Photodynamic Therapy and Its Role in Combined Modality Anticancer Treatment.

Technology in Cancer Research & Treatment http://tct.sagepub.com/

Photodynamic Therapy and Its Role in Combined Modality Anticancer Treatment N. Patrik Brodin, Chandan Guha and Wolfgang A. Tomé Technol Cancer Res Treat published online 5 November 2014 DOI: 10.1177/1533034614556192 The online version of this article can be found at: http://tct.sagepub.com/content/early/2014/11/03/1533034614556192

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Article

Photodynamic Therapy and Its Role in Combined Modality Anticancer Treatment

Technology in Cancer Research & Treatment 1–14 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1533034614556192 tct.sagepub.com

N. Patrik Brodin, PhD1,2, Chandan Guha, MD, PhD1,2, ´ PhD, FAAPM1,2 and Wolfgang A. Tome,

Abstract Photodynamic therapy (PDT) is a relatively new modality for anticancer treatment and although the interest has increased greatly in the recent years, it is still far from clinical routine. As PDT consists of administering a nontoxic photosensitizing chemical and subsequently illuminating the tumor with visible light, the treatment is not subject to dose-limiting toxicity, which is the case for established anticancer treatments like radiation therapy or chemotherapy. This makes PDT an attractive adjuvant therapy in a combined modality treatment regimen, as PDT provides an antitumor immune response through its ability to elicit the release of damage-associated molecular patterns and tumor antigens, thus providing an increased antitumor efficacy, potentially without increasing the risk of treatment-related toxicity. There is great interest in the elicited immune response after PDT and the potential of combining PDT with other forms of treatment to provide potent antitumor vaccines. This review summarizes recent studies investigating PDT as part of combined modality treatment, hopefully providing an accessible overview of the current knowledge that may act as a basis for new ideas or systematic evaluations of already promising results. Keywords photodynamic therapy, cancer, immunogenicity, combined-modality treatment Abbreviations 60 Co, cobalt-60; 137Cs, cesium-137; ATP, adenosine triphosphate; CY, cyclophosphamide; DAMPs, damage-associated molecular patterns; DCs, dendritic cells; ER, endoplasmic reticulum; HMGB1, high-mobility group box 1; HSP, heat-shock protein; ICD, immunogenic cell death; NSCLC, nonsmall cell lung cancer; PDT, photodynamic therapy; PS, photosensitizer; ROS, reactive oxygen species

Photodynamic Therapy In recent years, there has been increasing interest in the potential of using photodynamic therapy (PDT) to treat various types of cancers, either on its own or in combination with other anticancer treatments. The principle of PDT includes the administration of a photosensitizing drug and subsequently illuminating the target area with visible light corresponding to the absorbance wavelength of the photosensitizing drug, triggering a series of biological effects.1,2 Thus, there are 2 main components that govern the applicability of PDT in cancer therapy, the localization of the photosensitizing drug in tumor tissue and delivering the appropriate dose of light (near-infrared for most photosensitizing drugs) to that tissue. The characteristics of an ideal photosensitizing drug have been extensively described elsewhere,3-5 but a few key features include high-specific tumor uptake, negligible dark toxicity (biological effects without light

application), and a fairly long absorption wavelength as longer wavelengths allow deeper tissue penetration. Some of the most investigated photosensitizing drugs include hypericin,6-8 Photofrin,4,9 and 5-aminolevulinic acid.4,10 Alongside a wide variety of photosensitizing drugs, there

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Department of Radiation Oncology, Montefiore Medical Center, Bronx, NY, USA 2 Institute for Onco-Physics, Albert Einstein College of Medicine, Bronx, NY, USA

Received: March 15, 2014; Revised: July 23, 2014; Accepted: September 26, 2014. Corresponding Author: Wolfgang A. Tom´e, PhD, FAAPM, Institute for Onco-Physics, Albert Einstein College of Medicine, Block Building Room 106, 1300 Morris Park Ave, Bronx, NY 10461, USA. Email: [email protected]


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Technology in Cancer Research & Treatment

Figure 1. A ground state photosensitizer (PS) molecule in singlet state is excited by light boosting an electron into a higher energy orbital. The excited state reverts to the ground state via fluorescence or internal conversion (~nanoseconds) or the spin of the excited electron inverts resulting in an excited triplet state PS at lower energy, which is a relatively long-lived state (~microseconds) since the excitation energy is lost by phosphorescence in a spin-forbidden transfer directly back to the ground singlet state. The excited triplet state can then either transfer its energy to triplet molecular oxygen resulting in biologically reactive excited singlet state oxygen or transfer an electron to superoxide anions resulting in various reactive oxygen species (ROS) capable of causing substantial biological damage. The resulting biological effects of photodynamic therapy include cell death through different pathways and in many cases also a potent immune response involving both the innate and the adaptive immune system.

is a vast availability of light sources for PDT such as various types of lasers, broad wavelength band lamps, and light-emitting diodes.9,11 Figure 1 illustrates the photophysical and photochemical mechanisms that lead to the biological effects of PDT.

