FIGURE SUMMARY
Title

Heterogeneous Tumor Subpopulations Cooperate to Drive Invasion

Authors
Chapman, A., Fernandez Del Ama, L., Ferguson, J., Kamarashev, J., Wellbrock, C., Hurlstone, A.
Source
Full text @ Cell Rep.

Heterogeneity Results in Cooperative Invasion

(A) Western blot showing MITF expression in WM266-4 and 501mel cells.

(B) Homogeneous xenografts imaged at 1 (upper) and 4 days (lower) postinjection (dpi).

(C) Heterogeneous xenografts imaged at 1 (upper) and 4 dpi (lower).

Arrows indicate directions of invasion; arrowhead indicates autofluorescence.

(D) Section from engrafted embryo indicating primary tumor site (white dashed line) and infiltrating melanoma cells (white arrows). Scale bars represent 100 μm.

(E) Quantitation of invasion depicted in (A) and (B); mean ± SEM; Kruskal-Wallis test followed by Dunn’s multiple comparisons test; p < 0.01; n e 26 from three independent experiments.

MMP Inhibition Suppresses Cooperative Invasion

(A) Homogeneous (upper) or heterogeneous (bottom) xenografts were treated with either the vehicle control DMSO (left) or protease inhibitor cocktail (right). Scale bars represent 100 μm.

(B) Quantitation of 501mel invasion depicted in (A); mean ± SEM; Kruskal-Wallis test followed by Dunn’s multiple comparisons test; p < 0.01; n e 9 from three independent experiments.

(C) Quantitation of WM266-4 invasion depicted in (A); mean ± SEM; Kruskal-Wallis test followed by Dunn’s multiple comparisons test; p < 0.05; n e 13 from three independent experiments.

(D) Western blot showing MT1-MMP expression in WM266-4 transfected with either control or MT1-MMP specific siRNA.

(E) Quantitation of invasion of WM266-4 cells in homogeneous xenografts wherein WM266-4 cells have been transfected with either control or MT1-MMP-specific siRNA; mean ± SEM; Mann-Whitney test; n e 21 from three independent experiments.

(F) Quantitation of invasion of WM266-4 and 501mel in heterogeneous xenografts wherein WM266-4 cells have been transfected with either control or MT1-MMP specific siRNA; mean ± SEM; Kruskal-Wallis test followed by Dunn’s multiple comparisons test; p < 0.0001; n e 24 from three independent experiments.

A Diffusible Factor Emanating from 501mel Cells Modulates WM266-4 Cell Response to Protease Inhibitors

(A) Cartoon depicting experimental set-up, with WM266-4 spheroids being cocultured either with autologous cells or heterologous cells in porous transwells.

(B) Representative images of WM266-4 spheroids cocultured either with WM266-4 or 501mel cells in the presence of a cocktail of protease inhibitors.

ECM Proteins Correlate with Invasiveness

(A) Expression of ECM components collagen I and fibronectin in engrafted zebrafish 4 dpi; arrows indicate direction of invasion.

(B) Western blot showing collagen I and fibronectin expression in WM266-4 and 501mel cells.

(C) Collagen I (upper) and fibronectin (lower) in homogeneous compared to heterogeneous xenografts that are further treated with either DMSO or GM6001.

(D) Invasive WM266-4 and 501mel follow collagen I (upper) and fibronectin (lower) tracks radiating out from the tumor.

(E) Quantitation of ECM association. Cells were scored as being in touch “on” with collagen I or fibronectin strands or not “off.” Mean ± SEM; unpaired Student’s t test (collagen) and Mann-Whitney test (fibronectin); p < 0.0001; n e 18 from three independent experiments.

Fibronectin Is Essential for Cooperative Invasion; Invasive Primary Melanoma Cells Are Also Heterogeneous

(A) Western blot showing stable knockdown of fibronectin in WM266-4 GFP shFN cells. #1 and #2 are clones expressing independent shRNA targeting fibronectin. Control (con) cells express an irrelevant shRNA.

(B) Fibronectin associated with heterogeneous xenografts comprising either control WM266-4 cells (upper) or WM266-4 shFN#1 cells (lower).

(C) Quantitation of invasion of 501mel and WM266-4 cells from heterogeneous xenografts comprising either control WM266-4 cells, WM266-4 shFN#1, or WM266-4 shFN#2 cells. Mean ± SEM; Mann-Whitney test; p < 0.05, p < 0.001, p < 0.001; n e 18 from three independent experiments.

(D) MITF immunofluorescence in frozen sections of heterogeneous xenografts. Arrows indicate high and low MITF fluorescence intensity in invading cells.

(E) Model depicting the reciprocal interactions underlying cooperative invasion.

