Stromal Changes In The Aged Lung Induce An Emergence From Melanoma Dormancy

Ashani Weeraratna (  aweeraratna@jhu.edu ) Johns Hopkins University https://orcid.org/0000-0003-0448-6952 Mitchell Fane Johns Hopkins Stephen Douglass Johns Hopkins Gretchen Alicea Johns Hopkins Marie Webster Lankenau Institute of Medical Research Vito Rebecca Johns Hopkins Gloria Marino Johns Hopkins Yash Chhabra Johns Hopkins Filipe Almeida Wistar Institute Brett Ecker The Wistar Institute Thomas Beer Wistar Institute Hsin-Yao Tang Wistar Institute Andrew Kossenkov Wistar Institute David Speicher Wistar Institute Julio Aguirre-Ghiso Icahn School of Medicine at Mount Sinai

contrast to those previously observed in the skin, where the aged skin microenvironment decreased melanoma growth (Fig. 1A), while promoting local invasion 4 .
Given our previous data on skin broblasts driving changes in melanoma cell behavior 4 , we investigated whether lung broblasts similarly affected melanoma cell growth at the distal sites. We co-cultured melanoma cells with human skin or lung broblasts from young (< 35 years) or aged (> 55 years) healthy donors in a 3D collagen reconstruct ( Fig. 1D-E, Extended Data 1B-D). Consistent with our in vivo data ( Fig. 1A-C), melanoma cells in an aged lung broblast microenvironment increased proliferation compared to those in a young lung broblast environment ( Fig. 1D-E, Extended Data 1B-D). Conversely, melanoma cells in an aged skin broblast microenvironment proliferated more slowly when compared with young skin broblasts. Importantly, treatment of human melanoma cells ( Fig. 1F-G, Extended Data 2A-F) with conditioned media (CM) from young and aged lung or skin broblasts over a 10-day period in 2D culture phenocopied the results seen in 3D culture, suggesting that changes in secreted soluble factors are key in promoting these phenotypic differences.
Since aged lung broblasts induced melanoma cell proliferation in vitro ( Fig. 1D-G), we performed proteomic analysis on the secretome from healthy human young (< 35) and aged (> 55) lung broblasts (Fig. 1H). One of the most differentially secreted factors increased in the aged lung broblast secretome was sFRP1, a non-canonical Wnt antagonist [9][10][11] . This was intriguing, as previous analyses in aged skin broblasts revealed increased secretion of sFRP2, DKK3 and Wnt5A, which conversely promoted expression of the non-canonical Wnt signaling pathway in tumor cells, which was shown to be a prominent driver of a slower-growing, but invasive melanoma phenotype within primary tumors [4][5][6]12 .
Analysis of primary tumor lysate from young and aged mice in this study con rmed that indeed, aged mouse tumors have increased expression of the main effector protein in the non-canonical Wnt pathway, Wnt5A (Fig. 1I). Downstream pathways of Wnt5A signaling in melanoma, such as AXL 13 and slowcycling genes such as p21 and p27, were all increased in aged tumors in the skin (Fig. 1I). Conversely, well known targets of the proliferative axis within melanoma phenotype switching such as β-catenin, MITF and MART-1 3 , along with lesser known proliferative melanoma-associated markers including MER 14 and H2AFZ 15 , are all decreased in aged skin tumors (Fig. 1J), coinciding with the overall increased growth seen in these younger tumors (Fig. 1A). A recent observation within prostate cancer revealed that invasive pathways such as Wnt5A 16 and AXL 17 independently promote tumor cell dormancy, and potentially, survival within metastatic bone microenvironments following dissemination 18 . Subsequently, we analyzed other well-de ned markers of tumor dormancy in prostate and other cancer types, such as NR2F1, p-p38 and TGFB2 17,19 . All were increased in Wnt5A/AXL high slower-growing aged primary tumors in the skin (Fig. 1I), implicating Wnt5A and AXL in age-related signaling changes in melanoma dormancy.
