ISDoT: in situ decellularization of tissues for high-resolution imaging and proteomic analysis of native extracellular matrix.

The extracellular matrix (ECM) is a master regulator of cellular phenotype and behavior. It has a crucial role in both normal tissue homeostasis and disease pathology. Here we present a fast and efficient approach to enhance the study of ECM composition and structure. Termed in situ decellularization of tissues (ISDoT), it allows whole organs to be decellularized, leaving native ECM architecture intact. These three-dimensional decellularized tissues can be studied using high-resolution fluorescence and second harmonic imaging, and can be used for quantitative proteomic interrogation of the ECM. Our method is superior to other methods tested in its ability to preserve the structural integrity of the ECM, facilitate high-resolution imaging and quantitatively detect ECM proteins. In particular, we performed high-resolution sub-micron imaging of matrix topography in normal tissue and over the course of primary tumor development and progression to metastasis in mice, providing the first detailed imaging of the metastatic niche. These data show that cancer-driven ECM remodeling is organ specific, and that it is accompanied by comprehensive changes in ECM composition and topological structure. We also describe differing patterns of basement-membrane organization surrounding different types of blood vessels in healthy and diseased tissues. The ISDoT procedure allows for the study of native ECM structure under normal and pathological conditions in unprecedented detail.

Targeting ECM Disrupts Cancer Progression.

Metastatic complications are responsible for more than 90% of cancer-related deaths. The progression from an isolated tumor to disseminated metastatic disease is a multistep process, with each step involving intricate cross talk between the cancer cells and their non-cellular surroundings, the extracellular matrix (ECM). Many ECM proteins are significantly deregulated during the progression of cancer, causing both biochemical and biomechanical changes that together promote the metastatic cascade. In this review, the influence of several ECM proteins on these multiple steps of cancer spread is summarized. In addition, we highlight the promising (pre-)clinical data showing benefits of targeting these ECM macromolecules to prevent cancer progression.

Hypoxia and loss of PHD2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis.

Cancer-associated fibroblasts (CAFs) interact with tumour cells and promote growth and metastasis. Here, we show that CAF activation is reversible: chronic hypoxia deactivates CAFs, resulting in the loss of contractile force, reduced remodelling of the surrounding extracellular matrix and, ultimately, impaired CAF-mediated cancer cell invasion. Hypoxia inhibits prolyl hydroxylase domain protein 2 (PHD2), leading to hypoxia-inducible factor (HIF)-1α stabilisation, reduced expression of αSMA and periostin, and reduced myosin II activity. Loss of PHD2 in CAFs phenocopies the effects of hypoxia, which can be prevented by simultaneous depletion of HIF-1α. Treatment with the PHD inhibitor DMOG in an orthotopic breast cancer model significantly decreases spontaneous metastases to the lungs and liver, associated with decreased tumour stiffness and fibroblast activation. PHD2 depletion in CAFs co-injected with tumour cells similarly prevents CAF-induced metastasis to lungs and liver. Our data argue that reversion of CAFs towards a less active state is possible and could have important clinical implications.