Extended Data Fig. 5: WNT5A promotes in vivo metastatic dormancy.

a, TCGA analysis of relative mRNA expression of WNT5A in patient tumour samples comparing primary vs metastatic (distant mets only) tissue sites and presented as a violin plot, with quartiles represented by black lines and the median by the broken line (N=104, 68). A student’s two-way t-test was performed. P = 0.003 b Western blot analysis was performed on protein lysate from Yumm1.7 doxycycline (Dox) inducible shWnt5a mCherry melanoma cells comparing control and Dox (0.5ug/ml) treated cells over 48 h. HSP90 was used as a loading control. c Yumm1.7 Dox-inducible shWnt5a mCherry cells were seeded at a density of 2x10⁴ and assessed for proliferation at days 3-5 and 10 using a haemocytometer and further assessed using automated counting via NIS elements. Experiment was performed in biological triplicates with 3 technical replicates each. A two-way ANOVA was performed with a post-hoc Holm-Sidak’s multiple comparisons test for each time point. P-values were taken from day 10. Data were presented as the mean +/− the SEM for each time point. P < 0.0001. d Tumour volumes from young mice (N = 9 for Dox Day 21, N = 8 for Dox Day 3 and No Dox due to mouse death) subdermally injected with 2.5x10⁵ Yumm1.7 Dox inducible shWnt5a mCherry melanoma cells. Mice were treated with Dox (2 mg/ml in water) from day 3 or day 21 onwards, with tumours measured for 5 weeks. A two-way ANOVA was performed with a post-hoc Holm-Sidak’s multiple comparisons test for each time point. P-values were taken from day 37. Data were presented as the mean +/− the SEM for each time point. **** P < 0.0001, ** P = 0.0038. e, f 5 young mice per group were intradermally injected with 2.5x10⁵ Yumm1.7 Dox inducible shWnt5a mCherry melanoma cells. Mice were treated with Dox (2 mg/mL in water) from day 3 (Dox day 3) or day 21 onwards (Dox day 21), with tumours measured for 5 weeks. Lungs were taken, PFA embedded and underwent IHC analysis of mCherry positive melanoma cells. Representative images are displayed across three independent mice for mCherry positive cells and Ki-67 with the scale bar representing 100um. Given that the no Dox and Dox day 3 groups failed to produce larger lesions, these groups were assessed for the average number of single cell colonies per high powered frame (20 x) across entire lung sections and presented as mean +/- SEM. A student’s two-way t-test was performed. P = 0.0224. g Tumour volumes from 6 aged mice subdermally injected with 2.5x10⁵ Yumm1.7 Dox inducible WNT5A overexpressing mCherry melanoma cells. Mice were treated with Dox (2 mg/ml in water) from day 3 or day 21 onwards, with tumours measured for 5 weeks. A two-way ANOVA was performed with a post-hoc Holm-Sidak’s multiple comparisons test for each time point. P-values were taken from day 37. Data were presented as the mean +/− the SEM for each time point. * P = 0.0235 comparing No Dox with Dox Day 3. h Yumm1.7 Dox-inducible WNT5A mCherry cells were seeded at a density of 2x10⁴ and assessed for proliferation at days 3-5 and 10 using a haemocytometer and further assessed using automated counting via NIS elements. Experiment was performed in biological triplicates with 3 technical replicates each. A two-way ANOVA was performed with a post-hoc Holm-Sidak’s multiple comparisons test for each time point. P-values were taken from day 10. Data were presented as the mean +/− the SEM for each time point. P < 0.0001. i, j 5 Aged mice per group were subdermally injected with 2.5x10⁵ Yumm1.7 Dox inducible WNT5A overexpressing mCherry melanoma cells. Mice were treated with Dox (2 mg/ml in water) from day 3 (Dox day 3) and day 21 (Dox day 21) with tumours measured for 5 weeks. Lungs were taken, PFA embedded and underwent IHC analysis of mCherry positive melanoma cells. Representative images are displayed across three independent mice for mCherry positive cells and Ki-67. Given that the Dox day 3 and Dox day 21 groups failed to produce larger lesions, these groups were further assessed for the average number of single cell colonies per high powered frame (20 x) across entire lung sections and presented as mean +/− SEM. A student’s two-way t-test was performed. P = 0.001.