LRG1: an emerging player in disease pathogenesis.

The secreted glycoprotein leucine-rich α-2 glycoprotein 1 (LRG1) was first described as a key player in pathogenic ocular neovascularization almost a decade ago. Since then, an increasing number of publications have reported the involvement of LRG1 in multiple human conditions including cancer, diabetes, cardiovascular disease, neurological disease, and inflammatory disorders. The purpose of this review is to provide, for the first time, a comprehensive overview of the LRG1 literature considering its role in health and disease. Although LRG1 is constitutively expressed by hepatocytes and neutrophils, Lrg1-/- mice show no overt phenotypic abnormality suggesting that LRG1 is essentially redundant in development and homeostasis. However, emerging data are challenging this view by suggesting a novel role for LRG1 in innate immunity and preservation of tissue integrity. While our understanding of beneficial LRG1 functions in physiology remains limited, a consistent body of evidence shows that, in response to various inflammatory stimuli, LRG1 expression is induced and directly contributes to disease pathogenesis. Its potential role as a biomarker for the diagnosis, prognosis and monitoring of multiple conditions is widely discussed while dissecting the mechanisms underlying LRG1 pathogenic functions. Emphasis is given to the role that LRG1 plays as a vasculopathic factor where it disrupts the cellular interactions normally required for the formation and maintenance of mature vessels, thereby indirectly contributing to the establishment of a highly hypoxic and immunosuppressive microenvironment. In addition, LRG1 has also been reported to affect other cell types (including epithelial, immune, mesenchymal and cancer cells) mostly by modulating the TGFß signalling pathway in a context-dependent manner. Crucially, animal studies have shown that LRG1 inhibition, through gene deletion or a function-blocking antibody, is sufficient to attenuate disease progression. In view of this, and taking into consideration its role as an upstream modifier of TGFß signalling, LRG1 is suggested as a potentially important therapeutic target. While further investigations are needed to fill gaps in our current understanding of LRG1 function, the studies reviewed here confirm LRG1 as a pleiotropic and pathogenic signalling molecule providing a strong rationale for its use in the clinic as a biomarker and therapeutic target.

Increased levels of interleukin-6 exacerbate the dystrophic phenotype in mdx mice.

Duchenne muscular dystrophy (DMD) is characterized by progressive lethal muscle degeneration and chronic inflammatory response. The mdx mouse strain has served as the animal model for human DMD. However, while DMD patients undergo extensive necrosis, the affected muscles of adult mdx mice rapidly regenerates and regains structural and functional integrity. The basis for the mild effects observed in mice compared with the lethal consequences in humans remains unknown. In this study, we provide evidence that interleukin-6 (IL-6) is causally linked to the pathogenesis of muscular dystrophy. We report that forced expression of IL-6, in the adult mdx mice, recapitulates the severe phenotypic characteristics of DMD in humans. Increased levels of IL-6 exacerbate the dystrophic muscle phenotype, sustaining inflammatory response and repeated cycles of muscle degeneration and regeneration, leading to exhaustion of satellite cells. The mdx/IL6 mouse closely approximates the human disease and more faithfully recapitulates the disease progression in humans. This study promises to significantly advance our understanding of the pathogenic mechanisms that lead to DMD.

The disruptive role of LRG1 on the vasculature and perivascular microenvironment.

The establishment of new blood vessels, and their subsequent stabilization, is a critical process that facilitates tissue growth and organ development. Once established, vessels need to diversify to meet the specific needs of the local tissue and to maintain homeostasis. These processes are tightly regulated and fundamental to normal vessel and tissue function. The mechanisms that orchestrate angiogenesis and vessel maturation have been widely studied, with signaling crosstalk between endothelium and perivascular cells being identified as an essential component. In disease, however, new vessels develop abnormally, and existing vessels lose their specialization and function, which invariably contributes to disease progression. Despite considerable research into the vasculopathic mechanisms in disease, our knowledge remains incomplete. Accordingly, the identification of angiocrine and angiopathic molecules secreted by cells within the vascular microenvironment, and their effect on vessel behaviour, remains a major research objective. Over the last decade the secreted glycoprotein leucine-rich α-2 glycoprotein 1 (LRG1), has emerged as a significant vasculopathic molecule, stimulating defective angiogenesis, and destabilizing the existing vasculature mainly, but not uniquely, by altering both canonical and non-canonical TGF-ß signaling in a highly cell and context dependent manner. Whilst LRG1 does not possess any overt homeostatic role in vessel development and maintenance, growing evidence provides a compelling case for LRG1 playing a pleiotropic role in disrupting the vasculature in many disease settings. Thus, LRG1 has now been reported to damage vessels in various disorders including cancer, diabetes, chronic kidney disease, ocular disease, and lung disease and the signaling processes that drive this dysfunction are being defined. Moreover, therapeutic targeting of LRG1 has been widely proposed to re-establish a quiescent endothelium and normalized vasculature. In this review, we consider the current status of our understanding of the role of LRG1 in vascular pathology, and its potential as a therapeutic target.

