ABSTRACT
In several solid tumors such as breast cancer, prostate cancer, colorectal cancer or melanoma, tumor draining lymph nodes are the earliest tissues where colonization by tumor cells is detected. Lymph nodes act as sentinels of metastatic dissemination, the deadliest phase of tumor progression. Besides hematogenous dissemination, lymphatic spread of tumor cells has been demonstrated, adding more complexity to the mechanisms involved in metastasis. A network of blood and lymphatic vessels surrounds tumors providing routes for tumor soluble factors to mediate regional and long-distance effects. Additionally, extracellular vesicles (EVs), particularly small EVs/exosomes, have been shown to circulate through the blood and lymph, favoring the formation of pre-metastatic niches in the tumor-draining lymph nodes (TDLNs) and distant organs. In this review, we present an overview of the relevance of lymph node metastasis, the structural and immune changes occurring in TDLNs during tumor progression, and how extracellular vesicles contribute to modulating some of these alterations while promoting the formation of lymph node pre-metastatic niches.
Subject(s)
Extracellular Vesicles , Lymphatic Metastasis , Humans , Lymphatic Metastasis/pathology , Extracellular Vesicles/metabolism , Animals , Lymph Nodes/pathology , Lymph Nodes/metabolism , Neoplasms/pathology , Neoplasms/metabolismABSTRACT
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options, particularly with immune checkpoint inhibitors. Highly chemoresistant 'stem-like' cells, known as cancer stem cells (CSCs), are implicated in PDAC aggressiveness. Thus, comprehending how this subset of cells evades the immune system is crucial for advancing novel therapies. DESIGN: We used the KPC mouse model (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) and primary tumour cell lines to investigate putative CSC populations. Transcriptomic analyses were conducted to pinpoint new genes involved in immune evasion. Overexpressing and knockout cell lines were established with lentiviral vectors. Subsequent in vitro coculture assays, in vivo mouse and zebrafish tumorigenesis studies, and in silico database approaches were performed. RESULTS: Using the KPC mouse model, we functionally confirmed a population of cells marked by EpCAM, Sca-1 and CD133 as authentic CSCs and investigated their transcriptional profile. Immune evasion signatures/genes, notably the gene peptidoglycan recognition protein 1 (PGLYRP1), were significantly overexpressed in these CSCs. Modulating PGLYRP1 impacted CSC immune evasion, affecting their resistance to macrophage-mediated and T-cell-mediated killing and their tumourigenesis in immunocompetent mice. Mechanistically, tumour necrosis factor alpha (TNFα)-regulated PGLYRP1 expression interferes with the immune tumour microenvironment (TME) landscape, promoting myeloid cell-derived immunosuppression and activated T-cell death. Importantly, these findings were not only replicated in human models, but clinically, secreted PGLYRP1 levels were significantly elevated in patients with PDAC. CONCLUSIONS: This study establishes PGLYRP1 as a novel CSC-associated marker crucial for immune evasion, particularly against macrophage phagocytosis and T-cell killing, presenting it as a promising target for PDAC immunotherapy.
Subject(s)
Carcinoma, Pancreatic Ductal , Neoplastic Stem Cells , Pancreatic Neoplasms , Animals , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Mice , Neoplastic Stem Cells/immunology , Neoplastic Stem Cells/metabolism , Carcinoma, Pancreatic Ductal/immunology , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Humans , Cell Line, Tumor , Tumor Escape/immunology , Disease Models, Animal , Immune Evasion , Tumor Microenvironment/immunologyABSTRACT
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.
Subject(s)
Nerve Sheath Neoplasms , Neurofibrosarcoma , Humans , Biomarkers , Cell Line, Tumor , Endoglin/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , Nerve Sheath Neoplasms/drug therapy , Nerve Sheath Neoplasms/genetics , Nerve Sheath Neoplasms/metabolism , Signal TransductionABSTRACT
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.
Subject(s)
Extracellular Vesicles , Lymphatic Vessels , Melanoma , CD8-Positive T-Lymphocytes , Endothelial Cells/metabolism , Humans , Lymph Nodes , Lymphatic Metastasis/pathology , Lymphatic Vessels/pathology , Melanoma/metabolismABSTRACT
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.
ABSTRACT
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.
