decellularization-of-the-murine-cardiopulmonary-complex

Decellularization of the Murine Cardiopulmonary Complex

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and ...

Lund University

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.

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

Tissue regeneration requires dynamic cellular adaptation to the wound environment. It is currently unclear how this is orchestrated at the cellular level and how cell fate is affected by severe tissue damage. Here we dissect cell fate transitions during colonic regeneration in a mouse dextran sulfate sodium (DSS) colitis model, and we demonstrate that the epithelium is transiently reprogrammed into a primitive state. This is characterized by de novo expression of fetal markers as well as suppression of markers for adult stem and differentiated cells. The fate change is orchestrated by remodeling the extracellular matrix (ECM), increased FAK/Src signaling, and ultimately YAP/TAZ activation. In a defined cell culture system recapitulating the extracellular matrix remodeling observed in vivo, we show that a collagen 3D matrix supplemented with Wnt ligands is sufficient to sustain endogenous YAP/TAZ and induce conversion of cell fate. This provides a simple model for tissue regeneration, implicating cellular reprogramming as an essential element.

YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration.

Increased tissue stiffness is a classic characteristic of solid tumors. One of the major contributing factors is increased density of collagen fibers in the extracellular matrix (ECM). Here, we investigate how cancer cells biomechanically interact with and respond to the stiffness of the ECM. Probing the adaptability of cancer cells to altered ECM stiffness using optical tweezers-based microrheology and deformability cytometry, we find that only malignant cancer cells have the ability to adjust to collagen matrices of different densities. Employing microrheology on the biologically relevant spheroid invasion assay, we can furthermore demonstrate that, even within a cluster of cells of similar origin, there are differences in the intracellular biomechanical properties dependent on the cells' invasive behavior. We reveal a consistent increase of viscosity in cancer cells leading the invasion into the collagen matrices in comparison with cancer cells following in the stalk or remaining in the center of the spheroid. We hypothesize that this differential viscoelasticity might facilitate spheroid tip invasion through a dense matrix. These findings highlight the importance of the biomechanical interplay between cells and their microenvironment for tumor progression.

Cancer cells' ability to mechanically adjust to extracellular matrix stiffness correlates with their invasive potential.

Cancer-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment, although their origin and roles in shaping disease initiation, progression and treatment response remain unclear due to significant heterogeneity. Here, following a negative selection strategy combined with single-cell RNA sequencing of 768 transcriptomes of mesenchymal cells from a genetically engineered mouse model of breast cancer, we define three distinct subpopulations of CAFs. Validation at the transcriptional and protein level in several experimental models of cancer and human tumors reveal spatial separation of the CAF subclasses attributable to different origins, including the peri-vascular niche, the mammary fat pad and the transformed epithelium. Gene profiles for each CAF subtype correlate to distinctive functional programs and hold independent prognostic capability in clinical cohorts by association to metastatic disease. In conclusion, the improved resolution of the widely defined CAF population opens the possibility for biomarker-driven development of drugs for precision targeting of CAFs.

Spatially and functionally distinct subclasses of breast cancer-associated fibroblasts revealed by single cell RNA sequencing.

The extracellular matrix (ECM) is a major regulator of homeostasis and disease, yet the 3D structure of the ECM remains poorly understood because of limitations in ECM visualization. We recently developed an ECM-specialized method termed in situ decellularization of tissues (ISDoT) to isolate native 3D ECM scaffolds from whole organs in which ECM structure and composition are preserved. Here, we present detailed surgical instructions to facilitate decellularization of 33 different mouse tissues and details of validated antibodies that enable the visualization of 35 mouse ECM proteins. Through mapping of these ECM proteins, the structure of the ECM can be determined and tissue structures visualized in detail. In this study, perfusion decellularization is presented for bones, skeletal muscle, tongue, salivary glands, stomach, duodenum, jejunum/ileum, large intestines, mesentery, liver, gallbladder, pancreas, trachea, bronchi, lungs, kidneys, urinary bladder, ovaries, uterine horn, cervix, adrenal gland, heart, arteries, veins, capillaries, lymph nodes, spleen, peripheral nerves, eye, outer ear, mammary glands, skin, and subcutaneous tissue. Decellularization, immunostaining, and imaging take 4-5 d.

Decellularization and antibody staining of mouse tissues to map native extracellular matrix structures in 3D.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Decellularization and antibody staining of mouse tissues to map native extracellular matrix structures in 3D.