Taken together, while our previous data suggested that sFRP2 within the aged skin promotes noncanonical Wnt signaling to allow initial dissemination from the primary tumor 4 , this current data suggested that sFRP1 within the aged lung inhibits Wnt5A to induce reactivation and metastatic outgrowth after an initial period of dormancy. To test this hypothesis, we treated human melanoma cells with CM from 3 young or aged healthy human lung broblast donors. Treatment with aged lung broblast CM consistently reduced non-canonical Wnt5A signaling compared with young lung broblasts ( Fig. 2A, Extended Data 2G). Downstream dormancy associated pathways were also reduced, while proliferative pathways were increased ( Fig. 2A Supp 2G). Conversely, treatment with CM from aged skin broblasts induced the opposite effect on gene expression, promoting non-canonical Wnt5A/downstream dormancy pathways while inhibiting proliferative pathways (Fig. 2B, Extended Data 2H). Next, we treated melanoma cells with recombinant (r) sFRP1; we observed decreased Wnt5A and downstream dormancy markers AXL and p21, and increased proliferative markers MITF and MER (Fig. 2C, Extended Data 3A), as well as increased proliferation (Fig. 2D, Extended Data 3B). Furthermore, knockdown of sFRP1 from aged lung broblasts (Fig. 2E) ablated the previous growth phenotype and decreased proliferation of melanoma cells treated with CM relative to empty control (Fig. 2F, Extended Data 3C). To test this hypothesis in vivo, we intradermally injected aged mice with Yumm1.7 mCherry cells to form primary tumors. Tumors were grown for 3 weeks to allow single cell seeding within the lung, as shown in Fig. 1C, then treated with a neutralizing antibody against sFRP1 via Intraperitoneal (IP) injections to deplete its levels within the mouse until week 5. Depletion of sFRP1 reduced metastatic colony formation in the aged lungs compared with IgG treated mice ( Fig. 2G-H). Overall, we nd that aged lung broblasts promote phenotypic reactivation of dormant melanoma cells in the lung via increased secretion of sFRP1 ( Fig. 2I).
Given the importance of Wnt5A in prostate cancer dormancy 16 , coupled with it being a downstream antagonistic target of sFRP1, we chose to investigate it as a primary factor in melanoma dormancy. We rst strati ed patient melanoma samples (across both primary and metastatic) from the TCGA into the top and bottom 25th percentile of Wnt5A-expressing primary and metastatic tumors. Wnt5A highexpressing patient samples express a large number of well-de ned dormancy related genes [20][21][22] ( Fig. 3A). Importantly, many established proliferative melanoma markers correlated with Wnt5A low samples (Fig. 3B). Increasing Wnt5A expression in low-expressing human melanoma cells via lentiviral overexpression (Fig. 3C, Extended Data 4D) or treatment with rWnt5A (Extended Data 4I) increased many of these dormancy targets, while reducing proliferative markers and melanoma cell growth in vitro (Extended Data 4E-G) .
We next investigated whether Wnt5A expression promotes non-progressive, dormant disease in metastatic patients. We evaluated the TCGA dataset, rst looking at progression free interval (PFI) in metastatic patients only. We strati ed patients into those who progressed in < 3 years vs in > 3 years.
Patients who progressed in < 3 years were signi cantly older (Fig. 3D). Importantly, Wnt5A expression in metastatic samples was signi cantly higher in the younger patient cohort with progression in > 3 years (Fig. 3E). Analysis of these data via Kaplan Meier curves further shows that overall PFI probability is signi cantly higher in younger metastatic patients with lower Wnt5A levels (Extended Data 4A-B).
Examination of overall survival further shows that metastatic patients who did not survive up to 3 years were also signi cantly older (Fig. 3F) and had lower Wnt5A expression in metastatic sites (Fig. 3G). Overall survival probability signi cantly decreases in both aged patients and lower Wnt5A expression in metastatic samples (Fig. 3H). This was in direct contrast to our analysis of Wnt5A expression in patient primary tumors, which showed the opposite outcome, with higher Wnt5A expression within these primary tumors correlating with worst survival (Extended Data 4C). This speaks to the complexity of Wnt5A signaling within these different microenvironments and in the overall metastatic cascade. While our previous studies implicate the importance of Wnt5A in establishing an invasive phenotype 23 , dissemination 5 , and in therapeutic resistance 24 within the primary tumors, these new data suggested that in order to form competent, proliferative metastases at the distal site, Wnt5A needs to be down-regulated. This is further supported by the fact that Wnt5A expression is reduced in established metastatic tumor sites vs primary tumors in the TCGA (Fig. 3I) and other independent patient datasets (GSE15605) 25 (Extended Data 4H).
