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Published OnlineFirst March 14, 2017; DOI: 10.1158/1535-7163.MCT-16-0670

Large Molecule Therapeutics Molecular Cancer Therapeutics Maltotriose Conjugation to a Chlorin Derivative Enhances the Antitumor Effects of Photodynamic Therapy in Peritoneal Dissemination of Pancreatic Cancer Akihisa Kato1, Hiromi Kataoka1, Shigenobu Yano2, Kazuki Hayashi1, Noriyuki Hayashi1, Mamoru Tanaka1, Itaru Naitoh1, Tesshin Ban1, Katsuyuki Miyabe1, Hiromu Kondo1, Michihiro Yoshida1, Yasuaki Fujita1, Yasuki Hori1, Makoto Natsume1, Takashi Murakami3, Atsushi Narumi4, Akihiro Nomoto5, Aya Naiki-Ito6, Satoru Takahashi6, and Takashi Joh1

Abstract

Peritoneal dissemination is a major clinical issue associated occurred via apoptosis and reactive oxygen species generation, with dismal prognosis and poor quality of life for patients with whereas Mal3-chlorin alone did not cause any cytotoxicity in pancreatic cancer; however, no effective treatment strategies pancreatic cancer cells. Notably, using a peritoneal disseminat- have been established. Herein, we evaluated the effects of ed mice model, we demonstrated that Mal3-chlorin accumu- photodynamic therapy (PDT) with maltotriose-conjugated lated in xenograft tumors and suppressed both tumor growth chlorin (Mal3-chlorin) in culture and in a peritoneal dissem- and ascites formation with PDT. Furthermore, PDT with Mal3- inated mice model of pancreatic cancer. The Mal3-chlorin was chlorin induced robust apoptosis in peritoneal disseminated prepared as a water-soluble chlorin derivative conjugated with tumors, as indicated by immunohistochemistry. Taken togeth- four Mal3 molecules to improve cancer selectivity. In vitro, er, these ﬁndings implicate Mal3-chlorin as a potential next- Mal3-chlorin showed superior uptake into pancreatic cancer generation photosensitizer for PDT and the basis of a new cells compared with talaporﬁn, which is clinically used. More- strategy for managing peritoneal dissemination of pancreatic over, the strong cytotoxic effects of PDT with Mal3-chlorin cancer. Mol Cancer Ther; 16(6); 1124–32. 2017 AACR.

Introduction because the associated poor general condition of affected patients and problems in assessing scattered tumors due to Pancreatic cancer is a highly aggressive disease with a dismal ascites, jaundice, and ileus hamper the administration of stan- prognosis. The reported 5-year overall survival rate of patients dard treatment. The treatment of choice for patients with with pancreatic cancer is less than 5% (1), due mainly to the peritoneal dissemination is palliative systemic chemotherapy; frequently advanced stage at diagnosis, rapid tumor growth, however, its impact on the primary pancreatic cancer is limited and metastasis to distant organs such as the liver, lung and (3, 4). Thus, new methods of treating peritoneal dissemination peritoneum (2). Peritoneal dissemination of pancreatic cancer are needed. poses significant difficulties for both patients and clinicians One such approach, photodynamic therapy (PDT), is a promising, minimally invasive modality for cancer therapy that uses reactive oxygen species (ROS) generated by the photo- 1Department of Gastroenterology and Metabolism, Nagoya City University chemical reactions between a photosensitizer and irradiation 2 Graduate School of Medical Sciences, Nagoya, Japan. Graduate School of (5, 6). The anticancer effects of PDT are consequences of a low- Materials Science, Nara Institute of Science and Technology, Ikoma, Nara, Japan. to-moderately selective degree of photosensitizer taken up by 3Laboratory of Tumor Biology, Takasaki University of Health and Welfare, Takasaki, Japan. 4Department of Organic Materials Science, Graduate School malignant cells, direct cytotoxicity due to ROS, and severe of Organic Materials Science, Yamagata University, Yonezawa, Japan. 5Depart- tumorvasculardamagethatimpairsbloodsupplytothetreated ment of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture area (7–9). PDT also has several advantages over conventional University, Osaka, Japan. 6Department of Experimental Pathology and Tumor radiation and chemotherapy with respect to side effects and Biology, Nagoya City University Graduate School of Medical Sciences, Nagoya, damage to normal tissue. Japan. PDT using photofrin, a first-generation photosensitizer, is Note: Supplementary data for this article are available at Molecular Cancer widely used clinically (10, 11). More recently, PDT using a Therapeutics Online (http://mct.aacrjournals.org/). second-generation photosensitizer, talaporfin, has shown several Corresponding Author: Hiromi Kataoka, Nagoya City University Graduate advantages over PDT with photofrin, including deeper penetra- School of Medical Sciences, 1-Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya tion into tissue and less prolonged photosensitization (12, 13), 467-8601, Japan. Phone: 81-52-853-8211; Fax: 81-52-852-0952; E-mail: and has already been used to treat a wide range of cancers (14–16). [email protected] Nevertheless, PDT issues related to insufficient efficacy and skin doi: 10.1158/1535-7163.MCT-16-0670 photosensitivity remain unsolved, and the need remains for 2017 American Association for Cancer Research. photosensitizers with better cancer cell selectivity.

