Mitochondria-Targeted Photodynamic Therapy with a Galactodendritic

Oct 17, 2016 - Here, we report the rational design of a new third-generation photosensitizer (PS), a chlorin conjugated with galactodendritic units, C...
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Preclinical study on mitochondria-targeted photodynamic therapy using a galactodendritic chlorin for bladder cancer Patrícia Manuela Ribeiro Pereira, Sandrina Silva, Mafalda Bispo, Mónica da Rocha Zuzarte, Célia Gomes, Henrique Girão, José A. S. Cavaleiro, Carlos A. Fontes Ribeiro, Joao P. C. Tome, and Rosa Fernandes Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00519 • Publication Date (Web): 17 Oct 2016 Downloaded from http://pubs.acs.org on October 19, 2016

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Mitochondria-targeted photodynamic therapy with a galactodendritic chlorin to enhance cell death in resistant bladder cancer cells Patrícia M. R. Pereira,†,‡ Sandrina Silva,† Mafalda Bispo,† Mónica Zuzarte,‡ Célia Gomes,‡,⊥ Henrique Girão,‡ José A. S. Cavaleiro,† Carlos A. F. Ribeiro,‡ João P. C. Tomé*,†,§ and Rosa Fernandes*,‡,#,⊥ †

QOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal

‡

IBILI, Faculty of Medicine, University of Coimbra, 3000-548 Coimbra, Portugal

§

CQE, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal

#

CNC.IBILI, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal

⊥Center

of Investigation in Environment, Genetics and Oncobiology, 3001-301 Coimbra, Portugal

*Corresponding authors: Rosa Fernandes, Laboratory of Pharmacology and Experimental Therapeutics – IBILI, Faculty of Medicine of University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal. Phone: +351 239480072; Fax: +351 239480066; Email: [email protected] João Tomé, Centro de Química Estrutural, Departamento de Engenharia Química, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, nº 1, 1049-001 Lisboa, Portugal. Phone: +351 218419360; Fax: +351 218499242; Email: [email protected]

ABSTRACT: Here we report the rational design of a new third-generation photosensitizer (PS), a chlorin conjugated with galactodendritic units, ChlGal8, to improve the effectiveness of bladder cancer treatment. ChlGal8 shows better photochemical and -physical properties than a recently reported homologous porphyrin, PorGal8. In addition to inheriting excellent photostability, ability to generate singlet oxygen and to interact with the proteins galectin-1 and human serum albumin (HSA), ChlGal8 exhibits high absorption in the red region of the electromagnetic spectrum. In vitro studies of anticancer activity of ChlGal8 revealed that once this PS is taken up by UM-UC-3 bladder cancer cells, induces high cytotoxicity after a single dose of light irradiation. In HT-1376 bladder cancer cells resistant to therapy, enhanced photodynamic efficacy, both in vitro and in vivo, was achieved after a second light irradiation. The enhanced phototoxicity in HT1376 cancer cells seems to be due to ChlGal8 ability to accumulate in the mitochondria, via facilitative glucose transporter 1 (GLUT1), in the period between single and repeated irradiation. A photodynamic therapy (PDT) regimen by using an extra dose of light irradiation and ChlGal8 as PS, represents a promising strategy in treating resistant cancers in clinic.

INTRODUCTION Photodynamic Therapy (PDT) is a clinically approved, minimally invasive anticancer therapy which uses the localized delivery of light to activate a photosensitizing compound (photosensitizer, PS) previously accumulated in the 1, 2 tumor tissue. Upon irradiation, the light excited PS transfers its energy to molecular oxygen or to a biological substrate initiating type-II or type-I photochemical reactions, 3 respectively. Both photochemical reactions end up with the production of reactive oxygen species (ROS) which induce cell death by multifactorial mechanisms (immunologic, vas4 cular and cellular). However, the PDT efficiency could be improved when PSs reach and accumulate in hypersensitive 5 subcellular sites. The physicochemical properties of the PSs such as chemical structure, lipophilicity and charge have a key role on their accumulation in cell structures or organelles 6 such as plasma membranes, lysossomes and mitochondria. The mitochondrion is an attractive target for PDT due to its

central role in energy metabolism and regulation of apopto7, 8 sis. PSs able to target primarily mitochondria, such as cationic compounds or agents containing a mitochondrial localization peptide sequence have demonstrated enhanced pho9-11 totoxicity, through mitochondria-mediated apoptosis. However, a major limitation of the existing PS is the lack of high selectivity for cancer cells. The application of PDT for cancer treatment was first proposed in 1970s demonstrating in vivo PDT efficacy of hematoporphyrin derivatives. Findings on the chemical composition of hematoporphyrin derivatives have contribut12 ed to the approval of the first PS, Photofrin®. In fact, porphyrins are the most common PSs used in PDT for cancer treatment. Unfortunately, the porphyrin structures used for PDT are inherently hydrophobic and their longest-wave absorption band appears at around 630 nm with a low extinction coefficient.

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In the design of new PSs for cancer treatment, reduction of porphyrins to chlorins increases the extinction coefficient 13, 14 Particularly, perfluorophenylchlorin of the last Q band. (ChlF20) has demonstrated to be a useful core platform in the 15 development of new PSs. Based on our recent research, we envisaged that the conjugation of ChlF20 with dendritic units of galactose could improve not only the solubility of the PS in 16 an aqueous medium but also increase its specificity for tu17 mor tissues. The high local concentration of galactodendritic PSs in the tumor tissues is due to the possible interaction of galactose molecules with galactose-binding proteins (such as galectin-1 and glucose transporter 1 GLUT1) overexpressed in these tissues. In this work we synthesized a chlorin containing four dendritic units of galactose (ChlGal8). In order to enhance the efficacy of ChlGal8 in resistant cancer cells to a single light irradiation, we investigated a twofold illumination scheme with a dark interval. In this study, an unprecedentedly localization of ChlGal8 in the mitochondria after the first irradiation period resulted on high phototoxicity of resistant cancer cells.

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higher absorption at 650 nm, where tissue light penetration 3 is rather high, ChlGal8 could be advantageous for PDT when compared with the corresponding PorGal8.