To investigate this further, we generated Yumm1.7 mCherry Doxycycline (Dox) inducible cells that expressed sh-Wnt5A. Treatment with Dox (Dox, 0.5ug/ml) successfully reduced Wnt5A expression, while reducing downstream dormancy markers, increasing proliferative markers and overall proliferation ( Fig. 3J-K). We were interested in how uncoupling of Wnt5A across different timepoints in progression affected dissemination and metastatic dormancy. We formed intradermal tumors in our young c57BL6 mouse model, which were able to disseminate to the lungs e ciently but maintained dormant cell colonies in the lungs through week 5 (see Fig. 1C). We speci cally investigated temporal downregulation of Wnt5A starting at two different timepoints; 1) Beginning at day 3, to investigate Wnt5A ablation throughout the entirety of tumor progression (growth and dissemination) and; 2) Beginning at day 21, to investigate Wnt5A downregulation after cells had successfully disseminated and formed viable single dormant colonies within the lung (See Fig. 1C). Analysis of primary tumor growth reveals that inducible knockdown at either timepoint signi cantly increased overall growth (Fig. 3L), further establishing a role for Wnt5A in inducing a slower-growing cell within primary tumors. However, analysis of metastatic progression after 5 weeks revealed that only mice treated with Dox starting at day 21 formed large micrometastatic colonies, compared with non-treated mice, who predominantly contained single dormant cells (Fig. 3M, O). Interestingly, inducible knockdown of Wnt5A beginning at day 3 resulted in signi cantly fewer cells seeded in the lung compared with no-Dox treated mice (Fig. 3N). Overall, these new data demonstrate that non-canonical Wnt5A signaling is required for e cient dissemination from the primary tumor, and survival when seeding within the lung initially. Once these cells have successfully seeded as viable single dormant cells, temporal down-regulation of Wnt5A is able to promote reactivation, allowing metastatic outgrowth, even in the previously dormant young mouse lungs (Fig. 3P).
To further investigate the mechanisms underlying the complexity of dormancy vs outgrowth in aging, we next investigated the Tyro3, AXL and MER (TAM) family of tyrosine kinase receptors. Importantly, AXL is strongly correlated positively to Wnt5A in melanoma 26 and is involved in driving dormancy in metastatic prostate cancer 17 . The relationship between AXL with the other family members is very context speci c, as all three members are heavily co-expressed within immune cells and act in a redundant manner 27 . Conversly in melanoma, both MER and TYRO3 have been suggested to be differentially expressed to AXL and promote more of a proliferative phenotype 14,28 . Thus, we hypothesized that differential expression of AXL vs either MER or TYRO3 within melanoma cells may act to promote a dormancy-reactivation axis within metastatic tissues. To investigate this, we performed westernblot analysis of 3 previously characterized Wnt5A high human melanoma cell lines (FS4, FS5, 1205LU) vs 2 characterized Wnt5A low cell lines (FS13, FS14) (Fig. 4A). AXL strongly correlated positively with Wnt5A and these cells expressed the AXL ligand GAS6. Several established dormancy markers including p21, phospho-p38, and NR2F1 were also elevated in these Wnt5A/AXL high cells (Fig. 4A). While there was no consistent difference seen in TYRO3 expression across the cell lines, MER was inversely expressed with AXL and Wnt5A. These MER high expressing cell lines also displayed strong expression of the MER-speci c ligand PROS1 and the proliferative markers MITF, and MART1 (Fig. 4A).
To expand on this analysis in melanoma cells speci cally, we analyzed an RNA-seq dataset from the Cancer Cell Line Encyclopedia (CCLE) that contains over 60 human melanoma cell lines. We strati ed these into AXL high and AXL low subpopulations. Indeed, AXL high cells have signi cantly elevated levels of Wnt5A and dormancy markers ( Knockdown of AXL in Wnt5A high lines did not reduce Wnt5A, suggesting that AXL may be downstream of Wnt5A within this dormancy axis; however, MER and MITF were increased (Fig. 4G) alongside a modest increase in proliferation after 10 days (Fig. 4F). Conversely, shRNA-mediated knockdown of MER increased Wnt5A, AXL, and p21 expression, while reducing MITF (Fig. 4I, Extended Data 5A) and proliferation (Fig. 4H, Extended Data 5B). Given that MER knockdown increased Wnt5A/AXL signaling and proliferation, we hypothesized that MER promotes reactivation from metastatic dormancy. To investigate this, we created a Dox-inducible Yumm1.7 mCherry MER melanoma cell line. Inducible overexpression of MER decreased AXL and Wnt5A, while increasing MITF (Fig. 4K) and overall proliferation in vitro (Fig. 4J). Further validation of MER playing an important role in metastatic outgrowth came via analysis of the TCGA and other independent patient studies (GSE15605) 25 . MER expression was signi cantly increased in metastatic patient samples compared to primary tumors in both datasets ( Fig. 4L, Extended Data 5C) and was also increased in patients with stage III/IV disease relative to stage I/II (Extended Data 5D), suggesting an importance for driving metastatic growth and progression.
