Tumor-reactive antibodies evolve from non-binding and autoreactive precursors.

The tumor microenvironment hosts antibody-secreting cells (ASCs) associated with a favorable prognosis in several types of cancer. Patient-derived antibodies have diagnostic and therapeutic potential; yet, it remains unclear how antibodies gain autoreactivity and target tumors. Here, we found that somatic hypermutations (SHMs) promote antibody antitumor reactivity against surface autoantigens in high-grade serous ovarian carcinoma (HGSOC). Patient-derived tumor cells were frequently coated with IgGs. Intratumoral ASCs in HGSOC were both mutated and clonally expanded and produced tumor-reactive antibodies that targeted MMP14, which is abundantly expressed on the tumor cell surface. The reversion of monoclonal antibodies to their germline configuration revealed two types of classes: one dependent on SHMs for tumor binding and a second with germline-encoded autoreactivity. Thus, tumor-reactive autoantibodies are either naturally occurring or evolve through an antigen-driven selection process. These findings highlight the origin and potential applicability of autoantibodies directed at surface antigens for tumor targeting in cancer patients.

Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

Recycled melanoma-secreted melanosomes regulate tumor-associated macrophage diversification.

Extracellular vesicles (EVs) are important mediators of communication between cells. Here, we reveal a new mode of intercellular communication by melanosomes, large EVs secreted by melanocytes for melanin transport. Unlike small EVs, which are disintegrated within the receiver cell, melanosomes stay intact within them, gain a unique protein signature, and can then be further transferred to another cell as "second-hand" EVs. We show that melanoma-secreted melanosomes passaged through epidermal keratinocytes or dermal fibroblasts can be further engulfed by resident macrophages. This process leads to macrophage polarization into pro-tumor or pro-immune cell infiltration phenotypes. Melanosomes that are transferred through fibroblasts can carry AKT1, which induces VEGF secretion from macrophages in an mTOR-dependent manner, promoting angiogenesis and metastasis in vivo. In melanoma patients, macrophages that are co-localized with AKT1 are correlated with disease aggressiveness, and immunotherapy non-responders are enriched in macrophages containing melanosome markers. Our findings suggest that interactions mediated by second-hand extracellular vesicles contribute to the formation of the metastatic niche, and that blocking the melanosome cues of macrophage diversification could be helpful in halting melanoma progression.

The deacetylase Sirt1 is an essential regulator of Aire-mediated induction of central immunological tolerance.

Aire is a transcriptional regulator that induces the promiscuous expression of thousands of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs), a step critical for the induction of immunological self-tolerance. Studies have offered molecular insights into how Aire operates, but more comprehensive understanding of this process still remains elusive. Here we found abundant expression of the protein deacetylase Sirtuin-1 (Sirt1) in mature Aire(+) mTECs, wherein it was required for the expression of Aire-dependent TRA-encoding genes and the subsequent induction of immunological self-tolerance. Our study elucidates a previously unknown molecular mechanism for Aire-mediated transcriptional regulation and identifies a unique function for Sirt1 in preventing organ-specific autoimmunity.

Blockade of the protease ADAM17 ameliorates experimental pancreatitis.

