Small Molecule Therapeutics

Reduction of Muscle-Invasive Tumors byPhotodynamic Therapy withTetrahydroporphyrin-Tetratosylat in anOrthotopic Rat Bladder Cancer ModelMandy Berndt-Paetz1, Philipp Schulze1, Philipp C. Stenglein1, Annett Weimann1,Qiang Wang1, Lars-Christian Horn2, Yasser M. Riyad3, Jan Griebel3, Ralf Hermann4,Annegret Glasow5, Jens-Uwe Stolzenburg6, and Jochen Neuhaus1

Abstract

Photodynamic therapy (PDT) is a promising option forminimal-invasive treatment of bladder cancer. Efficacy ofPDT in muscle-invasive urothelial cancer is still hamperedby low tissue penetration of most photosensitizers due toshort excitation wavelength. The novel light reactive agenttetrahydroporphyrin-tetratosylat (THPTS) is excitable atnear-infrared (760 nm), allowing tissue penetration of up to15 mm. Here, we established an orthotopic rat bladder cancermodel and examined the effects of THPTS-PDT on tumorgrowth in vivo, and analyzed molecular mechanisms in vitro.We examined pharmacokinetics and subcellular localization,and evoked cell death mode in cultured rat urothelialcarcinoma cells (AY-27). We used female F344 Fischerrats for in vivo studies. Ten rats each were used for THPTS-PDTand light-only control. Bladders were evaluated by macro-

scopy and histology. Temperature-dependent THPTS uptakeresulted in endosomal/lysosomal localization. PDT (0–50 mmol/L THPTS; 10 J/cm2) induced early onset of apoptosisleading to dose-dependent cytotoxicity in AY-27 cells. Single-time transurethral THPTS-PDT (100 mmol/L THPTS; 10 J/cm2)in F344 rats led to significant reduction of muscle-invasivetumor number (2/10 vs. 7/10 in controls) and total tumorvolume (60% reduction) 2 weeks after PDT, while sparinghealthy tissue. Here, we report for the first time effective tumorgrowth control by PDT in vivo. THPTS is a promising newphotosensitizer with the advantage of higher therapeuticdepth and the potential of high-selective therapy in muscle-invasive urothelial cancer. This approach possibly allowsminimal-invasive bladder preserving treatment of bladdercancer without systemic side effects.

IntroductionBladder cancer is the ninth most common cancer world-

wide (1), with a mortality rate of 2.3 (GLOBOCAN 2012;http://www.globocan.iarc.fr/). Approximately 70% of cases areinitially diagnosed as non–muscle-invasive urothelial carcinomas(stage Ta, T1, CIS) and 30% as muscle-invasive urothelial carci-nomas. Non–muscle-invasive bladder cancer is usually treated bytransurethral resection of the tumor followed by instillation ofBacillus Calmette–Gu�erin or chemotherapy, respectively. In high-grade T1 tumors or after failure of first-line therapy, radical

cystectomy should prevent further progression (2). For muscle-invasive urothelial cancer (� pT2), radical cystectomy includinglymph node dissection with or without neoadjuvant chemother-apy is recommended (3). To date, there are no options of organpreserving therapies in advanced bladder cancer.

Photodynamic therapy (PDT) is a promising option for min-imal-invasive bladder preserving treatment of urothelial cancer.The principle of PDT is based on photoactivation of a nontoxiclight reactive agent, so-called photosensitizer (PS), by a specificwavelength (4). In the presence of tissue oxygen, photoactivationleads to intracellular oxidative stress by means of reactive oxygenspecies and singlet oxygen (1O2), which can destroy tumors bythree different effects: (i) tumor cell death (apoptosis/necrosis),(ii) coagulation of microvessels resulting in vascular shutdown(nutritional cutoff), and (iii) stimulation of the host immunesystem (5, 6). An ideal PS for tumor therapy has no dark toxicityand selectively accumulates in tumor cells (7). The photodynamicprinciple is implemented in diagnostics and therapies of severaltumor entities. Several systemically applied PSs (e.g., photofrin,haematoporphyrin derivates) have been used for PDT of super-ficial urothelial cancer and carcinoma in situ (CIS) (7–9). Systemicadministration of these drugs resulted in undesirable side effectssuch as severe skin photosensitization. Studies with locallyapplied 5-aminolevulinic acid (ALA) had complete response ratesup to 52%–60% in CIS patients without skin photosensitization,but caused bladder wall fibrosis due to high irradiation intensitiesused (10, 11). To date, no standard PDT for superficial urothelial

