Abstract
Disseminated cancer cells from primary tumours can seed in distal tissues, but may take several years to form overt metastases, a phenomenon that is termed tumour dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully characterize dormancy within melanoma. Here we show that the aged lung microenvironment facilitates a permissive niche for efficient outgrowth of dormant disseminated cancer cells—in contrast to the aged skin, in which age-related changes suppress melanoma growth but drive dissemination. These microenvironmental complexities can be explained by the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state1,2,3. It was previously shown that dermal fibroblasts promote phenotype switching in melanoma during ageing4,5,6,7,8. We now identify WNT5A as an activator of dormancy in melanoma disseminated cancer cells within the lung, which initially enables the efficient dissemination and seeding of melanoma cells in metastatic niches. Age-induced reprogramming of lung fibroblasts increases their secretion of the soluble WNT antagonist sFRP1, which inhibits WNT5A in melanoma cells and thereby enables efficient metastatic outgrowth. We also identify the tyrosine kinase receptors AXL and MER as promoting a dormancy-to-reactivation axis within melanoma cells. Overall, we find that age-induced changes in distal metastatic microenvironments promote the efficient reactivation of dormant melanoma cells in the lung.
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Data availability
Any data requested will be made available upon request. The mass spectrometry proteomics data have been deposited into the MassIVE (http://massive.ucsd.edu) and ProteomeXchange (http://www.proteomexchange.org) data repositories with accession numbers MSV000088977 and PXD032025, respectively.
Change history
07 August 2023
Editor’s Note: Readers are alerted that the reliability of some of the data presented in this manuscript is currently in question. Appropriate editorial action will be taken once this matter is resolved
References
Hoek, K. S. & Goding, C. R. Cancer stem cells versus phenotype-switching in melanoma. Pigment Cell Melanoma Res. 23, 746–759 (2010).
Fane, M. E., Chhabra, Y., Smith, A. G. & Sturm, R. A. BRN2, a POUerful driver of melanoma phenotype switching and metastasis. Pigment Cell Melanoma Res. 32, 9–24 (2019).
Kaur, A., Webster, M. R. & Weeraratna, A. T. In the Wnt-er of life: Wnt signalling in melanoma and ageing. Br. J. Cancer 115, 1273–1279 (2016).
Kaur, A. et al. sFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance. Nature 532, 250–254 (2016).
Webster, M. R. et al. Paradoxical role for wild-type p53 in driving therapy resistance in melanoma. Mol. Cell 77, 633–644 (2020).
Kaur, A. et al. Remodeling of the collagen matrix in aging skin promotes melanoma metastasis and affects immune cell motility. Cancer Discov. 9, 64–81 (2019).
Ecker, B. L. et al. Age-related changes in HAPLN1 increase lymphatic permeability and affect routes of melanoma metastasis. Cancer Discov. 9, 82–95 (2019).
Alicea, G. M. et al. Changes in aged fibroblast lipid metabolism induce age-dependent melanoma cell resistance to targeted therapy via the fatty acid transporter FATP2. Cancer Discov. 10, 1282–1295 (2020).
Pereira, C., Schaer, D. J., Bachli, E. B., Kurrer, M. O. & Schoedon, G. Wnt5A/CaMKII signaling contributes to the inflammatory response of macrophages and is a target for the antiinflammatory action of activated protein C and interleukin-10. Arterioscl. Thromb. Vasc. Biol. 28, 504–510 (2008).
Trevant, B. et al. Expression of secreted frizzled related protein 1, a Wnt antagonist, in brain, kidney, and skeleton is dispensable for normal embryonic development. J. Cell. Physiol. 217, 113–126 (2008).
Blakely, B. D. et al. Wnt5a regulates midbrain dopaminergic axon growth and guidance. PLoS ONE 6, e18373 (2011).
Webster, M. R. et al. Wnt5A promotes an adaptive, senescent-like stress response, while continuing to drive invasion in melanoma cells. Pigment Cell Melanoma Res. 28, 184–195 (2015).