Acute and chronic pancreatitis, the latter associated with fibrosis, are multifactorial inflammatory disorders and leading causes of gastrointestinal disease-related hospitalization. Despite the global health burden of pancreatitis, currently, there are no effective therapeutic agents. In this regard, the protease A Disintegrin And Metalloproteinase 17 (ADAM17) mediates inflammatory responses through shedding of bioactive inflammatory cytokines and mediators, including tumor necrosis factor α (TNFα) and the soluble interleukin (IL)-6 receptor (sIL-6R), the latter of which drives proinflammatory IL-6 trans-signaling. However, the role of ADAM17 in pancreatitis is unclear. To address this, Adam17ex/ex mice-which are homozygous for the hypomorphic Adam17ex allele resulting in marked reduction in ADAM17 expression-and their wild-type (WT) littermates were exposed to the cerulein-induced acute pancreatitis model, and acute (1-wk) and chronic (20-wk) pancreatitis models induced by the cigarette smoke carcinogen nicotine-derived nitrosamine ketone (NNK). Our data reveal that ADAM17 expression was up-regulated in pancreatic tissues of animal models of pancreatitis. Moreover, the genetic (Adam17ex/ex mice) and therapeutic (ADAM17 prodomain inhibitor [A17pro]) targeting of ADAM17 ameliorated experimental pancreatitis, which was associated with a reduction in the IL-6 trans-signaling/STAT3 axis. This led to reduced inflammatory cell infiltration, including T cells and neutrophils, as well as necrosis and fibrosis in the pancreas. Furthermore, up-regulation of the ADAM17/IL-6 trans-signaling/STAT3 axis was a feature of pancreatitis patients. Collectively, our findings indicate that the ADAM17 protease plays a pivotal role in the pathogenesis of pancreatitis, which could pave the way for devising novel therapeutic options to be deployed against this disease.

Integrated Metabolomics and Proteomics of Symptomatic and Early Presymptomatic States of Colitis.

Colitis has a multifactorial pathogenesis with a strong cross-talk among microbiota, hypoxia, and tissue metabolism. Here, we aimed to characterize the molecular signature of the disease in symptomatic and presymptomatic stages of the inflammatory process at the tissue and fecal level. The study is based on two different murine models for colitis, and HR-MAS NMR on "intact" colon tissues and LC-MS/MS on colon tissue extracts were used to derive untargeted metabolomics and proteomics information, respectively. Solution NMR was used to derive metabolomic profiles of the fecal extracts. By combining metabolomic and proteomic analyses of the tissues, we found increased anaerobic glycolysis, accompanied by an altered citric acid cycle and oxidative phosphorylation in inflamed colons; these changes associate with inflammation-induced hypoxia taking place in colon tissues. Different colitis states were also characterized by significantly different metabolomic profiles of fecal extracts, attributable to both the dysbiosis characteristic of colitis as well as the dysregulated tissue metabolism. Strong and distinctive tissue and fecal metabolomic signatures can be detected before the onset of symptoms. Therefore, untargeted metabolomics of tissues and fecal extracts provides a comprehensive picture of the changes accompanying the disease onset already at preclinical stages, highlighting the diagnostic potential of global metabolomics for inflammatory diseases.

The extracellular matrix protein agrin promotes heart regeneration in mice.

The adult mammalian heart is non-regenerative owing to the post-mitotic nature of cardiomyocytes. The neonatal mouse heart can regenerate, but only during the first week of life. Here we show that changes in the composition of the extracellular matrix during this week can affect cardiomyocyte growth and differentiation in mice. We identify agrin, a component of neonatal extracellular matrix, as required for the full regenerative capacity of neonatal mouse hearts. In vitro, recombinant agrin promotes the division of cardiomyocytes that are derived from mouse and human induced pluripotent stem cells through a mechanism that involves the disassembly of the dystrophin-glycoprotein complex, and Yap- and ERK-mediated signalling. In vivo, a single administration of agrin promotes cardiac regeneration in adult mice after myocardial infarction, although the degree of cardiomyocyte proliferation observed in this model suggests that there are additional therapeutic mechanisms. Together, our results uncover a new inducer of mammalian heart regeneration and highlight fundamental roles of the extracellular matrix in cardiac repair.

Emerging roles of ECM remodeling processes in cancer.

