Supplementary materials - Cancer Chemotherapy and Pharmacology Computed determination of the in vitro optimal chemocombinations of sphaeropsidin A with chemotherapeutic agents to combat melanomas Aude Ingels1, Carina Dinhof2,3, Abhishek D. Garg4 , Lucia Maddau5, Marco Masi6, Antonio Evidente6, Walter Berger 2,3 , Bieke Dejaegher7 and Véronique Mathieu1 1 Laboratoire de Cancérologie et Toxicologie Expérimentale, Université Libre de Bruxelles, Boulevard du Triomphe, Accès 2, 1050 Ixelles, Belgium. 2 Department of Medicine I, Institute of Cancer Research, Medical University Vienna, Spitalgasse 23, 1090 Vienna, Austria 3 Comprehensive Cancer Center, Medical University Vienna, Spitalgasse 23, 1090 Vienna, Austria 4 Laboratory for Cell Death Research and Therapy (CDRT), Department of Cellular and Molecular Medicine, KU Leuven University, Leuven, Belgium. 5 Dipartimento di Agraria, Sezione di Patologia vegetale ed Entomologia, Università degli Studi di Sassari, Viale Italia 39, 07100, Sassari, Italy. 6 Dipartimento di Scienze Chimiche, Universita’ di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Napoli, Italy. 7 Laboratoire d’Analyse Instrumentale et de Bioélectrochimie, Université Libre de Bruxelles, Boulevard du Triomphe, Accès 2, 1050 Ixelles, Belgium Correspondence to: Véronique Mathieu, MD, PhD Laboratoire de Cancérologie et Toxicologie Expérimentale – Faculté de Pharmacie – Université Libre de Bruxelles (ULB) Campus de la Plaine – Boulevard du Triomphe – 1050 Brussels – Belgium. Tel: +32 478 317 388 E-mail: [email protected] 1 Supplementary materials - Cancer Chemotherapy and Pharmacology Concentrations (µM) Cisplatin Sph A 0 0 N°Exp 1-3 Sph A 0 TMZ 0 4-6 6 0 6 0 7-9 0 100 0 1000 10-12 6 100 6 1000 13-15 0 50 0 500 16-18 6 50 6 500 19-21 3 0 3 0 22-24 3 100 3 1000 25-30 3 50 3 500 31 2 35 2 350 32 4 40 4 400 33 3 75 3 750 Table 1: Factorial experimental design performed to establish the model for sphaeropsidin A (Sph A)/ cisplatin and Sph A/ temozolomide (TMZ) combinations and tested on each cell line. 9 combinations of concentrations (µM) including the central point and 3 test points permit to build the model. 2 Supplementary materials - Cancer Chemotherapy and Pharmacology A B C Mean B16F10 ln(B16F10) 16.045 2.021 Standard Deviation 27.592 1.085 Minimum Maximum Centrum 2.076 0.731 100.000 4.605 51.038 2.668 Figs 1: Example of dissymmetric distribution of B16F10 dataset for sphaeropsidin A and cisplatin. Boxplot for B16F10 data before (A) and after logarithm transformation (B). Table C includes all absolute data visualized above. 3 Supplementary materials - Cancer Chemotherapy and Pharmacology Sph A and cisplatin Sph A and temozolomide Sph A and cisplatin Sph A and temozolomide Pre-treatment Y = 0.618 + 0.201 * X1 + 0.149 * X2 - 0.125 * (X1*X1) 0.135 * (X2*X2) - 0.038 * (X1*X2) Y= 0.319 + 0.251 * X1 + 0.096 * X2 - 0.010 * (X1*X1) 0.009 * (X2*X2) + 0.031 * (X1*X2) Co-treatment Y= 0.740 + 0.148 * X1 + 0.189 * X2 - 0.106 * (X1*X1) 0.156 * (X2*X2) - 0.105 * (X1*X2) Y= 0.631 + 0.252 * X1 + 0.093 * X2 - 0.212 * (X1*X1) 0.055 * (X2*X2) - 0.033 * (X1*X2) Table 2: The second-order polynomial equations built from experimental data to predict relationship between concentrations of each drug and their combined response. X1 and X2 are the coded values for [sphaeropsidin A (Sph A)] and [Cisplatin/Temozolomide], b0 is the intercept coefficient, b1/ b2 are the linear coefficients, b1-1/ b2-2 are the quadratic coefficients, and b1-2 is the factor interaction coefficient. 4 Supplementary materials - Cancer Chemotherapy and Pharmacology Pre-treatment Cisplatin Y optimal 75 0.7247 70 0.7245 80 0.7223 65 0.7215 70 0.7172 85 0.7172 75 0.7162 60 0.7159 65 0.7155 80 0.7126 B Co-treatment Sph A Cisplatin Y optimal 1 4 75 0.8149 2 5 70 0.8139 3 4 70 0.8138 4 5 65 0.8131 5 4 80 0.8128 6 5 75 0.8114 7 4 65 0.8096 8 5 60 0.8092 9 4 85 0.8076 10 5 80 0.8058 Pre-treatment Sph A TMZ Y optimal 1 6 1000 0.68 2 6 950 0.67 3 6 900 0.66 4 6 850 0.65 5 6 800 0.63 6 6 750 0.62 7 6 700 0.61 8 6 650 0.60 9 5 1000 0.59 10 6 600 0.59 D Co-treatment TMZ Y optimal 850 0.73 800 0.73 900 0.73 750 0.73 950 0.72 700 0.72 850 0.72 900 0.72 1000 0.72 650 0.72 A Sph A 1 5 2 5 3 5 4 5 5 6 6 5 7 6 8 5 9 6 10 6 C Sph A 1 5 2 5 3 5 4 5 5 5 6 5 7 4 8 4 9 5 10 5 Tables 3: Summary tables of the ten best combinations of compounds (in µM) ranked according to the best predicted response (Y optimal). Combinations of sphaeropsidin A (Sph A) with cisplatin in pre-treatment and co-treatment conditions are presented in A and B respectively. Combinations of Sph A with temozolomide (TMZ) in pre-treatment and cotreatment conditions are presented in C and D respectively. 5 Supplementary materials - Cancer Chemotherapy and Pharmacology Co-treatment with Sph A Pre-treatment with Sph A B Cisplatin A D Temozolomide C Figs 2: 2D contour plots obtained showing the isobole lines as a function of the levels of two factors: sphaeropsidin A (Sph A) and cisplatin (A-B) or temozolomide (C-D) concentrations. Two modes of sensitization are presented: pre-treatment (A-C) and cotreatment (B-D). 6