RESULTS AND DISCUSSION ChlGal8 exhibits excellent photo-physical and chemical properties for PDT. The ChlF20 was synthesized as previously described in the literature, by using 1,3-dipolar addition of tetraperfluorophenyl porphyrin (TPPF20) with 18, 19 azomethine ylides, and it was used as a core platform to synthesize the new galactodendritic chlorin (ChlGal8, Scheme 1). ChlGal8 was obtained in 70% yield by reacting ChlF20 with four equivalents of the previously reported galac16 todendritic unit in dry dimethylformamide (DMF), in the presence of an excess of N,N-Diisopropylethylamine 1 19 13 (DIPEA), and it was characterized by H, F and C NMR spectroscopy and mass spectrometry (Figures S1-4, see Supporting Information). The tetra-substitution of the para fluorine atoms in ChlF20 by the galactodendritic unit was sup19 ported by F NMR data, showing the absence of the signals corresponding to the Chl para fluorine atoms. Hydrolysis of the isopropylidene protective groups with a mixture of trifluoroacetic acid (TFA)/H2O (9:1, v:v) produced α/β mixtures of unprotected ChlGal8 in 87% yield (Scheme 1). Deprotection of the isopropylidene groups was confirmed by the com1 plete disappearance of the corresponding H resonances of the corresponding CH3 groups of the isopropylidenes (Figures S5,S6. see Supporting Information). Besides the NMR data, all structures were further confirmed by HRMS mass spectrometry (Figure S7, see Supporting Information). The steady-state fluorescence emission spectrum of ChlGal8 was acquired in DMF and the fluorescence quantum yield (ΦF) was determined (Table S1, see Supporting Information). These studies demonstrated that ChlGal8 has emission bands in the red spectral region at 653 nm and 714 nm. The electronic absorption spectra of ChlGal8 were measured in DMSO and their extinction coefficients (ɛ) in this solvent are summarized in Table S1, see Supporting Information. ChlGal8 at 4 µM (in DMSO) exhibit a very sharp Soret (around 410 nm) and Q bands (around 650 nm), which are typical of non-aggregated PSs (Figure 1b). ChlGal8 has a more -1 -1 than 50-fold higher extinction coefficient (6800 M .cm ) at the absorption maximum of 650 nm, than the corresponding previously reported porphyrin PorGal8 (Scheme S1, see Sup-1 -1 16 porting Information, 100 M .cm at 655 nm). Due to its

Scheme 1. General demonstration of the synthesis of ChlGal8. (i) DIPEA, DMF, 40 ºC, N2, 30 h, 70%; (ii) TFA/H2O (9:1 v/v), 87%. To determine the solubility of ChlGal8 in aqueous buffered solution, their absorption spectra were also acquired in PBS. In this solvent, ChlGal8 demonstrated a lowering intensity and a broadening of the Soret and Q bands, demonstrating reasonable water solubility (Figure 1b). The galactose-conjugate strictly follows the Beer-Lambert law at concentrations between 0 to 10 µM suggesting no aggregation in PBS at concentrations below 10 µM (Figure S8a,b see Supporting Information). To predict a priori the ability of ChlGal8 to diffuse across cancer cell membranes and to reach the inside of cancer cells, the partition coefficient value (log P, which is the log of the concentration of compounds in noctanol/H2O system) was determined using the classic shake 15 flask method. Data show that the values of log P are higher for PorGal8 when compared with ChlGal8 (Table S2, see Supporting Information), indicating that ChlGal8 has less water solubility when compared with the corresponding porphyrin PorGal8. The photostability of ChlGal8 was evaluated, by monitoring the intensity of Soret band of a PBS solution containing 4 µM ChlGal8, after irradiation with red light (>500 nm) at a -2 fluence rate of 150 mW.cm (Table S3, see Supporting Information). The results demonstrate that this galactose-

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conjugate is photostable when exposed to strong light power over the investigated irradiation period (30 min). The gener1 ation of singlet oxygen ( O2) by ChlGal8 was determined in DMF:H2O (9:1, v:v) using 1,3-diphenylisobenzofuran (DPBF) 1 1 as O2 scavenger. The well known O2 generator, TPPF20, was 20 1 used as reference. The ability of ChlGal8 to generate O2 was also compared with the corresponding porphyrin PorGal8. ChlGal8, PorGal8 and TPPF20 (both at 0.67 µM) were able to photo-oxidize DPBF (at 16.15 µM, Figure 1c). Under the conditions of the assay, ChlGal8 demonstrated higher ability to photo-oxidize DPBF when compared to the corresponding porphyrin PorGal8.

Figure 1. Characterization of ChlGal8. (a) Chemical structure of ChlGal8. (b) Electronic absorption spectra of ChlGal8 at 4 μM in DMSO and PBS. (c) Photo-oxidation of DPBF (16.15 µM) with or without ChlGal8, PorGal8 or TPPF20 at 0.67 µM, -2 after irradiation with red light (λ > 500 nm, 14 mW.cm ). The DPBF absorbance was recorded at 415 nm. Data are means ± s.e.m. of at least two independent experiments. (d) Fluorescence variation on the emission spectrum of 2 µM galectin-1 protein after addition of ChlGal8 (0-4 µM). Data are means ± s.e.m. of at least two independent experiments. ChlGal8 interacts with human serum albumin and galectin-1 proteins. To predict the in vivo pharmacokinetics and pharmacological properties of ChlGal8, its interaction with the most abundant plasma protein (human serum al16, 21 bumin, HSA) was studied by fluorescence spectroscopy. HSA is able to bind and transport anticancer drugs. Moreover, increased vascular permeability exhibited in solid tumors, leads to the accumulation of drug albumin conjugates in the tumor interstitium, affecting the unbound (free) drug quantity in the circulation, its distribution, metabolism, 22 elimination and biological activity. A quenching on the fluorescence emission (Figure S8c, see Supporting Information) of the single tryptophan residue present in the pro23 tein (Trp214, located in subdomain IIA of site I) occurred after addition of ChlGal8 (0 to 40 µM) to HSA samples (2 µM). The binding constant (Ka) and the number of binding -1 sites (n) for ChlGal8 were 3.28 x 104 M and 1.0, respectively. The value of n equal to one indicates that ChlGal8 binds HSA in the molar ratio of 1:1. We previously reported that the galactose-binding proteins overexpressed in bladder cancer cells, galectin-1 and GLUT1, have a key role on the uptake of galactodendritic 17 PSs. The addition of ChlGal8 (0 to 4 µM) to recombinant

