TiO₂ from a dual type I and II PDT agent to a predominantly type I photosensitizer, irrespective of the oxygen content.     

Cell-penetrating riboflavin conjugate for antitumor photodynamic therapy

Riboflavin (RF, vitamin B2) is an essential vitamin and has been considered as a promising natural photosensitizer for photodynamic therapy (PDT). However, further exploration of RF in antitumor application was limited by its poor cellular uptake. In this study, using cell-penetrating peptides Arg₈, (Cha-Arg)₃ and small molecule triphenylphosphine (TPP) as delivery compounds, three RF conjugates were prepared to increase the accumulation of RF in cells, termed as Arg₈-RF, (Cha-Arg)₃-RF and TPP-RF, respectively. Compared with TPP-RF and Arg₈-RF, (Cha-Arg)₃-RF exhibited better cell internalization and stronger cytotoxicity against HeLa cells upon exposure to blue light. Further researches proved that (Cha-Arg)₃-RF generated reactive oxygen species (ROS) under irradiation, which could indiscriminately destroy endogenous proteins and mitochondria, ultimately inducing cell death. This work provides a new approach to explore RF as a natural photosensitizer for antitumor photodynamic therapy.     

Photodynamic therapy with Pc 4 induces apoptosis of Candida albicans

The high prevalence of drug resistance necessitates the development of novel antifungal agents against infections caused by opportunistic fungal pathogens, such as Candida albicans. Elucidation of apoptosis in yeast-like fungi may provide a basis for future therapies. In mammalian cells, photodynamic therapy (PDT) has been demonstrated to generate reactive oxygen species, leading to immediate oxidative modifications of biological molecules and resulting in apoptotic cell death. In this report, we assess the in vitro cytotoxicity and mechanism of PDT, using the photosensitizer Pc 4, in planktonic C. albicans. Confocal image analysis confirmed that Pc 4 localizes to cytosolic organelles, including mitochondria. A colony formation assay showed that 1.0 μM Pc 4 followed by light at 2.0 J cm(-2) reduced cell survival by 4 logs. XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide) assay revealed that Pc 4-PDT impaired fungal metabolic activity, which was confirmed using the FUN-1 (2-chloro-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenylquinolinium iodide) fluorescence probe. Furthermore, we observed changes in nuclear morphology characteristic of apoptosis, which were substantiated by increased externalization of phosphatidylserine and DNA fragmentation following Pc 4-PDT. These data indicate that Pc 4-PDT can induce apoptosis in C. albicans. Therefore, a better understanding of the process will be helpful, as PDT may become a useful treatment option for candidiasis.     

(Metallo)porphyrins as Potent Phototoxic Anti‐Cancer Agents after Irradiation with Red Light

Novel photoactive (metallo)porphyrins were synthesised and characterised. When irradiated with light at a wavelength greater than 600 nm, these porphyrins act as photosensitisers and show high cytotoxicity towards two different human cancer cell lines with IC₅₀ values down to 0.4 μM . A paramagnetic copper(II) porphyrin is the first photosensitiser to display excellent phototoxicity, explained by the electron paramagnetic resonance (EPR) spin trapping of hydroxy radicals and experimentally confirmed by the discovery of elevated levels of reactive oxygen species (ROS) inside A2780 cells after irradiation with red light. This finding indicates that paramagnetic compounds should be considered for photodynamic therapy (PDT). Furthermore, an additive effect of cisplatin and a zinc porphyrin, both at subtherapeutic concentrations of 0.22 μm, was observed.     

Simultaneous and ultrarapid determination of reactive oxygen species and reduced glutathione in apoptotic leukemia cells by microchip electrophoresis

A microchip electrophoresis method coupled with laser-induced fluorescence (LIF) detection was established for simultaneous determination of two kinds of intracellular signaling molecules (reactive oxygen species, ROS, and reduced glutathione, GSH) related to apoptosis and oxidative stress. As the probe dihydrorhodamine-123 (DHR-123) can be converted intracellularly by ROS to the fluorescent rhodamine-123 (Rh-123), and the probe naphthalene-2,3-dicarboxaldehyde (NDA) can react quickly with GSH to produce a fluorescent adduct, rapid determination of Rh-123 and GSH was achieved on a glass microchip within 27 s using a 20 mM borate buffer (pH 9.2). The established method was tested to measure the intracellular ROS and GSH levels in acute promyelocytic leukemia (APL)-derived NB4 cells. An elevation of intracellular ROS and depletion of GSH were observed in apoptotic NB4 cells induced by arsenic trioxide (As(2)O(3)) at low concentration (1-2 microM). Buthionine sulfoximine (BSO), in combination with As(2)O(3) enhanced the decrease of reduced GSH to a great extent. The combined treatment of As(2)O(3) and hydrogen peroxide (H(2)O(2)) led to an inverse relationship between the concentrations of ROS and GSH obtained, showing the proposed method can readily evaluate the generation of ROS, which occurs simultaneously with the consumption of the inherent antioxidant.     

Reactive oxygen specie-induced photodynamic therapy activation by supramolecular strategy

Photodynamic therapy (PDT) agents may accumulate in skin and cause severe skin cytotoxicity. We report a pro-guest-based supramolecular strategy to selectively activate PDT in the reactive oxygen specie (ROS) overexpressed microenvironment, which is often existing in tumor and inflammatory tissues. PDT agents methylene blue (MB) and basic blue 17 (BB17) are used as model drugs. When encapsulated by acyclic cucurbit[n]uril (CB[n]), the efficacy of PDT agents is significantly inhibited. By contrast, in the presence of ROS (H₂O₂) and pro-guest, PDT agents are displaced and reactivated to show a dramatically enhanced PDT efficacy in cells.     

Therapeutic Enhancement of Verteporfin-mediated Photodynamic Therapy by mTOR Inhibitors

Photodynamic therapy (PDT) with photosensitizer verteporfin is a clinically approved vascular disrupting modality that is currently in clinical trial for cancer treatment. In this study, we evaluated PDT in combination with either mTORC1 inhibitor rapamycin or mTORC1/C2 dual inhibitor AZD2014 for therapeutic enhancement in SVEC endothelial cells. Verteporfin-PDT alone induced cell apoptosis by activating the intrinsic apoptotic pathway. However, it increased the expression of anti-apoptotic protein MCL-1 and the phosphorylation of S6, a downstream molecule of mTOR signaling. In contrast, mTOR inhibitors rapamycin and AZD2014 did not induce apoptosis in SVEC cells. They suppressed MCL-1 expression and S6 phosphorylation and imposed a potent inhibition on cell proliferation. PDT in combination with mTOR inhibitors activated the intrinsic apoptotic pathway and resulted in increased apoptosis. Combination treatments also led to sustained inhibition of cell proliferation. Although AZD2014 was more effective for cell growth inhibition and PDT enhancement than rapamycin at the higher concentrations examined in the study, both inhibitors effectively enhanced PDT response, suggesting that inhibition of mTORC1 is crucial for PDT enhancement. Our results indicate that mTOR inhibitors mechanistically cooperate with PDT for enhanced cell death and sustained growth inhibition, supporting a combination approach for therapeutic enhancement.