Immunogenic Cell Death and PDT Considerable research efforts have been undertaken to clarify the biological mechanisms responsible for the anticancer effectiveness of PDT. These mechanisms can generally be divided into direct antitumor effects and indirect effects related to an induced immunological response. The cell death mechanisms of direct PDT-induced cytotoxicity include apoptosis (programmed cell death), necrosis (unregulated cellular breakdown), and macroautophagy (degradation of cellular components by lysosomes).2,7,12 Direct effects can also include damage to tumor vasculature, which may however initiate subsequent tumor angiogenesis. 7,12,13 Which cell death

mechanism is responsible for the antitumor effects depends on the light dose and photosensitizer (PS) uptake. The amount of apoptotic versus necrotic cell death depends on the resulting reactive oxygen species (ROS) concentration within the cells after PDT.7 Photodynamic therapy has been shown to induce immunogenic cell death (ICD) that can trigger a considerable immune response further enhancing the antitumor effect.14 A detailed review on ICD was recently published by Kroemer et al15 where the authors provide the following operational definitions of ICD: Malignant cells are considered to undergo ICD in vitro if upon subsequent transplantation they provide the host with an immune response that protects against challenges with tumor cells of the same strain, that is, acting as a cancer vaccine. In vivo, cell death is defined as immunogenic if it triggers a response of the innate and adaptive immune system leading to antitumor effects that are a result of mechanisms dependent on the immune system.


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The innate immune response includes activation of the complement system with a strong inflammatory response and neutrophil infiltration, along with macrophage activation, maturation of dendritic cells (DCs), and increased natural killer cell activity.14,16 Adaptive immunity is mediated via B- and T cells. B cells produce antibodies that can in turn eliminate tumor cells. T-helper (T h) cells can both induce and suppress immune effects. On one hand, Th1 cells (CD4þT-betþ T-cells) activate and stimulate antitumor M1 macrophages as well as license CD8þ T-cells to become cytotoxic T-lymphocytes (CTLs) that kill cancer cells. While on the other hand Th2 cells (CD4þGATA-3þ Tcells) stimulate B cells into proliferation, induce B-cell antibody class switching as well as activate and stimulate protumor M2 macrophages. Additionally, regulatory T cells (Tregs; CD4þCD25þFoxP3þ T-cells) suppress the antitumor immune response through induction of anergy in Th1 cells and CTLs.12,14 Immunogenic cell death is triggered by the release of damage-associated molecular patterns (DAMPs), which are normally retained within the cell but act as danger signals in response to damage or stress by being translocated to the cell surface, secreted into the cytoplasm, or released extracellularly.16,17 The release of DAMPs prompts an efficient donation of tumor antigens priming the adaptive immune system to create antibodies and seek out the corresponding cancer cells. Damage-associated molecular patterns include calreticulin, heat shock proteins (HSPs) 70 and HSP90, high-mobility group box 1 (HMGB1), adenosine triphosphate (ATP), uric acid, interlukin 1a, DNA, and RNA.16 Thus, it has been shown that a robust approach to determine whether cancer cells subjected to a certain treatment undergo ICD is to measure subsequent calreticulin exposure, ATP secretion, and HMGB1 release.15 It has been shown that 2 essential components required to trigger the intracellular mechanisms resulting in ICD are endoplasmic reticulum (ER) stress and the generation of ROS.18 This is explained in further detail in the review by Krysko et al,18 but it has been extensively shown that both ER stress and ROS are required to elicit ICD. Interestingly, the strongest immune response is provided by treatments inducing focused primary ER stress rather than ER stress induced as a secondary effect through damage to other intracellular targets. This makes PDT an important candidate for ICDbased cancer therapy as hypericin-PDT has been shown to induce massive ROS production generating direct ER stress, as hypericin localizes within the ER.19,20 This suggests that the immune response resulting from hypericin-PDT will be more efficient than immune responses from treatments inducing secondary ER stress, as focused ROS-based ER stress has been shown to result in an increased number of emitted DAMPs as well as simplifying the DAMP trafficking pathways.18 In the following sections, we summarize the current preclinical, and in a few cases also clinical, evidence of the efficacy of PDT when used as part of combined modality anticancer treatment.

Photodynamic Therapy in Combination With Immunostimulants Since PDT has the potential of triggering a strong antitumor immune response, there has been great interest in potentiating this effect by stimulating various components of the immune system.21 This can be done by combining PDT with the administration of various immunostimulants to, for example, increase neutrophil infiltration, enhance tumor antigen presentation, upregulate T-cell activation, and suppress expression of Tregs. Immunostimulants can be administered intratumorally, intravenously, or even topically depending on the type of malignancy.21 Table 1 summarizes recent studies exploring different immunostimulatory agents and their effect on immune response when combined with PDT for various malignancies. Studies have been carried out on several different tumor models including lung cancer, colon cancer, squamous cell carcinomas, melanoma, and breast cancer. Many of the different strategies showed promising in vivo results with increased survival and reduction in tumor volumes. There were also some clear immunotherapeutic effects of these combination strategies showing effective rejection of tumor rechallenge or significant antitumoral effects of untreated contralateral tumors. Many of the studies used Photofrin as the PS agent, although some studies showed that the increase in therapeutic effect from combination therapy was independent of the choice of PS.26,27 As different studies addressed different mechanisms for enhancing tumor immunogenicity, there may also be potential for combining some of the strategies to further improve treatment results. This should of course be done with caution and considerable thought has to be given to how immunostimulants are administered, as systemic effects could result in severe autoimmune reactions.