Montagner, M. et al. Crosstalk with lung epithelial cells regulates Sfrp2-mediated latency in breast cancer dissemination. Nat. Cell Biol. 22, 289–296 (2020).
Ren, D. et al. Wnt5a induces and maintains prostate cancer cells dormancy in bone. J. Exp. Med. 216, 428–449 (2019).
Yumoto, K. et al. Axl is required for TGF-β2-induced dormancy of prostate cancer cells in the bone marrow. Sci. Rep. 6, 36520 (2016).
Lambert, A. W., Pattabiraman, D. R. & Weinberg, R. A. Emerging biological principles of metastasis. Cell 168, 670–691 (2017).
Sosa, M. S. et al. NR2F1 controls tumour cell dormancy via SOX9- and RARβ-driven quiescence programmes. Nat. Commun. 6, 6170 (2015).
Sosa, M. S., Bragado, P. & Aguirre-Ghiso, J. A. Mechanisms of disseminated cancer cell dormancy: an awakening field. Nat. Rev. Cancer 14, 611 (2014).
Gao, X. L., Zhang, M., Tang, Y. L. & Liang, X. H. Cancer cell dormancy: mechanisms and implications of cancer recurrence and metastasis. Onco. Targets Ther. 10, 5219–5228 (2017).
Gomis, R. R. & Gawrzak, S. Tumor cell dormancy. Mol. Oncol. 11, 62–78 (2017).
Weeraratna, A. T. et al. Wnt5a signaling directly affects cell motility and invasion of metastatic melanoma. Cancer Cell 1, 279–288 (2002).
Radaszkiewicz, T. et al. RNF43 inhibits WNT5A-driven signaling and suppresses melanoma invasion and resistance to the targeted therapy. eLife 10, e65759 (2021).
Luo, C. et al. H3K27me3-mediated PGC1α gene silencing promotes melanoma invasion through WNT5A and YAP. J. Clin. Invest. 130, 853–862 (2020).
O'Connell, M. P. et al. Hypoxia induces phenotypic plasticity and therapy resistance in melanoma via the tyrosine kinase receptors ROR1 and ROR2. Cancer Discov. 3, 1378–1393 (2013).
Sadeghi, R. S. et al. Wnt5a signaling induced phosphorylation increases APT1 activity and promotes melanoma metastatic behavior. eLife 7, e34362 (2018).
Wellbrock, C. & Arozarena, I. Microphthalmia-associated transcription factor in melanoma development and MAP-kinase pathway targeted therapy. Pigment Cell Melanoma Res. 28, 390–406 (2015).
Schoumacher, M. & Burbridge, M. Key roles of AXL and MER receptor tyrosine kinases in resistance to multiple anticancer therapies. Curr. Oncol. Rep. 19, 19 (2017).
Tworkoski, K. A. et al. MERTK controls melanoma cell migration and survival and differentially regulates cell behavior relative to AXL. Pigment Cell Melanoma Res. 26, 527–541 (2013).
Zhu, S. et al. A genomic screen identifies TYRO3 as a MITF regulator in melanoma. Proc. Natl Acad. Sci. USA 106, 17025–17030 (2009).
Lew, E. D. et al. Differential TAM receptor-ligand-phospholipid interactions delimit differential TAM bioactivities. eLife 3, e03385 (2014).
Davra, V., Kimani, S. G., Calianese, D. & Birge, R. B. Ligand activation of TAM family receptors—implications for tumor biology and therapeutic response. Cancers 8, 107 (2016).
Lemke, G. Biology of the TAM receptors. Cold Spring Harb. Perspect. Biol. 5, a009076 (2013).
Dutta, S. & Sengupta, P. Men and mice: relating their ages. Life Sci. 152, 244–248 (2016).
Grzelak, C. A. et al. Elimination of fluorescent protein immunogenicity permits modeling of metastasis in immune-competent settings. Cancer Cell 40, 1–2 (2022).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
Storey, J. D. & Tibshirani, R. Statistical significance for genomewide studies. Proc. Natl Acad. Sci. USA 100, 9440–9445 (2003).