1Department of Urology, Research Laboratories, University of Leipzig, Leipzig,Germany. 2Institute for Pathology, University of Leipzig, Leipzig, Germany.3Leibniz Institute of Surface Modification (IOM), Leipzig, Germany. 4WilhelmOstwald Institute for Physical and Theoretical Chemistry, Leipzig, Germany.5Department of Radiation Therapy, University of Leipzig, Leipzig, Germany.6Department of Urology, University Hospital Leipzig, Leipzig, Germany.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

M. Berndt-Paetz and P. Schulze contributed equally to this article.

CorrespondingAuthor:MandyBerndt-Paetz, University of Leipzig, Liebigstraße19, Building C, 04103 Leipzig, Germany. Phone: 49-341-9717654; Fax: 49-341-9717659; E-mail: mandy.berndt@medizin.uni-leipzig.de

doi: 10.1158/1535-7163.MCT-18-1194

�2019 American Association for Cancer Research.

MolecularCancerTherapeutics

www.aacrjournals.org 743

carcinomas could be developed, and PDT of muscle-invasivebladder cancer is hampered by the insufficient tissue penetrationdue to short excitation wavelength of the commonly usedPSs (12).

Tetrahydroporphyrin-tetratosylat (THPTS) is a novel water-soluble and positively charged PS. THPTS has an absorptionmaximum at 760.5 nm, which lies within the so-called photo-therapeutic window, thus allowing up to 15-mm tissue pene-tration (13, 14). THPTS strongly accumulates in tumor cellswith a tumor/normal tissue ratio from 1.4 to 25 (14). Phar-macokinetic examinations showed rapid clearance fromhealthy tissue (half-life 30 hours), which avoid generalizedskin photosensitivity. Reduced energy required for photoacti-vation minimizes tissue fibrosis following heat damage (14).Cytotoxic effects of THPTS-PDT have been described in differ-ent tumors (15–17), including human urothelial carcinomacells in vitro. THPTS-PDT on human bladder carcinoma cellsshowed significant tumor cell toxicity while sparing normalhuman detrusor myocytes. (18). To confirm our recent resultsof THPTS-PDT efficacy in human urothelial cancer cells, wehere evaluate the effects of THPTS-PDT in an orthotopic ratbladder cancer model (19). We applied THPTS to rat urothelialcancer cells (AY-27) in vitro to analyze pharmacokinetics, sub-cellular distribution, and cell death mechanisms; and we used aFischer F344 rat model to evaluate the effects of THPTS-PDT ontumor growth in vivo.

Materials and MethodsTHPTS

The near-infrared (760 nm) PS THPTS (C72H70N8O12S4;molecular weight: 1,367.66 Da; purity � 95%) was obtainedfrom TetraPDT GmbH. THPTS is a cationic bacteriochlorin der-ivate (Fig. 1), which shows no change in absorption spectrawithin

1 month when kept in aqueous solutions in the dark at 4�C (14).The physicochemical properties of THPTS have been extensivelydescribed in previous studies (14, 20).

Cell cultureRat AY-27 urothelial carcinoma cells are not commercially

available. Cells were kindly provided by Prof. F. Guillemin andDr. M.A. D'Hallewin (Centre de Lutte Contre le Cancer de Lor-raine, Nancy, France). The cell line was initially derived from anN-(4-[5-nitro-2-furyl]-2-thiazolyl) formamide (FANFT, 0.2%)-induced carcinoma of the urinary bladder in male F344 Fischerrats (Charles River; ref. 21). Cells were cultured in RPMI-1640w/ophenol red supplemented with 20% fetal calf serum (FCS), 5%penicillin/streptomycin, and200mmol/L glutamine (Biochrom).AY-27 were characterized as poorly differentiated cells of urothe-lial origin by cytokeratin staining (cytokeratin-pan, cytokeratin-7:positive; cytokeratin-13, cytokeratin-20: negative; SupplementaryFig. S1). Mycoplasma testing was performed monthly using PCRMycoplasma Test Kit (AppliChem GmbH).

Flow cytometry for pharmacokineticsUptake analyses were performed on a BD LSR II flow cytometer

(Becton-Dickinson). Prior tomeasurement AY-27 cells were incu-bated with THPTS (200 mmol/L, 2 hours) at 37�C or 4�C. THPTSfluorescencewasmonitored during 2hours using anoctagon laser(excitation: 488 nm–20mW) and a PE-Cy7-A filter set (detection:BP750/60 nm). Dead cells were excluded from analysis by pro-pidium iodide staining. Very small cells (probably representingcell detritus) and duplets were excluded in a two-step gatingstrategy using FSC-H/FSC-A scatters.

Subcellular localizationCells were cultured on collagen-A coated 35-cm dishes

with glass bottom (ibidi). For subcellular staining, AY-27 cells

Figure 1.

