Supplementary Figure Legends s11

1 SUPPLEMENTARY FIGURE LEGENDS

Supplementary Figure 1. Detection and characterization of HEp3 DTCs in lungs, BM and lymph nodes.

(a) Number of disseminated HEp3-GFP-tagged cells (DTCs - HEp3-GFP) found in live lungs suspensions and

BM flushes, 4 weeks after surgery of the primary tumour (PT). n=16 (BM), 35 (LU) mice per conditions. (b)

Upper panel: representative live fluorescence microscopy image of a lymph node 4 weeks after surgery of the

PT. Scale bar: 160 µm. Lower graph: number of disseminated HEp3-GFP cells in the lymph node of nude mice

4 weeks after PT surgery. n=15 mice. (c) BM was collected from nude mice, and single cell suspensions were prepared as described in methods. Q-PCR for Alu sequences was performed on genomic DNA extracted from the indicated number of HEp3 cells serially diluted into individual mice BM cell suspensions. Q-PCR for Alu sequences was used to generate a standard curve from 10 to 103 HEp3 cells / BM by plotting the AluCt- mGAPDHCt against the number of cells per bone marrow (n=3 BM samples per condition were assessed over

3 independent experiments) (d) Alu PCR was used to amplify Alu sequences in human (HEp3) DNA in BM cell suspensions from mice that carried HEp3 tumours and whose BM suspensions were apparently negative for

HEp3-GFP cells when examined under the fluorescence microscope. The standard curve shown in c was used to calculate the number of HEp3 cells in these BM. (n= 3 (1,4,7,9,10), n=2 (5, 6) Alu Ct values from independent qPCRs) see supplementary table S4 for the statistic source data. (e) Life fluorescence images from lungs at the time of surgery. (S= lung stroma). Scale bar: 160 µm. (f) Representative cleaved caspase-3

(C-C3) staining in PT-HEp3 (PT) (upper right panel), Lu-HEp3 (Lu, upper left panel) and BM-D1 (middle left panel) tumours. Scale bar: 40 µm. Representative P-H3 (right middle panel) and p27 (right bottom panel) staining in PT-HEp3 (PT) tumours. Scale bar: 40 µm. Arrows= positive cells. Lower left graph: Quantification of marker-positive cells. (n=100 cells assessed/section. 15 sections assessed from 3 different tumour/ condition).

FOV=field of view. (g) Serial passage of BM-D1 cells on CAMs. Generation matched primary tumour (PT)

HEp3 cells from the same animal were inoculated on CAM at (2x105 cells/animal) and transplanted weekly onto a new CAM (n=5 (PT), 6 (BM) tumour nodules). (h) Dormant (BM-D1) and tumorigenic (BM-T1) BM-HEp3 cells were injected subcutaneous (s.c.) in nude mice (5x105 cells / animal). Graph: time, in days, it took for the tumours to reach 100mm3. n=2 (BM-T1) 3 (BM-D1) mice, p=0.0878 by unpaired t test. See supplementary table S4 for the statistic source data. (i) Scheme adapted from (Kim, et al., 1998)25: 2.5*105 T-HEp3 GFP cells were inoculated on the chorioallantoic membrane (CAM) of d10 chicken embryos. 5 days after inoculation the 2 embryos were sacrificed. The BM, liver and lung were isolated and inspected under the fluorescence microscope for GFP cells. (j) Number of disseminated HEp3-GFP-tagged cells (DTCs - HEp3-GFP) found in the chicken embryo lungs, liver and BM 5 days after inoculation. n=10 (liver), 10 (lung), 7 (BM) Chicken embryos assessed. (k) Serial passage of BM-D2,-D3 and -D4 cells on CAMs for two weeks. n=8 (BM-D2, w1),

6 (BM-D3, w1), 8 (BM-D4, w1), 7 (PT, w1), 4 (BM-D2, w2), 3 (BM-D3, w2), 3, (BM-D4, w2) tumour nodules assessed in 2 independent experiments. See supplementary table S4 for the statistic source data. Data in c, d, f, g, h and k, represent mean s.e.m. In a, f, g and k, *p<0.05, **p<0.01 and ***p<0.001 by Mann Whitney test. In j *p<0.05, **p<0.01 and ***p<0.001 by One-way ANOVA-Bonferroni's multiple comparison test