Acknowledgements
We thank the Core Facilities of the Wistar Institute (supported by P30CA010815) and of the Johns Hopkins Kimmel Cancer Center (P30CA00697356). A.T.W., S.M.D., M.E.F. and G.M.A. are supported by R01CA174746 and R01CA207935. M.E.F., A.T.W. and J.A.A.-G. are also supported by a Team Science Award from the Melanoma Research Alliance. G.M.A., X.X., M.H. and A.T.W. are also supported by P01 CA114046. X.X. and M.H. are also supported by P50CA174523. M.R.W. is supported by R00CA208012. H.-Y.T. is supported by R50CA221838. V.W.R. is supported by K01CA245124. This work was supported in part by the Wistar Science Discovery Fund. A.T.W. is also supported by U01CA227550, R01CA232256, a Bloomberg Distinguished Professorship and the EV McCollum Endowed Chair. For samples from patients with melanoma, we thank L. M. Schuchter, T. C. Mitchell, R. K. Amaravadi, G. C. Karakousis, R. Elenitsas, C. Miller and M. E. Ming. We thank C. McQueen for editing of the final manuscript.
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Authors and Affiliations
Contributions
M.E.F. and A.T.W. conceived the study and designed experiments. J.A.A.-G. helped with the interpretation of all data and concepts underlying tumour dormancy. M.E.F. performed most of the experiments and was involved in most of the analyses. All tail-vein experiments (lung collection, handling and processing) were assisted by Y.C. and G.M.A. and the injections were performed by D.A.M. IHC experiments were assisted by Y.C. and G.M.A. Survival surgery was performed by G.M.A. and M.E.F. S.M.D., Y.C., G.M.A., V.W.R. and M.R.W. assisted with mouse experiments (mouse injections, tumour measurements, weighing mice and tissue collection). Sandwich reconstructions (Fig. 1i, j, Extended Data 1f–h) were performed by M.E.F. and F.A. G.E.M. assisted with WNT5A and AXL dox mouse experiments (Figs. 2g, h, 3m, n). B.L.E., D.J.Z. and L.H. assisted with the culture of primary fibroblasts and collection of conditioned medium. T.B., H.-Y.T. and D.W.S. performed proteomic analysis on fibroblasts, and A.K. performed statistical analysis and graphed the data. W.X. and X.X. provided tumour samples (FFPE) and expertise in the H&E staining and reading and analysis of stained human and mouse melanoma slides. M.H. and E.M.J. provided reagents. M.E.F. and A.T.W. wrote the original draft of the manuscript. J.A.A.-G., Y.C., G.M.A., S.M.D., V.W.R., M.H., E.M.J. and D.J.Z. contributed to editing of subsequent drafts. All authors discussed the results and commented on the manuscript.
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J.A.A.-G. is a scientific co-founder of, scientific advisory board member of, equity owner in and receives financial compensation as a consultant for HiberCell, a Mount Sinai spin-off company that is focused on therapeutics that prevent or delay the recurrence of cancer. A.T.W. is on the board of reGAIN Therapeutics. E.M.J. reports other support from Abmeta, personal fees from Genocea, personal fees from Achilles, personal fees from DragonFly, personal fees from Candel Therapeutics, other support from the Parker Institute, grants and other support from Lustgarten, personal fees from Carta, grants and other support from Genentech, grants and other support from AstroZeneca, personal fees from NextCure and grants and other support from Break Through Cancer outside of the submitted work.
