Human KIT+ myeloid cells facilitate visceral metastasis by melanoma
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ABSTRACT: Metastasis of melanoma significantly worsens prognosis and therefore therapeutic interventions that prevent, or slow metastasis could improve patient outcomes. Here, we show using humanized mice that colonization of distant visceral organs with melanoma is dependent upon a human CD33+CD11b+CD117+ progenitor cell subset comprising <4% of the human CD45+ leukocytes. Metastatic tumor-infiltrating CD33+ cells from patients and humanized (h)NSG-SGM3 mice showed converging transcriptional profiles. Single-cell RNAseq analysis identified a gene signature of a KIT/CD117 expressing CD33+ subset that correlated with decreased overall survival in TCGA melanoma samples. Thus, human CD33+CD11b+CD117+ myeloid cells represent a novel candidate biomarker as well as a therapeutic target for metastatic melanoma.
Project description:Neutrophils play critical roles in health and disease. Due to their very short half-life in blood and tissue, neutrophils are constantly replenished by bone marrow progenitors. Thus, a comprehensive understanding of bone marrow neutrophil development is of paramount importance to identify how neutrophil production is altered in disease. Recently, two novel human neutrophil progenitor populations were identified; ‘human neutrophil progenitor’ or ‘hNeP’ (Lin- CD66b+ CD117+ ) and ‘neutrophil precursor’ or ‘preNeu’ (Lin- CD66b+ CD15+ CD49d+ ). How these subsets fit into the neutrophil lineage is unclear. By using mass and flow cytometry, we show that hNeP are a heterogenous population containing a homogeneous progenitor subset termed ‘early neutrophil progenitor’ or ‘eNeP’ (Lin- CD66b+ CD117+ CD71+ ). Surface marker and RNA expression, together with the ability to form colonies in vitro and exclusively produce neutrophils in vivo in humanized NSG-SGM3 mouse transfer experiments indicate that eNeP are hierarchically the ‘earliest’ cells within preNeu. eNeP constitute ~0.14% of human bone marrow neutrophils, while preNeu constitute ~5% of bone marrow neutrophils. Furthermore, we have identified CD71 as a novel neutrophil surface marker associated with distinct early neutrophil developmental stages. Intriguingly, CD71+ characterizes proliferating neutrophils, which are expanded in the blood of melanoma and lung cancer patients and detectable in human lung tumors. Collectively, our findings i) identify CD117+ CD71+ eNeP as an early neutrophil progenitor population, ii) introduce a unified model of human neutrophil bone marrow development, iii) identify novel surface markers for distinct neutrophil developmental stages and iv) provide evidence for neutrophil progenitor expansion in cancer.
Project description:To identify biological differences between primitive and monocytic primary AML, we sorted ROS-low enriched LSC subfractions from phenotypically monocytic (CD45-bright/SSC-high/CD117-/CD11b+/CD68+/CD64+) and primitive AML (CD45-medium/SSC-low/CD117+/CD11b-/CD68-/CD64-) specimens and performed RNA-seq analysis.
Project description:Under the hypothesis that subset of parental cells may already harbor metastasis potential, which leads progression of metastasis, we investigated single-cell expression of parental B16 melanoma cell (B16F0) and its highly metastatic subclone (B16F10) (N=4,156) using single-cell RNA sequencing
Project description:Brain metastasis is a significant cause of morbidity and mortality in multiple cancer types and represents an unmet clinical need. The mechanisms that mediate metastatic cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the highest rate of brain metastasis among common cancer types, is an ideal model to study how cancer cells adapt to the brain parenchyma. We utilized pairs of brain metastasis-derived (BM) and non-brain metastasis-derived (NBM) melanoma short term cultures (STCs) obtained from the same patient. We performed TMT based multiplexed analysis of these cell lines using off-line fractionation to increase our proteomics coverage. Our unbiased proteomics analysis of these melanoma short-term cultures revealed that proteins implicated in neurodegenerative pathologies are differentially expressed in melanoma cells explanted from brain metastases compared to those derived from extracranial metastases. We showed that melanoma cells require amyloid beta for growth and survival in the brain parenchyma. Melanoma-secreted A beta activates surrounding astrocytes to a pro-metastatic, anti-inflammatory phenotype and prevents phagocytosis of melanoma by microglia. Finally, we demonstrate that pharmacological inhibition of Abeta decreases brain metastatic burden.
Project description:Uveal melanoma (UM) is an aggressive malignancy, in which nearly 50% of the patients die from metastatic disease. Formalin-fixed paraffin-embedded (FFPE) samples represent a valuable source of tumor tissue. Our aim was to investigate differential DNA methylation correlated to gene expression in relation to survival data.We sought to identify aberrant DNA methylation of genes that could be linked to metastatic disease and poor survival. Formalin-fixed parafin-embedded human uveal melanoma tumors; subset early metastasis (n=4) vs subset no metastasis (n=4) were examined by differential gene expression analysis (One-Way ANOVA) to identify 1536 transcripts (1394 up- and 142 down-regulated genes) (p<0.05, FC ≥1.5) .