Photodynamic Therapy in Combination With Established Anticancer Therapy The combination of PDT and established anticancer therapies such as ionizing radiation and/or chemotherapy provides an exciting platform for potential new treatment options, especially since PDT does not have the inherent dose-limiting toxicity of either radiation therapy or chemotherapy. The combination with ionizing radiation also includes the potential of certain PS agents to act as radiosensitizers.33 Since ionizing radiation causes cell death mainly via direct DNA damage, there are potential synergistic effects to be explored as PDT can cause DNA degradation leading to increased cell death as the DNA may lose its capacity to repair normally sublethal single-strand breaks.34 When it comes to combining chemotherapy with PDT, there are many possible ways of achieving a combined or synergistic effect, depending on the cell death mechanisms of the chemotherapeutic drug in question. Not only are the cell death mechanisms important but also the intracellular target of the chemotherapy agent, as this will likely affect how the combination with PDT affects the tumor cells. As PDT also affects


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Table 1. Studies Combining Photodynamic Therapy With Immunostimulants. Therapy Combination

In Vivo Tumor Model

Photosensitizer

Light Source and Dose

Main Results

5 mg/kg Photofrin at 24 Photofrin-PDT: a 150- Significantly more EMT6 mammary Glycated chitosan, W lamp with 630 + h before PDT for sarcoma and line 1 tumor-free mice at which stimulates the 10 nm filter delivered EMT6 tumors and lung adenocarcinoma >30 days when glyimmune system by 60 J/cm2 at 100 mW/ 0.1 mg/kg mTHPC subcutaneously cated chitosan was producing TNF-a and 24 h before PDT for implanted into added compared to IL-6 or T-cell uprecm2 line 1 lung tumors BALB/c mice Photofrin-PDT alone gulation and was mTHPC-PDT: a 652or mTHPC-PDT administered immenm laser delivered 30 alone diately after PDT at J/cm2 at 110 mW/ 0.5% or 1.5% cm2 concentration 10 mg/kg Photofrin A 150-W lamp with 630 Significant increase in Complement-activating Lewis lung carcinoma 24 h before PDT + 10 nm filter delivcells subcutaneously agents zymosan and tumor-free mice and ered 150 J/cm2 at 110 implanted in C57BL/6 heat-aggregated g C3 complement promice globulin (HAGG) tein levels with mW/cm2 administered intratuPhotofrin-PDT þ 0.5 morally directly after mg/mouse zymosan PDT or 1.25 mg/mouse HAGG Methylaminolevulinate Aktlite 128 lamp Phase II clinical trial of (1) At 1 year 65% of delivering 630 nm photosensitizer topical imiquimod, patients symptom light to a dose of cream topically which stimulates the free 50 J/cm2 applied 3 h before innate immune (2) More T-cells found PDT system through tollin biopsies of like receptors TLR7 responders and TLR8, for grade (3) More Treg-cells 2/3 vulval intraefound in pithelial neoplasia, nonresponders applied in the weeks prior to PDT 10 Hz pulsed laser light (1) Significant increase Hematoporphyrin NR-S1 squamous cell The neutrophil- and at 630 nm was used oligomers carcinoma transin survival and macrophageto deliver a dose of administered planted into the dordecrease in tumor activating strepto360 J/cm2 at 15 mJ/ intraperitoneally sum of C3H/HeNCrj volumes if OK-432 coccal preparation with 20 mg/kg at 48 mice given before PDT OK-432 was intratucm2 per pulse h before PDT (2) No effect if given morally injected after PDT either 3 h before or immediately after PDT Corresponding Six different EMT6 mammary (1) Percentage tumorDendritic cell and wavelengths of 630, photosensitizers; sarcoma cells were free mice increased macrophage 690, 671, 652, 732, Photofrin, BPD, zinc intramuscularly with BCG after activating Bacilus and 664 nm were phthalocyanine, transplanted to PDT, for all tested Calmette-Guerin delivered at 60-90 mTHPC (Foscan), BALB/c mice photosensitizers (BCG) administered mW/cm2 using a lutetium texaphyrin, (2) Substantial increase subtumorally either and NPe6 in immune memory 24 before or immetunable Xenon light T-cells in draining diately after PDT bulb source to diflymph nodes ferent light doses Corresponding Four different EMT6 mammary (1) MCWE immediately Administration of wavelengths of 630, photosensitizers: sarcoma cells were after PDT increased mycobacterium cell 690, 671, and 652 nm Photofrin, BPD, zinc intramuscularly percentage of wall extract were delivered at phthalocyanine, and transplanted to tumor-free mice (MCWE), which 100-130 mW/cm2 mTHPC (Foscan) BALB/cJ mice (2) Also increased stimulates humoralneutrophil and cell-mediated using a tunable infiltration MCWE immunity was perXenon light bulb before PDT had no formed subtumorally source to different effect at different time light doses points in relation to PDT

Reference Chen et al22

Korbelik et al23

Winters et al24

Uehara et al25

Korbelik et al26

Korbelik and Cecic27

(continued)


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Table 1. (continued) Therapy Combination

In Vivo Tumor Model

Squamous cell PDT was combined carcinoma (SCCVII) with vitamin D3cells were binding proteinsubcutaneously derived macrophageimplanted to C3H/ activating factor HeN (DBPMAF) at varying time points administered either intraperitoneally, intravenously, or peritumorally PDT was combined Wild-type CT26 colon with low-dose (50 carcinoma cells were mg/kg) cyclophosubcutaneously sphamide (CY) 2 days transplanted into before PDT to BALB/c mice. If surenhance antitumor viving is >90 days, immunity through mice were rechalCY-driven depletion lenged with CT26 of Tregs cells

Photosensitizer


Reference

A Xenon light bulb (1) Optimal treatment: Korbelik et al28 source was used to PDT þ DBPMAF deliver 150 J/cm2 of after 0, 4, 8, and 12 days 630 + 10 nm light at (2) 100% tumor-free 130 mW/cm2 mice compared to 25% with PDT alone (3) Result of the recruitment of activated macrophages to the tumor A diode laser was used (1) Low-dose CY prior Reginato et al29 Liposomal to deliver 690 nm benzoporphyrin to PDT substantially light at 100 mW/cm2 derivative (BPD) decreased tumor mono acid ring A at volumes and to a dose of 0.3 mg/mL was increased survival 120 J/cm2 intraperitoneally (2) Only if CY was injected 15 min administered at the before PDT time of rechallenge, the tumor was rejected Golab et al30 A He–Ne ion laser was (1) G-CSF þ PDT Photofrin was used to deliver administered showed increased 630 nm light at intraperitoneally number of 80 mW/cm2 to a 24 h before PDT neutrophils at 15 mg/kg (2) Significantly dose of 150 J/cm2 increased survival and decreased tumor volumes Photofrin was administered intravenously with 10 mg/kg at 24 h before PDT