Extracellular matrix (ECM) plays a central and dynamic role in the creation of tumor microenvironment. Herein we discuss the emerging biophysical and biochemical aspects of ECM buildup and proteolysis in cancer niche formation. Dysregulated ECM remodeling by cancer cells facilitate irreversible proteolysis and crosslinking, which in turn influence cell signaling, micro environmental cues, angiogenesis and tissue biomechanics. Further, we introduce the emerging roles of cancer microbiome in aberrant tumor ECM remodeling and membrane bound nano-sized vesicles called exosomes in creation of distant pre-metastatic niches. A detailed molecular and biophysical understanding of the ECM morphologies and its components such as key enzymes, structural and signaling molecules are critical in identifying the next generation of therapeutic and diagnostic targets in cancer.

The ADAM17 protease promotes tobacco smoke carcinogen-induced lung tumorigenesis.

Lung cancer is the leading cause of cancer-related mortality, with most cases attributed to tobacco smoking, in which nicotine-derived nitrosamine ketone (NNK) is the most potent lung carcinogen. The ADAM17 protease is responsible for the ectodomain shedding of many pro-tumorigenic cytokines, growth factors and receptors, and therefore is an attractive target in cancer. However, the role of ADAM17 in promoting tobacco smoke carcinogen-induced lung carcinogenesis is unknown. The hypomorphic Adam17ex/ex mice-characterized by reduced global ADAM17 expression-were backcrossed onto the NNK-sensitive pseudo-A/J background. CRISPR-driven and inhibitor-based (GW280264X, and ADAM17 prodomain) ADAM17 targeting was employed in the human lung adenocarcinoma cell lines A549 and NCI-H23. Human lung cancer biopsies were also used for analyses. The Adam17ex/ex mice displayed marked protection against NNK-induced lung adenocarcinoma. Specifically, the number and size of lung lesions in NNK-treated pseudo-A/J Adam17ex/ex mice were significantly reduced compared with wild-type littermate controls. This was associated with lower proliferative index throughout the lung epithelium. ADAM17 targeting in A549 and NCI-H23 cells led to reduced proliferative and colony-forming capacities. Notably, among select ADAM17 substrates, ADAM17 deficiency abrogated shedding of the soluble IL-6 receptor (sIL-6R), which coincided with the blockade of sIL-6R-mediated trans-signaling via ERK MAPK cascade. Furthermore, NNK upregulated phosphorylation of p38 MAPK, whose pharmacological inhibition suppressed ADAM17 threonine phosphorylation. Importantly, ADAM17 threonine phosphorylation was significantly upregulated in human lung adenocarcinoma with smoking history compared with their cancer-free controls. Our study identifies the ADAM17/sIL-6R/ERK MAPK axis as a candidate therapeutic strategy against tobacco smoke-associated lung carcinogenesis.

Intravital imaging of vascular anomalies and extracellular matrix remodeling in orthotopic pancreatic tumors.

Pancreatic cancers, both adenocarcinomas and endocrine tumors are characterized by varying levels of aberrant angiogenesis and fibrotic microenvironment. The difficulty to deliver drugs and treat the disease has been attributed in part to the vascular architecture and tissue/ECM density. Here we present longitudinal three-dimensional intravital imaging of vascular and tumor microenvironment remodeling in spontaneous transgenic tumors (RIP1-Tag2 insulinomas) and orthotopically injected tumors (KPC adenocarcinomas). Analysis of the data acquired in insulinomas revealed major differences in tumor blood vessel branching, fraction volume, number of branch points segments, vessel straightness and length compared to the normal tissue. The aggressive adenocarcinoma presented widespread peritumoral vascular remodeling and heterogeneous vascular distribution. Longitudinal imaging was used to acquire sequential vascular remodeling data during tumor progression. This work demonstrates the potential for using a pancreatic intravital imaging window for direct visualization of the tumor heterogenic microenvironments during tumor progression.

Exosomes as a storehouse of tissue remodeling proteases and mediators of cancer progression.