To investigate MER expression in metastatic reactivation in vivo, we used the Dox-inducible MER overexpressing Yumm1.7 mCherry cell line. These cells were implanted intradermally into our dormant young mouse model to form primary tumors. We again began treatment with Dox at either day 3 or day 21 to investigate the importance of MER expression at various timepoints throughout progression. Dox treatment at both timepoints increased overall tumor growth ( Fig. 4M), con rming that MER expression promotes a more proliferative phenotype. Induction of MER expression in dormant single cells seeded within the lung (starting at day 21) signi cantly increased metastatic outgrowth compared with nontreated mice, where cells remained largely as single dormant colonies ( Fig. 4N-O). MER induction beginning at day 3 decreased the number of cells able to seed (Fig. 4N), further highlighting the necessity for a Wnt5A high /MER low melanoma phenotype during early tumor development to allow dissemination from the primary tumor and survival during metastatic seeding (Fig. 3P).
We next created a Dox-inducible AXL mCherry Yumm1.7 melanoma cell line to investigate whether AXL could inhibit reactivation via inhibition of this MER phenotypic switch within metastatic aged models. Indeed, inducible activation of AXL in these cells decreased MER and MITF expression (Fig. 4P) as well as proliferation in vitro (Extended Data 5E); however, Wnt5A remained unchanged, further implicating AXL downstream of Wnt5A in this dormancy axis. AXL-inducible cells were implanted in the aged metastatic mouse model to form primary tumors and treated with Dox beginning at either day 3 or day 21. This aged mouse model has slow-growing primary tumors with higher AXL expression relative to younger mice under normal circusmtances (Fig. 1). Despite the already high level of AXL within these tumors, AXL induction was able to produce a modest decrease in tumor growth, but only upon treatment starting at day 3 (Fig. 4Q). AXL induction at days 3 and 21 signi cantly reduced colony formation in the aged lung and largely resulted in single cell colonies compared to the no Dox control (Fig. 4R-S). There was no difference in colony formation size between AXL induction at day 3 and 21, which we hypothesized was due to the fact that AXL expression is already higher in aged primary tumors, thus already allowing e cient dissemination and survival upon seeding. Overall, this data suggests a key role for this AXL/MER differentially regulated signaling pathway in producing a dormancy/reactivation axis downstream of Wnt5A.
Finally, we were interested in whether activation of this differential TAM axis by their respective primary ligands played a role in both dormancy and reactivation. GAS6 is the prominent ligand involved in activating AXL, although it has been shown to bind both MER and TYRO3 at a much lower a nity 29 . PROS1 is the prominent ligand for MER, but appears to be much more speci c compared to GAS6, and is unable to bind the AXL receptor in most contexts 30,31 . Analysis of ligand expression in the healthy lungs of young mice, which promote a dormant melanoma phenotype, reveals that GAS6 levels appeared to be higher in the majority of younger mouse lungs. Conversely, PROS1 levels were increased in the aged lung ( Fig. 5A). Furthermore, we nd that Wnt5A/AXL high cells secrete higher amounts of the GAS6 ligand into conditioned media in vitro, whereas MER high cells secrete higher amounts of PROS1, implicating the potential for paracrine-related modulation of neighboring melanoma cells in the microenvironment (Fig. 5B).
Treatment of Wnt5A/AXL high melanoma cells with rGAS6 consistently decreased melanoma cell proliferation across these cell lines (Fig. 5C, Extended Data 6A). However, Wnt5A/AXL low melanoma cells treated with GAS6 increased proliferation (Fig. 5D, Extended Data 6B). We hypothesized, given GAS6's promiscuity between the 3 different TAM family members, that it may actually be acting through the MER receptor in AXL low cell lines. Indeed, shRNA knockdown of MER in AXL low human melanoma cells, which increased AXL in previous experiments, instead decreased melanoma cell proliferation in response to rGAS6 treatment (Extended Data Fig. 6C); Conversely, sh-mediated knockdown of AXL in Wnt5A/AXL high cells treated with rGAS6 recues the phenotype and reverses the decreased proliferative response (Extended Data Fig. 6D). Treatment of Dox-inducible AXL melanoma cells upon AXL induction with rGAS6 decreased melanoma cell growth compared with untreated AXL-induced cells or with rGAS6 treatment in control cells (Fig. 5E). Protein expression analysis of these cells con rms that rGAS6 treatment of Yumm1.7 cells produces only a modest decrease in MER and MITF and a slight increase in Wnt5A (Fig. 5F). When AXL expression is induced via Dox treatment, administration of rGAS6 increases Wnt5A and produces a much more pronounced decrease in MER and MITF, suggesting that GAS6 regulation of melanoma cells relies heavily on whether they are in an AXL-or MER-high state. To investigate this in vivo, we formed primary tumors in our aged metastatic mouse model that had lower GAS6 levels in the lung. We began treatment of these mice with rGAS6 (IP injection) at 3 weeks to allow initial dissemination and seeding in the lung (Fig. 1C). We found that GAS6 treatment decreased metastatic colony formation after 5 weeks (Fig. 5G-H); however, this decrease was modest and larger micrometastatic colonies were still found in treated mice, as opposed to single cell colonies seen previously within the young lung. These data suggested that GAS6 is unable to fully inhibit the phenotypic switch towards reactivation in the aged lung microenvironment, likely due to competion with other soluble factors such as sFRP1.