Chemical structure. Structure formula of THPTS.

Berndt-Paetz et al.

Mol Cancer Ther; 18(4) April 2019 Molecular Cancer Therapeutics744

were treated with CellLight reagents BacMam 2.0 (Early/LateEndosomes-GFP), LysoTracker-Green or MitoTracker-Green(Thermo Fisher Scientific). After THPTS incubation (200 mmol/L,37�C, 2 hours), cells were washed and covered with calcium-free Krebs-Ringer solution and analyzed by confocal laserscanning microscopy (LSM 5 Pascal, Carl Zeiss).

Photodynamic therapy on cell culturesPrior to THPTS-PDT, cells were grown in 96-well plates

(Sarstedt). At 70% to 80% confluency, samples were treated2 hours with THPTS (0, 12.5, 25, 50, 100, and 200 mmol/L)and irradiated at 760 nm using a 760 � 5 nm diode laser(LAMI-Helios; TetraPDT GmbH). Gentle light dose of 10 J/cm2

was chosen based on its effective and dose-dependent cytotoxicityin initial irradiation experiments using various light doses (10, 30,and 60 J/cm2; Supplementary Fig. S2). Laser intensity was mea-sured and adjusted using a laser power meter (Laserstar). Afterirradiation, cells were incubated for indicated time periods andassayed for cytotoxic effects.

Time-lapse analysisCells were grown in 24-well plates (Greiner Bio-One). After

THPTS-PDT (12.5 mmol/L), 24-hour time-lapse recordings ofAY-27 cells were analyzed for membrane blebbing and nuclearcondensation using an EVOS XL digital inverted microscope(AMG).

Cell-based assaysCells were seeded into 96-well-plates. After irradiation, plates

were incubated for 2 and24hours. Cell survivalwas analyzedwiththe CellTiter-Blue Cell Viability Assay (ex/em: 560/590). Theapoptotic activity was measured with the Apo-ONE Homoge-neous Caspase-3/7 Assay (ex/em: 485/527) and the loss of mem-brane integrity was determined via measurement of lactate dehy-drogenase (LDH) release using the CytoTox-ONE HomogeneousMembrane Integrity Assay (ex/em: 560/590). Assays wereobtained from Promega and measured on a microplate reader(SpectraMax M5, Molecular Devices).

In vivo tumor inductionAll animal experiments were approved by the Landesdirektion

Sachsen (TVV 47/11) and complied with requirements of theARRIVE guidelines. Pilot study (n ¼ 11) was performed forestablishing the bladder cancermodel prior to treatment. Animalswere sacrificed after indicated time periods and bladders wereexcised (Table 1).

We followed the method of Xiao and colleagues (19, 22).Female F344 Fischer rats (150–180 g; Charles River Germany)were anesthetized i.p. with a mixture of 90 mg/kg Ketamine(Ketamin 10%,WDT) and 10mg/kg Xylazin (Rompun 2%, BayerHealthCare). Transurethral catheterizationwas donewith an 18Gcatheter (BD InsyteAutogard18GA). Bladder conditioning was

done by washing with 0.5 mL of 0.1 N HCl for 15 seconds and0.5 mL of 0.1 N NaOH for 15 seconds. Tumor inoculation wasdone by instillation of 2 � 106 AY-27 cells (in RPMI-1640, w/ophenol red, 20% FCS, 5% penicillin/streptomycin, 200 mmol/Lglutamine) for 60 minutes. The animals were kept for 10 daysunder standard conditions. Tramal (40 mg/kg; in drinking water;Gr€unenthal) was administered for pain management. Animalstemperature and body weight were checked daily. Ten days afterinstillation, animalswere sacrificed for pilot study or used for PDTtreatment.

Transurethral photodynamic therapyAnimals were anesthetized and catheterized as described

above. Bladders were instilled with THPTS (100 mmol/L in PBS)for 2 hours. Controls were instilled with PBS. Following rinsing,bladders were filled with 0.5 mL PBS and irradiated transureth-rally via a glass fiber at 760 nm� 5 nmwith light dose of 10 J/cm2

(LAMI-Helios, TetraPDT GmbH). After treatment, animals werekept for 14 days as described above. When applicable, tumorgrowth and treatment procedure was monitored by sonography(Venue 40, GE Healthcare).