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PDT with Mal3-Chlorin for Disseminated Pancreatic Cancer

We previously reported that PDT using a newly developed banks and revealed to be not misidentified with any other cell photosensitizer, glucose-conjugated chlorin (G-chlorin), exerted lines. These cell lines were certified to be free of fungal, bacterial, high photocytotoxicity compared with PDT with talaporfin (17, and mycoplasmal contaminations by the cell banks, and were 18). G-chlorin with its four glucose molecules (5,10,15,20-tetra- distributed to our institution. All cell lines were cultured in kis-(pentafluorophenyl)-2,3-[methano(N-methyl)iminometha- RPMI1640 medium (Wako Pure Chemical Industries Co. Ltd.) no] chlorin) is likely to elicit high photocytotoxicity in cancer supplemented with 10% FBS. Cells were cultured at 37Cin5% cells due to their tendency to accumulate glucose, called the CO2 humidified air, and experiments were carried out within 3 Warburg effect (19). Unfortunately, G-chlorin is insoluble in months of resuscitation. water, making it less suitable for clinical use. Furthermore, good water solubility ensures rapid clearance from the body, avoiding Flow cytometric analysis cutaneous phototoxicity. Pancreatic cancer cells were incubated with talaporfin(5mmol/L) We have therefore developed a straightforward strategy to or Mal3-chlorin (5 mmol/L) for 4 hours and washed once with improve the water solubility of chlorin derivatives by using a PBS. Cells were then harvested using 0.25% trypsin-EDTA (GIBCO) oligosaccharide, such as maltotriose (Mal3), called Mal3-chlorin and analyzed using a FACSCanto II Analyzer (BD Biosciences; and have reported its highly hydrophilicity and photocytotoxicity excitation; 405 nm, emission; 660 nm). At least 10,000 events were (20). In the present study, we investigated the efficacy of PDT with collected for each sample. a novel photosensitizer, Mal3-chlorin, in culture and in a mouse model of disseminated peritoneal pancreatic cancer using in vivo In vitro PDT fluorescence imaging to monitor sequentially. Pancreatic cancer cells (AsPC1, BxPC3, AsPC1/luc, and BxPC3/ luc) were incubated with photosensitizer in culture medium for 4 Materials and Methods hours. Cells were washed once with PBS, covered with PBS, and irradiated at 13.9 J/cm2 (intensity: 30.8 mW/cm2) of LED light Photosensitizers (Opto Code Corporation), which emits 660 nm wavelength. Talaporfin sodium (mono-l-aspartyl chlorin6, Laserphyrin) Following irradiation, the PBS in the wells was exchanged for was purchased from Meiji Seika (Tokyo, Japan; Fig. 1A). Mal - 3 medium supplemented with 10% FBS, and the cells were incu- chlorin (5,10,15,20-tetrakis-[4-(b-D-maltotriosylthio)-2,3,5,6- bated for the specified times before analyses. tetrafluorophenyl]-2,3-[methano-(N-methyl)iminomethano]- chlorin) was synthesized (Fig. 1B) and provided by Yamagata Cell viability assay University (Yamagata, Japan; ref. 20). Cell viability was analyzed by the WST-8 cell proliferation assay, with 5 103 cells per well of AsPC1, BxPC3, AsPC1/luc, Cell culture and BxPC3/luc incubated with photosensitizer for 4 hours, and Human pancreatic cancer cell lines, AsPC1 and BxPC3, were then irradiated as described in the preceding section. After incu- obtained from the ATCC on December 3, 2015, whereas the bation for 24 hours, cells were incubated for 2 hours with the Cell luciferase-transfected AsPC1/luc and BxPC3/luc lines were Counting Kit-8 (Dojindo) and absorption was measured with a obtained from the JCRB cell bank on December 1, 2015. Our microplate spectrophotometer (SPECTRA MAX340; Molecular cell lines were authenticated with STR-PCR profiling at the cell Devices). Cell viability was expressed as a percentage of untreated

