FIGS 1-3 NC3Rs F1000 submission 122987.xlsx

Data – tumour length and breadth (digital calipers) and mouse weight (digital scales) were collected daily and recorded in an Excel spreadsheet. <br> Tumour volume was estimated using the formula ((width*width)*length)/2 [Faustino-Rocha A, Oliveira PA, Pinho-Oliveira J, Teixeira-Guedes C, Soares-Maia R, da Costa RG, et al. Estimation of rat mammary tumor volume using caliper and ultrasonography measurements. Lab Anim (NY). 2013;42(6):217-24]. <br> Tumour length was also monitored, and mice in which tumour length reached 15mm (either individually or in total if tumours on both flanks) were sacrificed by a Schedule 1 method and blood, tissues and tumours harvested as appropriate for downstream analysis. <br> Mean, SD and SEM were calculated using Excel functions, and data were graphed in Excel. <br> Figure 1: Growth of a murine melanoma syngeneic graft in 8HUM mice and response to Dabrafenib treatment Adult female 8HUM_Rag2-/- mice (18-21w, n=5) were injected s.c. in one flank with 3.5 x 106 5555 murine melanoma cells, in 100 microlitresl ECM diluted 1:1 with DMEM. Tumours were allowed to establish and on day 5 after implantation daily treatment was commenced with either vehicle (0.5% (w/v) hydroxypropylmethylcellulose, 0.2% (v/v) Tween 80; closed circles) or dabrafenib methanesulfonate (in vehicle, open circles) suspended at 6.3mg/ml and administered at 5ml/kg, such that dabrafenib dose administered was 31.5mg/kg (arrow). Tumour measurements were taken three times weekly, then daily as required, and tumour volume calculated as detailed in Methods section. The study was terminated 15 days after implantation of cells. Data shown are mean tumour volume ± SEM. <br> <strong>Figure 2: Response of A375 human melanoma xenograft to dabrafenib treatment in 8HUM_Rag2-/-</strong> <strong>mice</strong> Adult female 8HUM mice (11-19w, n=3) were injected s.c. in both flanks with 4.4 x 106 A375 melanoma cells, in 100ml DMEM. Tumours were allowed to establish and on day 28 after implantation daily treatment was commenced with either vehicle (0.5% (w/v) hydroxypropylmethylcellulose, 0.2% (v/v) Tween-80; closed circles) or dabrafenib (in vehicle, open circles) suspended at 6.3mg/ml and administered at 5ml/kg, such that dabrafenib dose administered was 31.5mg/kg (arrow). Tumour measurements were taken three times weekly, then daily as required, and total volume of tumours on both flanks was calculated as detailed in Methods section. The study was terminated on d35 after implantation of cells, although one vehicle-treated animal had to be removed from the study on d29 as its total tumour size approached the maximum permitted under legislation. Data shown are mean tumour volume ± SEM. <br> <strong>Figure 3: Response of A375 human melanoma xenograft to trametinib treatment in 8HUM_Rag2-/-</strong> <strong>mice</strong> Adult female 8HUM mice (8-18w, n=3 or 4) were injected s.c. in one flank with 5 x 106 A375 melanoma cells, in 100ml DMEM. Tumours were allowed to establish and on day 22 after implantation daily treatment was commenced with either vehicle (0.5% (w/v) hydroxypropylmethylcellulose, 0.2% (v/v) Tween 80; closed circles) or trametinib (in vehicle, open circles) suspended at 0.07mg/ml and administered at 5ml/kg, such that trametinib dose administered was 0.35mg/kg (arrow). Tumour measurements were taken three times weekly, then daily as required, and tumour volume calculated as detailed in Methods section. Vehicle-treated mice were sacrificed on d30 after implantation of cells; trametinib treatment was discontinued (STOP) at that time for the drug-treated group to determine whether tumour re-growth would occur in the absence of drug. Data shown are mean tumour volume ± SEM.