Rapidly increasing scientific reports of exosomes and their biological effects have improved our understanding of their cellular sources and their cell-to-cell communication. These nano-sized vesicles act as potent carriers of regulatory bio-macromolecules and can induce regulatory functions by delivering them from its source to recipient cells. The details of their communication network are less understood. Recent studies have shown that apart from delivering its cargo to the cells, it can directly act on extracellular matrix (ECM) proteins and growth factors and can induce various remodeling events. More importantly, exosomes carry many surface-bound proteases, which can cleave different ECM proteins and carbohydrates and can shed cell surface receptors. These local extracellular events can modulate signaling cascades, which consequently influences the whole tissue and organ. This review aims to highlight the critical roles of exosomal proteases and their mechanistic insights within the cellular and extracellular environment.

Meprin ß induces activities of A disintegrin and metalloproteinases 9, 10, and 17 by specific prodomain cleavage.

Meprin ß is a membrane-bound metalloprotease involved in extracellular matrix assembly and inflammatory processes in health and disease. A disintegrin and metalloproteinase (ADAM)10 and ADAM17 are physiologic relevant sheddases of inactive promeprin ß, which influences its substrate repertoire and subsequent biologic functions. Proteomic analysis also revealed several ADAMs as putative meprin ß substrates. Here, we demonstrate specific N-terminal processing of ADAM9, 10, and 17 by meprin ß and identify cleavage sites within their prodomains. Because ADAM prodomains can act as specific inhibitors, we postulate a role for meprin ß in the regulation of ADAM activities. Indeed, prodomain cleavage by meprin ß caused increased ADAM protease activities, as observed by peptide-based cleavage assays and demonstrated by increased ectodomain shedding activity. Direct interaction of meprin ß and ADAM proteases could be shown by immunofluorescence microscopy and immunoprecipitation experiments. As demonstrated by a bacterial activator of meprin ß and additional measurement of TNF-α shedding on bone marrow-derived macrophages, meprin ß/ADAM protease interactions likely influence inflammatory conditions. Thus, we identified a novel proteolytic pathway of meprin ß with ADAM proteases to control protease activities at the cell surface as part of the protease web.-Wichert, R., Scharfenberg, F., Colmorgen, C., Koudelka, T., Schwarz, J., Wetzel, S., Potempa, B., Potempa, J., Bartsch, J. W., Sagi, I., Tholey, A., Saftig, P., Rose-John, S., Becker-Pauly, C. Meprin ß induces activities of A disintegrin and metalloproteinases 9, 10, and 17 by specific prodomain cleavage.

Differential tissue stiffness of body column facilitates locomotion of Hydra on solid substrates.

The bell-shaped members of the Cnidaria typically move around by swimming, whereas the Hydra polyp can perform locomotion on solid substrates in an aquatic environment. To address the biomechanics of locomotion on rigid substrates, we studied the 'somersaulting' locomotion in Hydra We applied atomic force microscopy to measure the local mechanical properties of Hydra's body column and identified the existence of differential Young's modulus between the shoulder region versus rest of the body column at 3:1 ratio. We show that somersaulting primarily depends on differential tissue stiffness of the body column and is explained by computational models that accurately recapitulate the mechanics involved in this process. We demonstrate that perturbation of the observed stiffness variation in the body column by modulating the extracellular matrix polymerization impairs the 'somersault' movement. These results provide a mechanistic basis for the evolutionary significance of differential extracellular matrix properties and tissue stiffness.

Demystifying the extracellular matrix and its proteolytic remodeling in the brain: structural and functional insights.

The extracellular matrix (ECM) plays diverse roles in several physiological and pathological conditions. In the brain, the ECM is unique both in its composition and in functions. Furthermore, almost all the cells in the central nervous system contribute to different aspects of this intricate structure. Brain ECM, enriched with proteoglycans and other small proteins, aggregate into distinct structures around neurons and oligodendrocytes. These special structures have cardinal functions in the normal functioning of the brain, such as learning, memory, and synapse regulation. In this review, we have compiled the current knowledge about the structure and function of important ECM molecules in the brain and their proteolytic remodeling by matrix metalloproteinases and other enzymes, highlighting the special structures they form. In particular, the proteoglycans in brain ECM, which are essential for several vital functions, are emphasized in detail.