human galectin-1 (2 µM) produced fluorescence quenching 24, 25 (Figure 1d) of the tryptophan residue (Trp68 present on the carbohydrate-recognition domain of the protein). The -1 values of Ka and n equal to 1.76 x 107 M and 1.5, respectively, as well as the dissociation constant (KD) equal to 3.79 ± 2.22 µM indicate that ChlGal8 has high interaction for galectin-1. The interaction of ChlGal8 with galectin-1 can be explained by carbohydrate interactions of the galactose units with the 25 carbohydrate-recognition domain of the protein, and hydrophobic interactions between the chlorin macrocycle and 24, 26, 27 the well-conserved hydrophobic core of the protein. ChlGal8 accumulates and induces phototoxicity in bladder cancer cells. Encouraged by the excellent photophysical and -chemical properties of ChlGal8, we tested the PDT efficacy of ChlGal8 in two bladder cancer cell lines derived from transitional cell carcinoma, HT-1376 and UM-UC3. Bladder cancer is a especially well suited for PDT, since bladder is an easily accessible organ for both intravesical 28 instillation and illumination. Cancer cell uptake of ChlGal8 (0.5 to 10 µM) for up to 4 h was evaluated by fluorescence spectroscopy. Both HT-1376 and UM-UC-3 cells took up ChlGal8 quite readily with similar uptake profile and a slower steady increase was observed after 1.5 h (Figure S9, see Supporting Information). We have previously reported that HT1376 and UM-UC-3 bladder cancer cells express high levels of the galactose-binding proteins GLUT1 and galectin-1 (Figure 17 S10, see Supporting Information), respectively. Therefore, it is envisaged that specific domains, enriched in these proteins, at the cell surface can promote the interaction with the galactodendritic units on the chlorin macrocycle of ChlGal8, mediating its cellular uptake. However, the mechanisms by which galactodendritic-PSs are accumulated inside cancer cells are not completely understood. The association of ChlGal8 with galactose-binding proteins can promote high local concentration of the PS around cancer cell membrane, which induces PS macrocycle partition into the membrane. Thus, uptake of ChlGal8 is also dependent on its relative hy29 drophobicity (Table S2, see Supporting Information). To evaluate the efficacy of PDT by ChlGal8 in bladder cancer cells, UM-UC-3 and HT-1376 cells were treated with ChlGal8 at various concentrations, and cells were then irradi-2 ated with red light (> 500 nm) at 2.5 mW.cm for 40 min (i.e. -2 6 J.cm ). No cytotoxicity was observed in cells incubated with ChlGal8 in the dark (Figure S11, see Supporting Information) for at least 72 h after treatment. However, when UM-UC-3 and HT-1376 bladder cancer cells were incubated with ChlGal8 and then irradiated there was an increased phototoxicity in a concentration-dependent manner (Figure S12, see Supporting Information). Although PDT with ChlGal8 is more active against UM-UC-3 cells than HT-1376 cells, both cell lines were unable to recover from the PDT-induced damage effects (up to 72 h). The degree of cytotoxic effect of PDT with ChlGal8 in the two cell lines can be partially explained by the different intracellular amount of ROS produced immediately after irradiation, as detected with the oxidationsensitive fluorescent dye H2DCFDA (Figure S13, see Supporting Information). Previous studies have demonstrated that HT-1376 cells exhibit a protective mechanism after PDT by 17 inducing an antioxidant response. The key role of microenvironment-related molecules, particularly the extracellular matrix-metalloproteinase inducer CD147 on the resistance of

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HT-1376 cancer cells to cisplatin-based chemotherapy has 30 been also reported. A second light irradiation enhances photodynamic efficacy of ChlGal8 in bladder cancer cells. The resistance of HT-1376 cancer cells to PDT with ChlGal8 prompted us to investigate the optimization of treatment regimens by repeated dose of light irradiation in order to enhance the effectiveness of PDT (Figure 2a). In this sense, cells were subject-2 -2 ed to two irradiations for 40 min at 2.5 mW.cm (6 J.cm ) with a time interval of 1.5 h. The phototoxicity was determined 24 h after treatment. Data show that the repeated PDT treatment was similar to the single PDT treatment in UM-UC-3 bladder cancer cells (Figure 2b). In HT-1376 bladder cancer cells, the repeated PDT treatment was shown to be more effective (Figure 2b).

Figure 2. Repeated PDT treatment with ChlGal8 improves phototoxicity in resistant HT-1376 bladder cancer cells. (a) Schematic illustration of single and repeated PDT experiments performed with ChlGal8 in bladder cancer cells. (b) Cytotoxicity was evaluated 24 h after single and repeated PDT with ChlGal8 in HT-1376 and UM-UC-3 cancer cells, using the MTT assay. Data are means ± s.e.m. of at least three independent experiments performed in triplicates. *P< 0.05, **P< 0.01, ***P< 0.001 compared to MTT reduction (%) at 24 h after PDT for control cells (untreated cells) using a $ $$ Student´s t test. P plasma membrane > cytosol (Figure S15, see Supporting Information, and Figure 3a). The distribution of ChlGal8 was similar in the subcellular fractions obtained before first and repeated PDT (Figure S15, see Supporting Information) in UM-UC-3 bladder cancer cells. In HT-1376 bladder cancer cells, there was an increase of ChlGal8 in the mitochondrial fraction obtained before repeated PDT (Figure 3a).

