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1.
J Invest Dermatol ; 2024 Jan 05.
Article in English | MEDLINE | ID: mdl-38185415

ABSTRACT

A substantial part of cutaneous malignant melanomas develops from benign nevi. However, the precise molecular events driving the transformation from benign to malignant melanoma are not well-understood. We used laser microdissection and mass spectrometry to analyze the proteomes of melanoma subtypes, including superficial spreading melanomas (n = 17), nodular melanomas (n = 17), and acral melanomas (n = 15). Furthermore, we compared the proteomes of nevi cells with those of melanoma cells within the same specimens (nevus-associated melanoma (n = 14)). In total, we quantified 7935 proteins. Despite the genomic and clinical differences of the melanoma subtypes, our analysis revealed relatively similar proteomes, except for the upregulation of proteins involved in immune activation in nodular melanomas versus acral melanomas. Examining nevus-associated melanoma versus nevi, we found 1725 differentially expressed proteins (false discovery rate < 0.05). Among these proteins were 140 that overlapped with cancer hallmarks, tumor suppressors, and regulators of metabolism and cell cycle. Pathway analysis indicated aberrant activation of the phosphoinositide 3-kinase-protein kinase B-mTOR pathways and the Hippo-YAP pathway. Using a classifier, we identified six proteins capable of distinguishing melanoma from nevi samples. Our study represents a comprehensive comparative analysis of the proteome in melanoma subtypes and associated nevi, offering insights into the biological behavior of these distinct entities.

2.
J Immunol ; 205(5): 1461-1472, 2020 09 01.
Article in English | MEDLINE | ID: mdl-32839214

ABSTRACT

Tumor-associated macrophages (TAMs) support tumor growth by suppressing the activity of tumor-infiltrating T cells. Consistently, TAMs are considered a major limitation for the efficacy of cancer immunotherapy. However, the molecular reason behind the acquisition of an immunosuppressive TAM phenotype is not fully clarified. During tumor growth, the extracellular matrix (ECM) is degraded and substituted with a tumor-specific collagen-rich ECM. The collagen density of this tumor ECM has been associated with poor patient prognosis but the reason for this is not well understood. In this study, we investigated whether the collagen density could modulate the immunosuppressive activity of TAMs. The murine macrophage cell line RAW 264.7 was three-dimensionally cultured in collagen matrices of low and high collagen densities mimicking healthy and tumor tissue, respectively. Collagen density did not affect proliferation or viability of the macrophages. However, whole-transcriptome analysis revealed a striking response to the surrounding collagen density, including the regulation of immune regulatory genes and genes encoding chemokines. These transcriptional changes were shown to be similar in murine bone marrow-derived macrophages and TAMs isolated from murine tumors. Strikingly, coculture assays with primary T cells showed that macrophages cultured in high-density collagen were less efficient at attracting cytotoxic T cells and capable of inhibiting T cell proliferation more than macrophages cultured in low-density collagen. Our study demonstrates that a high collagen density can instruct macrophages to acquire an immunosuppressive phenotype. This mechanism could reduce the efficacy of immunotherapy and explain the link between high collagen density and poor prognosis.


Subject(s)
Collagen/immunology , Immune Tolerance/immunology , Macrophages/immunology , Animals , Cell Line , Cell Proliferation/physiology , Cell Survival/immunology , Chemokines/immunology , Extracellular Matrix/immunology , Female , Gene Expression Profiling/methods , Immunotherapy/methods , Lymphocyte Activation/immunology , Mice , Mice, Inbred BALB C , RAW 264.7 Cells , Transcription, Genetic/immunology , Tumor Microenvironment/immunology
3.
J Immunother Cancer ; 7(1): 68, 2019 03 12.
Article in English | MEDLINE | ID: mdl-30867051

ABSTRACT

BACKGROUND: Tumor progression is accompanied by dramatic remodeling of the surrounding extracellular matrix leading to the formation of a tumor-specific ECM, which is often more collagen-rich and of increased stiffness. The altered ECM of the tumor supports cancer growth and metastasis, but it is unknown if this effect involves modulation of T cell activity. To investigate if a high-density tumor-specific ECM could influence the ability of T cells to kill cancer cells, we here studied how T cells respond to 3D culture in different collagen densities. METHODS: T cells cultured in 3D conditions surrounded by a high or low collagen density were imaged using confocal fluorescent microscopy. The effects of the different collagen densities on T cell proliferation, survival, and differentiation were examined using flow cytometry. Cancer cell proliferation in similar 3D conditions was also measured. Triple-negative breast cancer specimens were analyzed for the number of infiltrating CD8+ T cells and for the collagen density. Whole-transcriptome analyses were applied to investigate in detail the effects of collagen density on T cells. Computational analyses were used to identify transcription factors involved in the collagen density-induced gene regulation. Observed changes were confirmed by qRT-PCR analysis. RESULTS: T cell proliferation was significantly reduced in a high-density matrix compared to a low-density matrix and prolonged culture in a high-density matrix led to a higher ratio of CD4+ to CD8+ T cells. The proliferation of cancer cells was unaffected by the surrounding collagen-density. Consistently, we observed a reduction in the number of infiltrating CD8+ T-cells in mammary tumors with high collagen-density indicating that collagen-density has a role in regulating T cell abundance in human breast cancer. Whole-transcriptome analysis of 3D-cultured T cells revealed that a high-density matrix induces downregulation of cytotoxic activity markers and upregulation of regulatory T cell markers. These transcriptional changes were predicted to involve autocrine TGF-ß signaling and they were accompanied by an impaired ability of tumor-infiltrating T cells to kill autologous cancer cells. CONCLUSIONS: Our study identifies a new immune modulatory mechanism, which could be essential for suppression of T cell activity in the tumor microenvironment.


Subject(s)
Collagen/metabolism , Lymphocyte Activation/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Neoplasms/etiology , Neoplasms/metabolism , Tumor Microenvironment/immunology , Cell Line, Tumor , Cell Proliferation , Cells, Cultured , Extracellular Matrix , Gene Expression Profiling , Humans , Immunomodulation , Lymphocyte Activation/genetics , Lymphocytes, Tumor-Infiltrating/pathology , Neoplasms/pathology , Tumor Microenvironment/genetics
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