Detection of isolated protein-bound metal ions by single-particle cryo-STEM.

Metal ions play essential roles in many aspects of biological chemistry. Detecting their presence and location in proteins and cells is important for understanding biological function. Conventional structural methods such as X-ray crystallography and cryo-transmission electron microscopy can identify metal atoms on protein only if the protein structure is solved to atomic resolution. We demonstrate here the detection of isolated atoms of Zn and Fe on ferritin, using cryogenic annular dark-field scanning transmission electron microscopy (cryo-STEM) coupled with single-particle 3D reconstructions. Zn atoms are found in a pattern that matches precisely their location at the ferroxidase sites determined earlier by X-ray crystallography. By contrast, the Fe distribution is smeared along an arc corresponding to the proposed path from the ferroxidase sites to the mineral nucleation sites along the twofold axes. In this case the single-particle reconstruction is interpreted as a probability distribution function based on the average of individual locations. These results establish conditions for detection of isolated metal atoms in the broader context of electron cryo-microscopy and tomography.

Enhanced Collagen Deposition in the Duodenum of Patients with Hyaline Fibromatosis Syndrome and Protein Losing Enteropathy.

Hyaline fibromatosis syndrome (HFS), resulting from ANTXR2 mutations, is an ultra-rare disease that causes intestinal lymphangiectasia and protein-losing enteropathy (PLE). The mechanisms leading to the gastrointestinal phenotype in these patients are not well defined. We present two patients with congenital diarrhea, severe PLE and unique clinical features resulting from deleterious ANTXR2 mutations. Intestinal organoids were generated from one of the patients, along with CRISPR-Cas9 ANTXR2 knockout, and compared with organoids from two healthy controls. The ANTXR2-deficient organoids displayed normal growth and polarity, compared to controls. Using an anthrax-toxin assay we showed that the c.155C>T mutation causes loss-of-function of ANTXR2 protein. An intrinsic defect of monolayer formation in patient-derived or ANTXR2KO organoids was not apparent, suggesting normal epithelial function. However, electron microscopy and second harmonic generation imaging showed abnormal collagen deposition in duodenal samples of these patients. Specifically, collagen VI, which is known to bind ANTXR2, was highly expressed in the duodenum of these patients. In conclusion, despite resistance to anthrax-toxin, epithelial cell function, and specifically monolayer formation, is intact in patients with HFS. Nevertheless, loss of ANTXR2-mediated signaling leads to collagen VI accumulation in the duodenum and abnormal extracellular matrix composition, which likely plays a role in development of PLE.

Early disruption of the alveolar-capillary barrier in a ricin-induced ARDS mouse model: neutrophil-dependent and -independent impairment of junction proteins.

Irrespective of its diverse etiologies, acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) leads to increased permeability of the alveolar-capillary barrier, which in turn promotes edema formation and respiratory failure. We investigated the mechanism of ALI/ARDS lung hyperpermeability triggered by pulmonary exposure of mice to the highly toxic plant-derived toxin ricin. One prominent hallmark of ricin-mediated pulmonary intoxication is the rapid and massive influx of neutrophils to the lungs, where they contribute to the developing inflammation yet may also cause tissue damage, thereby promoting ricin-mediated morbidity. Here we show that pulmonary exposure of mice to ricin results in the rapid diminution of the junction proteins VE-cadherin, claudin 5, and connexin 43, belonging, respectively, to the adherens, tight, and gap junction protein families. Depletion of neutrophils in ricin-intoxicated mice attenuated the damage caused to these junction proteins, alleviated pulmonary edema, and significantly postponed the time to death of the intoxicated mice. Inhibition of matrix metalloproteinase (MMP) activity recapitulated the response to neutrophil depletion observed in ricin-intoxicated mice and was associated with decreased insult to the junction proteins and alveolar-capillary barrier. However, neutrophil-mediated MMP activity was not the sole mechanism responsible for pulmonary hyperpermeability, as exemplified by the ricin-mediated disruption of claudin 18, via a neutrophil-independent mechanism involving tyrosine phosphorylation. This in-depth study of the early stage mechanisms governing pulmonary tissue integrity during ALI/ARDS is expected to facilitate the tailoring of novel therapeutic approaches for the treatment of these diseases.