While GAS6 shows promiscuous regulation of cell growth based on the adopted cell phenotype, PROS1 is thought to have very little receptor interaction with AXL. We showed previously that even in Wnt5A/AXL high melanoma cell lines in vitro, MER expression was still maintained at detectable levels (Fig. 4A). Based on this, we hypothesized that PROS1 may act to speci cally promote increased growth and reactivation regardless of cell phenotype. Indeed, treatment of either Wnt5A/AXL high or MER high human and mouse melanoma cells signi cantly increased melanoma growth (Fig. 5J, Extended Data 6E-H); shRNA mediated knockdown of MER ablates this effect (Extended Data 6I). Protein expression analysis of rPROS1 treated Yumm1.7 cells shows that not only did PROS1 decrease Wnt5A and AXL expression, but further increased MER and MITF (Fig. 5I). To examine whether the PROS1 ligand could facilitate reactivation of dormant melanoma cells within our dormant young mouse model where PROS1 levels were low in the lung, we implanted primary tumors in young mice using Yumm 1.7 mCherry cells. Tumors were grown for 3 weeks to allow single cell seeding in the lung (see Fig. 1C). Mice were then treated with r-PROS1 every two days via IP. Treatment with r-PROS1 signi cantly increased metastatic colonies in the previously dormant young lung microenvironment at 5 weeks, compared with a PBS control (Fig. 5K,L).
Overall, these data reveal an unexpected complexity in the role of Wnt signaling in melanoma cell metastasis that is regulated by aging (Fig. 5M). We show that Wnt5A promotes initial dissemination of the tumor cells as previously de ned, but then acts to maintain them in a dormant state to allow survival and adaptation in the lung microenvironment. This state is maintained until age-related changes in the distal site microenvironment induce an emergence from tumor dormancy. We speci cally show that sFRP1 is secreted at higher levels by aged-lung broblasts and decreases Wnt5A expression to allow reactivation from dormancy in the aged mouse lung. We further de ne an AXL-MER dormancyreactivation axis downstream of Wnt5A. MER expression is required to induce a phenotypic downregulation of the Wnt5A/AXL high dormant state to allow age-induced metastatic reactivation and outgrowth, whereas maintaining AXL expression inhibits reactivation. While we nd that GAS6-mediated regulation of this axis is promiscuous, the MER-mediated ligand PROS1 is increased in the aged lung microenvironment and secreted in a paracrine manner by MER high reactivated cells and is shown to be capable of driving reactivation and metastatic outgrowth in the previously dormant young lung microenvironment (Fig. 5M). These observations may explain the predisposition of elderly patients to aggressive metastatic melanoma and identify the role of aging as a critical step in in the emergence of tumor cells from dormancy. Further, these data, and other data from our laboratory and others suggest that treating patients according to age may have bene t. For example, we have recently shown that lipid uptake by melanoma cells in an aged, but not young, microenvironment drives resistance to BRAF/MEK inhibitors, which can be overcome by targeting the fatty acid transporter (FATP2) responsible 8  streptomycin. Cell lines were cultured at 37°C in 5% CO 2 and the medium was replaced as required. Cell stocks were ngerprinted using an AmpFLSTR Identi ler PCR Ampli cation Kit from Life Technologies at The Wistar Institute Genomics Facility. Although it is desirable to compare the pro le with the tissue or patient of origin, our cell lines were established over the course of 40 years, long before acquisition of normal control DNA was routinely performed. However, each short tandem repeat pro le is compared with our internal database of over 200 melanoma cell lines, as well as control lines, such as HeLa and 293T. Short tandem repeat pro les are available upon request. Cell culture supernatants were tested for mycoplasma using a Lonza MycoAlert assay at the University of Pennsylvania Cell Center Services.
Proliferation Assays 2x10 4 melanoma cells were seeded into a six well plate in triplicate. Cells were then counted at days 3, 5 and 10 post-seeding using a haemocytometer and NIS elements automated software. The total number of cells were analyzed using GraphPad Prism software, presented as the mean +/-the SEM. The 3D reconstruct proliferation assay was performed by seeding young or aged broblasts into a top and bottom collagen layer (collagen I, Gibco A1048301) and GFP melanoma cells in a middle layer at a density of 2x10 5 in a 24 well plate. The cells were imaged using the Nikon TE automated microscope and cell number per eld was quanti ed using NIS elements, with the data represented in GraphPad Prism as the mean +/-the SEM.