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Extended data figures and tables
Extended Data Fig. 1 Aged lung versus skin fibroblasts promote opposing melanoma phenotypes.
a, b, 5 Young (8 week) and aged (>52 week) C57BL6 mice per group were subdermally injected with 2.5x105 Yumm1.7 mCherry-labelled mouse melanoma cells. Lungs were collected and PFA embedded from 5 tumour bearing young and aged mice at week 3 and 5. IHC was performed using an mCherry antibody. Lung sections were analysed and quantified 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 = 0.3064 for 3 weeks and P = 0.2011 for 5 weeks. c, d, e 2.5 x105 Yumm1.7 melanoma cells were intradermally implanted into 3 C57BL6 aged mice (>52 weeks) per group. Tumours were left to grow for three weeks. Mice underwent survival surgery to allow excision of the primary tumour. After 5 total weeks, Lungs were taken, PFA embedded and underwent IHC analysis for the number of mCherry positive colonies (>10 mCherry positive cells per lesion) per section (p = 0.2879) along with the average number of single cell colonies per high powered frame (20 x) across entire lung sections (p = 0.081) presented as mean +/− SEM. A student’s two-way t-test was performed with P < 0.05 designated as significant. Representative images were displayed across three independent mouse lung samples for mCherry positive cells and Ki-67 across subsequent sections with the scale bar representing 100um. f, g, h Collagen sandwich reconstructions were formed with healthy human lung or skin fibroblasts from young (>35) or aged (> 55) patients in the top and bottom layer. The middle layer contained FS4 GFP human melanoma cells seeded at the same density at day 0. GFP positive melanoma cells were imaged using an automated Nikon TI and an average count per field was quantified using imaging software NIS elements at day 4. Representative images are displayed for each condition (N = 3 independent wells in triplicate). A student’s two-way t-test was performed. P = 0.0432 for FS4 lung and P = 0.0088 for FS4 skin. i, j Western blot analysis was performed on primary tumour protein lysate from 5 young and 5 aged mice investigating dormancy and proliferative associated expression. HSP90 was used as a loading control.
Extended Data Fig. 2 Aged lung conditioned medium promotes melanoma outgrowth in vitro.
a, b, FS13 c, d FS4 e, f FS14 g, h and 1205Lu GFP melanoma cells were seeded at a density of 2x104. Cells were then grown over a 10-day period in conditioned medium from young or aged lung or skin fibroblasts and underwent counting using a haemocytometer at days 3, 5 and 10 and were further assessed via automated counting using 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. ****= P < 0.0001. ** P = 0.0084 for Fig. 2d. Data were presented as the mean +/− the SEM for each time point.
Extended Data Fig. 3 sFRP1 promotes melanoma outgrowth.
a, b, Protein lysate was collected from 1205Lu human melanoma cells treated with young (under 35) or aged (over 55) lung or skin fibroblast conditioned medium over a 5-day period. Western blot analysis was performed examining NR2F1, H2AFZ, Filamin A, Cleaved Filamin A, ROR2 and ROR1. HSP90 was used as a loading control. c, d Protein lysate was collected from FS4 human melanoma cells treated with young or aged lung or skin fibroblast conditioned medium over a 5-day period. Western blot analysis was performed examining dormant (WNT5A, AXL, p21, p27 P-p38. p-PKC, NR2f1, Filamin A, Cleaved Filamin A, ROR2) and proliferative (β-catenin, H2AFZ, MER, MITF, ROR1) melanoma markers. HSP90 was used as a loading control. e FS4 melanoma cells were treated with 500ng/ml of recombinant sFRP1, and protein expression was assessed after 5 days via western blot. HSP90 was used as a loading control. f Proliferation of the above cells was assessed by seeding them at a density of 2x104. Cells were then grown over a 10-day period while being treated with 500 ng of r-sFRP1 or a PBS control (Experiment was performed in biological triplicates with 3 technical replicates each) every 2 days and assessed for proliferation at days 3-5 and 10 using a haemocytometer and further assessed using automated counting via NIS elements. 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 g Aged lung human fibroblasts were transduced with lentiviral sh-sFRP1 or scrambled control vectors. Conditioned medium was taken from fibroblasts after 72 h and FS4 melanoma cells were then grown in this conditioned medium or a DMEM control medium, with proliferation assessed over 10 days. 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. ****= P < 0.0001 for Aged conditioned medium compared to both Aged conditioned medium in SHsFRP1 and DMEM conditions. Data were presented as the mean +/− the SEM for each time point. h Protein lysate was collected from the lungs of 5 young and 5 aged healthy mouse tissues. Western blot analysis was performed looking at sFRP1. HSP90 was used as a loading control. i Protein lysate was taken from healthy human young vs aged lung fibroblasts and a 1205Lu human melanoma cell line. Western blot analysis was performed examining sFRP1 and sFRP2 expression and HSP90 was used as a loading control. j Protein lysate was taken from two independent young lung fibroblasts, treated with conditioned medium from intrinsically WNT5A-low and WNT5A-high melanoma cells for 48 h and was compared with two independent aged lung fibroblasts for sFRP1 expression. HSP90 was used as a loading control.