Bladder processing and evaluationThe bladders were excised and opened ventrally by a median

incision. Bladders were fixed (buffered formaldehyde 4% solu-tion, pH 7.2; VWR) for 24 hours. After macroscopy, paraffin-embedded bladders were serially sectioned (7 mm; Supplemen-tary Fig. S3). Every 20th sectionwas stained by hematoxylin/eosin(H&E) and analyzed microscopically for tumor staging. There-fore, current TNM classification of malignant tumors (TNM;ref. 23) was slightly modified to our needs (SupplementaryTable S1). H&E-stained slides were scanned (EPSON PerfectionV750 Pro, Epson) to determine tumor size. Tumor volume wascalculated from tumor area of manually selected regions on thehistologic sections using ImageJ and the spacing of the serialsections.

Data analysis and statisticsComplete data analysis was performed using Prism 6.0

(GraphPad Software Inc.) statistical software. In vitro resultspresent data from at least three independent experiments. Differ-ences were analyzed by c2 test, Wilcoxon signed-rank test andMann–Whitney test. P � 0.05 was considered statisticallysignificant.

ResultsPharmacokinetics, subcellular localization, and cellular effects

Cellular uptake of THPTS into rat AY-27 urothelial cancercells was analyzed by flow cytometry after incubation with200 mmol/L THPTS at 37�C. THPTS uptake was highlytemperature dependent. At 37�C uptake was rapid, startedwithin 1 minute and increased linearly during 2 hours ofincubation. THPTS uptake was significantly decreased toalmost nonexistent at 4�C (Fig. 2A and B). Localization ofTHPTS in endosomes and lysosomes, but not mitochondria,was observed using confocal laser microscopy, revealing colo-calization of labels after treatment with cell compartmentstains (Fig. 2C–K).

Cytotoxicity and cell death mechanisms were analyzedafter THPTS treatment and irradiation with a dose of 10 J/cm2.

Table 1. Trial schedule. Inoculation with 2 � 106 AY-27 cells diluted in culturemedium w/o phenol red; local application of 100 mmol/L THPTS (in PBS) andirradiation (10 J/cm2)

Group n Day 0 Day 10 Day 24

Pilot study 11 Inoculation a

PDT 10 Inoculation Local THPTS application and irradiation a

Control 10 Inoculation Local PBS application and irradiation a

aSacrificed animals by carbon dioxide insufflation.

Effective Bladder Tumor Growth Control by PDT with THPTS

www.aacrjournals.org Mol Cancer Ther; 18(4) April 2019 745

Figure 2.

Pharmacokinetics and subcellular localization. A and B, Cellular uptake of THPTS (200 mmol/L) into AY-27 cells was monitored by flow cytometry during 2-hourincubation at 37�C or 4�C; THPTS enhancement is indicated as mean fluorescence intensity (MFI); n¼ 3. C–E, THPTS did not localize to mitochondria: C,MitoTracker-Green; D, THPTS; E,merged. F–H, Localization of THPTS in endosomes: F, CellLight Endosome-GFP; G, THPTS; H,merged. I–K, Localization ofTHPTS in lysosomes: I, LysoTracker-Green; J, THPTS; K,merged. Arrows indicate regions of interest (overlay/no overlay). Scale bar, 10 mm.

Berndt-Paetz et al.

Mol Cancer Ther; 18(4) April 2019 Molecular Cancer Therapeutics746

Time-lapse analyses (Fig. 3) demonstrated early onset of apopto-sis by induction of membrane blebbing within 5 minutes afterTHPTS-PDTof AY-27 cells. Amaximumof blebbingwas seen after10 minutes (Fig. 3B and C). Cell detachment was observedimmediately after PDT (Fig. 3D and E). Significant nuclear con-densation was seen as early as 4 hours after irradiation andincreased steadily during 24 hours of observation (Fig. 3G andH). Cytotoxic effects were additionally determined by cell-basedassays 2 and 24 hours after PDT using various THPTS concentra-tions. THPTS-PDT led to a significant dose-dependent decrease ofAY-27 cell survival, resulting in 82% loss of viability 2 hours afterPDT (25 mmol/L THPTS) and 97% after 24 hours (25 mmol/LTHPTS). Caspase-3/7 activity was significantly increased after 2hourswith amaximumat 12.5mmol/L. Release of LDH indicatingloss of membrane integrity significantly increased 24 hours afterPDT with a maximum at 25 mmol/L THPTS. At that point,apoptotic caspase-3/7 activity was not significantly changed (Sup-plementary Fig. S4).

In vivo tumor growth controlPilot study (n ¼ 11) was performed for establishing the ortho-

topic bladder cancer model in Fischer rats prior to treatment.Animals were sacrificed after indicated time periods and bladderswere excised (Table 1).