Western Blot
Cell lines were plated and lysed with RIPA buffer supplemented with protease and phosphatase inhibitors.
Total protein lysate was quanti ed using a Pierce TM BCA assay kit (# 23225, Thermo Fisher Scienti c) and 50mg of protein was prepared in sample buffer, heated and loaded into NuPAGE™ 4-12% Bis-Tris Protein Gels (#NP0321BOX, Thermo Fisher Scienti c) and ran at 160 volts. Proteins were then transferred onto PVDF membrane using the iBlot system (Invitrogen), and blocked in 5% milk/TBST for 1 hour.
Primary antibodies were diluted in 5% milk/TBST and incubated at 4°C overnight. The membranes were washed in TBST and probed with the corresponding HRP-conjugated secondary antibody at 0.2µg/ml. Proteins were visualized using, ECL prime (Amersham, Uppsala, Sweden) and detected using Doxycycline shRNA Wnt5A (Clone Id: V2THS_172016) TRIPZ system was purchased from Dharmacon. The Wnt5A overexpression cells was driven by the cloned vector pLU-EF1Wnt5A-iCherry. Lentiviral production was performed according to the protocol suggested by the Broad Institute. Brie y, 293T cells were at 70% con uency and co-transfected with shRNA plasmid and the lentiviral packaging plasmids (pCMV-dR8.74psPAX2, pMD2.G for 2 nd generation, pMDLg/pRRE, pRSV/REV and PMD2.G for 3 rd generation). Appropriate empty vector and scrambled controls were created for Overexpressing and shRNA constructs respectively. Cells were transduced with lentivirus for 48 hours, and then treated with appropriate antibiotic selection (puro, hygro, blast) with previously established kill curves for each cell line or were sorted for their selection marker (mCherry or GFP). Tumor sizes for each experiment were measured every 2-4 days using digital calipers, and tumor volumes were calculated using the following formula: volume = 0.5 x (length x width 2 ). Mice were euthanized after 5 weeks or when a group reached 1500 mm 3 , and tumor and lung tissue was preserved. Half of the tissue was embedded in para n and the other half was ash frozen and processed for protein analysis. All reagents injected in live mice were tested for endotoxin levels at University of Pennsylvania Cell Center Services using The Associates of Cape Cod LAL test.

Immunohistochemistry (IHC)
Mouse tumor and lung sections were para n embedded and sectioned. Para n embedded sections were rehydrated through a series of xylene and different concentrations of alcohol, which was followed with a rinse in water and washed in PBS. Slides were put with an antigen retrieval buffer (#3300, Vector Labs, Burlingame, CA) and steamed for 20 minutes. Slides were then blocked in a peroxide blocking buffer (#TA060H2O2Q, Thermo Scienti c) for 15 minutes, followed by protein block (#TA-060-UB, Thermo Scienti c) for 5 minutes and incubated with the primary antibody of interest which was prepared in antibody diluent (S0809, Dako). Slides were put in a humidi ed chamber at 4C overnight. Samples were washed with PBS and incubated in biotinylated anti-rabbit (#ab64256 Abcam), followed by streptavidin-HRP solution at room temperature for 20 minutes (#TS-060-HR Thermo Scienti c). Samples were then washed with PBS and incubated with AEC (3-Amino-9Ethyl-I-Carboazole) chromogen for the appropriate amount of time after optimization (#TA060SA, Thermo Scienti c). Slides were then washed with water and incubated in Mayer's hematoxylin (MHS1, Sigma) for 1 minute, rinsed with water, and mounted in Aquamount (#143905, Thermo Scienti c). Lungs were assessed for localization of mCherry positive melanoma cells using a Nikon eclipse 80I digital. The number of mCherry cells per high power eld (HPF) per lung section was assessed using automated NIS elements software and was analyzed using GraphPad Prism.

