Endoglin, a Novel Biomarker and Therapeutical Target to Prevent Malignant Peripheral Nerve Sheath Tumor Growth and Metastasis.

PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive soft-tissue sarcomas that lack effective treatments, underscoring the urgent need to uncover novel mediators of MPNST pathogenesis that may serve as potential therapeutic targets. Tumor angiogenesis is considered a critical event in MPNST transformation and progression. Here, we have investigated whether endoglin (ENG), a TGFß coreceptor with a crucial role in angiogenesis, could be a novel therapeutic target in MPNSTs. EXPERIMENTAL DESIGN: ENG expression was evaluated in human peripheral nerve sheath tumor tissues and plasma samples. Effects of tumor cell-specific ENG expression on gene expression, signaling pathway activation and in vivo MPNST growth and metastasis, were investigated. The efficacy of ENG targeting in monotherapy or in combination with MEK inhibition was analyzed in xenograft models. RESULTS: ENG expression was found to be upregulated in both human MPNST tumor tissues and plasma-circulating small extracellular vesicles. We demonstrated that ENG modulates Smad1/5 and MAPK/ERK pathway activation and pro-angiogenic and pro-metastatic gene expression in MPNST cells and plays an active role in tumor growth and metastasis in vivo. Targeting with ENG-neutralizing antibodies (TRC105/M1043) decreased MPNST growth and metastasis in xenograft models by reducing tumor cell proliferation and angiogenesis. Moreover, combination of anti-ENG therapy with MEK inhibition effectively reduced tumor cell growth and angiogenesis. CONCLUSIONS: Our data unveil a tumor-promoting function of ENG in MPNSTs and support the use of this protein as a novel biomarker and a promising therapeutic target for this disease.

Melanoma-derived extracellular vesicles mediate lymphatic remodelling and impair tumour immunity in draining lymph nodes.

Tumour-draining lymph nodes (LNs) undergo massive remodelling including expansion of the lymphatic sinuses, a process that has been linked to lymphatic metastasis by creation of a pre-metastatic niche. However, the signals leading to these changes have not been completely understood. Here, we found that extracellular vesicles (EVs) derived from melanoma cells are rapidly transported by lymphatic vessels to draining LNs, where they selectively interact with lymphatic endothelial cells (LECs) as well as medullary sinus macrophages. Interestingly, uptake of melanoma EVs by LN-resident LECs was partly dependent on lymphatic VCAM-1 expression, and induced transcriptional changes as well as proliferation of those cells. Furthermore, melanoma EVs shuttled tumour antigens to LN LECs for cross-presentation on MHC-I, resulting in apoptosis induction in antigen-specific CD8+ T cells. In conclusion, our data identify EV-mediated melanoma-LN LEC communication as a new pathway involved in tumour progression and tumour immune inhibition, suggesting that EV uptake or effector mechanisms in LECs might represent a new target for melanoma therapy.

Extracellular vesicles and melanoma: New perspectives on tumor microenvironment and metastasis.

Secreted extracellular vesicles (EVs) are lipid bilayer particles without functional nucleus naturally released from cells which constitute an intercellular communication system. There is a broad spectrum of vesicles shed by cells based on their physical properties such as size (small EVs and large EVs), biogenesis, cargo and functions, which provide an increasingly heterogenous landscape. In addition, they are involved in multiple physiological and pathological processes. In cancer, EV release is opted by tumor cells as a beneficial process for tumor progression. Cutaneous melanoma is a cancer that originates from the melanocyte lineage and shows a favorable prognosis at early stages. However, when melanoma cells acquire invasive capacity, it constitutes the most aggressive and deadly skin cancer. In this context, extracellular vesicles have been shown their relevance in facilitating melanoma progression through the modulation of the microenvironment and metastatic spreading. In agreement with the melanosome secretory capacity of melanocytes, melanoma cells display an enhanced EV shedding activity that has contributed to the utility of melanoma models for unravelling EV cargo and functions within a cancer scenario. In this review, we provide an in-depth overview of the characteristics of melanoma-derived EVs and their role in melanoma progression highlighting key advances and remaining open questions in the field.

Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through an NGFR-dependent mechanism.

Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines were enriched in nerve growth factor receptor (NGFR, p75NTR), spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK kinase, nuclear factor (NF)-κB activation and intracellular adhesion molecule (ICAM)-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to that in matched primary tumors, and the frequency of NGFR+ metastatic melanoma cells in lymph nodes correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs, reinforcing lymph node pre-metastatic niche formation and metastasis.

Specific Activation of the CD271 Intracellular Domain in Combination with Chemotherapy or Targeted Therapy Inhibits Melanoma Progression.

CD271 (NGFR) is a neurotrophin receptor that belongs to the tumor necrosis receptor (TNFR) family. Upon ligand binding, CD271 can mediate either survival or cell death. Although the role of CD271 as a marker of tumor-initiating cells is still a matter of debate, its role in melanoma progression has been well documented. Moreover, CD271 has been shown to be upregulated after exposure to both chemotherapy and targeted therapy. In this study, we demonstrate that activation of CD271 by a short ß-amyloid-derived peptide (Aß(25-35)) in combination with either chemotherapy or MAPK inhibitors induces apoptosis in 2D and 3D cultures of eight melanoma cell lines. This combinatorial treatment significantly reduced metastasis in a zebrafish xenograft model and led to significantly decreased tumor volume in mice. Administration of Aß(25-35) in ex vivo tumors from immunotherapy- and targeted therapy-resistant patients significantly reduced proliferation of melanoma cells, showing that activation of CD271 can overcome drug resistance. Aß(25-35) was specific to CD271-expressing cells and induced CD271 cleavage and phosphorylation of JNK (pJNK). The direct protein-protein interaction of pJNK with CD271 led to PARP1 cleavage, p53 and caspase activation, and pJNK-dependent cell death. Aß(25-35) also mediated mitochondrial reactive oxygen species (mROS) accumulation, which induced CD271 overexpression. Finally, CD271 upregulation inhibited mROS production, revealing the presence of a negative feedback loop in mROS regulation. These results indicate that targeting CD271 can activate cell death pathways to inhibit melanoma progression and potentially overcome resistance to targeted therapy. SIGNIFICANCE: The discovery of a means to specifically activate the CD271 death domain reveals unknown pathways mediated by the receptor and highlights new treatment possibilities for melanoma.

Physiological models for in vivo imaging and targeting the lymphatic system: Nanoparticles and extracellular vesicles.

Imaging of the lymphatic vasculature has gained great attention in various fields, not only because lymphatic vessels act as a key draining system in the body, but also for their implication in autoimmune diseases, organ transplant, inflammation and cancer. Thus, neolymphangiogenesis, or the generation of new lymphatics, is typically an early event in the development of multiple tumor types, particularly in aggressive ones such as malignant melanoma. Still, the understanding of how lymphatic endothelial cells get activated at distal (pre)metastatic niches and their impact on therapy is still unclear. Addressing these questions is of particular interest in the case of immune modulators, because endothelial cells may favor or halt inflammatory processes depending on the cellular context. Therefore, there is great interest in visualizing the lymphatic vasculature in vivo. Here, we review imaging tools and mouse models used to analyze the lymphatic vasculature during tumor progression. We also discuss therapeutic approaches based on nanomedicines to target the lymphatic system and the potential use of extracellular vesicles to track and target sentinel lymph nodes. Finally, we summarize main pre-clinical models developed to visualize the lymphatic vasculature in vivo, discussing their applications with a particular focus in metastatic melanoma.

CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells.

Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells.

Postlymphadenectomy Analysis of Exosomes from Lymphatic Exudate/Exudative Seroma of Melanoma Patients.

Circulating extracellular vesicles in biofluids have become an interesting approach to analyse disease biomarkers. There are multiple methods for isolation of extracellular vesicles, though differential ultracentrifugation is still considered as the gold-standard isolation technique for exosomes. Furthermore, exosomes purified by this method have been demonstrated to display functional activity in vitro and in vivo and exhibit great versatility for subsequent analysis including proteomics, electron microscopy, mass spectrometry, or nucleic acid analysis. Here, we describe the method for isolation of exosomes from lymphatic exudate (seroma) obtained postlymphadenectomy for liquid biopsy approaches.

Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence.