Bladder tumor induction was done according to Xiao andcolleagues (19). In the pilot study, all 11 rats developed anurothelial cell carcinoma (Fig. 4). Intravesical global AY-27administration resulted in solid tumors 10 days after inocu-lation (Fig. 4A and C). H&E staining revealed pT1 tumorsinvading the subepithelial connective tissue (Fig. 4B and D)with total tumor volume ranging from 0.02 to 0.48 mm3

(mean 0.10 mm3). Relative tumor volume normalized tobladder wall volume ranged from 0.40% to 6.51% (mean,1.77%; Fig. 4F). Minimal variations of animal body weightcomplied with legal requirements for establishment of ananimal model (Fig. 4E). Animal numbers of treatment studieswere determined by power analysis (G�-Power V.3.1; Univer-sity of D€usseldorf, Germany). Because adequate group size of10 rats was confirmed (power ¼ 0.970), animal numbers wereminimized in respect to the 3R principle (24). Treatment wasdone 10 days after tumor induction by global THPTS admin-istration (PBS in controls) and transurethral light application.Bladders were extracted 14 days after treatment and evaluatedby macroscopy and histology. There were no irradiation-related deaths in the groups.On dissection, bladderswere coveredby intraperitoneal fat and no perforations were observed. Inaddition, there was no macroscopic evidence of abscess in thekidneys to suggest serious local infections. Histology of serial

Figure 3.

Cytotoxic effects of in vitro THPTS-PDT by time-lapse analysis of cell morphology. AY-27 cells were treated with 12.5 mmol/L THPTS and 10 J/cm2 irradiation.A–C, Early onset of apoptosis by induction of membrane blebbing.D and E, Cell detachment immediately after irradiation. F–H, Nuclear condensation increasedsteadily during 24 hours. Meanþ SD; � , P < 0.05, Mann–Whitney test; n¼ 5.

Effective Bladder Tumor Growth Control by PDT with THPTS

www.aacrjournals.org Mol Cancer Ther; 18(4) April 2019 747

sections allowed quantification of bladder tumor volumes(Supplementary Table S2). Treatment with THPTS-PDT led tosignificantly reduced numbers of � pT2 tumors (P ¼ 0.024; c2

test). In the treatment group, 2 of 10 animals had muscle-invasive (� pT2a) tumors and 8 of 10 animals exhibited non–muscle-invasive bladder cancer (pT1). In contrast, in thecontrol group, 7 of 10 animals showed � pT2a tumors and3 of 10 non–muscle-invasive tumors. Quantification revealedsignificant reduction of tumor volume in the THPTS-PDTtreatment group (Fig. 5A). Relative tumor volume was reducedfrom 17.42% � 4.43% to 6.99% � 4.07% (mean � SD;P ¼ 0.023; Mann–Whitney test). Relative tumor volume of� pT2 classified tumors was reduced from 13.79% � 4.68% to4.51% � 4.13% (mean � SD; P ¼ 0.034; Mann–Whitney test).Interestingly, pT1 tumors were not significantly reduced involume.

Photodynamic effects on tissue architecture are shownin Fig. 5B andC. Therewas fragmentary damage of the urotheliumin the tumor area with regions of complete urothelial destructionand superficial ulceration. Tumor-surrounding areas showedacute inflammation by infiltration with mononucleated roundcells. Extensive full-thickness damage of the bladder wall was notseen. There were no signs indicating phototoxic side effects (e.g.,fibrosis, inflammation) in theflanking normal urothelium,whichconsisted of 3 to 6 tightly opposed cell layers. The lamina propriacontained loose connective tissue with isolated cellular compo-nents and a few vascular structures extending to the underlyingmuscle layers (Fig. 5C).

DiscussionPDT could be a promising alternative in bladder preserving

treatment of bladder cancer. Due to selective tumoricidalphototoxicity with minor side effects in clinical experience,research concentrated on the prodrug ALA/hexaminolevulinate(HAL) as possible photosensitizers (25, 26). The major dis-advantage of these agents is low therapeutic invasion depth.The present study aimed to evaluate the near-infrared excitableTHPTS for treatment of invasive bladder carcinoma in anorthotopic rat model.

Investigations in cultured rat AY-27 cells gave insights intocellular mechanisms of THPTS-PDT. Cellular THPTS accumu-lation is comparable to results on human TCC in our previousstudy (18). As in human urothelial carcinoma cells, THPTSpharmacokinetics showed strong dependency on temperaturewith linear uptake at 37�C and almost no uptake at 4�C,speaking in favor of an active uptake mechanism (Fig. 2A andB). Also, THPTS colocalized to endosome and lysosometrackers, but did not show mitochondrial accumulation(Fig. 2C–K). This indicates cell type–specific subcellulartransport mechanisms, because THPTS preferentially accumu-lated in mitochondria in retinoblastoma cells (16). Cytotoxiceffects of PDT could be due to direct release of lysosomalenzymes after illumination (27). THPTS-PDT led to earlyonset of apoptosis as demonstrated in time lapse (Fig. 3) andcytotoxicity assays (Supplementary Fig. S4), which is in linewith findings in human urothelial cancer cells (18). Therefore,

Figure 4.