TCGA and CCLE Analysis
RNA-Seq of the TCGA and the Cancer Cell Line Encyclopedia (CCLE) data was downloaded from the TCGA database using cBioportal (http://www.cbioportal.org). Individual gene expression values for genes of interest were retrieved as normalized RNA-Seq by Expectation Maximization (RSEM) read counts processed through the TCGA and CCLE cBioportal. The data was then transformed to represent a standardized Z-score for each gene and further transformed such that genes of interest (Wnt5A, AXL, MER) were separated into two groups based on the top 25 percentile and bottom 25 percentile of gene expression within the melanoma patient samples from the TCGA dataset (N=~120) t and the top and bottom 50 th percentile for melanoma cells within the CCLE (N=~34). A students t-test (unpaired) was then performed on each gene in the data set to individually analyze the difference in average Z-score in low vs. high percentile melanoma samples. Data is presented as a heatmap based on high (red, 0.5) and low (blue, -0.5) z-scores and associated p-value. Signi cance was taken at p<0.05. Overall survival and progression free interval was obtained from the TCGA dataset. Metastatic patient samples only (Distant and lymph) were strati ed into those that survived in under 3 years vs greater then 3 years and data was analyzed using a students T-test and present as the mean +/-SEM. Kaplan Meier probability/survival plots were also performed on metastatic patient samples.

Proteomics
Fibroblasts were plated at an equal number and grown to 70~ con uency in a 10 cm dish. They were washed one time with PBS and 5 times with serum free media, and then grown for 16 hours in 10 mls of serum free media. Conditioned media was collected, centrifuge at low speed to remove cells and debris, then centrifuge through a 0.22 um lter. Protease inhibitors (PMSF, Pepstatin A, and Leupeptin) were added and samples at this time were ash frozen at -80 o C until ready. Fibroblasts from the 10cm dish were disassociated and cell number (haemocytometer) and protein concentration was determined using a Pierce BCA assay (Thermo Fisher). CM was then concentrated using a 3K MWCO ultra ltration concentrator. Equal protein concentrations and media was assessed via colloidal Coomassie staining on a gel and BCA assay and proportional volumes were loaded on a preparative gel. Lanes for each sample were excised, digested with trypsin and analysed by TMT10plex with peptide fractionation. Raw protein intensity levels were log2 transformed and undetected intensities were oored to a minimum detected intensity across all proteins and samples. Unpaired students t-test was performed to estimate signi cance of difference between conditions and false discovery rate was estimated 33 . Proteins that passed FDR<5% were considered signi cant and top changed proteins with nominal p<0.05 were also reported on a scatter plot.

Statistical Analysis
For in vitro studies, a students t-test or Mann Whitney was performed for two-group comparisons. Estimate of variance was performed and unequal variances for the t-test were adjusted accordingly using Welch's correction. Multiple comparisons were performed using ANOVA or Kruskal-Wallis test with posthoc Holm-Sidak's adjusted p-values. The indicated sample size for each in vivo study was designed to have 80% power at a two-sided alpha of 0.05 to detect a difference of large effect size of about 1.5 between two groups on a continuous measurement. GraphPad/Prism 8 was used for plotting graphs and statistical analysis. Signi cance was designated as follows: *, p<0.05; **, p<0.01; ***, p<0.001, p<0.0001, ****.  Figure 1 The aged lung microenvironment promotes e cient lung metastasis via aged broblasts. A Young (8 week) and aged (<52 week) c57BL6 mice were subdermally injected with 2.5x105 Yumm1.7 mCherry murine melanoma cells and tumors were measured over 36 days. N=5. B-C Lungs were collected and PFA embedded from tumor bearing young and aged mice at weeks 3 and 5 and IHC was performed using an mCherry antibody. Total number of positive cells were counted and averaged per high powered eld (20x) and presented as mean +/-SEM, with representative images displayed (N=5). D-E Collagen sandwich reconstructs were formed with healthy human lung or skin broblasts from young (>35) or aged (<55) patients in the top and bottom layer. The middle layer contained 1205lu GFP human melanoma cells seeded at the same density on day 0. GFP positive melanoma cells were imaged using an automated Nikon TI and an average count per eld was quanti ed using imaging software NIS elements at day 4. F-G 1205lu GFP melanoma cells were seeded at a density of 2x104. Cells were then grown over a 10 day period in conditioned media from young or aged lung or skin broblasts and underwent cell counting at days 3,5 and 10. H Proteomic analysis was performed on two combined young (>35) vs aged (<55) healthy human lung broblasts in triplicate. Unpaired t-test was performed to estimate signi cance of difference between conditions and false discovery rate was estimated. Proteins that passed FDR<5% were considered signi cant.I-J Western blot analysis was performed on tumor protein lysate from 5 young and 5 aged mice investigating dormancy and proliferative associated expression. HSP90 was used as a loading control.

Figure 2
The aged lung broblast secretome promotes metastatic melanoma outgrowth via sFRP1 secretion. A-B Protein lysate was collected from 1205lu human melanoma cells treated with young or aged lung or skin broblast conditioned media over a 5-day period. Western blot analysis was performed examining dormant (Wnt5A, AXL, NR2F1, p21, p27 P-p38) and proliferative (H2AFZ, MER, MITF) melanoma markers.
