Colorectal Cancer Growth Retardation Through Induction of Apoptosis, Using an Optimized Synergistic Cocktail of Axitinib, Erlotinib, and Dasatinib

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Article Colorectal Cancer Growth Retardation through Induction of Apoptosis, Using an Optimized Synergistic Cocktail of Axitinib, Erlotinib, and Dasatinib

Robert H. Berndsen 1,2, Nathalie Swier 2, Judy R. van Beijnum 2 and Patrycja Nowak-Sliwinska 1,3,*

axitinib erlotinib dasatinib 0 μM 0 μM 0 μM 0.16 μM 0.2 μM 0.002 μM 120 1.6 μM 2 μM 0.02 μM 100 6.4 μM 8 μM 0.1 μM 80 16 μM 20 μM 0.2 μM 60 40 20 0 metabolicactivity(%ctrl) CI

SW620 0.1 0.3 0.1 ODC 0.1 0.1 0.2 0.1 0.1 0.2 >1 0.3 0.1 >1 >1 >1 >1 6.4 - 8 - 0 - 6.4 0 - 8 - 0.1 1.6 - 2 - 0 0.02 0- 2 - HT29 0.3 0.4 0.6 0.2 16 - 20 0 0.1 0.4 0.4 0.3 0.2 0.2 0.5 0.2 >1 >1 >1 0.8 0 -20 - 0.2 16 - 0 - 0.2 0 - 16 - 0.1 - 8 6.4 0.1 - 0 6.4 1.6 2- - 0.02 1.6 0- - 0.02

DLD-1 0.1 0.6 0.4 0.4 0.2 0.5 0.4 0.3 0.3 >1 >1 0.2 0.6 0.8 0.8 >1 0 - 0.2 - 0.002 0.16 - 0 - 0.002 - 0 - 0.16 0.16 - 0.2 - 0.00 - 0.2 - 0.16

0.16 - 0.2 - 0.002 Figure S1. Combinations of axitinib, erlotinib and dasatinib synergistically inhibit colorectal cancer cell metabolic activity in a dose dependent manner. Metabolic activity of 2- and 3-drug combinations at multiple dose levels in CRC (SW620, HT29, DLD-1) cell lines. Color coded drug doses used include axitinib 0.16, 1.6, 6.4 and 16 μM, erlotinib 0.2, 2, 8 and 20 μM and dasatinib 0.002, 0.02, 0.1 and 0.2 μM and indicate dose reductions based on drug doses used on our previous study [1]. CI indicates Combination Index that was assessed with Compusyn software using the Chou-Talalay method [2]. CI values are showed below the graph. CI < 0.9 indicates synergism, CI = 0.9 indicates an additive effect and CI > 1 indicates antagonism. Metabolic activity was assessed after 72h of drug administration by the CellTiter-Glo® luminescence assay and represented as a percentage of control. Cells treated with 0.1% DMSO were used as control. Error bars indicate SEM.

Cancers 2019, 11, 1878; doi:10.3390/cancers11121878 www.mdpi.com/journal/cancers Cancers 2019, 11, 1878 2 of 4

axitinib erlotinib dasatinib 120 0 μM 0 μM 0 μM 100 6.4 μM 8 μM 0.1 μM 80 16 μM 20 μM 0.2 μM 60 40 20 0 metabolic(%ctrl) activity CI SW620 0.1 0.1 0.1 0.1 0.1 0.1 0.3 0.2 0.1 0.3 0.1 0.1 16 - 8 - 0 - 8 - 16 0 - 8 0.2 6.4- 20 0 0 - 20 0.1 16 - 8 0.2 16 - 8 0.1 16 - 0 0.1 6.4 - 0 0.2 6.4 - 8 0.2

HT29 0.2 0.1 0.316 - 20 - 0.1 0.4 0.1 0.1 0.4 0.3 0.4 0.5 0.3 0.3 6.4- 20 - 0.2 6.4- 20 - 0.1 DLD-1 0.2 0.2 0.3 0.3 0.2 0.3 0.5 0.6 0.4 0.9 0.4 0.2

Figure S2. Variation in dose ratios of axitinib, erlotinib and dasatinib combinations results in similar inhibition of colorectal cancer cell metabolic activity. Metabolic activity of varying dose ratios of 2- and 3-drug in CRC (SW620, HT29, DLD-1) cell lines. Color coded drug doses include axitinib 6.4 and 16 μM, erlotinib 8 and 20 μM and dasatinib 0.1 and 0.2 μM and are based on our previous study[1]. CI indicates Combination Index that was assessed with Compusyn software using the Chou-Talalay method [2]. CI values are showed below the graph. CI < 0.9 indicates synergism, CI = 0.9 indicates an additive effect and CI > 1 indicates antagonism. Metabolic activity was assessed after 72 h of drug administration by the CellTiter-Glo® luminescence assay and represented as a percentage of control. Cells treated with 0.1% DMSO were used as a control. Error bars indicate SEM.