Colon carcinoma C26 Granulocyte colonycells and Lewis lung stimulating factor (Gcarcinoma cells were CSF), which causes transplanted into the enhanced neutrophil footpad of BALB/c production was and (C57BL/6xDBA/ intratumorally admi2)F1 mice, nistered prior to PDT twice daily for 5 respectively days Colon carcinoma C26 Photofrin was Immature dendritic administered cells were injected cells (DCs) obtained intraperitoneally 24 into the footpad of from mouse bone h before PDT at 10 BALB/c mice marrow were mg/kg administered intratumorally 1 h after PDT BALB/c mice were Immature dendritic injected with C26 cells (DCs) obtained colon carcinoma from mouse bone cells, and C57BL/6 marrow were mice were injected subcutaneously intratumorally on 4 inoculated with B16 separate days melanoma cells following PDT

Main Results

Jalili et al31 (1) PDT þ immature DCs showed DC maturation and increased IL-12 (2) Significant inhibition of contralateral untreated C26 tumors A diode laser was used (1) PDT þ immature ATX-S10 Na(II) Saji et al32 to deliver 670 nm photosensitizer was DCs resulted in light to a dose of administered 5 h increased survival for 150 J/cm2 before PDT at C26 and B16 tumors 5 mg/kg (2) Adoptive transfer of splenocytes from treated mice inhibited C26 tumor growth in naive mice A He–Ne laser was used to deliver 630 nm light at 80 mW/cm2 to a dose of 90 J/cm2

Abbreviations: IL interleukin; mTHPC, meta-tetra(hydroxyphenyl)chlorine; PDT, photodynamic therapy; TNF, tumor necrosis factor; Tregs, regulatory T cells.

the cell membrane permeability, adding it as an adjuvant to chemotherapy may increase the deliverability of cytotoxic drugs. Some chemotherapy drugs can act both as a cytotoxic agent and as a PS, providing the option to illuminate the tumor tissue after chemotherapy administration in order to elicit a potentially synergistic treatment effect.35 The interest in this combination therapy does not only lie in enhancing the

antitumor effects but also in the ability to reduce the risk of severe side effects by reducing the required chemotherapy dose.36 Tables 2 and 3 summarize the experiments and results of several recent studies combining PDT with either chemotherapy or ionizing radiation. Most of the studies combining ionizing radiation with PDT showed an additive antitumor effect of


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Table 2. Studies Combining Photodynamic Therapy With Ionizing Radiation.

Therapy Combination

In Vitro Tumor Model

In Vivo Tumor Model Photosensitizer

60

Ehrlich ascites carcinoma cells HPde photosensitizer was injected were inoculated intraperitoneally 3 h intraperitoneally to BALB/c before exclusion of mice, tumor cells were then intraperitoneal cells excluded from the at concentrations of intraperitoneum, treated 10-60 mg/kg with PDT and radiation, and then inoculated into healthy BALB/c mice Cells were incubated PDT was combined with Squamous cell for 4 h prior to carcinoma cell lines ionizing radiation at 1.2 treatment with 1 V134, V175, and Gy/min with a 137Cs mmol/L/L 5SCC-61 were culsource delivered either aminolevulinic acid tured and used in 24 h before or 24 h (5-ALA) the exponential after PDT growth phase A

Co source was used to deliver different doses of ionizing radiation in combination with PDT at different time sequence

PDT was combined with Nonsmall cell lung 60 cancer lines A549, Co ionizing radiation SKMES1, NCIH460, at 1.4-1.9 Gy/min to a and NCIH23 were dose of 2 or 7 Gy either used along with 15-30 min before PDT normal human or 15-30 min after PDT fibroblasts

Clinical case study of 4 patients with Bowen disease where fractionated electron beam therapy was applied to 12 Gy, 30 min after PDT Human MCF-7 breast PDT and chemotherapy cancer cells were were combined with used concurrent 100 kV xray treatment at 1.1 Gy/ min to a dose of 4 Gy


Main Results

Reference

A tungsten lamp (1) HPde acts both as a Luksiene was used to et al33 photosensitizer deliver light and a between 370 radiosensitizer and 680 nm at (2) Additive effect on 30 mW/cm2 for tumor growth, independent on 90-180 s treatment order

A tungsten(1) Tumor cells halogen lamp accumulated in the with a 400-750 G2/M phase after nm filter was ionizing radiation used to deliver (2) Additive effect on light at 50 mW/ survival with cm2 for 3 min combination therapy, independent of order An LED light Cells were incubated (1) Less than additive source was used for 24 h before effect on cell to deliver 20 treatment with 2.5 survival, mW/cm2 light mg/mL Photofrin irrespective of treatment order for 100 or 400 s (2) Significantly less depending on than additive cell line cytotoxic effect on normal fibroblasts A topical solution of 5- Laser light at 630 All lesions disappeared nm was aminolevulinic acid following delivered in 4 (5-ALA) was applied combination fractions 2-3 at 20% concentration therapy and no days apart to a 4-6 h before recurrences after total dose of treatment 14-month average 200 J/cm2 follow-up 1 mmol/L mitoxantrone A Lumacare PDT, chemotherapy, LC122-A fiber was used as a photoand 100 kV x-rays optic probe and radiosensitizer, had a potent cytodelivered 10 J/ and cells were toxic effect on cm2 at fluence incubated 90 min MCF-7 cells, before treatment exceeding that of an rate 75 mW/ additive effect cm2