Subject(s)
Extracellular Vesicles , Melanoma , Animals , Endothelial Cells/metabolism , Extracellular Vesicles/metabolism , Humans , Lymphangiogenesis/physiology , Lymphatic Metastasis , Melanoma/metabolism , Mice , Nerve Tissue Proteins , Receptors, Nerve Growth Factor/genetics , Tumor MicroenvironmentABSTRACT
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.
Subject(s)
Amyloid beta-Peptides/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Melanoma/drug therapy , Molecular Targeted Therapy , Nerve Tissue Proteins/agonists , Receptors, Nerve Growth Factor/agonists , Animals , Apoptosis , Cell Proliferation , Drug Therapy, Combination , Female , Humans , Melanoma/metabolism , Melanoma/pathology , Mice , Mice, Nude , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , ZebrafishABSTRACT
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.
Subject(s)
Extracellular Vesicles/metabolism , Lymphatic System/diagnostic imaging , Nanoparticle Drug Delivery System , Animals , Extracellular Vesicles/pathology , Humans , Lymph Nodes/diagnostic imaging , Lymph Nodes/drug effects , Lymph Nodes/pathology , Lymphatic System/drug effects , Lymphatic System/pathology , Lymphatic Vessels/diagnostic imaging , Lymphatic Vessels/drug effects , Lymphatic Vessels/pathology , Sentinel Lymph Node/diagnostic imaging , Sentinel Lymph Node/pathologyABSTRACT
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.
Subject(s)
Extracellular Vesicles/metabolism , Melanoma/metabolism , Tetraspanin 29/metabolism , Cell Line , Humans , Melanoma/genetics , Mitophagy/genetics , Mitophagy/physiology , Secretory Vesicles/metabolism , Tetraspanin 29/analysis , Tetraspanin 29/antagonists & inhibitors , Tetraspanin 30/analysis , Tetraspanins/analysis , Tetraspanins/genetics , Tetraspanins/metabolismABSTRACT
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.
Subject(s)
Exosomes/metabolism , Lymph Node Excision , Lymphatic Vessels/metabolism , Melanoma/metabolism , Melanoma/surgery , HumansABSTRACT
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.
Subject(s)
Extracellular Vesicles/metabolism , Fibroblasts/metabolism , NF-kappa B/metabolism , Plasma , Plasmodium vivax/physiology , Reticulocytes/metabolism , Spleen/metabolism , Animals , Cell Adhesion , Cell-Derived Microparticles , Disease Models, Animal , Extracellular Vesicles/parasitology , Fibroblasts/pathology , Host-Parasite Interactions/physiology , Humans , Intercellular Adhesion Molecule-1/metabolism , Malaria, Vivax/parasitology , Male , Mice , Mice, Inbred C57BL , Microvessels/parasitology , Proteomics , Reticulocytes/parasitology , Spleen/pathologyABSTRACT
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.
Subject(s)
Disease Progression , Extracellular Vesicles/metabolism , Exudates and Transudates/metabolism , Melanoma/genetics , Melanoma/metabolism , Mutation , Proto-Oncogene Proteins B-raf/genetics , Seroma/metabolism , Skin Neoplasms/genetics , Skin Neoplasms/metabolism , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/metabolism , Cell Line, Tumor , Cohort Studies , Disease-Free Survival , Drainage , Exosomes/metabolism , Female , Humans , Lymphatic Metastasis , Male , Melanoma/pathology , Middle Aged , Proteomics/methods , Skin Neoplasms/pathologyABSTRACT
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.
Subject(s)
Lung Neoplasms/immunology , Lung/immunology , Melanoma/immunology , Neutrophil Infiltration/immunology , Neutrophils/immunology , Animals , Female , Granulocyte Colony-Stimulating Factor/genetics , Granulocyte Colony-Stimulating Factor/immunology , Interleukin-23/genetics , Interleukin-23/immunology , Lung/pathology , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Macrophages/immunology , Macrophages/pathology , Male , Melanoma/genetics , Melanoma/pathology , Mice , Mice, Knockout , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/immunology , Neutrophils/pathology , Transcription, Genetic/immunologyABSTRACT
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.
Subject(s)
Macrophages/physiology , Phagocytosis/physiology , Animals , Female , Interleukin-1beta/metabolism , Lectins, C-Type/metabolism , Male , Mannose Receptor , Mannose-Binding Lectins/metabolism , Mice , Mice, Inbred C57BL , Opsonin Proteins/physiology , Receptors, Cell Surface/metabolism , Transcription Factors/physiologyABSTRACT
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.