Plasmodium vivax is the most widely distributed human malaria parasite. Previous studies have shown that circulating microparticles during P. vivax acute attacks are indirectly associated with severity. Extracellular vesicles (EVs) are therefore major components of circulating plasma holding insights into pathological processes. Here, we demonstrate that plasma-derived EVs from Plasmodium vivax patients (PvEVs) are preferentially uptaken by human spleen fibroblasts (hSFs) as compared to the uptake of EVs from healthy individuals. Moreover, this uptake induces specific upregulation of ICAM-1 associated with the translocation of NF-kB to the nucleus. After this uptake, P. vivax-infected reticulocytes obtained from patients show specific adhesion properties to hSFs, reversed by inhibiting NF-kB translocation to the nucleus. Together, these data provide physiological EV-based insights into the mechanisms of human malaria pathology and support the existence of P. vivax-adherent parasite subpopulations in the microvasculature of the human spleen.

Use of extracellular vesicles from lymphatic drainage as surrogate markers of melanoma progression and BRAF V600E mutation.

Liquid biopsies from cancer patients have the potential to improve diagnosis and prognosis. The assessment of surrogate markers of tumor progression in circulating extracellular vesicles could be a powerful non-invasive approach in this setting. We have characterized extracellular vesicles purified from the lymphatic drainage also known as exudative seroma (ES) of stage III melanoma patients obtained after lymphadenectomy. Proteomic analysis showed that seroma-derived exosomes are enriched in proteins resembling melanoma progression. In addition, we found that the BRAFV600E mutation can be detected in ES-derived extracellular vesicles and its detection correlated with patients at risk of relapse.

Neutrophils instruct homeostatic and pathological states in naive tissues.

Immune protection relies on the capacity of neutrophils to infiltrate challenged tissues. Naive tissues, in contrast, are believed to remain free of these cells and protected from their toxic cargo. Here, we show that neutrophils are endowed with the capacity to infiltrate multiple tissues in the steady-state, a process that follows tissue-specific dynamics. By focusing in two particular tissues, the intestine and the lungs, we find that neutrophils infiltrating the intestine are engulfed by resident macrophages, resulting in repression of Il23 transcription, reduced G-CSF in plasma, and reinforced activity of distant bone marrow niches. In contrast, diurnal accumulation of neutrophils within the pulmonary vasculature influenced circadian transcription in the lungs. Neutrophil-influenced transcripts in this organ were associated with carcinogenesis and migration. Consistently, we found that neutrophils dictated the diurnal patterns of lung invasion by melanoma cells. Homeostatic infiltration of tissues unveils a facet of neutrophil biology that supports organ function, but can also instigate pathological states.

Phagocytosis imprints heterogeneity in tissue-resident macrophages.

Tissue-resident macrophages display varying phenotypic and functional properties that are largely specified by their local environment. One of these functions, phagocytosis, mediates the natural disposal of billions of cells, but its mechanisms and consequences within living tissues are poorly defined. Using a parabiosis-based strategy, we identified and isolated macrophages from multiple tissues as they phagocytosed blood-borne cellular material. Phagocytosis was circadianally regulated and mediated by distinct repertoires of receptors, opsonins, and transcription factors in macrophages from each tissue. Although the tissue of residence defined the core signature of macrophages, phagocytosis imprinted a distinct antiinflammatory profile. Phagocytic macrophages expressed CD206, displayed blunted expression of Il1b, and supported tissue homeostasis. Thus, phagocytosis is a source of macrophage heterogeneity that acts together with tissue-derived factors to preserve homeostasis.

Melanosomes foster a tumour niche by activating CAFs.

Extracellular vesicles, such as exosomes, are important effectors in the formation of tumour-fostering niches. Pigmented melanosomes are now shown to have a relevant role in establishing a tumour niche in primary melanoma by reprogramming dermal fibroblasts into cancer-associated fibroblasts through the transfer of miR-211.

Microenvironmental hCAP-18/LL-37 promotes pancreatic ductal adenocarcinoma by activating its cancer stem cell compartment.