Supplementary Figure 2. DEC2 and p53 expression in HNSCC and breast cancer cell lines and in

HNSCC patient samples. (a) Lysates from 4T1, 4T07 and F3II cell lines were probed by immunoblot (IB) for the indicated antigens. Numbers on top of blots = [ERK/p38] ratio quantification (b-c) QPCR for DEC2 (b) and p53 (c) mRNAs in the indicated cell lines after 24 h in culture, n=6 RNA samples per condition were assessed over 3 independent experiments, error bars denote s.e.m., *p<0.05, **p<0.01, ***p<0.001 by Mann Whitney test. (d) Left panel: p38 immunoblot in scrambled (scr) or p38-siRNA transfected BM-D1 cells. Right and middle panels: qPCR for DEC2 and p53 mRNA in BM-D1 cells 24 h after transfection with the indicated siRNAs. n=2 RNA samples (middle panel) 3 RNA samples (right panel) per condition were assessed over 2 independent experiments, *p<0.05 by Mann Whitney test. See supplementary table S4 for the statistic source data. (e) IB anti-HA (left panel) and anti-V5 (right panel) in Lu-HEp3 cells transfected with GFP, p38 constitutively active construct (p38-CA-HA) or a DEC2-V5 construct. (f) P-H3 (left column) and Cleaved

Caspase-3 (C-C3) (right column) immunohistochemistry in Lu-HEp3 cells transfected with vector (upper panels) or a p38 constitutively active construct (p38-CA-HA) (lower panels). Scale bar: 40 µm.

Quantification of marker-positive cells is shown in the graphs on the right panels. Y axis shows the number of positive cells (for either Cleaved Caspase-3 (C-C3) or P-H3) per field of view (FOV). Graph: mean ± s.e.m.

(n=100 cells assessed/section. 15 sections assessed from 3 different tumour/ condition) **p<0.01 by Mann

Whitney test. (g) Representative DEC2 staining in primary tumours (upper row) and lymph node metastasis

(lower row) from 4 HNSCC patients. (S= stroma, T=primary tumour, M=Metastasis). Scale bar: 120 µm. 3 Supplementary Figure 3. Effect of pharmacologic and genetic p38 inhibition on HEp3 DTC and metastasis burden. (a) Representative images of Vimentin (Vim), p27, P-H3 and Cytokeratin8/18 (CK) staining in solitary liver (left column) and BM DTCs (right column). Arrows = marker positive cells. Scale bar:

40 µm. (b) Tumour volume after 5 days inoculation on CAMs of T-HEp3 cells transfected with either scrambled

(scr) or p38 siRNA. n=4 tumour nodules per condition, p=0.3830 by Mann Whitney test. (c) IB for p38 in T-

HEp3 cells transfected with either scrambled (scr) or p38 siRNA. (d) Representative fluorescence intravital images of liver (upper panels) DTCs in chicken embryos 5 days after inoculation of T-HEp3-GFP cells transfected with scrambled (scr) (left panels) or p38 (right panels) siRNA. Scale bar: 160µm. Lower Graph: quantification of HEp3 DTCs in chicken embryo livers 5 days after inoculation of T-HEp3-GFP cells transfected with scrambled (scr) or p38 siRNAs. n=7 chicken embryo livers analyzed per condition/ 2 independent experiments. p=0.2610 by Mann Whitney test. (e) Left panel: Representative image of Vimentin staining on a

T-HEp3 tumour. Scale bar: 40 µm. Right panel: IB for Vimentin in T-HEp3 (T) and D-HEp3 (D) lysates.