Extended Data Fig. 4 WNT5A regulates dormant pathways and slows proliferation.
a, b, Five aged mice per group were intradermally injected with 2.5x105 Yumm1.7 mCherry melanoma cells. After three weeks of tumour growth, mice were treated with 1 mg/kg of a neutralizing sFRP1 or an IgG control antibody via IP injection every 4 days. Representative images are displayed across three independent mice for mCherry positive cells and Ki-67. The number of single cell (>10 mCherry positive cells) was assessed at week 5 and quantified per section and presented as mean +/− SEM. Scale bar represents 100 um. A student’s two-way t-test was performed. P = 0.244 c Protein expression analysis was performed on WNT5A low human melanoma samples (FS13, FS14) that underwent lentiviral induced overexpression of WNT5A. HSP90 was used as a loading control. d, e FS13 and FS14 WNT5A overexpressing mCherry human melanoma cells were seeded at a density of 2x104. Cells were then grown over a 10-day period 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 for both conditions. f FS13 GFP melanoma cells were seeded at a density 2x104. Cells were treated with 200ng of r-WNT5A every 2 days 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 g Western blot analysis was performed on protein lysates from FS13 melanoma cells treated with 200ng of recombinant WNT5A for five days. HSP90 was used as a loading control.
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 (2ug/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 ug/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 ug/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.
Extended Data Fig. 6 Role of MER and AXL in metastatic dormancy and reactivation.
a, b, Analysis of melanoma cell lines from the Cancer Cell line Encyclopedia (CCLE) dataset were performed on human samples stratified into the top (AXL high) and bottom (AXL low) 50th percentile of AXL expression (N = 30) and presented as a heat map of the average Z-score. An unpaired two-way t-test was performed on each condition, investigating previously established dormancy and proliferative genes, with P < 0.05 considered significant. c, d Analysis of MER high and MER low cells from the CCLE as described above. e TCGA analysis of relative mRNA expression of MER in patient tumour samples comparing primary vs metastatic (distant metastases 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 = 0.0023. f 1205Lu shAXL and control human mCherry melanoma cells were seeded at a density of 2x104. Cells were then grown over a 10-day period 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 = 0.0160 (AXL SH1) and P = 0.340 (AXL SH2) when compared with the negative control group. g Protein expression analysis was performed on protein lysates of lentiviral induce shRNA knockdown of MER in FS14 human melanoma cells. HSP90 was used as a loading control. h, i FS13 and FS14 shMER and control human mCherry melanoma cells were seeded at a density of 2x104. Cells were then grown over a 10-day period 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 triplicate 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 for all groups compared with the negative controls. j Protein expression analysis was performed on protein lysates of Yumm1.7 Dox inducible MER overexpressing mCherry mouse melanoma cells treated with Dox (0.5ug/ml) vs a No Dox control for 48 h. HSP90 was used as a loading control. k Yumm1.7 Dox inducible MER overexpressing mCherry mouse melanoma cells were seeded at a density of 2x104. Cells were then grown over a 10-day period 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 triplicate 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. l TCGA analysis of relative mRNA expression of MER in patient samples stratified into stage I/II vs. Stage III/IV and presented as a violin plot, with quartiles