Figure 1. Chemical structure and uptake of

Mal3-chlorin. A, Chemical structure of talaporﬁn. B, Synthesis of Mal3- chlorin by conjugation of four maltotriose molecules to a chlorin derivative. C, Uptake of the photosensitizers in pancreatic cancer cells (AsPC1/luc) was estimated by ﬂow cytometric analysis.

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Figure 2.

Cytotoxic effects of PDT with Mal3-chlorin through apoptosis and ROS generation. A, Representative microscopic images of AsPC1/luc cells treated with (24 hours after PDT) or without Mal3-chlorin–mediated PDT (original magniﬁcation, 200). B, Cell viability in AsPC1/luc and BxPC3/luc cells treated by Mal3-chlorin with or without irradiation. C, Caspase-3/7 activity in AsPC1/luc cells after PDT. Values are means SD, n ¼ 4. D and E, ROS production photos (C) and data (D) were

detected by DCFH-DA with (1 hour after PDT) or without Mal3-chlorin–mediated PDT. Data are means SD values from three independent experiments. F, H2O2 concentrations in AsPC1/luc cells after PDT. Values are means SD, n ¼ 4 in each; , P < 0.05; , P < 0.01; and , P < 0.001 compared with non-treated cells.

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PDT with Mal3-Chlorin for Disseminated Pancreatic Cancer