Figure 3. ChlGal8 amount increases in the mitochondrial fraction in the time between single and repeated PDT. (a) Distribution of ChlGal8 in the subcellular fractions “nucleus (N) plus cell debris”, mitochondria (MT), plasma membrane (PM) and cytosol (Cy) of HT-1376 cancer cells, obtained before single and repeated PDT treatments. Data are means ± s.e.m. of at least three independent experiments performed in triplicates. * P< 0.05, **P< 0.01 compared to uptake (nmol PS/mg protein) of ChlGal8 before single PDT for the respective subcellular fraction using a Student´s t test. (b,c) Western blot analysis of lamin B (nuclear marker), GLUT1, β-actin (cytosol marker), Tom20 (mitochondrial marker) and ZO-1 (plasma membrane marker) in the subcellular fractions of HT-1376 bladder cancer cells, obtained before (b) single and (c) repeated PDT. The increase of ChlGal8 in the mitochondrial fraction of HT-1376 cells was accompanied with a decrease of this PS in the “nucleus plus cell debris” fraction. Interestingly, Western blot analysis of GLUT1 demonstrated that in the period of time between single and repeated PDT there is an increase of GLUT1 protein in the mitochondrial fraction of HT-1376 cells (Figure 3c). In control experiments where HT-1376 bladder cancer cells were incubated with PBS and then repeated PDT was performed, no changes were observed in GLUT1 protein

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present in the mitochondria-enriched fraction (Figure S16, see Supporting Information). To determine whether the preferential accumulation of ChlGal8 in the mitochondria before repeated PDT is involved on the increment of mitochondrial ROS production and further enhancement of phototoxicity, additional experiments were performed with the mitochondria-targeted superoxide scavenger (MitoTEMPO) and with the fluorescent mitochondrial probe for highly reactive species, the mitochondria 32 peroxy yellow 1 (MitoPY1). Overall, we found that phototoxicity was attenuated when PDT assays were performed in HT-1376 cells incubated with MitoTEMPO (Figure S17a, see Supporting Information). Additionally, the increase in phototoxicity after repeated PDT with ChlGal8 in HT-1376 cells demonstrated to be dependent on the production of mitochondrial ROS (namely hydrogen peroxide), as detected with MitoPY1 (Figure S17b, see Supporting Information). ChlGal8 promotes translocation of GLUT1 to the mitochondria after a single irradiation and induces high phototoxicity after repeated irradiation. GLUT1 is ubiqui33 tously expressed in most mammalian cells. Although the role of GLUT1 in the mitochondria is not completely clear, it is speculated that GLUT1 is mostly involved on the transport of the oxidized form of vitamin C, the dehydroascorbic 34 acid. Once inside mitochondria, dehydroascorbic acid is 33 reduced to ascorbic acid that quenches ROS, inhibits mitochondrial membrane depolarization and protects mitochon34 dria from oxidative injury. To determine whether GLUT1 plays a role in PDT efficacy with ChlGal8, small interfering RNA (siRNA) was used to knockdown GLUT1 within HT-1376 cancer cells (Figure S18a, see Supporting Information). No significant differences were detected on the uptake of ChlGal8 by HT-1376 cells after knockdown of GLUT1 protein (Figure S18b, see Supporting Information). As we did not achieve a full protein knockdown, the amount of GLUT1 present in cancer cells after treatment with siRNA could be enough to mediate ChlGal8uptake in HT-1376 cancer cells .Interestingly, knockdown of GLUT1 strongly reduced both ROS production and phototoxicity after repeated irradiation (Figure S18c,d see Supporting Information). Furthermore, we showed that GLUT1 siRNA prevented the irradiation-induced increase of ChlGal8 in the mitochondrial fraction of HT-1376 cells (Figure S19, see Supporting Information). These results collectively point to an antioxidant response of HT-1376 cancer cells after single PDT treatment, which stimulates the translocation of GLUT1 to the mitochondria. As a first stage, it is expected that GLUT1 translocation to the mitochondria contributes to a protective response against ROS-mediated damage. However, upon accumulation of ChlGal8 in mitochondria, if a repeated PDT treatment occurs, this PS is highly efficient in inducing phototoxicity. Repeated light irradiation treatment with ChlGal8 induces ultrastructural alterations in mitochondria of bladder cancer cells. Mitochondria have been recognized as a key target for the initiation of apoptosis in PDT. ChlGal8bound to mitochondria can generate large amounts of ROS after irradiation, which can lead to mitochondrial damage triggering cell death signaling cascades and the mi35 tochondria-dependent apoptosis. Ultrastructural alterations in mitochondria of HT-1376 cells were evident after PDT with ChlGal8 (Figure S20, see Supporting Information). TEM images of control cells display normal mitochondria with evi-

dent cristae and intact membrane. After single and repeated PDT there are alterations in this organelle shape. Particularly, after repeated PDT treatment the mitochondria are smaller with mitochondrial cristae and membrane disruption. Repeated light irradiation treatment with ChlGal8 improves the in vivo photodynamic efficacy against HT1376 tumors inoculated in nude mice. Given that ChlGal8 was demonstrated to be a powerful PS in in vitro assays, we next conducted a proof-of-concept study to demonstrate its increased efficacy after a repeated irradiation treatment in a xenograft tumor. Subcutaneous xenograft HT-1376 tumors were used to evaluate the PDT efficacy of ChlGal8 after single and repeated treatments (Figure 4a).

Figure 4. Repeated PDT treatment with ChlGal8 improves the in vivo photodynamic efficacy against HT-1376 tumors inoculated in nude mice. (a) Schematic illustration of single and repeated PDT experiments performed in vivo with ChlGal8. (b) Tumor volumes in control mice, and mice treated with single and repeated PDT at days 1-15 after treatment. Values are means ± s.e.m. (n = 6 mice per group). In single PDT treatment, the HT-1376 tumors were treated with a single dose of light (>500 nm) with a fluence rate of -2 -2 30 mW.cm for 20 min, i.e. 36 J.cm ) at 1.5 h after intra-1 tumoral injection of ChlGal8 (3.33 µmol.kg ). In repeated PDT treatment, the tumors were irradiated for an additional -2 time (36 J.cm ) at 40 min after the first PDT session. As illustrated in Figure 4b, a single intratumoral administration of ChlGal8 and exposure to repeated photo-irradiation was significantly more efficacious in the inhibition of tumor growth than single shot photoirradiation. The tumor growth of mice treated with single treatment was slightly suppressed after PDT, however, accelerated after 6 days and exhibited almost no difference to the control group at the endpoint. CONCLUSIONS In summary, we have successfully developed a galactodendritic chlorin which exhibits excellent properties as PS: i) water solubility, ii) interaction with galectin-1 protein 36, 37 which is overexpressed in many tumor types, iii) ability 1 to generate O2, iv) photostability and v) interaction with the most abundant protein of blood plasma (HSA). Importantly, ChlGal8 exhibits a more than 50-fold increase in the extinction coefficient at the lowest-energy Q band relative to the 16 previously reported porphyrin PorGal8. In vitro, a single dose of ChlGal8 and irradiation induced high photosensitivity in UM-UC-3 bladder cancer cells sensitive to therapy. We further elucidated, using both in vitro and in vivo assays, that a repeated irradiation treatment (instead of the traditional single irradiation treatment) could improve phototoxicity with ChlGal8 in HT-1376 bladder cancer cells resistant to PDT. The particular ability of ChlGal8 to be accumulated in