Tumor induction in the pilot study. All rats developed a urothelial cell carcinoma. A and B,Minimum tumor load (pT1); macroscopic view (A) and histology (B).C and D,Maximum tumor load (pT1); macroscopic view (C) and histology (D). B and D, Scan of slides and close-up (2.5x); O tumor area; � , attached fat. E, Animalweight complies with legal requirements for establishment of an animal model (mean� SD). F, Total tumor volume and relative tumor volume (normalized tobladder volume; mean� SD); n¼ 11. Scale bar, 5 mm.

Berndt-Paetz et al.

Mol Cancer Ther; 18(4) April 2019 Molecular Cancer Therapeutics748

AY-27 cells are suitable to investigate THPTS-PDT efficacy inbladder cancer.

We established the orthotopic rat model initially described in1999 by Xiao and colleagues (19). Various trial setups resulted in80% to 100% tumor induction (19, 22, 28). We could showtumor induction in 11 of 11 rats (all pT1) 10 days after instillationof 2 � 106 AY-27 cells (Fig. 4). To overcome tumor volumevariations observed in pilot study, we developed a strict methodfor studies in the treatment groups. Accurate evaluationwith serialsections (7 mm) and examination of every 20th section was notachieved in any former studies (e.g., serial sections with only twostains; ref. 29).

Animal studies confirmed the in vitro results and indicatetumoricidal phototoxicity and high tumor selectivity of THPTSin vivo. Laser light exposure after intravesical THPTS applicationdid not cause skin photosensitization, irradiation-relateddeaths, or extensive full-thickness bladder damage. Other sec-ond-generation PSs (e.g., ALA/HAL) showed comparable tumorselectivity and low bladder wall phototoxicity in the F344bladder cancer model. For instance, there were no deaths orhistopathologic changes in the bladder muscular layer afterintravesical ALA-PDT (30). In addition, selective urothelialcancer necrosis could be achieved with ALA/HAL-PDT andHypericin-PDT without major effects to the underlying mus-culature in F344 (31, 32). However, these PSs have a thera-peutic absorption maximum at shorter wavelengths, a lessfavorable characteristic for deep tumoricidal action. Therefore,research concentrated on PDT of superficial urothelial cancer.Due to its absorption maximum at 760.5 nm, THPTS-PDTcould overcome these limitations of low tissue penetration.In the present study, we evaluated THPTS-PDT in vivo. Ourfindings are in accordance with previous in vivo results ofeffective THPTS-PDT on heterotopic cholangiocarcinoma inSCID mice (13). Additionally, selective necrosis with aninvasion depth of 13 mm could be induced by THPTS-PDTon heterotopic C26 colon carcinoma in BALB/c mice (14).

Here, we revealed significant reduction of total tumorvolume and � pT2 tumor number after singular intravesicalTHPTS-PDT on AY-27–induced bladder tumors in animmunocompetent animal host without side effects (e.g., skinphotosensitization, bladder shrinkage; Fig. 5). Reasons forlacking complete tumor remission could be numerous. Futureresearch should focus on optimizing drug and light dose andshould explore repeated treatment regimens resembling currentclinical standards to improve treatment effects. Nevertheless,this is the first time PDT was successfully evaluated for treat-ment of muscle-invasive bladder cancer. Effective reduction tonon–muscle-invasive bladder cancer was shown, which mightoffer bladder-preserving treatment option by transurethralresection.

Our results support the idea of THPTS-PDT as a promisingalternative in antitumor therapies. We confirmed cell cultureresults in human urothelial carcinoma cell lines for rat cells. Forthe first time, its possible therapeutic use was shown in animmunocompetent animal model. THPTS-PDT may overcomemajor problems of currently used photosensitizers and mightoffer a new treatment strategy in advanced bladder cancertherapy.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L.-C. Horn, J. NeuhausDevelopment of methodology: M. Berndt-Paetz, P. Schulze, A. Weimann,R. Hermann, J. NeuhausAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Berndt-Paetz, P. Schulze, P.C. Stenglein,A. Weimann, J. NeuhausAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):M. Berndt-Paetz, P. Schulze, P.C. Stenglein, Q.Wang,L.-C. Horn, J. Griebel, J. Neuhaus

Figure 5.