control cells and the half maximal (50%) inhibitory concentra- ed the relative fluorescence intensity by the autofluorescence tion (IC50) was calculated. intensity to compare relative fluorescence intensity ratios of the tested photosensitizers in the target tissue. Caspase-3/7 assay Apoptosis in the AsPC1/luc cells was assessed using the Cas- In vivo PDT and bioluminescence imaging pase-Glo3/7 Assay Kit (Promega). AsPC1/luc cells were treated One week after injection of the cells, bioluminescence was with photosensitizers (0.25–1 mmol/L) at 37 C for 4 hours, and measured using a multifunctional in vivo imaging system (IVIS; irradiated. Analyses were performed at 4 hours after PDT by MIIS, Molecular Devices Japan) to confirm the cell implantations. adding 100 mL of caspase-3/7 reagent to each well, mixing, and The mice were randomly allocated to three groups. Then, the mice then incubating for 1 hour at room temperature. Luminescence were given an intraperitoneal injection with photosensitizer was measured using a Lu mat LB 9507 instrument (EG&G BERT- (talaporfin and Mal3-chlorin) at 1.25 mmol/kg in saline. Four HOLD), and the data were normalized against the average value of hours later, the mice were irradiated with 660-nm LED light (Opto nontreated cells. Relative luminescence units (RLU) of the cas- Code Corporation) at a dose of 13.9 J/cm2. This light device was pase-3/7 activity are expressed as mean SD. placed just above the abdomen so that the irradiation spot on the abdominal skin was 1.77 cm2 (diameter 1.5 cm), thus covering Estimation of cellular ROS production the lower abdomen and avoiding the liver. Treatment was per- AsPC1/luc cells were treated with or without Mal3-chlorin formed on day 0 and 7, as shown in Fig. 4A. To visualize the (0.25–1 mmol/L) for 4 hours with or without subsequent irradi- peritoneal tumors in mice, IVIS was performed as follows. The 2 ation (13.9 J/cm ). At 1 hour after irradiation, cells were incubated mice were anesthetized and subsequently intraperitoneally 0 0 with 100 mg/mL 2 ,7 -dichlorofluorescein-diacetate (DCFH-DA, injected with D-luciferin (150 mg/g body weight, Wako, Ltd.) in Sigma) at 37 C for 20 minutes, in the dark. The cells were washed D-PBS. Two minutes later, a monochrome photograph was two times with warm PBS and viewed with a fluorescence micro- acquired followed immediately by bioluminescence acquisition scope (BZ-9000; Keyence). The fluorescence intensity scores of for 20 seconds and in vivo fluorescence imaging. From this, total imaged cells were evaluated using the associated software (21). luminescence flux from the region of tumor on days 0, 7, 14 was Furthermore, H2O2 released into media collected from AsPC1/luc quantified using MetaMorph-MIIS software (Molecular Devices cells with or without PDT was determined using ROS-Glo H2O2 Japan) to monitor tumor growth. The bioluminescence images assay (Promega). Cells were incubated with each photosensitizer were exported as false-colored images using matched visualiza- (0.25–2 mmol/L) for 4 hours and irradiated. At 1 hour after PDT, tion scales. At the end of the experiment on day 21, mice were cells were incubated in a H2O2 substrate solution at 37 C for 3 sacrificed and ascites volumes were measured. Another in vivo hours and then luciferin detection reagent at room temperature experiment was also conducted on the mice peritoneal tumor for 1 hour. Luminescence was measured using a Lu mat LB 9507 tissue to analyze apoptosis, whereby PDT with each photosensi- instrument (EG&G BERTHOLD) and the RLU of H2O2 concen- tizer was administered only on day 14 followed by excision of the trations are expressed as mean SD, relative to the average value tumor tissue on day 15 and fixation in 10% phosphate-buffered of nontreated cells. formalin.

Animals and peritoneal dissemination model Immunohistochemical analysis of apoptosis of pancreatic cancer For immunohistochemistry, the sections were immunostained Female nude mice (BALB/c Slc-nu/nu) aged 4 weeks and as previously described (22). In brief, apoptotic cells were weighing 15 to 20 g were obtained from Japan SLC and acclimated detected by terminal deoxy nucleotidyl transferase-mediated to the laboratory for 1 week. They were housed at five animals per dUTP nick end labeling (TUNEL) assay as well as cleaved cas- cage on pulp-chip bedding in an air-conditioned animal room at pase-3 IHC. TUNEL assays were performed using an In Situ 22 2C and 55 5% humidity. All mice were maintained under Apoptosis Detection Kit from Takara (Otsu, Japan). For detection specific pathogen-free conditions with a 12-hours light/dark cycle. of cleaved caspase-3, deparaffinized sections of tumor from mice To prepare the in vivo peritoneal dissemination model, AsPC1/luc peritoneum were incubated with 1:100 diluted cleaved caspase-3 cells were injected intraperitoneally with 1 106 cells in 200 mL antibody (Cell Signaling Technology). Antibody binding was PBS. All animal experiments were performed under protocols visualized by a conventional immunostaining method using an approved by the Institutional Animal Care and Use Committee of autoimmunostaining apparatus (HX System, Ventana). Nagoya City University Graduate School of Medical Sciences. Statistical analysis fi In vivo spectrophotometric analysis The statistical signi cance of differences was determined using t c2 The accumulation of photosensitizer in the in vivo models was the Dunnett's test, Student test, or the test as appropriate. fi P < examined by optical-filter imaging (Longpass Filter VL0470, cut- Differences were considered statistically signi cant at 0.05. on; 470 nm, Asahi Spectra Inc.) and using a semiconductor laser Data are expressed as mean SD. with a VLD-M1 spectrometer (M&M Co., Ltd.) that emitted a laser Table 1. The IC for pancreatic cancer with PDT using talaporfin and Mal - light with a peak wavelength of 405 1 nm and a light output of 50 3 chlorin 140 mW. The spectrometer and its accessory software (BW-Spec IC50 (mmol/L) V3.24; B&W TEK, Inc.), used to analyze the spectrum waveform, AsPC1 AsPC1/luc BxPC3 BxPC3/luc fl revealed an amplitude peak (relative uorescent intensity) at 505 Talaporfin 16.59 12.02 11.92 11.23 fl nm for auto uorescence and 655 nm for photosensitizers. The Mal3-chlorin 1.51 0.45 2.24 0.66 relative intensities of the photosensitizers were also measured NOTE: Talaporfin, talaporfin sodium; Mal3-chlorin, maltotriose-conjugated spectrophotometrically. To reduce measurement error, we divid- chlorin; IC50, the half maximal (50%) inhibitory concentration.

