Periodic Reporting for period 2 - METANICHE (Regulation of bone metastases by age-associated angiocrine signals) Reporting period: 2020-07-01 to 2021-12-31 Summary of the context and overall objectives of the project Blood and lymphatic vessels provide supportive niche microenvironments for immune cells and maintain tissue functions. Ageing is associated with a decline in tissue and immune system functions. However, ageing of the vasculature and its contribution to tissue physiology is poorly understood. Here, we will interrogate the age-dependent changes in vasculature.Bone-marrow vascular niches support bone and blood progenitors. Vascular niches in bone are diverse, and distinct blood vessel subtypes differentially regulate blood and bone cells. Blood vessels in bone show remarkable changes with age. Further, age-dependent changes in bone vasculature lead to ageing of bone and blood progenitors. However, how blood vessels regulate the fate and behaviour of cancer cells in bone remains elusive. Also, how the age-dependent vascular changes impact bone metastasis is unknown. In this project, we interrogate the relationship and interactions of different vascular niches with cancer cells during bone metastasis. We have developed an ultrafast method for single cell-resolution panoptic multicolour 3D imaging of intact bones to enable this. This enables us to analyse the spatial interactions of cancer cells in bones at an unprecedented level. Finally, we will analyse the age-dependent changes in these interactions of cancer cells with the blood vessels in bone. This project will contribute to the development of therapeutic approaches to manage bone metastasis. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far 1. Identification of pericyte to fibroblast differentiation and endothelial attrition as a hallmark of vascular and tissue ageingBlood vessels provide supportive microenvironments for various cell types to maintain tissue homeostasis and organ function. Ageing is associated with alterations in tissue homeostasis and a decline in tissue functions. However, ageing of the vasculature and how it contributes to changes in tissue physiology is poorly understood. Here, we show age-related changes in vascular microenvironments. We found an age-dependent decline of capillary, artery and pericyte numbers and an increase in fibroblast abundances in mice and humans. Vascular attrition precedes the cellular hallmarks of ageing such as senescence and mitochondrial dysfunction. Endothelial cell-specific genetic experiments confirm that vascular perturbations are sufficient to stimulate cellular changes coupled with ageing. Further, genetic lineage tracing experiments reveal differentiation of pericytes to fibroblasts upon ageing. Finally, gene expression and functional analysis demonstrated that age-related molecular changes in the endothelium dictate pericyte to fibroblast differentiation. We find the contribution of pericyte-derived fibroblasts to the age-related increase in fibrosis. These findings unearth vascular attrition marked by pericyte to fibroblast differentiation as a primary hallmark of ageing tissues (Chen et al, Science Advances 2021). To facilitate further research, we provide a freely available resource of >1500 3D maps highlighting organ-specific and age-related features of vascular microenvironments http://homeros.kennedy.ox.ac.uk/pub/chen-et-al-2021-3dOrgans2. Identification of gap junction communication protein Gja1 as a driver of endothelial cell ageingFurther, the comprehensive analysis of endocrine tissues shows that angiogenesis and beta-cell expansion in the pancreas during ageing showed molecularly and a functionally distinct age-dependent subset of Endothelial Cells (ECs). This EC subset support pancreatic -cells, and their decline with ageing caused by gap junction protein Gja1 led to the reduction of -cell proliferation, while their genetic or pharmacological reactivation allowed the restoration of beta-cell expansion (Chen et al, EMBO Journal 2021). These results provide a proof-of-concept for understanding age-related vascular changes for vascular targeting to restore endocrine tissue function. Towards this, our open image datasets provide a wealth of spatial information in endocrine tissues for download and exploration. http://homeros.kennedy.ox.ac.uk/pub/chen-et-al-2020-3dEndocrine3. Identification of endothelial and perivascular factors regulating quiescence of disseminated tumour cells in boneBone marrow provides supportive microenvironments for long-lived Hematopoietic Stem Cells (HSCs), Mesenchymal Stem Cells (MSCs), and certain immune cells. Skeletal ageing is associated with a decline in HSC function and increased incidences of bone metastasis. However, the role of the ageing bone marrow microenvironment in regulating stem and cancer cell behaviour remains elusive. Here, we find that the aged bone marrow microenvironment perturbs the quiescence of HSCs and MSCs, and metastatic cancer cells in bone. In particular, the aged bone marrow microenvironment exhibits a decline in secreted factors that induce and maintain quiescence. Both radiation and chemotherapy induce bone-specific upregulation of quiescence promoting secreted factors. Cell-specific secretome screening identified the involvement of pericytes in promoting quiescent microenvironments in bone in response to radiation and chemotherapy. Administration of PDGFR inhibitor alongside radiation or chemotherapy led to the decline in quiescent cancer cells, and an increase in the susceptibility to these therapeutic strategies (Singh et al JCI Insight 2019). Thus, our study provides a framework for targeting pericytes to manipulate the bone marrow microenvironment in therapeutic interventions to manage bone metastasis. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) In the skeletal system, blood vessels play a central role in maintaining microenvironments required for regulating osteogenesis and hematopoiesis during development and homoeostasis. 3D imaging of transparent tissues is essential for analyzing vascular structures, tissue architecture and spatial distributions. However, imaging of whole bones remains technically challenging due to their calcified nature. The current immunolabelling and clearing methods for whole bones are limited, complicated, and time-consuming. Overcoming these limitations, we developed an efficient method that enables rapid immunolabelling and clearing for high-resolution 3D imaging of intact bones and calcified murine and human tissues. We are using this technique to characterize the bone marrow microenvironment. We are now using this method to characterise the bone marrow environment. We are also investigating the location of dormant tumour cells in bone and their location response to chemotherapy and radiation administration.