Supplementary Figure 4. TGFβ1 & 2 expressions and signalling in HEp3-derived cell lines. (a) Affymetrix array values for the expression of TGFβ2 mRNA in D-HEp3, T-HEp3 and R-HEp3 (D-HEp3 cells that resumed growth after a prolonged dormancy ~ 2 months). (b) D-HEp3 cells were treated with a TGF receptor I inhibitor

(LY-364947, 5 µM) for 2, 4 and 6 h in serum free conditions and whole cell lysates were immunoblotted for P- p38 and p38. (c) qPCR analysis of DEC2 mRNA expression in D-HEp3 cells treated with the TGFRI inhibitor

(LY-364947, 5 µM) for 24 h in serum free conditions. n=6 RNA samples per condition were assessed over 3 independent experiments, error bars denote s.e.m., *p<0.05 by Mann Whitney test. (d) D-HEp3 cells were treated with the TGFRI inhibitor (LY-364947, 5 µM) for 24 h and then inoculated on CAM (2x105cells / animal) and then treated with the LY-364947, 5 µM on the CAM every 24 h. 4 days later tumours were minced and the number D-HEp3 cells quantified in collagenase suspensions. Graph: number of cells/tumour, n=6 tumour nodule assessed per condition, p=0.0011 by Mann Whitney test. (e) qPCR for DEC2 in 4T1 cells treated with

BM CM for 24 h. n=6 RNA samples per condition were assessed over 3 independent experiments. Error bars denote s.e.m., *p<0.05 by Mann Whitney test (f) Representative cleaved caspase-3 (C-C3) staining in T-HEp3 tumours after treatment with either serum free, lung CM or BM CM for 4 days in vivo. Scale bar: 40 µm. Lower right graph: quantification of Cleaved Caspase-3 (C-C3) positive cells. Y axis: number of C-C3 positive cells 4 per field of view (FOV), mean ± s.e.m. (n=100 cells assessed/section. 15 sections assessed from 3 different tumour/ condition). (g) Top panel: IB against TGF1 after control IgG or anti-TGF1 IgG immunodepletion of the BM CM. Lower panel: Tumour growth of T-HEp3 cells treated with full (IP-IgG) or TGF1 immunodepleted

(IP-TGF1) BM CM for 5 days in vivo, n=6 (SF), 8 (IP-IgG), 9 (IP-TGFb1) tumour nodules assessed, *p<0.05 by One-way ANOVA-Bonferroni's multiple comparison test. Right panel: IB showing p38 activation after treatment of T-HEp3 cells with full (IP-IgG) or TGF1 depleted (IP-TGF1) BM CM for 24 h.

Supplementary Figure 5. TGFRIII knockdown effect on TGF2 signalling. (a) DEC2 mRNA levels in 4T1 and F3II cell lines after treatment with 2 ng/ml TGF2 for 24 h in SF media. n=6 RNA samples per condition were assessed over 3 independent experiments. Error bars denote s.e.m., *p<0.05 by Mann Whitney test. (b)

Q-PCR for TGFβ2 mRNA expression in BM-D1 cells 24 h after transfection with TGF2 siRNAs. n=6 RNA samples per condition were assessed over 3 independent experiments. Error bars denote s.e.m., *p<0.05 by

Mann Whitney test. (c) Graph: number of 4T1 tumour spheres in control and TGF2 (2 ng/ml) treated cultures after 7 days (n= 6 wells per condition were assessed for tumour spheres/ 3 different experiments) Right column panels: representative images of control (upper panel) and TGF2 treated (lower panel) 4T1 tumour spheres. Scale bar: 100 µm. (d-e) Effect of SB203580 (5 µM) or LY364947 (5 µM) treatment in 4T07 tumour spheres cultures. The graph shows the number of tumour spheres as compared to control cells after 7 days (d) and the number of cells per sphere (e) (n= 6 wells per condition were assessed for tumour spheres / 3 different experiments). Error bars denote s.e.m., **p<0.01 by Mann Whitney test. Representative images of 4T07 tumour spheres in control, 5 µM SB203580 (SB) and 5 µM LY-364947 (LY) treated cultures after 7 days. Scale bar: 100 µm. (f) Affymetrix array values for the expression of Type I, II and III TGFβ receptors mRNA in T-

HEp3 cells. (g) qPCR for TGF receptor III (TGFβR3) in T-HEp3 cells treated with 2 ng/ml TGF2 for 24 h in the presence or absence of TGF receptor III siRNA (siTGFβR3), n=6 RNA samples per condition were assessed over 3 independent experiments. Error bars denote s.e.m., *p<0.05 by Mann Whitney test (h) IB for the indicated antigens on T-HEp3 cell lysates after treatment with 2 ng/ml TGFβ2 for 24 h in SF media and inhibition of TGFβ receptor III (TGFβR3).