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Results cell death. However, neither Mal3-chlorin nor light applied alone caused any toxicity in AsPC1/luc cells. Similar results Pancreatic cancer cells take up Mal -chlorin more efficiently 3 were obtained with the other cell lines (AsPC1, BxPC3, and than talaporfin BxPC3/luc; data not shown). Furthermore, neither Mal - We first examined the uptake of talaporfin and Mal -chlorin in 3 3 chlorin nor talaporfin without irradiation induced any cyto- AsPC1/luc cells using flow cytometric analysis, and then mea- toxicity (Fig. 2B; Supplementary Fig. S1, respectively). We next sured intensity of the characteristic red fluorescence at the single- examined the IC of talaporfinandMal-chlorin at 24 hours cell level. The cells treated with Mal -chlorin showed a stronger 50 3 3 after PDT. As summarized in Table 1, PDT with Mal -chlorin signal (mean intensity: 1,545) than both the nontreated cells 3 induced cell death with 5 to 27 times more cytotoxicity to all (mean intensity: 44) and the talaporfin-treated cells (mean inten- cancer cells than PDT with talaporfin. At the same time, in sity: 80), indicated that more Mal -chlorin was taken up by cancer 3 AsPC1/luc cells, we confirmed that the luciferase luminescence cells compared with talaporfin (Fig. 1C). intensities reduced in parallel with cell viability by luciferase assay (Supplementary Fig. S2).

The high cytotoxic effects of PDT with Mal3-chlorin on Moreover, the level of apoptosis after PDT with Mal3-chlorin pancreatic cancer cells were induced through apoptosis and was increased in a dose-dependent manner compared with after ROS generation PDT with talaporﬁn, whereas neither Mal3-chlorin nor talaporﬁn We next evaluated the cell death induced by PDT with Mal3- alone induced apoptosis (Fig. 2C). To further explore these chlorin.AsshowninFig.2A,PDTwithMal3-chlorin induced cytotoxic effects, we then analyzed the levels of intracellular ROS

Figure 3.

Accumulation of Mal3-chlorin in mice disseminated peritoneal tumors. A–C, Representative disseminated peritoneal tumors in the mice 2 weeks after AsPC1/luc cell implantation. A, Bioluminescence imaging with in vivo imaging system; white light image (B)andﬂuorescence image (C). Arrows indicate disseminated peritoneal tumors, and red ﬂuorescence aligns with the peritoneal tumor. D, Images of excised tumor tissue and some organs in each group (top, white light image;

bottom, fluorescence image). E, The fluorescence intensity ratio in tumors and some organs of talaporfin- and Mal3-chlorin–mediated PDT groups was calculated and shown relative to that in control mice.