Allman et al37

Sharma et al38

Nakano et al39

Sazgarnia et al35

Abbreviations: 60Co, cobalt-60; 137Cs, cesium-137; HPde, hematoporphyrin dimethyl ether; LED, light emitting diode; MCF-7, Michigan Cancer Foundation 7; PDT, photodynamic therapy; SCC-61, squamous cell carcinoma 61.

the 2 treatments, when either a cobalt-60 (60Co) or cesium-137 (137Cs) source was used. There was, however, a potent increase in cytotoxicity shown in 1 study of human MCF-7 breast cancer cells receiving PDT and treatment with 100 kV x-rays.35 Interestingly, the studies investigating the order in which PDT and ionizing radiation were administered found that the effect of combination therapy was independent on which treatment preceded the other.33,37,38 As for the combination with chemotherapy, many alternatives have been explored and one quite interesting finding was

the highly increased treatment efficacy of drug-resistant murine leukemia and human uterine sarcoma cell lines, when PDT was added.36,46 In these cases, PDT might prove a promising addition or even alternative to chemotherapy. It was also shown that PDT in combination with cisplatin resulted in a considerably increased in vitro and in vivo antitumor effect, which may prove very useful considering the often dose-limiting toxicity of cisplatin.40,41 One study also examined the combination of PDT, chemotherapy, and adoptive immunotherapy with splenic lymphocytes, adding a further dimension to the combination


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A 405-nm laser was used Following gefitinib incubation, to treat cells at 10 mW/ cells were incubated with 1 cm2 to a light dose of mmol/L 5-aminolevulinic acid (5-ALA) for 6 h prior to PDT either 0.6, 1.8, or 3.0 J/ cm2

Laser light at 630 nm was The CNE-2 cells were Cells were incubated with 5used to deliver 1, 3, or aminolevulinic acid (5-ALA) at also transplanted 6.25 J/cm2 to CNE-2 1 mmol/L/L for 6 h before subcutaneously to PDT, and the mice were BALB/c nude mice cells and 100 J/cm2 at administered 5-ALA at 100 after PVP3 100 mW/cm2 to the mg/kg 3 h prior to PDT incubation mice The NOZ cells were Cells were incubated for 24 h A semiconductor laser was used to illuminate cells with 20 mg/mL talaporfin also transplanted with 664 + 2 nm light sodium (TPS) subcutaneously to to 12 J/cm2 and mice to photosensitizer. The mice BALB/cANcrj mice were injected with 5 mg/kg a dose of 60 J/cm2 TPS at 2 h before PDT

Human glioma cell lines Gefitinib, which can inhibit U87MG, U118MG, A172, ABCG2 protein-mediated and T98G were used efflux of porphyrin out from malignant cells, was evaluated at different concentrations in vitro in combination with PDT

The apoptosis-inducing protein Nasopharyngeal carcinoma CNE-2 cells were used apoptin was tested in combination with PDT via PVP3 plasmid administration

Human biliary cancer 5-FU, gemcitabine, oxaliplatin (NOZ) cells were and ciscultured diamminedichloroplatinum chemotherapy in combination with PDT in vitro and gemcitabine and oxaliplatin in combined with PDT in vivo

Cells were incubated with pheophorbide a (Pa) photosensitizer 2 h before PDT

The effect of combined PDT and The multidrug-resistant human uterine sarcoma doxorubicin of varying cell line MES-SA/Dx5 was concentration (4-16 mmol/L) used on a multidrug-resistant cell line was evaluated in vitro

A quartz-halogen lamp was used to illuminate the cells with 610 nm light at 70 mW/cm2 to a dose of 84 J/cm2

A 632-nm laser was used AMC-HN3 cells were In vitro: cells were incubated The potential enhancement in Human head and neck to illuminate cells to 6.0 with 5-aminolevulnic acid (5also subcutasquamous cell carcinoma cytotoxicity when J/cm2 and in vivo ALA) at 25 or 50 mg/mL fro neously implanted (AMC-HN3) cells were administering cisplatin (6.25 24 h in BALB/c/nu/nu used mg/mL in vitro and 2 mg/kg in tumors to 132 J/cm2 In vivo: 375 mg/kg 5-ALA was mice vivo) along with the administered 6 h before PDT photosensitizer prior to PDT was evaluated After incubation with cisplatin, A 635-nm laser was used HeLa cells were used as a HeLa cells subjected to to deliver a light dose of cells were incubated with 5human cervical cancer combination treatment with 5 J/cm2 aminolevulininc acid (5-ALA) model 24 h incubation with lowfor 4 h prior to PDT dose cisplatin followed by PDT, evaluated through cell viability and cell death mechanisms

Therapy Combination

Table 3. Studies Combining Photodynamic Therapy With Chemotherapy.

Wei et al41

(1) Combination treatment with cisplatin doses >1 mg/L synergistically enhanced cytotoxicity (2) Increased apoptosis rate, related to upregulation of p53 and changes in p21, Bcl-2, and Bax expression (1) Synergistic effect on cytotoxicity from combined doxorubicin þ PDT mediated by intracellular ROS generation (2) Synergistic effect seen only in the multidrugresistant line (1) Gefitinib þ PDT resulted in dose-dependent reduction of the surviving glioma fraction (2) Effect due to decreased ABCG2 expression and subsequent increase in intracellular porphyrin levels Apoptin gene therapy þ PDT resulted in significantly stronger antitumor effects in vitro and in vivo compared to monotherapies (1) Significant increase in tumor necrotic area and apoptosis-positive cells (2) Synergistic cytotoxicity increase from oxaliplatin and gemcitabine þ PDT

(continued)

Nonaka et al44

Fang et al43

Sun et al42

Cheung et al36

Ahn et al40

Reference

(1) Combined 5-ALA PDT and cisplatin increased cytotoxicity (2) More efficient against tumor recurrence

Main Results

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mTHPC (Foscan) was Hybrid DBA/2x administered BALB/c mice were postchemotherapy and 24 h intraperitoneally prior to PDT at 0.3 mg/kg injected with L1210 murine leukemia cells

Reference

Abbreviations: ABCG2, adenosine triphosphate-binding cassette subfamily G member 2; mTHPC, meta-tetra(hydroxyphenyl)chlorine; PDT, photodynamic therapy; PVP, polyvinylpyrrolidone; ROS, reactive oxygen species.