Persistent Chemiluminescent Glow of Phenoxy-dioxetane Luminophore Enables Unique CRET-Based Detection of Proteases.

Chemiluminescence is being considered an effective imaging modality as it offers low background and high sensitivity. Recent discovery by our group has led to development of new phenoxy-dioxetane chemiluminescence luminophores, which are highly bright under physiological conditions. However, the current scope of probes based on these luminophores is limited, as they can only be turned on by phenol protecting group removal. Here we present a new chemiluminescence resonance energy transfer (CRET) system, Glow-CRET, in which light emission is triggered by proteolytic cleavage of a peptide substrate that links a dioxetane luminophore and a quencher. In order to compose such system, a new phenoxy-dioxetane luminophore, 7-HC-CL, was developed. This luminophore exhibits intense and persistent glow chemiluminescence; it undergoes very slow chemiexcitation, and it has the highest chemiluminescence quantum yield ever reported under physiological conditions. Based on 7-HC-CL, a Glow-CRET probe for matrix metalloproteinases, MMP-CL, was synthesized. Incubation of MMP-CL with its cognate protease resulted in 160-fold increase in chemiluminescence signal. MMP-CL was also able to detect matrix metalloproteinase activity in cancer cells with significantly higher signal-to-background ratio than an analogous fluorescence resonance energy transfer (FRET)-based probe. This work is expected to open new horizons in chemiluminescence imaging, as it enables to use the dioxetanes in ways that had not been possible. We anticipate that 7-HC-CL and future derivatives will be utilized not only for the construction of further Glow-CRET probes, but also for other applications, such as chemiluminescence tagging of proteins.

Distinct biological events generated by ECM proteolysis by two homologous collagenases.

It is well established that the expression profiles of multiple and possibly redundant matrix-remodeling proteases (e.g., collagenases) differ strongly in health, disease, and development. Although enzymatic redundancy might be inferred from their close similarity in structure, their in vivo activity can lead to extremely diverse tissue-remodeling outcomes. We observed that proteolysis of collagen-rich natural extracellular matrix (ECM), performed uniquely by individual homologous proteases, leads to distinct events that eventually affect overall ECM morphology, viscoelastic properties, and molecular composition. We revealed striking differences in the motility and signaling patterns, morphology, and gene-expression profiles of cells interacting with natural collagen-rich ECM degraded by different collagenases. Thus, in contrast to previous notions, matrix-remodeling systems are not redundant and give rise to precise ECM-cell crosstalk. Because ECM proteolysis is an abundant biochemical process that is critical for tissue homoeostasis, these results improve our fundamental understanding its complexity and its impact on cell behavior.

Matrix metalloproteinases: The sculptors of chronic cutaneous wounds.

Cutaneous wound healing is a complex mechanism with multiple processes orchestrating harmoniously for structural and functional restoration of the damaged tissue. Chronic non-healing wounds plagued with infection create a major healthcare burden and is one of the most frustrating clinical problems. Chronic wounds are manifested by prolonged inflammation, defective re-epithelialization and haphazard remodeling. Matrix metalloproteinases (MMPs) are zinc dependent enzymes that play cardinal functions in wound healing. Understanding the pathological events mediated by MMPs during wound healing may pave way in identifying novel drug targets for chronic wounds. Here, we discuss the functions and skewed regulation of different MMPs during infection and chronic tissue repair. This review also points out the potential of MMPs and their inhibitors as therapeutic agents in treating chronic wounds during distinct phases of the wound healing. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.