Leucine-rich alpha-2-glycoprotein 1 (LRG1) as a novel ADC target.

Leucine-rich alpha-2-glycoprotein 1 (LRG1) is present abundantly in the microenvironment of many tumours where it contributes to vascular dysfunction, which impedes the delivery of therapeutics. In this work we demonstrate that LRG1 is predominantly a non-internalising protein. We report the development of a novel antibody-drug conjugate (ADC) comprising the anti-LRG1 hinge-stabilised IgG4 monoclonal antibody Magacizumab coupled to the anti-mitotic payload monomethyl auristatin E (MMAE) via a cleavable dipeptide linker using the site-selective disulfide rebridging dibromopyridazinedione (diBrPD) scaffold. It is demonstrated that this ADC retains binding post-modification, is stable in serum and effective in in vitro cell studies. We show that the extracellular LRG1-targeting ADC provides an increase in survival in vivo when compared against antibody alone and similar anti-tumour activity when compared against standard chemotherapy, but without undesired side-effects. LRG1 targeting through this ADC presents a novel and effective proof-of-concept en route to improving the efficacy of cancer therapeutics.

LRG1 destabilizes tumor vessels and restricts immunotherapeutic potency.

BACKGROUND: A poorly functioning tumor vasculature is pro-oncogenic and may impede the delivery of therapeutics. Normalizing the vasculature, therefore, may be beneficial. We previously reported that the secreted glycoprotein leucine-rich α-2-glycoprotein 1 (LRG1) contributes to pathogenic neovascularization. Here, we investigate whether LRG1 in tumors is vasculopathic and whether its inhibition has therapeutic utility. METHODS: Tumor growth and vascular structure were analyzed in subcutaneous and genetically engineered mouse models in wild-type and Lrg1 knockout mice. The effects of LRG1 antibody blockade as monotherapy, or in combination with co-therapies, on vascular function, tumor growth, and infiltrated lymphocytes were investigated. FINDINGS: In mouse models of cancer, Lrg1 expression was induced in tumor endothelial cells, consistent with an increase in protein expression in human cancers. The expression of LRG1 affected tumor progression as Lrg1 gene deletion, or treatment with a LRG1 function-blocking antibody, inhibited tumor growth and improved survival. Inhibition of LRG1 increased endothelial cell pericyte coverage and improved vascular function, resulting in enhanced efficacy of cisplatin chemotherapy, adoptive T cell therapy, and immune checkpoint inhibition (anti-PD1) therapy. With immunotherapy, LRG1 inhibition led to a significant shift in the tumor microenvironment from being predominantly immune silent to immune active. CONCLUSIONS: LRG1 drives vascular abnormalization, and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics. FUNDING: Wellcome Trust (206413/B/17/Z), UKRI/MRC (G1000466, MR/N006410/1, MC/PC/14118, and MR/L008742/1), BHF (PG/16/50/32182), Health and Care Research Wales (CA05), CRUK (C42412/A24416 and A17196), ERC (ColonCan 311301 and AngioMature 787181), and DFG (CRC1366).

Temporal multi-omics identifies LRG1 as a vascular niche instructor of metastasis.