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the mitochondria in the period of time between single and repeated PDT treatment provides new insights into the optimization of treatment regimens by fractionated dosing of light irradiation to achieve enhanced PDT efficacy (Figure S21, see Supporting Information). EXPERIMENTAL PROCEDURES Synthesis of the photosensitizers PorGal8, ChlF20 and ChlGal8. PorGal8 and ChlF20 were synthesized as previously 16, 18, 19 reported and details are described in Supporting Information. ChlGal8 synthesis was performed into two steps, conjugation of ChlF20 with four equivalents of the previously 16 reported galactodendritic unit and cleavage of the isopropylidene acetals in a mixture of trifluoroacetic acid (TFA)/water (H2O) (9:1 v/v). ChlGal8 characterization – Nuclear Magnetic Resonance (NMR) spectroscopy, mass spectrometry, photophysical and -chemical assays – was performed as described in detail in Supporting Information. Stock solutions of the PSs were prepared at a concentration of 2 mM in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St Louis, MO, USA). Working solutions were freshly prepared by phosphate-buffered saline (PBS)-dilution of the stock solution. Cells culture. Human bladder cancer cell lines UM-UC-3 and HT-1376 derived from high-grade transitional cell carcinoma were obtained from the American Type Culture Collection (ATCC®, Manassas, VA, USA). Cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Sig-1 ma) supplemented with 2 g.L sodium bicarbonate (Sigma), 2 -1 mM.L glutamine (Sigma), 10% (v/v) of heat-inactivated Fetal Bovine Serum (FBS; Life Technologies, Carlsbad, CA, USA) -1 and antibiotic/antimicotic containing 100 units.mL penicil-1 -1 lin, 100 µg.mL streptomycin and 0.25 µg.mL amphotericin B (Sigma). UM-UC-3 and HT-1376 cells were seeded at a den4 5 sity of 3 x 10 and 4 x 10 cells per well in 96- and 6-well culture plates (Orange Scientific, Braine-l'Alleud, Belgium), respectively. Twenty-four hours after plating, cells were incubated with the desired concentrations of ChlGal8 in the dark for the indicated period of time. Cellular uptake of ChlGal8. After incubation with ChlGal8 in the dark, UM-UC-3 and HT-1376 cells were immediately washed with PBS buffer and mechanically scrapped in 1% (m/v) sodium dodecyl sulfate (SDS; Sigma) in PBS buffer at pH 7.0. ChlGal8 intracellular concentration was determined by spectrofluorimetry using a microplate reader (Synergy HT, Biotek, Winooski, VT, USA) with the excitation and emission filters set at 360±40 nm and 645±40 nm, respectively, and the results were normalized for protein concentration (determined by bicinchoninic acid reagent; Pierce, Rockford, IL, USA). Single and repeated PDT on cells. Photodynamic irradiation was carried out in fresh culture medium, in the absence of PS, covering UM-UC-3 and HT-1376 cell monolayers with RPMI medium and exposing them to light > 500 nm delivered by the illumination system LC-122 LumaCare at 2.5 -2 -2 mW.cm for 40 min (i.e. 6 J.cm ). Single PDT was performed by incubation of bladder cancer cells for 1.5 h with the desired concentration of PS and then irradiated with red light -2 at 2.5 mW.cm for 40 min. Repeated PDT was performed 1.5 h after single photo-irradiation of the cells with red light at -2 2.5 mW.cm for 40 min. Sham-irradiated cells, used as controls, consisted of cells kept in the dark for the same dura-

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tions and under the same environmental conditions as the irradiated cells. In all treatments, triplicate wells were established under each experimental condition, and each experiment was repeated at least three times. MTT assay. Cell metabolic activity after ChlGal8 incubation in the dark, irradiation, or both was determined 24, 48 or 72 h after treatments by measuring the ability of bladder cancer cells to reduce 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl-tetrazolium bromide (MTT, Sigma), to a colored formazan using a microplate reader (Synergy HT). The data were expressed in percentage of control (i.e. optical density of formazan from cells not exposed to ChlGal8). IC50 values (i.e. concentration of ChlGal8 required to reduce cell viability by 50% as compared to the control cells) were calculated using non-linear regression analysis to fit dose-response curves in GraphPad Prism 5.0 software (La Jolla, CA, USA). Detection of intracellular Reactive Oxygen Species (ROS) generation. Immediately after irradiation or shamirradiation, cancer cells were washed twice with PBS and incubated with either 2 or 5 μM of 2′,7′dichlorodihydrofluorescein diacetate (H2DCFDA; Invitrogen Life Technologies, Carlsbad, CA, USA) or MitoPY1 (Sigma) for an additional 1 h period, at 37 ºC, protected from light. After incubation, cells were washed with PBS and mechanically scrapped in 1% (m/v) SDS solution in PBS (pH 7.0). DCF (oxidized H2DCFDA) fluorescence was determined using a microtiter plate reader (Synergy HT) with the excitation and emission filters set at 485±20 nm and 528±20 nm, respectively. For the MitoPY1 probe, the excitation and emission filters used were 485±20 nm and 590±35 nm, respectively. Protein concentration was determined using the Pierce® BCA Protein Assay Kit. Redox quenching studies. Before single or repeated PDT, culture medium containing 10 µM of MitoTEMPO (Sigma) was added to cell monolayers. The effect of MitoTEMPO on cell viability or ROS generation was evaluated using the aforementioned assays. Transfection assays. GLUT1 was depleted in HT-1376 human bladder cancer cells using a pool of three targetspecific 20-25 nucleotides siRNA (Santa Cruz Biotechnology, 17 Inc., Santa Cruz, CA, USA), as previously reported. HT-1376 cells were transfected in 6- or 96-well culture plates, at 6080% confluence, with GLUT1 hsiRNA. Cells were also transfected with a scrambled siRNA in parallel as controls. For each transfection, cells were treated for 5 h with 2.4 µM of siRNA in transfection medium (Santa Cruz) containing 0.5 -2 µL.cm of transfection reagent (Santa Cruz). After incubation, complete media was added and the cells were incubated for 24 h. GLUT1 downregulation was evaluated 24 h posttransfection by Western blot. The uptake and PDT experiments were performed 24 h post-transfection with GLUT1 hsiRNA. Cellular fractionation. Cellular fractionation was per31 formed as previously described with minor modifications. After single and repeated PDT, the cells were washed twice with ice-cold PBS and lysed with ice-cold hypotonic buffer (10 mM NaCl, 1.5 mM MgCl2 and 10 mM Tris-HCl at pH 7.5). The cell lysates were transferred to microtubes with the help of a rubber policeman (cell scrapper) and incubated on ice during 10 min. The cell suspension was then transferred into a Dounce homogenizer (Thomas Scientific, NJ, USA). To disrupt the membranes, 40 up-and-down strokes were per-