represented by black lines and the median by the broken line (N = 219, 197). A student’s two-way t-test was performed. P = 0.0025. m Tumour volumes from young mice (N = 9 for Dox Day 21, N = 8 for Dox Day 3, N = 7 for No Dox due to mouse deaths) subdermally injected with 2.5x105 Yumm1.7 Dox inducible MER mCherry melanoma cells. Mice were treated with Dox (2 ug/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 31. Data were presented as the mean +/− the SEM for each time point. *** P = 0.0004 comparing No Dox with Dox Day 3. * P = 0.0281 comparing No Dox with Dox Day 21. n Western blot analysis was performed on protein lysate from Yumm1.7 Dox-inducible AXL overexpressing mCherry melanoma cells comparing control and Dox (0.5ug/ml) treated cells over 48 h. HSP90 was used as a loading control. o Yumm1.7 Dox inducible AXL overexpressing mCherry mouse melanoma cells were seeded at a density of 2x104. Cells were then grown over a 10-day period 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 = 0.0272. p Tumour volumes from aged mice (N = 8 for No Dox, N = 5 for Dox Day 3 and N = 7 for Dox Day 21 due to mouse deaths) per group were subdermally injected with 2.5x105 Yumm1.7 Dox inducible AXL mCherry melanoma cells. Mice were treated with Dox (2 ug/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 36. **** P < 0.0001 for comparisons between No Dox with both Dox Day 3 and Dox Day 21. Data were presented as the mean +/- the SEM for each time point.
Extended Data Fig. 7 MER expression is inversely correlated with WNT5A and AXL and is higher in lung metastases.
a–d, IHC analysis was performed on matched human primary melanoma tumours and lung metastases for MER, WNT5A and AXL. Scale bar represents 100um with representative images displayed for each patient sample.
Extended Data Fig. 8 MER promotes emergence from metastatic dormancy whereas AXL induces dormancy.
a, Five young mice per group were intradermally injected with 2.5x105 Yumm1.7 Dox inducible MER overexpressing 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 100 um. b, c 5 aged mice per group were intradermally injected with 2.5x105 Yumm1.7 Dox inducible AXL overexpressing 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 100 um. Given that both Dox 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 = 0.5240.
Extended Data Fig. 9 GAS6 regulates proliferation in a context-specific manner.
a, b, FS4 and FS13 human GFP melanoma cells were seeded at a density of 2x104. Cells were then grown over a 10-day period, while being treated with 500ng of r-GAS6 every 2 days 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 = 0.033 for 9A, P = 0.0205 for 9B c, d. FS14 control vs SH MER and 1205Lu control vs shAXL human melanoma cells were seeded at a density of 2x104. Cells were treated with 500ng of r-GAS6 vs a PBS control every two days 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. P-values were taken from day 10. **** P < 0.0001 shMER + GAS6 compared to control, ** P = 0.0013 Control vs Control + GAS6, * P = 0.0238 shMER vs shMER + GAS6. **** P < 0.0001 Control + GAS6 vs Control, * P = 0.0161 Control + GAS6 vs shAXL + GAS6. e, f Aged mice were intradermally injected with 2.5x105 Yumm1.7 mCherry melanoma cells (5 mice for PBS, 4 mice for GAS6 treatment due to one early death). After three weeks of tumour growth, mice were subsequently treated with 500 ng of rGAS6 or PBS via IP injection every 2 days. After 5 total weeks, Lungs were taken, PFA embedded and underwent IHC analysis of mCherry positive melanoma cells. Both groups were further assessed for the average number of single cell colonies (<10 mCherry-positive cells) 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 = 0441. Representative images are displayed across three independent mice for mCherry positive cells and Ki-67 with the scale bar representing 100 um.