OBJECTIVES: The tumour stroma/microenvironment not only provides structural support for tumour development, but more importantly it provides cues to cancer stem cells (CSCs) that regulate their self-renewal and metastatic potential. This is certainly true for pancreatic ductal adenocarcinomas (PDAC), where tumour-associated fibroblasts, pancreatic stellate cells and immune cells create an abundant paracrine niche for CSCs via microenvironment-secreted factors. Thus understanding the role that tumour stroma cells play in PDAC development and CSC biology is of utmost importance. DESIGN: Microarray analyses, tumour microarray immunohistochemical assays, in vitro co-culture experiments, recombinant protein treatment approaches and in vivo intervention studies were performed to understand the role that the immunomodulatory cationic antimicrobial peptide 18/LL-37 (hCAP-18/LL-37) plays in PDAC biology. RESULTS: We found that hCAP-18/LL-37 was strongly expressed in the stroma of advanced primary and secondary PDAC tumours and is secreted by immune cells of the stroma (eg, tumour-associated macrophages) in response to tumour growth factor-ß1 and particularly CSC-secreted Nodal/ActivinA. Treatment of pancreatic CSCs with recombinant LL-37 increased pluripotency-associated gene expression, self-renewal, invasion and tumourigenicity via formyl peptide receptor 2 (FPR2)- and P2X purinoceptor 7 receptor (P2X7R)-dependent mechanisms, which could be reversed by inhibiting these receptors. Importantly, in a genetically engineered mouse model of K-Ras-driven pancreatic tumourigenesis, we also showed that tumour formation was inhibited by either reconstituting these mice with bone marrow from cathelicidin-related antimicrobial peptide (ie, murine homologue of hCAP-18/LL-37) knockout mice or by pharmacologically inhibiting FPR2 and P2X7R. CONCLUSIONS: Thus, hCAP-18/LL-37 represents a previously unrecognised PDAC microenvironment factor that plays a critical role in pancreatic CSC-mediated tumourigenesis.

Stem cells & pancreatic cancer.

It is now well established that human pancreatic ductal adenocarcinoma (PDAC) contains a subset of cells with self-renewal capabilities and subsequent exclusive in vivo tumorigenic capacity as assessed by limiting dilution tumorigenic transplantation assays into immunodeficient mice. These cells are considered pancreatic cancer stem cells (CSCs) and are able to form tumors indistinguishable from parental ones. Furthermore they display strong chemotherapy resistance and are implicated in tumor relapses and metastatic spread. Important next steps for advancing the field of pancreatic CSC research include the identification and characterization of CSCs in the unperturbed in vivo setting. This has been achieved just recently for other solid tumors such as glioblastoma using clonal analysis after lineage tracing in mice [1]. In vivo imaging of CSCs during tumor development should not only provide new insights into the in vivo features of CSCs, but also help to further unravel the influence of the stroma on CSC biology. Comprehensive studies of the tumor heterogeneity with respect to the coexistence of different clones potentially generated by distinct population of CSCs that are evolving by stochastic cell fate decisions may actually unite the CSC concept and the model of clonal evolution for pancreatic cancer. Eventually, the design of specific therapies against CSCs should open new alleys to improve survival of patients with PDAC. Combined therapies targeting CSCs and their progenies as well as the supportive stroma may represent the most promising approach for the future treatment of patients with PDAC.

The thyroid hormone receptors as tumor suppressors.

In addition to the well-known role of the thyroid hormone receptors (TRs) in growth, development and metabolism, there is increasing evidence that they have profound effects on cell proliferation and malignant transformation. TRs repress transcriptional induction of cyclin D1 by the ras oncogene and block transformation and tumor formation by Ras-transformed fibroblasts in nude mice. Mutant receptors that do not bind coactivators are able to display these actions, whereas receptors defective in corepressors binding are unable to antagonize the responses to the ras oncogene. Furthermore, expression of TRß1 in hepatocarcinoma and breast cancer cells abolishes anchorage-independent growth and migration, blocks responses to growth factors and represses expression of prometastatic genes, reducing tumor growth and strongly inhibiting invasiveness, extravasation and metastasis formation in euthyroid mice. By contrast, when cells are inoculated into hypothyroid host, tumor growth is retarded, but tumors are more invasive and metastatic growth is enhanced. Increased aggressiveness and tumor growth retardation was also observed with parental cells that do not express TRs, showing that changes secondary to hypothyroidism can modulate tumor progression and metastatic growth independently of the presence of TRs on the tumor cells. Finally, increased malignancy of skin tumors is found in mice lacking TRs, further demonstrating the role of these receptors as inhibitors of tumor progression and suggesting that they represent a potential therapeutic target in cancer.