Effects of in vivo THPTS-PDT. A,Phototoxicity on bladder carcinoma.Tumor volume was determined relative tovolume of bladder wall. Total tumorvolume (%) was significantly reduced intherapy group; pT1 tumor volume (%) withno significant difference;�pT2 tumorvolume (%) significantly reduced intherapy group; meanþ SD; � , P�0.05;Mann–Whitney test; n¼ 10. B and C,Photodynamic effects on healthy bladdertissue. Healthy urothelium (�),submucosal tissue (#), and bladdersmooth muscle (þ) not affected byTHPTS-PDT and in direct contact tourothelial tumor (o) with surroundinginflammation (x; x, artifact). Scale bar,500 mm.

Effective Bladder Tumor Growth Control by PDT with THPTS

www.aacrjournals.org Mol Cancer Ther; 18(4) April 2019 749

Writing, review, and/or revision of the manuscript: M. Berndt-Paetz,P. Schulze, L.-C. Horn, Y.M. Riyad, J. Griebel, A. Glasow, J.-U. Stolzenburg,J. NeuhausAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. NeuhausStudy supervision: J. Neuhaus

AcknowledgmentsWe thank Prof. F. Guillemin and Dr. M.A. D'Hallewin (Centre de Lutte le

Cancer de Lorraine, Nancy, France) for providing the rat urothelial cancer cell

line AY-27. The study was supported by the Deutsche Forschungsgemeinschaft(grant: NE425/6-1 to J. Neuhaus).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 19, 2018; revised December 19, 2018; accepted February18, 2019; published first March 1, 2019.

References1. Antoni S, Ferlay J, Soerjomataram I, ZnaorA, Jemal A, Bray F. Bladder cancer

incidence and mortality: a global overview and recent trends. Eur Urol2017;71:96–108.

2. Babjuk M, Burger M, Zigeuner R, Shariat SF, van Rhijn BW, Comp�erat E,et al. EAU guidelines on non-muscle-invasive urothelial carcinoma of thebladder: update 2013. Eur Urol 2013;64:639–53.

3. Witjes JA, Comp�erat E, CowanNC, SantisMde,GakisG, Lebret T, et al. EAUguidelines on muscle-invasive and metastatic bladder cancer: summary ofthe 2013 guidelines. Eur Urol 2014;65:778–92.

4. Ackroyd R, Kelty C, Brown N, Reed M. The history of photodetection andphotodynamic therapy. Photochem Photobiol 2001;74:656–69.

5. Castano AP, Mroz P, Hamblin MR. Photodynamic therapy and anti-tumorimmunity. Nat Rev Cancer 2006;6:535–45.

6. Mroz P, Hashimi JT, Huang Y, Lange N, Hamblin MR. Stimulation of anti-tumor immunity by photodynamic therapy. Expert Rev Clin Immunol.2011;7:75–91.

7. Jichlinski P, Leisinger HJ. Photodynamic therapy in superficial bladdercancer: past, present and future. Urol Res 2001;29:396–405.

8. Walther MM, Delaney TF, Smith PD, Friauf WS, Thomas GF, Shawker TH,et al. Phase I trial of photodynamic therapy in the treatment of recurrentsuperficial transitional cell carcinoma of the bladder. Urology 1997;50:199–206.

9. Nseyo UO, Shumaker B, Klein EA, Sutherland K. Photodynamic therapyusing porfimer sodium as an alternative to cystectomy in patients withrefractory transitional cell carcinoma in situ of the bladder. BladderPhotofrin Study Group. J Urol 1998;160:39–44.

10. Berger AP, Steiner H, Stenzl A, Akkad T, Bartsch G, Holtl L. Photodynamictherapy with intravesical instillation of 5-aminolevulinic acid for patientswith recurrent superficial bladder cancer: a single-center study. Urology2003;61:338–41.

11. Skyrme RJ, French AJ, Datta SN, Allman R, Mason MD, Matthews PN. Aphase-1 study of sequential mitomycin C and 5-aminolaevulinic acid-mediated photodynamic therapy in recurrent superficial bladder carcino-ma. BJU Int 2005;95:1206–10.

12. Yoon I, Li JZ, Shim YK. Advance in photosensitizers and light delivery forphotodynamic therapy. Clin Endos 2013;46:7–23.

13. Oertel M, Schastak SI, Tannapfel A, Hermann R, Sack U, M€ossner J, et al.Novel bacteriochlorine for high tissue-penetration: photodynamic prop-erties in human biliary tract cancer cells in vitro and in a mouse tumourmodel. J Photochem Photobiol B 2003;71:1–10.

14. Schastak S, Jean B, Handzel R, Kostenich G, Hermann R, Sack U, et al.Improved pharmacokinetics, biodistribution and necrosis in vivo using anew near infra-red photosensitizer: tetrahydroporphyrin tetratosylat.J Photochem Photobiol B 2005;78:203–13.

