The role of galectin-1 in in vitro and in vivo photodynamic therapy with a galactodendritic porphyrin  Patrícia M.R. Pereira, Sandrina Silva, José S. Ramalho, Célia M. Gomes, Henrique Girão, José A.S. Cavaleiro, Carlos A.F. Ribeiro, João P.C. Tomé, Rosa Fernandes  European Journal of Cancer  Volume 68, Pages 60-69 (November 2016) DOI: 10.1016/j.ejca.2016.08.018 Copyright © 2016 Elsevier Ltd Terms and Conditions

Fig. 1 Hypothetic illustration of phototoxicity of PorGal8 (a porphyrin with dendritic units of galactose) in in vitro and in vivo biological models containing high levels of galectin-1 protein. European Journal of Cancer 2016 68, 60-69DOI: (10.1016/j.ejca.2016.08.018) Copyright © 2016 Elsevier Ltd Terms and Conditions

Fig. 2 Galectin-1 plays a role in the uptake of PorGal8 (a porphyrin with dendritic units of galactose) by HT-1376 and UM-UC-3 bladder cancer cells. (A) Fluorescence variation on the emission spectra of galectin-1 after addition of PorGal8. Data are means ± standard error of the mean (SEM) of two independent experiments. (B) Intracellular uptake of PorGal8 by HT-1376 and UM-UC-3 bladder cancer cells. Data are means ± SEM of at least three independent experiments performed in triplicates. (C) Western blotting analysis of galectin-1 protein in HT-1376 and UM-UC-3 bladder cancer cells. β-actin was used as a loading control. (D) Knockdown of galectin-1 in UM-UC-3 bladder cancer cells as determined by Western blotting 48 h post-transfection. (E) Intracellular uptake of PorGal8 by UM-UC-3 bladder cancer cells transfected with galectin-1 small-interfering RNA (siRNA). **P < 0.01 compared to non-transfected control cells using a Student's t test. Data are means ± SEM of at least three independent experiments performed in triplicates. European Journal of Cancer 2016 68, 60-69DOI: (10.1016/j.ejca.2016.08.018) Copyright © 2016 Elsevier Ltd Terms and Conditions

Fig. 3 Photodynamic therapy (PDT) with PorGal8 (a porphyrin with dendritic units of galactose) induces cytotoxicity in HT-1376 and UM-UC-3 bladder cancer cells. (A and B) Cytotoxicity 24 h after PDT with PorGal8 was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and trypan blue staining assays, respectively. *P < 0.05, **P < 0.01, ***P < 0.001 compared to MTT reduction (%) or excluded trypan blue (%) at 24 h after PDT for the respective control using a Student's t test. Data are means ± standard error of the mean (SEM) of at least three independent experiments performed in triplicates. (C) Photocytotoxic effects after PDT with PorGal8 in UM-UC-3 cells transfected with galectin-1 small-interfering RNA (siRNA). *P < 0.05, **P < 0.01, ***P < 0.001 compared to MTT reduction (%) at 24 h after PDT for the respective non-transfected cells using a Student's t test. Data are means ± SEM of at least three independent experiments performed in triplicates. (D) Quantification of terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL)-positive cells at 24 h after PDT with PorGal8 obtained from counts of randomly chosen microscopic fields. **P < 0.01 compared to control cells and $P < 0.05 compared to TUNEL-positive HT-1376 cells at 24 h after PDT using the multiple pairwise Kruskal–Wallis analysis of variance. European Journal of Cancer 2016 68, 60-69DOI: (10.1016/j.ejca.2016.08.018) Copyright © 2016 Elsevier Ltd Terms and Conditions

Fig. 4 PorGal8 (a porphyrin with dendritic units of galactose) generates reactive oxygen species (ROS) after photodynamic therapy (PDT). (A) Quantification and (B) representative fluorescence images of dichlorofluorescein (DCF) increase (as a measure of ROS production) in HT-1376 and UM-UC-3, after PDT with PorGal8. Scale bars, 20 μm. **P < 0.01 compared to control cells using a Student's t test. Data are means ± standard error of the mean (SEM) of at least three independent experiments performed in triplicates. (C) Phototoxicity after PDT with PorGal8 in the presence of 50 nM of ROS quenchers (sodium azide, histidine and cysteine) in HT-1376 and UM-UC-3 cells. Cytotoxicity was assessed 24 h after treatment using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The percentage of cytotoxicity was calculated relatively to control cells (untreated cells). ***P < 0.001 compared to MTT reduction (%) 24 h after PDT with PorGal8 using a Student's t test. Data are means ± SEM of at least three independent experiments performed in triplicates. (D) Oxygen consumption in HT-1376 and UM-UC-3 cells after PDT with PorGal8. *P < 0.05 and $$P < 0.01compared to control cells and HT-1376 cells after PDT, respectively, using the multiple pairwise Kruskal–Wallis analysis of variance. Data are means ± SEM of at least three independent experiments performed in triplicates. European Journal of Cancer 2016 68, 60-69DOI: (10.1016/j.ejca.2016.08.018) Copyright © 2016 Elsevier Ltd Terms and Conditions

Fig. 5 PorGal8 (a porphyrin with dendritic units of galactose) induces changes on the pattern of filamentous actin (F-actin) staining of bladder cancer cells after photodynamic therapy (PDT). (A and B) Representative fluorescence images of F-actin (stained with Phalloidin-TRICT, green) in cancer cells before and at 30 min and 24 h after PDT with PorGal8, with 4′,6-diamidino-2-phenylindole (DAPI) staining the nucleus (blue). Scale bars, 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article). European Journal of Cancer 2016 68, 60-69DOI: (10.1016/j.ejca.2016.08.018) Copyright © 2016 Elsevier Ltd Terms and Conditions

Fig. 6 PorGal8 (a porphyrin with dendritic units of galactose) has an anti-cancer photodynamic effect on UM-UC-3luc+ tumours inoculated in nude mice. (A) Tumour growth inhibition by photodynamic therapy (PDT) with PorGal8 in UM-UC-3luc+ tumours. *P < 0.05, **P < 0.01 compared to the control group using a Student's t test. Data are means ± standard error of the mean (n ≥ 6 mice in each group). (B) Representative photographs of dissected tumours in control group and animals treated with PDT. Scale bars, 5 mm. Tumour volumes in untreated mice and mice treated with PDT at 12 d post PDT with PorGal8. **P < 0.01 compared to the control group using a Student's t test. (C) Representative fluorescence images of Ki-67 protein in UM-UC-3luc+ tumours of control and treated group. Scale bars, 20 μm. (D) Western blotting analysis of E-cadherin protein in UM-UC-3luc+ tumours in control and treated group. GAPDH was blotted as loading control. (E) Representative fluorescence images of Phalloidin-TRICT in control and treated group. Scale bars, 20 μm. European Journal of Cancer 2016 68, 60-69DOI: (10.1016/j.ejca.2016.08.018) Copyright © 2016 Elsevier Ltd Terms and Conditions