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formed. The pellet containing the nuclei, intact cells and large membrane fragments was obtained after centrifugation at 1,300 g for 10 min at 4 ºC. The supernatant was recovered and transferred into clean microtubes which were then centrifuged at 17,000 g during 20 min at 4º C to obtain the mitochondrial fraction. The pellet rich in plasma membrane and the supernatant rich in cytosolic fraction were obtained after centrifugation at 20,000 g during 2h. After centrifugations, the pellets were resuspended in 100 µL of ice-cold hypotonic buffer. The cellular fractions were used for determination of ChlGal8 fluorescence and for protein concentration determination, followed by denaturation with Laemmli buffer. Western Blot. Total cellular extracts or cellular fractions of UM-UC-3 and HT-1376 cells were obtained as described in detail in Supporting Information or above, respectively. Primary antibody probing was performed with rabbit antiGalectin1 1:1,000 (Abcam, Cambridge, UK), rabbit anti-GLUT1 1:1,000 (Chemicon, Boston, MA, USA), mouse anti-Lamin B 1:100 (Oncogene Research Products, Cambridge, MA), mouse anti β-actin 1:20,000 (Sigma), rabbit anti-Tom20 (Santa Cruz Biotechnology) and rabbit anti ZO-1 (Invitrogen Life Technologies) antibodies. After washing, the membranes were probed with secondary anti-goat, anti-rabbit or anti-mouse IgG-HRP-linked antibodies 1:10,000 (Bio-Rad). The fluorescent signals were detected by enhanced chemiluminiscence (ECL) substrate using an imaging system (VersaDoc 4000 MP, Bio-Rad) followed by densitometric analysis. Transmission Electron Microscopy (TEM). After single and repeated PDT treatment, pellet cells were obtained by centrifugation at 3,000 g for 5min. Supernatant was discarded and pellet cells were fixed with 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer (pH 7.2) supplemented with 1 mM calcium chloride for 2h. Following rinsing in cacodylate buffer, post-fixation and contrast enhancement was performed using 1% osmium tetroxide followed by 1% aqueous uranyl acetate, for 1 h. After rinsing in distilled water, samples were dehydrated in a graded ethanol series (30–100%), impregnated and embedded using an Epoxy embedding kit (Fluka Analytical). Ultrathin sections were mounted on copper grids and stained with lead citrate 0.2%, for 7min. Observations were carried out on a FEI-Tecnai G2 Spirit Bio Twin at 80kV. Mice. Male Swiss nude (nu/nu) mice were purchased from Charles River (National Cancer Institute Frederick) and maintained under specific-pathogen-free conditions in the animal care facility of IBILI, Faculty of Medicine, University of Coimbra (Coimbra, Portugal). We used a total of 24 animals in the experiments (divided into 4 experimental groups, n = 6 mice per group) which were 5 to 8 weeks of age and weighed 20-25 g at the start of the experiments. In vivo study design. Based on previous studies, describing HT-1376 bladder cancer cells to develop a xenograft tumor model (please see Supporting Information for additional details about the development of the xenograft tumor mod38 el), we determined the sample size using G*Power 3.1 for all 39 power calculations. We required groups of at least 6 mice to achieve appropriate power for anticancer analysis (effect size: a 50% decrease in tumor volume at 5 days after treatment with PDT; standard deviation: 80; significance level: 5%; desired power of the experiment: 80%). When the volume (please see Supporting Information for additional detail about assessment of tumor volume) of xenografts reached

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approximately 50 mm (normally 10 days after cells inoculation), the tumor-bearing mice were randomly divided into the following 4 groups (n = 6 per group): -2

Group I) no ChlGal8 injection and PDT at 36 J.cm (i.e. 30 -2 mW.cm for 20 min, negative control); -1

Group II) 3.33 µmol.kg of ChlGal8 injected intratumorally and no PDT (mice treated with ChlGal8 in darkness); -2

Group III) PDT at 36 J.cm performed 1.5 h after intra-1 tumoral injection of 3.33 µmol.kg of ChlGal8 (single PDT treatment group); -2