A CYFIP2 SYK LCK EPHB2 SW620 PI3K/AKT signaling GNG4 ITPR2 FYN EFNB2 STC2 LGALS1 VIM Wnt signaling Apelin signaling GAL DPYSL3 SDC3 TNC NOTUM NES FGFR signaling Hippo signaling MDK FGF19 SAT1 GAS6 TUBB3 Angiogenesis

0123456 212 FGF3 FGFR1 SERPINE1 TMSB4X -Log(p-value) Log2(FC) SW620 vs HT29

PDGFA SPRY2

HT29 B Axon guidance Actin cytoskeleton EPH-Ephrin ECM-receptor interaction 214 Log2(FC) PI3K/AKT signaling HT29 vs SW620 Integrin signaling GCNT3 MUC5AC GALNT14 EGFR signaling

0 1 2 3 4 5 6 ST3GAL1 ST3GAL4 NOTCH2 -Log(p-value)

MMP7 CD44 ERRFI1 AGO2 ABLIM1 CTSH AP1M2

KITLG EGFR SRC YES1 ABLIM3 FTL LYZ

PTPRF IRS1 PVR PTK2 ARHGAP35 ASAP1 SDCBP IMPDH1

FAM49B TRAPPC9 AREG EREG ITGA3 EFNB1 EPHB4

PDIA6 SERPINA1 CD59 SLC44A2 ITGAV ITGA2 AGRN EXT1

PRSS23 TMED3 CD55 TMBIM1 ITGB6 LRP10

Figure S3. Differential gene expression analysis of HT29 and SW620. RNAseq data were analysed using DESeq2 in R for differentially expressed genes in SW620 (A) and HT29 (B). Gene lists were entered in String (String-db.org) and analysed for protein-protein interaction networks (confidence levels 0.7 and 0.9 for SW620 and HT29, respectively). Unconnected nodes were left out of the graphical representation. Nodes are color coded according to expression difference. Furthermore, gene lists 2 Cancers 2019, 11, 1878 3 of 4

were entered in Enrichr (http://amp.pharm.mssm.edu/Enrichr/) for enrichment analysis on ontologies and pathways. Significant and relevant (>5 genes per group) features are represented in the bar graphs ordered by p-value.

Figure S4. Hematoxylin and eosin staining of SW620 and HT29 CAM tumors treated with axitinib, erlotinib and dasatinib drug combination. Representative images of H&E stained tumor sections. Scale bars indicate 100 μm.

3 Cancers 2019, 11, 1878 4 of 4

Table S1. Comparison of the metabolic activity of SDC axitinib, erlotinib and dasatinib on CRC cells, PBMCs and HDFa.

SW620 HT29 DLD-1 PBMC [1] HDFa [2] SDC 15.2 ± 0.7 26.4 ± 1.8 13.8 ± 1.7 43.0 ± 2.2 48.0 ± 3.6 Metabolic activity was assessed after 72 h of drug administration by the CellTiter-Glo luminescence assay and represented as a percentage of control.

Table S2. Overview of methods used to grow tumors on the chicken chorioallantoic membrane (CAM) model.

Method CRC cell line

SW 620 H T29 D LD -1

H ig h tu m o r ta k e 5 x 106 C R C c e lls in 4 5 µL Matrigel

5 x 106 CRC cells in 45 µL Geltrex

2.5 x 106 CRC c e lls in 2 5 µ L G e ltre x

1 x 106 CRC cells in 25 µL Geltrex

1 x 106 CRC cells in 25 µ L G e ltr e x + 25 µL CM

1x 106 C RC cells in hanging drop spheroid

1 x 106 CRC + 0.5 x 106 H D F a in 2 5 µ L G e ltr e x

0.5 x 106 CRC c e lls in 2 5 µ L G e ltre x L o w tu m o r ta k e

Green areas indicate the best tested method in regard to tumor take, survival of embryo’s and tumor size. Red area’s indicate the opposite. White spaces indicate that the method was not evaluated.

References

1. Weiss, A, Berndsen, R.H.; Ding, X; Ho, C.-M.; Dyson, P.J.; van den Bergh, H.; Griffioen, A.W.; Nowak- Sliwinska, P. A Streamlined Search Technology for Identification of Synergistic Drug Combinations. Sci. Rep. 2015, 5, 14508. 2. Chou TC. Drug Combination Studies and their Synergy Quantification Using the Chou-Talalay Method. Cancer Res. 2010, 70, 440–446.