The polytherapy combination of Navelbine or cisplatin chemotherapy followed by PDT, and by adoptive immunotherapy with splenic lymphocytes from PDTtreated mice was tested

PDT was combined with 5 mg/ kg Adriamycin to investigate increased antitumor effects through potential enhanced apoptosis and inhibited tumor angiogenesis

Main Results

Tong et al45 (1) Adriamycin þ PDT resulted in significantly reduced tumor volumes (2) Also considerable increase in survival compared to separate Adriamycin or PDT (1) Chemotherapy-resistant Diez et al46 LBR-D160 and LBRV160 cell lines were sensitive to 5-ALA PDT (2) No increase in treatment efficacy from combination therapy A continuous wave dye (1) Chemotherapy, PDT and Canti et al47 laser was used to adoptive immunotherapy deliver 670 nm light at successfully treated this 100 mW/cm2 to a dose aggressive metastatic tumor of 100 J/cm2 (2) PDT or chemotherapy alone showed no survival advantage over control


Laser light at 690 nm was The photosensitizer 4T1 mammary used to deliver 120 J/ benzoporphyrin derivate carcinoma cells cm2 to the tumors monoacid ring (BPD-MA) was were intravenously injected 24 h subcutaneously before PDT at 1 mg/kg transplanted to the right flank of BALB/ c mice Fluorescent lamps at 400Cells were incubated for 4 h Vincristine-resistant LBRThe effect of combining 700 nm were used to prior to PDT with 1 mmol/L V160 cells, doxorubicindoxorubicin or vincristine illuminate cells for 0, 5, 5-aminolevulinic acid (5-ALA) resistant LBR-D160 cells, with PDT in the treatment of 10, or 20 min and sensitive LBR-murine sensitive or resistant murine leukemic cells were leukemia cells was tested tested

Therapy Combination

Table 3. (continued)

Brodin et al

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approach.47 This triple combination proved effective in treating an aggressive murine leukemia model in vivo, where either PDT alone or chemotherapy alone was ineffective. Although very promising, great care should be taken when attempting to translate this into a clinical alternative as the addition of each new treatment regimen potentially increases the risk of severe, and possibly systemic, treatment-related toxicity.

Photodynamic Therapy in Combination With Experimental Anticancer Therapy There are several anticancer treatment strategies that are currently not well established in the clinic, used on an experimental basis, or are still being tested in the laboratory. Two such strategies are hyperthermia and focused ultrasound (sometimes referred to as sonodynamic therapy). In relation to PDT, hyperthermia can cause damage to the tumor vasculature that would affect angiogenesis and, just like PDT, damage the cell membranes, which could allow for a synergistic effect between the 2 treatments.48 As the use of focused ultrasound in the management of cancer is fairly new, the combination between PDT and ultrasound has only started to be explored in the last few years. The main hypothesis suggesting that this combination may yield a considerable antitumor effect is based on the fact that there are PS agents that can be triggered by both light energy and ultrasound energy, which should potentiate the response.49 Tables 4 and 5 summarize the results of studies performed to evaluate the potential antitumor efficacy of PDT in combination with either hyperthermia or focused ultrasound. Photodynamic therapy in combination with hyperthermia has been shown to yield a strong direct cytotoxic response but also to prompt a considerable antitumor immune response.13,50 Some studies have shown that the timing between therapies is of critical importance and that hyperthermia applied after PDT is more effective than the reversed order.13,48 There also appears to be a considerable dose–response effect dependent both on the light dose and on the length and temperature of hyperthermia,53,54 suggesting that there is a need for systematically evaluating this dose–response relationship, in vitro and in vivo, to obtain the optimal treatment parameters for different cancer types. Although very few studies have been conducted so far, the experience of PDT combined with focused ultrasound suggests that this combination can provide a strong and synergistic antitumor response. Interestingly, some studies found that the response was most pronounced when ultrasound preceded PDT, and it has been suggested that this is caused by increased ROS generation due to enhanced PS uptake as a result of ultrasound-induced increase in cell membrane permeability through sonoporation.49,55 This also suggests that a combination of focused ultrasound, PDT, and chemotherapy may be promising as both PS and chemotherapy drug uptake can be enhanced.

Combination Therapy and Tumor Hypoxia Tumor hypoxia presents a major challenge in cancer therapy, and hypoxic tumors are often much harder to treat than welloxygenated tumors, something that is a common problem in, for example, radiation therapy of head and neck tumors. In PDT, hypoxia is clearly a substantial problem as the lack of oxygen in the tumor cells leads to significantly less ROS generation. To achieve an efficient tumor response under hypoxic conditions, combination therapy would have to be optimized to promote some form of tumor reoxygenation. It is often the center of the tumor that is the most hypoxic, and in this case, one option could be to apply fractionated radiation therapy to effectively treat the oxygenated outer parts of the tumor. This would eventually lead to some reoxygenation of previously hypoxic tumor cells that are still viable, and at this point radiation therapy could be combined with PDT to effectively treat the previously hypoxic parts of the tumor. Another option would be to combine PDT with hyperthermia, which has been shown to target tumor vasculature, which can subsequently initiate tumor angiogenesis, leading to reoxygenation of tumor tissue.12,13 Such an approach should, however, be explored with caution since increased tumor angiogenesis can lead to an increase in tumor growth. All in all, the effects of a hypoxic tumor environment will impact the effectiveness of combination therapy involving PDT and should be considered when evaluating different treatment options.