Project description:Genome wide DNA methylation profiling of CD11b+CD33+ cells after treatment with different cytokines. The Illumina Infinium MethylationEPIC Beadchip was used to obtain DNA methylation profiles across approximately 850,000 CpGs in CD11b+CD33+ cells isolated from PBMCs treated in vitro for 4 days with TNF-alpha, IFN-alpha, IFN-gamma or control.
Project description:Malignant melanoma is characterized by frequent metastasis, however specific changes that regulate this process have not been clearly delineated. Although it is well known that Wnt signaling is frequently dysregulated in melanoma, the functional implications of this observation are unclear. By modulating beta-catenin levels in a mouse model of melanoma that is based on melanocyte-specific Pten loss and BrafV600E mutation, we demonstrate that beta-catenin is a central mediator of melanoma metastasis to lymph node and lung. In addition to altering metastasis, beta-catenin levels control tumor differentiation and regulate both MAPK/Erk and PI3K/Akt signaling. Highly metastatic tumors with beta-catenin stabilization are very similar to a subset of human melanomas; together these findings establish Wnt signaling as a metastasis regulator in melanoma.
Project description:Malignant melanoma is characterized by frequent metastasis, however specific changes that regulate this process have not been clearly delineated. Although it is well known that Wnt signaling is frequently dysregulated in melanoma, the functional implications of this observation are unclear. By modulating beta-catenin levels in a mouse model of melanoma that is based on melanocyte-specific Pten loss and BrafV600E mutation, we demonstrate that beta-catenin is a central mediator of melanoma metastasis to lymph node and lung. In addition to altering metastasis, beta-catenin levels control tumor differentiation and regulate both MAPK/Erk and PI3K/Akt signaling. Highly metastatic tumors with beta-catenin stabilization are very similar to a subset of human melanomas; together these findings establish Wnt signaling as a metastasis regulator in melanoma. MoGene-1_0-st-v1: Four samples total. Two biological replicates of uncultured Pten/Braf murine melanomas and two biological replicates of uncultured Pten/Braf/Bcat-STA murine melanomas. MoEx-1_0-st-v1: Two samples total. Dissociated tumor and FACS-enriched Pten/Braf and Pten/Braf/Bcat-STA murine melanoma.
Project description:Secreted extracellular vesicles are known to influence the tumor microenvironment and promote metastasis. In this work, we have analyzed the involvement of extracellular vesicles in the establishment of lymph node pre-metastatic niches by melanoma cells. We found that small extracellular vesicles (sEVs) derived from highly metastatic melanoma cell lines spread broadly through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Melanoma-derived sEVs induce lymphangiogenesis, a hallmark of pre-metastatic niche formation, in vitro and in lymphoreporter mice in vivo. We found that neural growth factor receptor (NGFR) is secreted in melanoma-derived small extracellular vesicles and shuttled to lymphatic endothelial cells, inducing lymphangiogenesis and tumor cell adhesion through the activation of ERK and NF-B pathways and ICAM1 expression. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased melanoma lymph node metastasis and extended the survival. Importantly, analysis of NGFR expression in lymph node metastases and matched primary tumors shows that levels of MITF+NGFR+ lymph node metastatic cells are correlated with disease outcome. Our data support the idea that NGFR secreted in sEVs favors lymph node pre-metastatic niche formation and lymph node metastasis in melanoma
Project description:Secreted extracellular vesicles are known to influence the tumor microenvironment and promote metastasis. In this work, we have analyzed the involvement of extracellular vesicles in establishing the lymph node pre-metastatic niche by melanoma cells. We found that small extracellular vesicles (sEVs) derived from highly metastatic melanoma cell lines spread broadly through the lymphatic system and are taken up by lymphatic endothelial cells reinforcing lymph node metastasis. Melanoma-derived sEVs induce lymphangiogenesis, a hallmark of pre-metastatic niche formation, in vitro and in lymphoreporter mice in vivo. Analysis of involved factors demonstrated that the neural growth factor receptor (NGFR) is secreted in melanoma-derived small extracellular vesicles and shuttled to lymphatic endothelial cells inducing lymphangiogenesis and tumor cell adhesion through the activation of ERK and NF-B pathways and ICAM1 expression. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased melanoma lymph node metastasis and extended mice survival. Importantly, analysis of NGFR expression in lymph node metastases and matched primary tumors shows that levels of MITF+NGFR+ lymph node metastatic cells are correlated with disease outcome. Our data support that NGFR is secreted in sEVs favoring lymph node pre-metastatic niche formation and lymph node metastasis in melanoma.