Group IV) PDT at 36 J.cm performed 40 min after single PDT treatment (repeated PDT treatment group). On day 0 (4-5 pm), the animals of negative control, single PDT treatment group and repeated PDT treatment group -1 were anesthetized (2 mg.kg intraperitoneal cocktail of a 2:1 -1 50 mg.mL ketamine in 2.5% chlorpromazine) and injected -1 intratumorally with 3.33 µmol.kg ChlGal8 (freshly prepared in PBS with a percentage of DMSO of 0.3%) by using a sterile syringe with a 21-gauge needle. On the same day, 1.5 h after injection of ChlGal8 the tumors of animals, from single and repeated PDT treatment groups, were light irradiated. Light irradiation (white light 400-800 nm) was performed with the illumination system LC-122 LumaCare) with light > 500 nm. The tumors of mice from repeated PDT treatment group were light irradiated for a second time, 40 min after the first irradiation time. PDT treatments were performed with mice under a heat pad to maintain body heat and only the tumor areas were irradiated. As the PDT experiments were performed with mice outside of microbiological safety cabinets; simple measures (use of latex gloves and face masks, disinfection of the working bench with 90% alcohol for 3 min) were taken to prevent infections. One day after treatments, the mice were monitored daily and the tumor volume was measured as described in Supporting Information. Early endpoints were applied during the experiments to increase the precision of the results and to decrease non40 specific systemic effects. The mice were euthanized (cervical dislocation)and subjected to necropsy examination when the tumor volume of the control and single PDT treatment 3 groups reached approximately 500 mm (normally at days 9 and 11 after treatment, respectively), when ulcerating lesions occurred, when an animal had lost > 10% of its body weight, or when a mouse appeared seriously ill. On Day 15, all remaining animals were sacrificed and subjected to a gross necropsy examination. The observations and assessments of the treated groups (single and repeated PDT treatment groups) were compared with the negative control groups (negative control groups I and II). Statistical analysis. G*Power 3.1 was used for all power 39 calculations. Statistical analyses were carried out using a statistics program (GraphPad Prism; GraphPad Software). Student’s t-test was used to compare the treatment effects with that of control. P-value was considered at the 5% level of significance to deduce inference of the significance of the data. All graphs and statistics were prepared using the GraphPad Prism 5.0 software.

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ASSOCIATED CONTENT Experimental details for the synthesis and characterization of galactodendritic chlorin ChlGal8, photo-physical and chemical properties, and in vitro and in vivo studies. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT Thanks are due to FCT/MEC for the financial support to QOPNA (FCTUID/QUI/00062/2013), IBILI (FCTUID/NEU/04539/2013) and CQE (FCTUID/QUI/0100/2013) research units, through national funds and where applicable cofinanced by the FEDER, within the PT2020 Partnership Agreement. This work was also supported by ACIMAGO (ref. 12/12). The authors also acknowledge FCT for the postdoctoral and doctoral research grants SFRH/BPD/64812/2009 (to SS) and SFRH/BD/85941/2012 (to PMRP), respectively.

ABBREVIATIONS PDT, photodynamic therapy; PS, photosensitizer; ROS, reactive oxygen species; GLUT1, glucose transporter 1; nuclear magnetic resonance, NMR; human serum albumin, HSA; small interfering RNA; siRNA.

REFERENCES 1. Celli, J. P., Spring, B. Q., Rizvi, I., Evans, C. L., Samkoe, K. S., Verma, S., Pogue, B. W., and Hasan, T. (2010) Imaging and photodynamic therapy: mechanisms, monitoring, and optimization. Chem. Rev. 110, 2795-2838. 2. Dolmans, D. E. J. G. J., Fukumura, D., and Jain, R. K. (2003) Photodynamic therapy for cancer. Nat. Rev. Cancer. 3, 380-387. 3. Agostinis, P., Berg, K., Cengel, K. A., Foster, T. H., Girotti, A. W., Gollnick, S. O., Hahn, S. M., Hamblin, M. R., Juzeniene, A., Kessel, D., et al. (2011) Photodynamic therapy of cancer: An update. CA Cancer J. Clin. 61, 250-281. 4. Castano, A. P., Mroz, P., and Hamblin, M. R. (2006) Photodynamic therapy and anti-tumour immunity. Nat. Rev. Cancer. 6, 535-545. 5. Oleinick, N. L., Morris, R. L., and Belichenko, T. (2002) The role of apoptosis in response to photodynamic therapy: what, where, why, and how. Photochem. Photobiol. Sci. 1, 1-21. 6. Rosenkranz, A. A., Jans, D. A., and Sobolev, A. S. (2000) Targeted intracellular delivery of photosensitizers to enhance photodynamic efficiency. Immunol. Cell Biol. 78, 452-464. 7. Szewczyk, A., and Wojtczak, L. (2002) Mitochondria as a pharmacological target. Pharmacol. Rev. 54, 101-127. 8. Pathania, D., Millard, M., and Neamati, N. (2009) Opportunities in discovery and delivery of anticancer drugs targeting mitochondria and cancer cell metabolism. Adv. Drug Deliv. Rev. 61, 1250-1275.

