ADAMTS proteases and the tumor immune microenvironment: Lessons from substrates and pathologies.

The relationship of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteases with inflammatory processes was anticipated since their discovery. Although knowledge of these extracellular proteases in different contexts continues to grow, many questions remain unanswered. In this review, we summarize the most important studies of ADAMTSs and their substrates in inflammation and in the immune system of non-oncological disorders. In addition, we update the findings on cancer and highlight their emerging role in the tumor immune microenvironment. Although the overall functions of extracellular molecules are known to be modulated by proteolysis, specific activities attributed to intact proteins and cleaved fragments in the context of inflammation are still subject to debate. A better understanding of ADAMTS activities will help to elucidate their contribution to the immune phenotype and to open up new therapeutic and diagnostic possibilities.

Extracellular Protease ADAMTS1 Is Required at Early Stages of Human Uveal Melanoma Development by Inducing Stemness and Endothelial-Like Features on Tumor Cells.

Extracellular matrix remodeling within the tumor microenvironment has been recognized as a relevant dynamic framework during tumor growth. However, research on proteases that trigger this remodeling keeps revealing a wide range of actions including both pro- and anti-tumorigenic. The extracellular protease ADAMTS1 exemplifies this dual role. In this work, we first confirmed a positive correlation of ADAMTS1 with endothelial-like phenotype of human melanoma cells together with the finding of associated signatures, including key genes such as endothelial CDH5. Using a CRISPR-Cas9 approach, we observed that the inhibition of ADAMTS1 in an aggressive uveal melanoma model compromised its endothelial-like properties, and more importantly, caused a robust blockade on the progression of tumor xenografts. Although vasculature emerged affected in ADAMTS1-deficient tumors, the most relevant action implied the downregulation of endothelial CDH5 in tumor cells, in association with stemness markers. Indeed, melanoma sphere assays also revealed a deficient commitment to form spheres in the absence of ADAMTS1, directly correlating with stemness markers and, remarkably, also with CDH5. Finally, taking advantage of advanced bioinformatics tools and available public data of uveal melanomas, we disclosed new prognosis factors, including endothelial elements and ADAMTS proteases. Our findings support the key role of ADAMTS proteases for uveal melanoma development since earlier stages, modulating the complex crosstalk between extracellular matrix and the induction of stemness and endothelial-like features. To our knowledge, this is the first report that supports the development of therapeutic targets on the extracellular matrix to overcome uveal melanoma.

ADAMTS1 Supports Endothelial Plasticity of Glioblastoma Cells with Relevance for Glioma Progression.

Gliomas in general and the more advanced glioblastomas (GBM) in particular are the most usual tumors of the central nervous system with poor prognosis. GBM patients develop resistance to distinct therapies, in part due to the existence of tumor cell subpopulations with stem-like properties that participate in trans-differentiation events. Within the complex tumor microenvironment, the involvement of extracellular proteases remains poorly understood. The extracellular protease ADAMTS1 has already been reported to contribute to the plasticity of cancer cells. Accordingly, this basic knowledge and the current availability of massive sequencing data from human gliomas, reinforced the development of this work. We first performed an in silico study of ADAMTS1 and endothelial markers in human gliomas, providing the basis to further assess these molecules in several primary glioblastoma-initiating cells and established GBM cells with the ability to acquire an endothelial-like phenotype. Using a co-culture approach of endothelial and GBM cells, we noticed a relevant function of ADAMTS1 in GBM cells leading the organization of endothelial-like networks and, even more significantly, we found a blockade of the formation of tumor-spheres and a deficient response to hypoxia in the absence of ADAMTS1. Our data support a chief role of this protease modulating the phenotypic plasticity of GBM.

Inhibition of ADAMTS1 Expression by Lentiviral CRISPR/Cas9 Gene Editing Technology.

The continuous improvement of gene editing tools has allowed a major revolution in biological sciences. Although a variety of gain and loss-of-function approaches have been widely used for the last decades, some limitations arose from non-specific targeting or lack of complete inhibition of the gene of interest. CRISPR/Cas9 editing technology introduced new and significant advantages because it can directly modify the gene of interest and completely blocks its expression.In the context of cancer studies, the heterogeneity of the tumor microenvironment requires comprehensive approaches to unveil the contribution of multiple genes. For example, a deeper understanding of the biology of proteases such as ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 motifs) will improve our perspective of complex phenomena affected by extracellular matrix remodeling, including embryonic development, angiogenesis, immune infiltration, metastasis, and tumor plasticity. Here, we present a method using CRISPR/Cas9 technology to inhibit the expression of the representative ADAMTS1 in cancer cells. Following the first steps of gene edition, we pursue further selection of silenced cells and provide a detailed description of sequence analysis and validation assays. This method leads to inactivation of ADAMTS1 in cancer cells, providing a relevant biological tool that will allow subsequent in vivo and in vitro ADAMTS1 functional analysis.

ADAMTS1 protease is required for a balanced immune cell repertoire and tumour inflammatory response.

Recent advances have emphasized the relevance of studying the extracellular microenvironment given its main contribution to tissue homeostasis and disease. Within this complex scenario, we have studied the extracellular protease ADAMTS1 (a disintegrin and metalloprotease with thrombospondin motif 1), implicated in vascularization and development, with reported anti- and pro-tumorigenic activities. In this work we performed a detailed study of the vasculature and substrates in adult organs of wild type and Adamts1-deficient mice. In addition to the expected alterations of organs like kidney, heart and aorta, we found that the lack of ADAMTS1 differently affects lymphocyte and myeloid populations in the spleen and bone marrow. The study of the substrate versican also revealed its alteration in the absence of the protease. With such premises, we challenged our mice with subcutaneous B16F1 syngeneic tumours and closely evaluated the immune repertoire in the tumours but also in the distant spleen and bone marrow. Our results confirmed a pro-inflammatory landscape in the absence of ADAMTS1, correlating with tumour blockade, supporting its novel role as a modulator of the immune cell response.

Stroma-derived but not tumor ADAMTS1 is a main driver of tumor growth and metastasis.

The matrix metalloprotease ADAMTS1 (A Disintegrin And Metalloprotease with ThromboSpondin repeats 1) has been involved in tumorigenesis although its contributions appeared ambiguous. To understand the multifaceted actions of this protease, it is still required a deeper knowledge of its implication in heterogeneous tumor-stroma interactions. Using a syngeneic B16F1 melanoma model in wild type and ADAMTS1 knockout mice we found distinct stroma versus tumor functions for this protease. Genetic deletion of ADAMTS1 in the host microenvironment resulted in a drastic decrease of tumor growth and metastasis. However, the downregulation of tumor ADAMTS1 did not uncover relevant effects. Reduced tumors in ADAMTS1 KO mice displayed a paradoxical increase in vascular density and vascular-related genes; a detailed characterization revealed an impaired vasculature, along with a minor infiltration of macrophages. In addition, ex-vivo assays supported a chief role for ADAMTS1 in vascular sprouting, and melanoma xenografts showed a relevant induction of its expression in stroma compartments. These findings provide the first genetic evidence that supports the pro-tumorigenic role of stromal ADAMTS1.