15. Schastak S, Yafai Y, Geyer W, Kostenich G, Orenstein A, Wiedemann P.Initiation of apoptosis by photodynamic therapy using a novel positivelycharged and water-soluble near infra-red photosensitizer and white lightirradiation. Methods Find Exp Clin Pharmacol 2008;30:17–23.

16. Walther J, Schastak S, Dukic-Stefanovic S, Wiedemann P, Neuhaus J,Claudepierre T. Efficient photodynamic therapy onhuman retinoblastomacell lines. PLoS ONE 2014;9:e87453.

17. Hambsch P, Istomin YP, Tzerkovsky DA, Patties I, Neuhaus J, Kortmann R,et al. Efficient cell death induction in human glioblastoma cells byphotodynamic treatment with tetrahydroporphyrin-tetratosylat (THPTS)and ionizing irradiation. Oncotarget 2017;8:72411–23.

18. Berndt-PaetzM,WeimannA, SiegerN, Schastak S, RiyadYM,Griebel J, et al.Tetrahydroporphyrin-tetratosylat (THPTS): a near-infraredphotosensitizerfor targeted and efficient photodynamic therapy (PDT) of human bladdercarcinoma. An in vitro study. Photodiag Photodynamic Ther 2017;18:244–51.

19. Xiao Z, McCallum TJ, Brown KM, Miller GG, Halls SB, Parney I, et al.Characterization of a novel transplantable orthotopic rat bladder transi-tional cell tumour model. Br J Cancer 1999;81:638–46.

20. Riyad YM, Naumov S, Schastak S, Griebel J, Kahnt A, H€aupl T, et al.Chemical modification of a tetrapyrrole-type photosensitizer: tuningapplication and photochemical action beyond the singlet oxygen channel.J Phys Chem B 2014;118:11646–58.

21. Cohen SM, Yang JP, Jacobs JB, Arai M, Fukushima S, Friedell GH. Trans-plantation and cell culture of rat urinary bladder carcinoma. Invest Urol1981;19:136–41.

22. El Khatib S, Berrahmoune S, Leroux A, Bezdetnaya L, Guillemin F,D'Hallewin M. A novel orthotopic bladder tumor model with predict-able localization of a solitary tumor. Cancer Biol Ther 2006;5:1327–31.

23. Greene FL, Compton CC, Fritz AG, Shah JP, Winchester DP. AJCC cancerstaging atlas. New York: Springer New York; 2006.

24. Russell WMS, Burch RL. The principle of human experimental techniques.London: Methuen; 1959.

25. Waidelich R, BeyerW, Kn€uchel R, SteppH, Baumgartner R, Schr€oder J, et al.Whole bladder photodynamic therapy with 5-aminolevulinic acid using awhite light source. Urology 2003;61:332–37.

26. Hungerhuber E, Stepp H, Kriegmair M, Stief C, Hofstetter A, Hartmann A,et al. Seven years' experience with 5-aminolevulinic acid in detection oftransitional cell carcinoma of the bladder. Urology 2007;69:260–4.

27. Berg K, Moan J. Lysosomes and microtubules as targets for photoche-motherapy of cancer. Photochem Photobiol 1997;65:403–9.

28. Miyazaki K, Morimoto Y, Nishiyama N, Satoh H, TanakaM, Shinomiya N,et al. Preconditioning methods influence tumor property in an orthotopicbladder urothelial carcinoma rat model. Mol Clin Oncol 2014;2:65–70.

29. Francois A, Salvadori A, Bressenot A, Bezdetnaya L, Guillemin F,D'Hallewin MA. How to avoid local side effects of bladder photody-namic therapy: impact of the fluence rate. J Urol 2013;190:731–6.

30. Xiao Z, Brown K, Tulip J, Moore RB. Whole bladder photodynamictherapy for orthotopic superficial bladder cancer in rats: a study ofintravenous and intravesical administration of photosensitizers. J Urol2003;169:352–6.

31. Gr€onlund-Pakkanen S, Wahlfors J, Talja M, Kosma V, Pakkanen TM,Ala-Opas M, et al. The effect of photodynamic therapy on rat urinarybladder with orthotopic urothelial carcinoma. BJU Int 2003;92:125–30.

32. KamuhabwaAA,RoskamsT,D'HallewinM,Baert L, vanPoppelH, deWittePA. Whole bladder wall photodynamic therapy of transitional cell carci-noma rat bladder tumors using intravesically administered hypericin. Int JCancer 2003;107:460–7.

Mol Cancer Ther; 18(4) April 2019 Molecular Cancer Therapeutics750

Berndt-Paetz et al.