The STRIPAK complex has been linked to a variety of biological processes taking place during embryogenesis and development, but its role in cancer has only just started to be defined. Here, we expand on previous work indicating a role for the scaffolding protein STRIP1 in cancer cell migration and metastasis. We show that cell cycle arrest and decreased proliferation are seen upon loss of STRIP1 in MDA-MB-231 cells due to the induction of cyclin dependent kinase inhibitors, including p21 and p27. We demonstrate that p21 and p27 induction is observed in a subpopulation of cells having low DNA damage response and that the p21/γH2AX ratio within single cells can be rescued by depleting MST3&4 kinases. While the loss of STRIP1 decreases cell proliferation and tumor growth, cells treated with low dosage of chemotherapeutics  paradoxically escape therapy-induced senescence and begin to proliferate after recovery. This corroborates with already known research on the dual role of p21 and indicates that STRIP1 also plays a contradictory role in breast cancer, suppressing tumor growth, but once treated with chemotherapeutics, allowing for possible recurrence and decreased patient survival.

The STRIPAK Complex Regulates Response to Chemotherapy Through p21 and p27.

The use of in vitro cell cultures is a powerful tool for obtaining key insights into the behaviour and response of cells to interventions in normal and disease situations. Unlike in vivo settings, in vitro experiments allow a fine-tuned control of a range of microenvironmental elements independently within an isolated setting. The recent expansion in the use of three-dimensional (3D) in vitro assays has created a number of representative tools to study cell behaviour in a more physiologically 3D relevant microenvironment. Complex 3D in vitro models that can recapitulate human tissue biology are essential for understanding the pathophysiology of disease.

The Mini-Organo: A rapid high-throughput 3D coculture organotypic assay for oncology screening and drug development.

The basement membrane (BM) is a special type of extracellular matrix and presents the major barrier cancer cells have to overcome multiple times to form metastases. Here we show that BM stiffness is a major determinant of metastases formation in several tissues and identify netrin-4 (Net4) as a key regulator of BM stiffness. Mechanistically, our biophysical and functional analyses in combination with mathematical simulations show that Net4 softens the mechanical properties of native BMs by opening laminin node complexes, decreasing cancer cell potential to transmigrate this barrier despite creating bigger pores. Our results therefore reveal that BM stiffness is dominant over pore size, and that the mechanical properties of 'normal' BMs determine metastases formation and patient survival independent of cancer-mediated alterations. Thus, identifying individual Net4 protein levels within native BMs in major metastatic organs may have the potential to define patient survival even before tumour formation. The ratio of Net4 to laminin molecules determines BM stiffness, such that the more Net4, the softer the BM, thereby decreasing cancer cell invasion activity.

Basement membrane stiffness determines metastases formation.

Clinical implementation of anti-stromal therapies in pancreatic cancer has been delayed by unanticipated tumor-restraining properties of the desmoplastic stroma. In confronting these challenges, Chen et al. demonstrate in this issue of Cancer Cell that fibroblast-specific deletion of collagen I, in the background of oncogenic Kras-induced spontaneous murine pancreatic ductal adenocarcinoma, enhances immune suppression and accelerates progression of disease.

Pancreatic cancer is suppressed by fibroblast-derived collagen I.

Cancer-associated fibroblasts (CAFs) comprise a heterogeneous population of stromal cells within the tumour microenvironment. CAFs exhibit both tumour-promoting and tumour-suppressing functions, making them exciting targets for improving cancer treatments. Careful isolation, identification, and characterisation of CAF heterogeneity is thus necessary for ex vivo validation and future implementation of CAF-targeted strategies in cancer.

Article	Year
ISDoT: in situ decellularization of tissues for high-resolution imaging and proteomic analysis of native extracellular matrix. Nature medicine\| PubMed ID: 28604702	2017
YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration. Cell stem cell\| PubMed ID: 29249464	2018
Cancer cells' ability to mechanically adjust to extracellular matrix stiffness correlates with their invasive potential. Molecular biology of the cell\| PubMed ID: 30091653	2018
Spatially and functionally distinct subclasses of breast cancer-associated fibroblasts revealed by single cell RNA sequencing. Nature communications\| PubMed ID: 30514914	2018
Decellularization and antibody staining of mouse tissues to map native extracellular matrix structures in 3D. Nature protocols\| PubMed ID: 31705125	2019
Author Correction: Decellularization and antibody staining of mouse tissues to map native extracellular matrix structures in 3D. Nature protocols\| PubMed ID: 32203488	2020
The STRIPAK Complex Regulates Response to Chemotherapy Through p21 and p27. Frontiers in cell and developmental biology\| PubMed ID: 32258031	2020
The Mini-Organo: A rapid high-throughput 3D coculture organotypic assay for oncology screening and drug development. Cancer reports (Hoboken, N.J.)\| PubMed ID: 32671954	2020
Basement membrane stiffness determines metastases formation. Nature materials\| PubMed ID: 33495631	2021
Pancreatic cancer is suppressed by fibroblast-derived collagen I. Cancer cell\| PubMed ID: 33711271	2021
Deciphering the temporal heterogeneity of cancer-associated fibroblast subpopulations in breast cancer. Journal of experimental & clinical cancer research : CR\| PubMed ID: 34016130	2021

Chris D. Madsen

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