Extended Data Fig. 10 PROS1 promotes melanoma growth.
a–e, 1205Lu (P < 0.0001), FS4 (P < 0.0054), FS13 (P = 0.0015), FS14 (P < 0.0001) and Yumm1.7 (P < 0.0001). fluorescent melanoma cells were treated every two days with 500ng of r-PROS1 every 2 days vs a PBS control 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 and P < 0.05 was designated significant. Data were presented as the mean +/− the SEM for each time point. f FS14 shMER knockdown cells and control cells were seeded at a density of 2x104. Cells were then grown over a 10-day period, while being treated with 500 ng of r-PROS1 every two days 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 for control + PROS1 vs all other treatment groups. g Protein lysate from Yumm1.7 cells treated with 500 ng of r-PROS1 for 48 h underwent western blot analysis. HSP90 was used as a loading control. h, i Young mice were intradermally injected with 2.5x105 Yumm1.7 mCherry melanoma cells (5 mice per group). After three weeks of tumour growth, mice were subsequently treated with 500 ng of rPROS1 or PBS via IP injection every 2 days. After 5 total weeks, Lungs were taken, PFA embedded and underwent IHC analysis of mCherry positive melanoma cells. Both groups were further assessed for the average number of single cell colonies (<10 mCherry positive cells) per high powered frame (20 x) across entire lung sections and presented as mean +/− SEM. A student’s two-way t-test was performed. Representative images are displayed across three independent mice for mCherry positive cells and Ki-67 with the scale bar representing 100 um. P = 0.8757. j 5 young mice per group were intradermally injected with 2.5x105 Yumm1.7 mCherry melanoma cells. After three weeks of tumour growth, mice were subsequently treated with 500 ng of r-PROS1, r-SFRP1, a combination of both or PBS via IP injection every 2 days. After 5 total weeks, lungs were taken, PFA embedded and subjected to IHC analysis of mCherry positive melanoma cells. Representative images were displayed for mCherry and Ki-67 with the scale bar representing 100 um.
Extended Data Fig. 11 Aged liver fibroblasts express sFRP1 and are growth permissive for melanoma cells.
a, Five young (8 week) and five aged (>52 week) C57BL6 mice were intradermally injected with 2.5x105 Yumm1.7 parental mouse melanoma cells and tumours were measured over 36 days. Lungs were collected and PFA embedded from 5 tumour bearing young and aged mice at weeks 3 and 5 and IHC was performed using MITF, KI-67 and H&E. Representative images are displayed across three independent aged mice for MITF positive cells and Ki-67 with the scale bar representing 100 um. b Data were generated using the Human Protein Atlas looking at RNA expression of sFRP1 in specific cell types across various tissue types. Green circles indicate high levels of expression, grey circles low expression and empty spaces no expression. c Western blot was performed on protein lysate taken from the healthy liver of young (<8 weeks) and aged (>52 weeks) C57BL6 mice. GAPDH was used as a loading control. d Protein lysate from one healthy human young (<35) and aged (>55) liver fibroblast. HSP90 was used as a loading control. e, f Collagen sandwich reconstructions were formed with healthy human Liver fibroblasts from young (< 35) or aged (> 55) patients in the top and bottom layer. The middle layer contained FS4 or 1205Lu GFP human melanoma cells seeded at the same density at day 0. GFP positive melanoma cells were imaged using an automated Nikon TI and an average count per field was quantified using imaging software NIS elements at day 4. Representative images are displayed for each condition (N = 3 independent wells in triplicate). A student’s two-way t-test was performed. 1205Lu P = 0.0001, FS4 P = 0.0055. g, h 1205Lu and FS4 GFP melanoma cells were seeded at a density of 2x104. Cells were then grown over a 10-day period in conditioned medium from young or aged lung or skin fibroblasts and underwent counting using a haemocytometer at days 3, 5 and 10 and were further assessed via automated counting using 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. ** P = 0.0018, **** P < 0.0001. Data were presented as the mean +/− the SEM for each time point.
Supplementary information
Supplementary Information
The supplementary information contains all of the full western blots for each panel of each figure.
Tumour growth volumes
The supplementary information also contains the excel files with the raw data for each of the tumour growth curves for all in vivo experiments.
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Fane, M.E., Chhabra, Y., Alicea, G.M. et al. Stromal changes in the aged lung induce an emergence from melanoma dormancy. Nature 606, 396–405 (2022). https://doi.org/10.1038/s41586-022-04774-2
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DOI: https://doi.org/10.1038/s41586-022-04774-2
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