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in AsPC1/luc cells using the DCFH-DA assay and found signifi- Similarly, following an intraperitoneal injection of 1.25 mmol/ cantly elevated ROS intensities after PDT with Mal3-chlorin kg talaporfinorMal3-chlorin, tumor tissue and some organs in (Fig. 2D and E). Likewise, the H2O2 assay also showed dose- each group were excised and observed under white light and dependent increases in ROS generations after PDT with Mal3- fluorescence imaging (Fig. 3D). We then measured the biodis- chlorin that were higher than the increases observed after PDT tribution of talaporfinandMal3-chlorin in xenograft models by with talaporfin (Fig. 2F). spectrophotometry. Notable accumulation of Mal3-chlorin in the tumor tissue was detected (Fig. 3E), while among the organ

Mal3-chlorin accumulates in xenograft tumors of tissues, Mal3-chlorin tended to accumulate in liver and stom- pancreatic cancer ach, although at a much lower level than those in tumor. The We examined the ability of Mal3-chlorin to accumulate in stomach accumulation was measured at the lower stomach due xenograft tumors established by subcutaneously implanting to the strong autofluorescence of the upper stomach. AsPC1/luc pancreatic cancer cells into mice. At 2 weeks after the cell injections, we first confirmed successful cell implanta- PDT with Mal3-chlorin suppressed tumor progression in a tion by bioluminescence imaging (Fig. 3A). Then, the perito- mouse model of disseminated peritoneal pancreatic cancer neal cavities were washed with saline and imaged under white To investigate the antitumor effects of PDT with Mal3- light (Fig. 3B), followed by fluorescence imaging of the same chlorin on intraperitoneal dissemination in vivo,eachPDTwas view at 4 hours after intraperitoneal injection of 1.25 mmol/kg performed on a pancreatic cancer model established by intra- Mal3-chlorin. We observed the characteristic red fluorescence of peritoneal implantation of AsPC1/luc cells into nude mice, Mal3-chlorin in the disseminated peritoneal tumors (Fig. 3C). as shown in Fig. 4A. All animals remained healthy throughout

Figure 4.

Treatment effects of PDT with Mal3-chlorin in a mouse model of disseminated peritoneal pancreatic cancer. A, The treatment regimen is shown. Bioluminescence images were obtained at day 0, 7, and 14. B, Representative bioluminescence imaging of AsPC1/luc peritoneal tumors at day 14 in control mice (left), talaporﬁn-mediated PDT mice (middle), and Mal3-chlorin-mediated PDT mice (right). C, Quantitative analysis of bioluminescence dynamics in each group (n ¼ 7 per group); , P < 0.05. D, Volumes of ascites in each group of mice.

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Figure 5. IHC and TUNEL indices of peritoneal tumors. A, Representative IHC staining for TUNEL and cleaved caspase-3 in peritoneal tumor tissues of each group; bars, 100 mm. B, TUNEL indices in peritoneal tumors form each group (n ¼ 3 in each; , P < 0.05; , P < 0.01).

