Whole-body imaging of lymphovascular niches identifies pre-metastatic roles of midkine.

Cutaneous melanoma is a type of cancer with an inherent potential for lymph node colonization, which is generally preceded by neolymphangiogenesis. However, sentinel lymph node removal does not necessarily extend the overall survival of patients with melanoma. Moreover, lymphatic vessels collapse and become dysfunctional as melanomas progress. Therefore, it is unclear whether (and how) lymphangiogenesis contributes to visceral metastasis. Soluble and vesicle-associated proteins secreted by tumours and/or their stroma have been proposed to condition pre-metastatic sites in patients with melanoma. Still, the identities and prognostic value of lymphangiogenic mediators remain unclear. Moreover, our understanding of lymphangiogenesis (in melanomas and other tumour types) is limited by the paucity of mouse models for live imaging of distal pre-metastatic niches. Injectable lymphatic tracers have been developed, but their limited diffusion precludes whole-body imaging at visceral sites. Vascular endothelial growth factor receptor 3 (VEGFR3) is an attractive 'lymphoreporter' because its expression is strongly downregulated in normal adult lymphatic endothelial cells, but is activated in pathological situations such as inflammation and cancer. Here, we exploit this inducibility of VEGFR3 to engineer mouse melanoma models for whole-body imaging of metastasis generated by human cells, clinical biopsies or endogenously deregulated oncogenic pathways. This strategy revealed early induction of distal pre-metastatic niches uncoupled from lymphangiogenesis at primary lesions. Analyses of the melanoma secretome and validation in clinical specimens showed that the heparin-binding factor midkine is a systemic inducer of neo-lymphangiogenesis that defines patient prognosis. This role of midkine was linked to a paracrine activation of the mTOR pathway in lymphatic endothelial cells. These data support the use of VEGFR3 reporter mice as a 'MetAlert' discovery platform for drivers and inhibitors of metastasis.

Atherosclerotic Pre-Conditioning Affects the Paracrine Role of Circulating Angiogenic Cells Ex-Vivo.

In atherosclerosis, circulating angiogenic cells (CAC), also known as early endothelial progenitor cells (eEPC), are thought to participate mainly in a paracrine fashion by promoting the recruitment of other cell populations such as late EPC, or endothelial colony-forming cells (ECFC), to the injured areas. There, ECFC replace the damaged endothelium, promoting neovascularization. However, despite their regenerative role, the number and function of EPC are severely affected under pathological conditions, being essential to further understand how these cells react to such environments in order to implement their use in regenerative cell therapies. Herein, we evaluated the effect of direct incubation ex vivo of healthy CAC with the secretome of atherosclerotic arteries. By using a quantitative proteomics approach, 194 altered proteins were identified in the secretome of pre-conditioned CAC, many of them related to inhibition of angiogenesis (e.g., endostatin, thrombospondin-1, fibulins) and cell migration. Functional assays corroborated that healthy CAC released factors enhanced ECFC angiogenesis, but, after atherosclerotic pre-conditioning, the secretome of pre-stimulated CAC negatively affected ECFC migration, as well as their ability to form tubules on a basement membrane matrix assay. Overall, we have shown here, for the first time, the effect of atherosclerotic factors over the paracrine role of CAC ex vivo. The increased release of angiogenic inhibitors by CAC in response to atherosclerotic factors induced an angiogenic switch, by blocking ECFC ability to form tubules in response to pre-conditioned CAC. Thus, we confirmed here that the angiogenic role of CAC is highly affected by the atherosclerotic environment.

Urea Artifacts Interfere with Immuno-Purification of Lysine Acetylation.

Comprehensive analysis of post-translational modifications (PTMs) often depends on the purification of modified peptides prior to LC-MS/MS. The implementation of these enrichment methods requires thorough knowledge of the experimental conditions to achieve optimal selectivity and sensitivity. In this regard, large-scale analysis of lysine acetylation, a key PTM for multiple cellular processes, makes use of monoclonal pan-antibodies designed against this moiety. We report that the immuno-purification of lysine-acetylated peptides is hampered by the copurification of lysine carbamylated peptides, a frequent urea artifact. This specific interaction can be explained by the similar chemical structures of lysine acetylation and lysine carbamylation. As an alternative, we propose a sample preparation protocol based on sodium deoxycholate that eliminates these artifacts and dramatically improves the selectivity and sensitivity of this immuno-purification assay.

On the Statistical Significance of Compressed Ratios in Isobaric Labeling: A Cross-Platform Comparison.

Isobaric labeling is gaining popularity in proteomics due to its multiplexing capacity. However, copeptide fragmentation introduces a bias that undermines its accuracy. Several strategies have been shown to partially and, in some cases, completely solve this issue. However, it is still not clear how ratio compression affects the ability to identify a protein's change of abundance as statistically significant. Here, by using the "two proteomes" approach (E. coli lysates with fixed 2.5 ratios in the presence or absence of human lysates acting as the background interference) and manipulating isolation width values, we were able to model isobaric data with different levels of accuracy and precision in three types of mass spectrometers: LTQ Orbitrap Velos, Impact, and Q Exactive. We determined the influence of these variables on the statistical significance of the distorted ratios and compared them to the ratios measured without impurities. Our results confirm previous findings1-4 regarding the importance of optimizing acquisition parameters in each instrument in order to minimize interference without compromising precision and identification. We also show that, under these experimental conditions, the inclusion of a second replicate increases statistical sensitivity 2-3-fold and counterbalances to a large extent the issue of ratio compression.

