Fig. 4.

DKK3 promotes the pro-tumorigenic behaviour of CAFs in vivo. a Cartoon describing the experimental set-up used to assess the tumour-promoting potential of CAFs in vivo by subcutaneous co-injection of TS1 murine cancer cells and indicated CAFs. b Graph showing the volumes of tumours in syngeneic mice (FVB/n) at the indicated days post-injection. Lines represent mean ± SEM (n = 7 or more individual tumours). c Survival curves for tumours as described in (b), representing the percentage of animals alive at the indicated time point after injection. Individual p values are shown. d Representative images showing Masson’s trichrome, fibronectin (FN), laminin and αSMA staining of indicated tumours at day 14 post-injection. Scale bars, 100 µm. e Charts show quantification of positive areas relative to total areas for stainings in (d). Bars indicate mean ± SEM (n = 4 individual tumours except for WT-CAF tumours that n = 5). f Representative images of intravital imaging of tumours generated by co-injection of TS1 cancer cells (red) with CAF-WT or CAF-KO.9 CAFs (green) in CD-1 nude mice at day 20. Collagen fibres (blue) were imaged by SHG. Asterisk indicates areas of cancer cell invasion at the tumour border. Scale bars, 100 µm. Tukey boxplot shows collagen content as calculated by SHG intensity, n = 24 or more fields of view at the tumour border from 3 independent tumours. g Representative time-lapse images from intravital imaging of TS1 (red) and CAF-WT (green) tumours in CD-1 nude mice. Collagen fibres (blue) were imaged by SHG. Zoom-up areas with cancer cell movement associated to a CAF (Area 1, asterisk), cancer cell movement towards a CAF-enriched area (Area 1, arrow) and CAF movement (Area 2, hashtag) are also shown. Scale bar, 50 µm. h Graph showing analysis of cancer cell and CAF movement by intravital imaging. Bars indicate average number of moving cells per field of view ± SEM (CAF-WT, n = 23 fields of view from 3 independent tumours; CAF-KO, n = 14 fields of view from 2 independent tumours). For all graphs, *P < 0.05; **P < 0.01; ***P < 0.001; #P < 0.0001; n.s., non-significant