the experimental period, and there was no significant differ- strategies. Against this background, PDT is emerging as a clinically ence in the mean body weight among the groups at the end promising locoregional therapy for pancreatic cancer that could of the study (Supplementary Fig. S3). We detected the inhib- also provide aggressive treatment for peritoneal dissemination in itoryeffectofPDTwithMal3-chlorin on intraperitoneal dis- partnership with chemotherapy. Such therapy could also be semination with bioluminescence imaging (Fig. 4B), although useful to control ascites resulting in improved quality of life there was no effect on abdominal skin (Supplementary during palliative treatments. Supporting this promise, we dem- Fig. S4). Numeric evaluations revealed that PDT with Mal3- onstrated herein a suppressive effect of PDT with Mal3-chlorin in a chlorin significantly suppressed tumor growth compared with mouse model of disseminated peritoneal pancreatic cancer, based control treatment (P ¼ 0.036) and tended to suppress it more on in vivo luminescent imaging and measurement of ascites than PDT with talaporfin(P ¼ 0.074; Fig. 4C). We also volume. We could not elucidate the median survival time in the observed that PDT with Mal3-chlorin tended to inhibit the scope of this study within the protocols approved by the Institu- incidence of ascites formation (Supplementary Table S1) and tional Animal Care and Use Committee. However, we expect that the mean volume of ascites compared with mice in the control PDT with Mal3-chlorin could improve survival rates in such and PDT with talaporfingroup(P ¼ 0.066 and P ¼ 0.159, experimental models compared to untreated mice due to the respectively; Fig. 4D). reduced volumes of both peritoneal tumor and ascites. At clinical sites, the tumoritropic characteristics of photosensitizers become crucial in reducing the damage to adjacent PDT with Mal -chlorin induced apoptosis in peritoneal 3 normal tissue. There have been many attempts to develop new disseminated tumors photosensitizers that show preferential accumulation within Peritoneal tumor tissues from the PDT with the Mal -chlorin 3 the target tumor tissue through combinations with various group showed some chromatin-condensed single cells, therefore active targeting approaches, such as conjugation with peptides TUNEL assays were performed and positivity was found in such or antibodies (23–26), incorporation within liposomes (27, dead lesions (Fig. 5A). PDT with Mal -chlorin significantly 3 28), and encapsulation within polymeric nanoparticles (29– increased the TUNEL apoptotic indices compared with control 32). To initiate accumulation in the target tumor, G-chlorin was treatment or PDT with talaporfin (Fig. 5B), and additional immu- initially synthesized by linking glucose to the photosensitizer nohistochemistry revealed some TUNEL-positive cells expressing chlorine based on tumors consuming higher levels of glucose cleaved caspase-3 (Fig. 5A). In contrast, cleaved caspase-3–pos- than normal cells and indeed, G-chlorin showed improved itive cells were scarcely found in peritoneal tumors of control and cancer cell targeting compared to chlorin and other photosen- PDT with talaporfin-treated tumors. sitizers (5, 17, 18). For clinical use, we recently developed Mal3- chlorin introduced water-solubility. As for the 1O generation Discussion 2 efficiency and photocytotoxicity, Mal3-chlorin was proved to The poor outcome of pancreatic cancer is due, at least in part, to have a very high performance comparable to that of G-chlorin. peritoneal dissemination (3, 4). In pancreatic cancer patients with In addition, the crucial water-soluble advantage of Mal3-chlorin peritoneal dissemination, the lack of effective chemotherapeutic allows its injection without the use of toxic organic solvents, and targeted agents highlight the urgent need for novel treatment with hydrophilicity similar to that of talaporfin (20).