Zebrafish embryo melanoma xenografts. (Related to Figure 1.)

(A) A 48 hour post fertilisation zebrafish showing the pericardial cavity (white circle) as the site of xenograft injection. (B) GFP labelled WM266-4 melanoma cells injected into the pericardial cavity form a tumour-like mass capable of local invasion. Dashed line indicates plane of section in (C). (C) A cryo-section through the tumour like mass shown in (B) was labelled with DAPI (nuclei) and melanoma cells visualised through GFP fluorescence (weak autofluorescence is apparent at certain sites). The mass engrafted onto the body wall of the pericardial cavity. (D) UACC62 and 888mel homogeneous and heterogeneous xenografts. (E) Quantification of 888mel invasion depicted in (D) normalised to UACC62 invasion; mean ± SEM; Mann-Whitney test; **** = p < 0.0001; N e 26 from 3 independent experiments. (F) Western blot showing protein levels of MITF, MT1-MMP and fibronectin in UACC62 and 888mel cells. Scale bars = 100 μm.

Follower-leader behaviour underlies co-operative invasion in heterogeneous xenografts— a crucial role for MT1-MMP. (Related to Figure 2.)

(A) Quantification of file leader; mean ± SEM; Mann-Whitney test; * = p<0.05; N e 26 from 3 independent experiments. (B) Video time-lapse stills showing WM266-4 ‘leader’ cells and 501mel ‘follower’ cells (white arrows); scale bar = 50 μm. (C) Relative MMP expression determined by RT-qPCR from 3 independent experiments. Mean ± SEM; unpaired Student’s T-test; ** = p <0.01; *** = p < 0.001; **** = p < 0.0001. (D) Quantification of invasion of WM266-4 cells in homogeneous treated with either the vehicle control DMSO or GM6001; mean ± SEM; Mann-Whitney test; N ≥ 14 from 3 independent experiments. (E) Quantification of invasion of WM266-4 and 501mel cells in heterogeneous xenografts treated with either the vehicle control DMSO or GM6001; mean ± SEM; Kruskal-Wallis test followed by Dunn’s multiple comparisons test; ** = p<0.01; N e 17 from 3 independent experiments. (F) Representative images of WM266-4 cells transfected with either control or MT1-MMP specific siRNA in homogeneous and heterogeneous xenografts.

Characterisation of matrix deposition in xenografts. (Related to Figure 4.) (A) Cryosections of homogeneous or heterogenous xenografts treated either with the vehicle control DMSO, or the pan-MMP inhibitor GM6001 and stained for fibronectin (upper panels) and collagen I (lower panels). (B) Quantification of fibronectin fluorescence intensity volume normalised to WM266-4 (GFP) volume of homogeneous and heterogeneous xenografts; mean ± SEM; one-way ANOVA followed by Tukey’s multiple comparisons test; N ≥ 9 from 3 independent experiments. (C) Quantification of collagen I fluorescence intensity volume normalised to WM266-4 (GFP) volume of homogeneous and heterogeneous xenografts; mean ± SEM; Kruskal-Wallis followed by Dunn’s multiple comparisons test N e 9 from 3 independent experiments. (D) Western blot showing collagen I or fibronectin expression in either WM266-4 cells (middle panels) or 501mel (right panels) co-cultured either with autologous cells (hom) or heterologous cells (het; as depicted in the cartoon, left). Furthermore, cells were either treated with DMSO or a cocktail of protease inhibitors. (E) Wholemount immunofluorescence labelling of both fibronectin and collagen I in homogeneous WM266-4 xenografts at 4 dpi. Scale bars = 50 μm.

Fibronectin is not required for homogeneous WM226-4 cell invasion and collagen I deposition is independent of fibronectin. (Related to Figure 5.)

(A) Wholemount immunofluorescence labelling of fibronectin in homogeneous xenografts of control (upper panel) and shFN#1 WM266-4 cells (lower panel). Mean ± SEM; unpaired Student’s T-test; N ≥ 15 from 3 independent experiments. (B) Wholemount immunofluorescence labelling of fibronectin and collagen I in homogeneous xenografts of control (top panel) and shFN#1 (bottom panel) WM266-4 cells. Scale bars = 100 μm. (C) Western blot showing fibronectin and collagen I expression in control and shFN#1 WM266-4 cells. (D) Relative invasion quantification of (A).

Invading cells are heterogeneous. (Related to Figure 5.)

MITF expression in cells during human melanoma invasion. Boxed area is magnified and reveals heterogeneous MITF expression. (B) Images courtesy of the Human Protein Atlas http:// www.proteinatlas.org/ENSG00000187098/cancer/melanoma (Uhlen et al., 2010).

Acknowledgments
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