Summary and Conclusions A considerable amount of preclinical evidence has been gathered regarding the combination of PDT and other forms of anticancer treatment, and this seems to be increasing rapidly. In this review, we have shown that the potential of using PDT as part of combination therapy is being investigated from many different directions, with promising results in terms of direct cytotoxicity as well as prompting a strong antitumor immune response. A promising option that has been explored is the addition of low-dose cyclophosphamide (CY) prior to PDT in order to deplete the number of Tregs.29 This combination has led to a strong antitumor response, and more importantly tumor rechallenges were only resisted in mice receiving a second CY administration at the time of rechallenge. This shows the importance of Treg depletion and the potential of combining this not only with PDT but possibly with other anticancer treatments as well. Another promising option is anticancer vaccination using immature DCs that have been treated with PDT.31,32 This DC priming elicited substantial immune responses, and there was even a clear growth inhibition of C26 tumors in naive mice that received splenocytes from mice treated with PDT and immature DC injections. Adding a further dimension to this approach, Canti et al tested the combination of PDT and chemotherapy, plus adoptive immunotherapy with splenic lymphocytes from PDTtreated mice.47 This resulted in successful treatment of an


10



Main Results

Abbreviations: AC, alternating current; HSP70, heat-shock protein 70; IFN-g, interferon g.

A temperature-controlled incubator was used to deliver hyperthermia at different temperatures concurrently with PDT

Hyperthermia was applied simultaneously with PDT using a hot plate heating cells to 43 C + 0.5 C during treatment to evaluate potential increase in treatment efficacy

Magnetoliposomes loaded with photosensitizer-based complex were used to evaluate the combined treatment of PDT and magnetohyperthermia using an AC magnetic field at 1 MHz

Infrared light hyperthermia (940-1600 nm) at 310 mW/cm2 to a dose of 437.5 J/cm2 was combined with PDT to enhance treatment efficacy deep within the tumor

Tumor vaccines were generated through a combination of PDT and hyperthermia, which was applied at 41 C for 1 h during cell incubation

RIF-1 tumor cells were Hypericin was administered subcutaneously intravenously 6 h before PDT illumination at 5 mg/kg implanted into the hind leg of C3H/Km mice

Laser light at 595 nm was used (i) Combination therapy lead to to deliver a dose of 120 J/ increased damage to tumor cm2 at a fluence rate of 100 vasculature mW/cm2 (ii) Most pronounced effect of interaction was the increased direct cytotoxicity Colon carcinoma C26 cells were treated with PDT and Cells were incubated in medium Laser light at 630 nm was used (1) PDT þ hyperthermia hyperthermia and then subjected to freeze/thawing to containing 10 or 30 mg/mL to treat the cells for 20 min resulted in significantly create the tumor vaccine. Balb/c mice were to a dose of 5 J/cm2 hematoporphyrin monomethyl smaller tumor volumes subcutaneously injected with C26 cells to form tumor (HMME) for 12 h prior to PDT (2) Also higher expression of models CD8þ T cells, IFN-g and HSP70, indicating immune response BALB/cAjcl mice were 5-Aminolevulinic acid (5-ALA) was A linear polarized near(1) PDT þ hyperthermia led to subcutaneously administered subcutaneously 3 h infrared irradiator was significant tumor growth implanted with before PDT at 250 mg/kg used to deliver 50 J/cm2 of inhibition human squamous 580-740 nm light at 35 (2) PDT or hyperthermia alone cell carcinoma cells mW/cm2 had no effect on these tumors with progression in the deep layer Pigmented melanoma B16-F10 Liposomes were loaded with a zinc Laser light was used to deliver (1) Applied separately PDT was cells were incubated with the phthalocyanine (ZnPc)-based 670 nm light at 84 mW/ more effective than magnetoliposomes complex, and cells were incubated cm2 to doses between 0.5 magnetohyperthermia and 2 J/cm2 at a concentration of 0.25 mg/mL (2) Combined treatment for 3 h before PDT reduced the B16-F10 cell viability to about half that of PDT alone Human osteosarcoma cell lines Cells were incubated for 6 h before A near infrared halogen lamp (1) PDT alone was effective HOSM-1 and HOSM-2 were PDT with medium containing 0.2 was used to deliver 580against HOSM-2 cells used mmol/L 5-aminolevulinic acid 740 nm light at 42 mW/ (2) Reduction in cell survival of 2 hexylester (5-hALA) cm to doses ranging from HOSM-1 cells was only seen 10 to 80 J/cm2 with combination therapy, from 40 J/cm2, 16 min heat and upward Cells were incubated in either 100 or An optical fiber was used to (1) Substantial cytotoxicity at Human grade IV glioblastoma 500 mg/mL 5-aminolevulinic acid deliver 635 nm light at 25 spheroids (ACBT) and rat 49 C and light doses 2 (5-ALA) for 4 h before PDT mW/cm to doses ranging >25 J/cm2 BT4C rat glioma cells (2) Between 40 C and 46 C from 12 to 100 J/cm2 there was a highly synergistic effect of PDT þ hyperthermia on surviving fraction

Photosensitizer

Hyperthermia consisting of a 43 C water bath was applied immediately after PDT for 1 h

In Vivo Tumor Model


Therapy Combination

Table 4. Studies Combining Photodynamic Therapy With Hyperthermia.