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Talaporfin, a second-generation photosensitizer, shows im- microenvironment compared to tumors from human patients, proved efficacy compared with first-generation photosensitizers including in the vasculature, stromal cells, and immune cells (12). Now, our in vivo results indicate that Mal3-chlorin–mediated (43, 44). We therefore plan to further investigate the effects shown PDT tends to be more effective than talaporfin-mediated PDT at a here in a future study using a spontaneous peritoneal dissemi- low photosensitizer concentration (1.25 mmol/kg). Although we nated model. could not find the effects of PDT with talaporfin, we speculate that Collectively, our results indicated that PDT with Mal3-chlorin talaporfin required over five times more dose (>6.25 mmol/kg) could effectively suppress the growth of peritoneal disseminated than the dose which was used in the present study, according to a xenograft mouse models of human pancreatic cancer cells previous study (33, 34). This indicated that Mal3-chlorin was through apoptosis without observable damage to the adjacent more efficiently taken up by pancreatic cancer cells than talapor- normal tissue. Furthermore, the newly developed water-soluble fin. In support of this, previous PDT experiments with photo- Mal3-chlorin had superior cancer cell selectivity without skin sensitizers including talaporfin used irradiation doses of >100 J/ phototoxicity (Supplementary Fig. S4). Thus, Mal3-chlorin stands cm2 in carcinoma xenografts models (33, 35, 36), whereas in as a candidate for the next-generation photosensitizers and our this study, PDT with Mal3-chlorin showed antitumor effects findings may offer new opportunities for the clinical use of PDT in in vitro and in vivo at a low irradiation dose of 13.9 J/cm2. The treating peritoneal dissemination of pancreatic cancer. high cancer cell selectivity of Mal3-chlorin could, therefore, sub- stantially reduce the total energy of light irradiation needed in Disclosure of Potential Conflicts of Interest PDT treatment. No potential conflicts of interest were disclosed. Glucose transporter 1 (GLUT1) is believed to maintain basal glucose transport in most cell types (37, 38) and is predominant Authors' Contributions in many types of cancer, including pancreatic cancer (39). We Conception and design: A. Kato, H. Kataoka, M. Natsume investigated the correlation of GLUT1 to the uptake of Mal3- Development of methodology: A. Kato, K. Hayashi, N. Hayashi, M. Yoshida chlorin using a pharmacological GLUT1 inhibitor, WZB117 (40), Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A. Kato, M. Yoshida, S. Takahashi and found that WZB117 inhibited the uptake of Mal3-chlorin (Supplementary Fig. S5). The experiment also hinted at an Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Kato, M. Yoshida, Y. Fujita, M. Natsume, involvement for GLUT1 in the mechanism by which Mal -chlorin 3 A. Naiki-Ito is taken up by cancer cells, although what underlies such a Writing, review, and/or revision of the manuscript: A. Kato, H. Kataoka, mechanism remains unclear. In addition, several recent reports S. Yano, K. Miyabe, H. Kondo, Y. Hori showed that mutated KRAS caused higher glucose uptake and Administrative, technical, or material support (i.e., reporting or organizing accumulation possibly by upregulation of GLUT1 (41, 42). KRAS data, constructing databases): A. Kato, S. Yano, M. Tanaka, I. Naitoh, T. Ban, mutations occur in more than 90% of pancreatic cancers, thus G- T. Murakami, A. Nomoto Study supervision: H. Kataoka, T. Joh chlorin and Mal -chlorin might produce more effects in pancre- 3 Other (synthesis of PDT photosensitizer): A. Narumi atic cancer than in other cancers expressing wild-type KRAS. Importantly, Mal -chlorin might have additional, prospective 3 Grant Support value as a photodynamic diagnosis (PDD) reagent to detect and This study was partially supported by JSPS KAKENHI grant numbers discriminate tumor locations due to its high cancer cell selectivity. in vitro fi 26460947, the Translational Research Network Program of the Japan Agency Our ndings revealed dose- and cell number-dependent for Medical Research and Development, AMED, 2015–2016 (to H. Kataoka), increases in fluorescence intensities with Mal3-chlorin (Supple- JSPS KAKENHI grant numbers 19350031, 25288028, the Japan–German mentary Fig. S6). However, it is currently too difficult to evaluate Exchange Program supported by the JSPS and the Deutsche Forschungsge- the correlation between tumor fluorescence and either the dose of meinschaft (DFG; to S. Yano), and Pancreas Research Foundation of Japan (to A. Kato). Mal3-chlorin or tumor size in vivo because of mice interindividual The costs of publication of this article were defrayed in part by the payment of differences in microvascular construction and tumor microenvi- page charges. This article must therefore be hereby marked advertisement in ronment. Further studies are therefore needed to establish the accordance with 18 U.S.C. Section 1734 solely to indicate this fact. usefulness of Mal3-chlorin in PDD. It should also be noted that the xenograft peritoneal cancer Received October 14, 2016; revised November 29, 2016; accepted March 8, mouse model used in the present study shows differences in 2017; published OnlineFirst March 14, 2017.

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1132 Mol Cancer Ther; 16(6) June 2017 Molecular Cancer Therapeutics

Maltotriose Conjugation to a Chlorin Derivative Enhances the Antitumor Effects of Photodynamic Therapy in Peritoneal Dissemination of Pancreatic Cancer

Akihisa Kato, Hiromi Kataoka, Shigenobu Yano, et al.

Mol Cancer Ther 2017;16:1124-1132. Published OnlineFirst March 14, 2017.

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