Live imaging of neolymphangiogenesis identifies acute antimetastatic roles of dsRNA mimics.

Long-range communication between tumor cells and the lymphatic vasculature defines competency for metastasis in different cancer types, particularly in melanoma. Nevertheless, the discovery of selective blockers of lymphovascular niches has been compromised by the paucity of experimental systems for whole-body analyses of tumor progression. Here, we exploit immunocompetent and immunodeficient mouse models for live imaging of Vegfr3-driven neolymphangiogenesis, as a versatile platform for drug screening in vivo. Spatiotemporal analyses of autochthonous melanomas and patient-derived xenografts identified double-stranded RNA mimics (dsRNA nanoplexes) as potent inhibitors of neolymphangiogenesis, metastasis, and post-surgical disease relapse. Mechanistically, dsRNA nanoplexes were found to exert a rapid dual action in tumor cells and in their associated lymphatic vasculature, involving the transcriptional repression of the lymphatic drivers Midkine and Vegfr3, respectively. This suppressive function was mediated by a cell-autonomous type I interferon signaling and was not shared by FDA-approved antimelanoma treatments. These results reveal an alternative strategy for targeting the tumor cell-lymphatic crosstalk and underscore the power of Vegfr3-lymphoreporters for pharmacological testing in otherwise aggressive cancers.

Midkine rewires the melanoma microenvironment toward a tolerogenic and immune-resistant state.

An open question in aggressive cancers such as melanoma is how malignant cells can shift the immune system to pro-tumorigenic functions. Here we identify midkine (MDK) as a melanoma-secreted driver of an inflamed, but immune evasive, microenvironment that defines poor patient prognosis and resistance to immune checkpoint blockade. Mechanistically, MDK was found to control the transcriptome of melanoma cells, allowing for coordinated activation of nuclear factor-κB and downregulation of interferon-associated pathways. The resulting MDK-modulated secretome educated macrophages towards tolerant phenotypes that promoted CD8+ T cell dysfunction. In contrast, genetic targeting of MDK sensitized melanoma cells to anti-PD-1/anti-PD-L1 treatment. Emphasizing the translational relevance of these findings, the expression profile of MDK-depleted tumors was enriched in key indicators of a good response to immune checkpoint blockers in independent patient cohorts. Together, these data reveal that MDK acts as an internal modulator of autocrine and paracrine signals that maintain immune suppression in aggressive melanomas.

The RNA Polymerase II Factor RPAP1 Is Critical for Mediator-Driven Transcription and Cell Identity.

The RNA polymerase II-associated protein 1 (RPAP1) is conserved across metazoa and required for stem cell differentiation in plants; however, very little is known about its mechanism of action or its role in mammalian cells. Here, we report that RPAP1 is essential for the expression of cell identity genes and for cell viability. Depletion of RPAP1 triggers cell de-differentiation, facilitates reprogramming toward pluripotency, and impairs differentiation. Mechanistically, we show that RPAP1 is essential for the interaction between RNA polymerase II (RNA Pol II) and Mediator, as well as for the recruitment of important regulators, such as the Mediator-specific RNA Pol II factor Gdown1 and the C-terminal domain (CTD) phosphatase RPAP2. In agreement, depletion of RPAP1 diminishes the loading of total and Ser5-phosphorylated RNA Pol II on many genes, with super-enhancer-driven genes among the most significantly downregulated. We conclude that Mediator/RPAP1/RNA Pol II is an ancient module, conserved from plants to mammals, critical for establishing and maintaining cell identity.

Integrative analysis of miRNA and mRNA expression profiles in pheochromocytoma and paraganglioma identifies genotype-specific markers and potentially regulated pathways.

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare neuroendocrine neoplasias of neural crest origin that can be part of several inherited syndromes. Although their mRNA profiles are known to depend on genetic background, a number of questions related to tumor biology and clinical behavior remain unanswered. As microRNAs (miRNAs) are key players in the modulation of gene expression, their comprehensive analysis could resolve some of these issues. Through characterization of miRNA profiles in 69 frozen tumors with germline mutations in the genes SDHD, SDHB, VHL, RET, NF1, TMEM127, and MAX, we identified miRNA signatures specific to, as well as common among, the genetic groups of PCCs/PGLs. miRNA expression profiles were validated in an independent series of 30 composed of VHL-, SDHB-, SDHD-, and RET-related formalin-fixed paraffin-embedded PCC/PGL samples using quantitative real-time PCR. Upregulation of miR-210 in VHL- and SDHB-related PCCs/PGLs was verified, while miR-137 and miR-382 were confirmed as generally upregulated in PCCs/PGLs (except in MAX-related tumors). Also, we confirmed overexpression of miR-133b as VHL-specific miRNAs, miR-488 and miR-885-5p as RET-specific miRNAs, and miR-183 and miR-96 as SDHB-specific miRNAs. To determine the potential roles miRNAs play in PCC/PGL pathogenesis, we performed bioinformatic integration and pathway analysis using matched mRNA profiling data that indicated a common enrichment of pathways associated with neuronal and neuroendocrine-like differentiation. We demonstrated that miR-183 and/or miR-96 impede NGF-induced differentiation in PC12 cells. Finally, global proteomic analysis in SDHB and MAX tumors allowed us to determine that miRNA regulation occurs primarily through mRNA degradation in PCCs/PGLs, which partially confirmed our miRNA-mRNA integration results.