Densitometry analysis was performed relative to the loading control HSP90, and was normalized to control (C) cells. C Yumm1.7 and 1205lu melanoma cells were treated with recombinant sFRP1 every two days and protein expression was assessed after 5 days via western blot. D Proliferation was assessed in these cells over 10 days of treatment. E Aged-lung broblasts were transduced with lentiviral sh-sFRP1 (SH) or scrambled control (NEG) vectors. Westernblot analysis was performed on protein lysates. F Conditioned media (CM) was taken from SH and control (shNEG) broblasts after 72 hours. 1205lu melanoma cells were initially seeded at 2x104 and were grown in CM, or with regular media (DMEM) for 10 days with proliferation assessed. G-H Aged mice were subdermally injected with 2.5x105 Yumm1.7 mCherry melanoma cells. After three weeks of tumor growth, mice were treated with 1mg/kg of a neutralizing sFRP1 or an IgG control antibody via IP injection every 4 days and lung metastasis was assessed at week 5 via total number of positive cells averaged per high powered eld (20x). Data was presented as mean +/-SEM, with representative images displayed (N=6). I Comparative diagram displaying key differences in the metastatic cascade between young and aged patients. A switch from sFRP2 to sFRP1 secretion by aged broblasts in the aged skin and lung respectively promotes e cient metastatic progression in aged models Figure 3 Temporal downregulation of Wnt5A promotes metastatic melanoma outgrowth in previously dormant microenvironments. A-B Analysis of the melanoma TCGA dataset (all samples) was performed on human samples strati ed into the top (Wnt5A high) and bottom (Wnt5a low) 25th percentile of Wnt5A expression and presented as a heat map of the average Z-score. Unpaired t-test was performed to estimate signi cance of difference between conditions and P<0.05 was considered signi cant. C Protein expression analysis was performed on Wnt5A low human melanoma samples (FS13, FS14) that underwent lentiviral induced overexpression of Wnt5A (OE). Densitometry was performed against HSP90, which was used as a loading control and values were normalized to control (C) for each line. D-E TCGA analysis was performed on metastatic (distant and lymph mets only) patient samples strati ed by progression free index (PFI) for samples that progressed in under 3 years vs those that took longer than 3 years or did not progress. Patient age and relative Wnt5A mRNA expression was assessed.  Age-induced differential AXL/MER axis promotes a dormancy and reactivation axis during metastatic melanoma. A Protein expression analysis of 3 Wnt5A high (FS4, FS5, 1205lu) and 2 Wnt5A low (FS13, FS14) human melanoma samples. HSP 90 was used as a loading control. B-C Analysis of melanoma cell lines from the Cancer Cell line Encyclopedia (CCLE) dataset was performed on human samples strati ed into the top (AXL high) and bottom (AXL low) 50th percentile of MER expression (N=30) and presented as a heat map of the average Z-score. An unpaired t-test was performed on each condition, investigating previously established dormancy and proliferative genes, with P<0.05 considered signi cant. D-E Analysis of MER high and MER low cells from the CCLE as described. F 1205lu shAXL and control human melanoma cells were seeded at a density of 2x104. Cells were then grown over a 10-day period and proliferation was assessed. G Westernblot analysis was performed on these shAXL vs control lysates. Densitometry was performed against HSP90, which was used as a loading control and values were normalized to control (C) for each line H-I Proliferation assay and western blot assays as described on shMER FS13 and control human melanoma cells. J-K Western blot and proliferation assay as described on Dox inducible MER Yumm1.7 melanoma cells vs non-dox control cells. L TCGA analysis of relative mRNA expression of MER in patient tumor samples comparing primary vs metastatic (distant and lymph mets) tissue sites. M Tumor volumes from young mice subdermally injected with 2.5x105 Yumm1.7 DOX inducible MER mCherry melanoma cells. Mice were treated with DOX (2ug/ml in water) from day 3 or day 21 onwards and compared with a no dox control and were measured over 31 days. N-O IHC analysis of mCherry positive melanoma cells in the lungs of these mice was assessed after 5 weeks of total tumor growth via an average count per high powered eld, with representative images displayed for each (N=5). Data was presented as mean +/-SEM. P Western blot analysis of on Dox inducible AXL Yumm1.7 melanoma cells vs non-dox control cells. Q Tumor volumes from aged mice subdermally injected with 2.5x105 Yumm1.7 DOX inducible AXL mCherry melanoma cells. Mice were treated with DOX (2ug/ml in water) from day 3 or day 21 onwards and compared with a no dox control and were measured over 36 days. R-S IHC analysis of mCherry positive melanoma cells in the lungs of these mice as described above. Figure 5