Hirschberg et al54

Yanase et al53

Bolfarini et al52

Yanase et al51

He et al50

Chen et al13

Reference

11



Rat C6 glioma cells were used

White randombred rats were subcutaneously implanted with rat C6 glioma cells

In Vivo Tumor Model

Abbreviations: PDT, photodynamic therapy; ROS, reactive oxygen species.

Combination therapy consisted of ultrasound delivered prior to PDT at 1 MHz and either 0.4, 0.7, or 1.0 W/cm2 for 10 min

Combination therapy was evaluated with ultrasound at 1 MHz and 0.5 W/cm2 for 90 s delivered prior to PDT

Continuous-wave ultrasound at 1 Human breast cancer MDA-MB-231 cells MHz and 0.36 W/cm2 for 1 min were used was used in combination with PDT at different time points

Continuous-wave ultrasound at 1 Murine 4T1 mammary carcinoma cells were MHz and 0.36 W/cm2 for 1 min used in combination with PDT to increase cell membrane permeability and enhance intracellular photosensitizer uptake

Therapy Combination

Table 5. Studies Combining Photodynamic Therapy With Focused Ultrasound. Light Source and Dose

Main Results

A 650-nm laser was used to illu- (1) Significant enhancement in Cells were incubated with minate cells to 1.2 J/cm2 at chlorin e6 photosensitizer antitumor efficacy with at 1 mg/mL for 4 h prior to combined PDT and 10.4 mW/cm2 PDT ultrasound (2) Suggested mechanism: increased ROS generation via loss of cell membrane potential and caspasedependent apoptosis Laser light at 650 nm was used to (1) Cell survival significantly Cells were incubated with illuminate tumor cells to a chlorin e6 photosensitizer reduced with dose of 1.2 J/cm2 at 1.3 mW/ at 1 mg/mL for 4 h prior to combination therapy PDT (2) Slightly more effective cm2 when ultrasound preceded PDT, likely due to enhanced cell membrane permeability Tumor cells were incubated Laser light at 630 nm was used to (1) Ultrasound þ PDT illuminate cells to doses of 20, for 4 h prior to PDT with effectively killed C6 40, 80, 120, 160, 200, and 240 10 mg/mL glioma cells synergistically J/cm2 at a fluence rate of 100 (2) 80 J/cm2 was resulted in hematoporphyrin (HMME) mW/cm2 the highest apoptotic rate Chlorin e6 conjugated with Laser light at 661 nm was used to Tumor necrosis area deliver a dose of either 50 or polyvinyl pyrolidone significantly increased in rat 100 J/cm2 at 170 mW/cm2 (Photolon) was injected glioma models with intravenously 2.5 h prior ultrasound þ Photolonto PDT at 2.5 mg/kg PDT

Photosensitizer

Tserkovsky et al57

Li et al56

Wang et al55

Li et al49

Reference

12


aggressive metastatic murine leukemia model, a quite promising result, especially considering the many potential extensions or alterations in this therapy combination, including also other treatment modalities. Yet a further interesting result was obtained when combining PDT with 60Co ionizing radiation for various nonsmall cell lung cancer (NSCLC) lines.38 Using this combination of therapeutic agents, a less synergistic effect on normal lung fibroblasts compared to the NSCLC cells was found, suggesting a potential increase in the therapeutic window using this approach. Yanase et al used hyperthermia in combination with PDT in order to more effectively treat deep-seated tumors.51 Their results showed promising antitumor effects from combination therapy, where neither PDT nor hyperthermia alone was an effective treatment. The inefficiency of treating deep-seated tumors is an inherent difficulty in PDT as most of the PS absorbance wavelengths do not penetrate far into tissue. Thus combination with hyperthermia seems to be one promising alternative, although combinations with ionizing radiation or chemotherapy could also provide a way to circumvent this problem. The use of focused ultrasound to increase cell membrane permeability seems to be an effective method to increase intratumoral ROS generation and substantially enhance the antitumor efficacy of PDT.49,55 This approach holds great potential as neither PDT nor ultrasound is inherently dose-limiting treatments, so applying this combination also as an adjuvant to chemo- or radiation therapy may be a very interesting option.

Future Perspectives With the availability of many different photosensitizing drugs and the large number of possible treatment combinations, there is a need for systematically evaluating the optimal dose– response combinations and making sure the risks of increasing treatment-related toxicities are low. This need for systematic evaluation is further highlighted by the heterogeneity in PDT parameters among the studies included in this review. Taking the in vivo experiments as an example, the drug concentrations ranged from 0.1 to 250 mg/kg and light doses from 50 to 360 J/cm2, with large variation even between studies using the same PS. The ideal PDT setting in a combination therapy approach will therefore have to be assessed by systematically investigating different photosensitizing agents, concentrations, and light doses to find the optimal parameters for a certain indication. This will require a great deal of work, and the results will likely not be directly transferable between different tumor types. The ideal combination therapy involving PDT will be one that can reach deep-seated tumors, show synergistic cytotoxic efficacy, promote a strong antitumor immune response, enhance tumor-specific drug uptake, and reoxygenate hypoxic tumor tissue. Based on this review, it is not straightforward to conclude which of the combination approaches comes closest to the ideal situation, but it will likely require the combination of several different treatment modalities.

Despite the fact that many of the suggested strategies may never reach clinical applicability, and the ideal combination therapy has yet to be found, PDT as part of combination anticancer treatment shows great potential and promise based on the current preclinical evidence. Authors’ Note Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work has been supported in part by R01 EB009040 from the United States National Institutes of Health (NIH).

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