Page 8 of 10

9. Zhao, Z. X., Chan, P. S., Li, H. G., Wong, K. L., Wong, R. N. S., Mak, N. K., Zhang, J., Tam, H. L., Wong, W. Y., Kwong, D. W. J., et al. (2012) Highly selective mitochondria-targeting amphiphilic silicon(IV) phthalocyanines with axially ligated Rhodannine B for photodynamic therapy. Inorg. Chem. 51, 812821. 10. Xu, J., Zeng, F., Wu, H., Hu, C., and Wu, S. (2014) Enhanced photodynamic efficiency achieved via a dual-targeted strategy based on photosensitizer/micelle structure. Biomacromolecules. 15, 4249-4259. 11. Zhang, S., Yang, L., Ling, X., Shao, P., Wang, X., Edwards, W. B., and Bai, M. (2015) Tumor mitochondria-targeted photodynamic therapy with a translocator protein (TSPO)specific photosensitizer. Acta Biomater. 12. Allison, R. R., and Sibata, C. H. (2010) Oncologic photodynamic therapy photosensitizers: A clinical review. Photodiagnosis Photodyn. Ther. 7, 61-75. 13. Senge, M. O., and Brandt, J. C. (2011) Temoporfin (Foscan®, 5,10,15,20-Tetra(m-hydroxyphenyl)chlorin)-a secondgeneration photosensitizer. Photochem. Photobiol. 87, 12401296. 14. Lu, K., He, C., and Lin, W. (2015) A chlorin-based nanoscale metal-organic framework for photodynamic therapy of colon cancers. J. Am. Chem. Soc. 137, 7600-7603. 15. Singh, S., Aggarwal, A., Thompson, S., Tomé, J. P. C., Zhu, X. C., Samaroo, D., Vinodu, M., Gao, R. M., and Drain, C. M. (2010) Synthesis and photophysical properties of thioglycosylated chlorins, isobacteriochlorins, and bacteriochlorins for bioimaging and diagnostics. Bioconjugate Chem. 21, 2136-2146. 16. Silva, S., Pereira, P. M. R., Silva, P., Paz, F. A. A., Faustino, M. A. F., Cavaleiro, J. A. S., and Tomé, J. P. C. (2012) Porphyrin and phthalocyanine glycodendritic conjugates: synthesis, photophysical and photochemical properties. Chem. Commun. 48, 3608-3610. 17. Pereira, P. M. R., Silva, S., Cavaleiro, J. A. S., Ribeiro, C. A. F., Tomé, J. P. C., and Fernandes, R. (2014) Galactodendritic phthalocyanine targets carbohydrate-binding proteins enhancing photodynamic therapy. PLoS One. 9. 18. Silva, A. M. G., Tomé, A. C., Neves, M. G. P. M. S., Silva, A. M. S., and Cavaleiro, J. A. S. (2005) 1,3-dipolar cycloaddition reactions of porphyrins with azomethine ylides. J. Org. Chem. 70, 2306-2314. 19. Silva, A. M. G., Tomé, A. C., Neves, M. G. P. M. S., Silva, A. M. S., and Cavaleiro, J. A. S. (1999) mesoTetraarylporphyrins as dipolarophiles in 1,3-dipolar cycloaddition reactions. Chem. Commun. 1767-1768. 20. Williams, A. T. R., Winfield, S. A., and Miller, J. N. (1983) Relative fluorescence quantum yields using a computercontrolled luminescence spectrometer. Analyst. 108, 1067-1071. 21. Zhang, Y.-Z., Zhou, B., Zhang, X.-P., Huang, P., Li, C.H., and Liu, Y. (2009) Interaction of malachite green with bovine serum albumin: Determination of the binding mechanism and binding site by spectroscopic methods. J. Hazard. Mater. 163, 1345-1352. 22. Yang, F., Zhang, Y., and Liang, H. (2014) Interactive association of drugs binding to Human Serum Albumin. Int. J. Mol. Sci. 15, 3580-3595. 23. He, X. M., and Carter, D. C. (1992) Atomic-structure and chemistry of human serum-albumin. Nature. 358, 209-215. 24. López-Lucendo, M. F., Solís, D., André, S., Hirabayashi, J., Kasai, K., Kaltner, H., Gabius, H. J., and Romero, A. (2004) Growth-regulatory human galectin-1: Crystallographic characterisation of the structural changes induced by single-site mutations and their impact on the thermodynamics of ligand binding. J. Mol. Biol. 343, 957-970. 25. Iglesias, M. M., Rabinovich, G. A., Ivanovic, V., Sotomayor, C., and Wolfenstein-Todel, C. (1998) Galectin-1 from

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Bioconjugate Chemistry

ovine placenta - Amino-acid sequence, physicochemical properties and implications in T-cell death. Eur. J. Biochem. 252, 400407. 26. D'Auria, S., Petrova, L., John, C., Russev, G., Varriale, A., and Bogoeva, V. (2009) Tumor-specific protein human galectin-1 interacts with anticancer agents. Mol. Biosyst. 5, 1331-1336. 27. Gegg, C. V., Roberts, D. D., Segel, I. H., and Etzler, M. E. (1992) Characterization of the adenine binding-sites of two Dolichos Biflorus lectins. Biochemistry. 31, 6938-6942. 28. Dhallewin, M. A., Baert, L., Marijnissen, J. P. A., and Star, W. M. (1992) Whole bladder wall photodynamic therapy with in situ light dosimetry for carcinoma in situ of the bladder. J. Urol. 148, 1152-1155. 29. Mishra, P. P., Patel, S., and Datta, A. (2006) Effect of increased hydrophobicity on the binding of two model amphiphilic chlorin drugs for photodynamic therapy with blood plasma and its components. J. Phys. Chem. B. 110, 21238-21244. 30. Afonso, J., Santos, L. L., Miranda-Gonçalves, V., Morais, A., Amaro, T., Longatto-Filho, A., and Baltazar, F. (2014) CD147 and MCT1-potential partners in bladder cancer aggressiveness and cisplatin resistance. Mol. Carcinogen. 54, 1451-1466. 31. Rangel, R., Dobroff, A. S., Guzman-Rojas, L., Salmeron, C. C., Gelovani, J. G., Sidman, R. L., Pasqualini, R., and Arap, W. (2013) Targeting mammalian organelles with internalizing phage (iPhage) libraries. Nat. Protoc. 8, 1916-1939. 32. Dickinson, B. C., Srikun, D., and Chang, C. J. (2010) Mitochondrial-targeted fluorescent probes for reactive oxygen species. Curr. Opin. Chem. Biol. 14, 50-56. 33. Vatassery, G. T., Smith, W. E., Quach, H. T., and Lai, J. C. K. (1995) In vitro oxidation of vitamin E, vitamin C, thiols and cholesterol in rat brain mitochondria incubated with free radicals. Neurochem. Int. 26, 527-535. 34. Sagun, K. C., Cárcamo, J. M., and Golde, D. W. (2005) Vitamin C enters mitochondria via facilitative glucose transporter 1 (GLUT1) and confers mitochondrial protection against oxidative injury. Faseb J. 19, 1657-1667. 35. Green, D. R., and Kroemer, G. (2004) The pathophysiology of mitochondrial cell death. Science. 305, 626-629. 36. Liu, F.-T., and Rabinovich, G. A. (2005) Galectins as modulators of tumour progression. Nat. Rev. Cancer. 5, 29-41. 37. Thijssen, V. L., Heusschen, R., Caers, J., and Griffioen, A. W. (2015) Galectin expression in cancer diagnosis and prognosis: A systematic review. BBA - Rev. Cancer. 1855, 235-247. 38. Amit, D., and Hochberg, A. (2010) Development of targeted therapy for bladder cancer mediated by a double promoter plasmid expressing diphtheria toxin under the control of H19 and IGF2-P4 regulatory sequences. J. Transl. Med. 8. 39. Faul, F., Erdfelder, E., Buchner, A., and Lang, A. G. (2009) Statistical power analyses using G*Power 3.1: Tests for correlation and regression analyses. Behav. Res. Methods. 41, 1149-1160. 40. Workman, P., Aboagye, E. O., Balkwill, F., Balmain, A., Bruder, G., Chaplin, D. J., Double, J. A., Everitt, J., Farningham, D. A. H., Glennie, M. J., et al. (2010) Guidelines for the welfare and use of animals in cancer research. Brit. J. Cancer. 102, 15551577.

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