Metastasis is the primary cause of cancer-related mortality. Tumor cell interactions with cells of the vessel wall are decisive and potentially rate-limiting for metastasis. The molecular nature of this cross-talk is, beyond candidate gene approaches, hitherto poorly understood. Using endothelial cell (EC) bulk and single-cell transcriptomics in combination with serum proteomics, we traced the evolution of the metastatic vascular niche in surgical models of lung metastasis. Temporal multiomics revealed that primary tumors systemically reprogram the body's vascular endothelium to perturb homeostasis and to precondition the vascular niche for metastatic growth. The vasculature with its enormous surface thereby serves as amplifier of tumor-induced instructive signals. Comparative analysis of lung EC gene expression and secretome identified the transforming growth factor­ß (TGFß) pathway specifier LRG1, leucine-rich alpha-2-glycoprotein 1, as an early instructor of metastasis. In the presence of a primary tumor, ECs systemically up-regulated LRG1 in a signal transducer and activator of transcription 3 (STAT3)­dependent manner. A meta-analysis of retrospective clinical studies revealed a corresponding up-regulation of LRG1 concentrations in the serum of patients with cancer. Functionally, systemic up-regulation of LRG1 promoted metastasis in mice by increasing the number of prometastatic neural/glial antigen 2 (NG2)+ perivascular cells. In turn, genetic deletion of Lrg1 hampered growth of lung metastasis. Postsurgical adjuvant administration of an LRG1-neutralizing antibody delayed metastatic growth and increased overall survival. This study has established a systems map of early primary tumor-induced vascular changes and identified LRG1 as a therapeutic target for metastasis.

Non-Invasive Longitudinal Bioluminescence Imaging of Human Mesoangioblasts in Bioengineered Esophagi.

IMPACT STATEMENT: Methodologies for incorporation of cells into tissue-engineered grafts, particularly at the later preclinical stages, are suboptimal and non-validated, and monitoring cell fate within scaffolds cultured in bioreactors and in vivo is challenging. In this study, we demonstrate how bioluminescence imaging (BLI) can overcome these difficulties and allow quantitative cell tracking at multiple stages of the bioengineering preclinical pipeline. Our robust bioluminescence-based approach allowed reproducible longitudinal monitoring of mesoangioblast localization and survival in 2D/3D tissue culture, in organ-scale bioreactors, and in vivo. Our findings will encourage the use of BLI in tissue engineering studies, improving the overall quality of cell-scaffold interaction research.

Multi-stage bioengineering of a layered oesophagus with in vitro expanded muscle and epithelial adult progenitors.

A tissue engineered oesophagus could overcome limitations associated with oesophageal substitution. Combining decellularized scaffolds with patient-derived cells shows promise for regeneration of tissue defects. In this proof-of-principle study, a two-stage approach for generation of a bio-artificial oesophageal graft addresses some major challenges in organ engineering, namely: (i) development of multi-strata tubular structures, (ii) appropriate re-population/maturation of constructs before transplantation, (iii) cryopreservation of bio-engineered organs and (iv) in vivo pre-vascularization. The graft comprises decellularized rat oesophagus homogeneously re-populated with mesoangioblasts and fibroblasts for the muscle layer. The oesophageal muscle reaches organised maturation after dynamic culture in a bioreactor and functional integration with neural crest stem cells. Grafts are pre-vascularised in vivo in the omentum prior to mucosa reconstitution with expanded epithelial progenitors. Overall, our optimised two-stage approach produces a fully re-populated, structurally organized and pre-vascularized oesophageal substitute, which could become an alternative to current oesophageal substitutes.

Long-term cryopreservation of decellularised oesophagi for tissue engineering clinical application.

Oesophageal tissue engineering is a therapeutic alternative when oesophageal replacement is required. Decellularised scaffolds are ideal as they are derived from tissue-specific extracellular matrix and are non-immunogenic. However, appropriate preservation may significantly affect scaffold behaviour. Here we aim to prove that an effective method for short- and long-term preservation can be applied to tissue engineered products allowing their translation to clinical application. Rabbit oesophagi were decellularised using the detergent-enzymatic treatment (DET), a combination of deionised water, sodium deoxycholate and DNase-I. Samples were stored in phosphate-buffered saline solution at 4°C (4°C) or slow cooled in medium with 10% Me2SO at -1°C/min followed by storage in liquid nitrogen (SCM). Structural and functional analyses were performed prior to and after 2 and 4 weeks and 3 and 6 months of storage under each condition. Efficient decellularisation was achieved after 2 cycles of DET as determined with histology and DNA quantification, with preservation of the ECM. Only the SCM method, commonly used for cell storage, maintained the architecture and biomechanical properties of the scaffold up to 6 months. On the contrary, 4°C method was effective for short-term storage but led to a progressive distortion and degradation of the tissue architecture at the following time points. Efficient storage allows a timely use of decellularised oesophagi, essential for clinical translation. Here we describe that slow cooling with cryoprotectant solution in liquid nitrogen vapour leads to reliable long-term storage of decellularised oesophageal scaffolds for tissue engineering purposes.