Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At ...the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies.
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•PACT: passive tissue clearing and immunolabeling protocol for intact thick organs•RIMS: compatible storage and imaging media preserves fluorescent markers over months•Single-molecule FISH compatible 3D phenotyping enabled for thick tissue samples•PARS: whole-body clearing and phenotyping compatible with endogenous fluorescence
Methodology for rendering whole organs and bodies optically transparent and macromolecule-permeable while maintaining intact connectivity allows tissue visualization, immunolabeling, and RNA FISH at single-cell resolution and should be broadly applicable to extend imaging to anatomy that was previously optically inaccessible.
White blood cell (WBC) count is a valuable metric for assisting with diagnosis or prognosis of various diseases such as coronary heart disease, type 2 diabetes, or infection. Counting WBCs can be ...done either manually or automatically. Automatic methods are capable of counting a large number of cells to give a statistically more accurate reading of the WBC count of a sample, but the specialized equipment tends to be expensive. Manual methods are inexpensive since they only involve a conventional light microscope setup. However, it is more laborious and error-prone because the small field-of-view (FOV) of the microscope necessitates mechanical scanning of a specimen for counting an adequate number of WBCs. Here, we investigate the use of Fourier ptychographic microscopy (FPM) to bypass these issues of the manual methods. With a 2x objective, FPM can provide a FOV of 120 mm2 with enhanced resolution comparable to that of a 20x objective, which is adequate for non-differentially counting WBCs in just one FOV. A specialist was able to count the WBCs in FPM images with 100% accuracy compared to the count as determined from conventional microscope images. An automatic counting algorithm was also developed to identify WBCs from FPM's captured images with 95% accuracy, paving the way for a cost-effective WBC counting setup with the advantages of both the automatic and manual counting methods.
A blood-based, low cost alternative to radiation intensive CT and PET imaging is critically needed for cancer prognosis and management of its treatment. "Liquid biopsies" of circulating tumor cells ...(CTCs) from a relatively non-invasive blood draw are particularly ideal, as they can be repeated regularly to provide up to date molecular information about the cancer, which would also open up key opportunities for personalized therapies. Beyond solely diagnostic applications, CTCs are also a subject of interest for drug development and cancer research. In this paper, we adapt a technology previously introduced, combining the use of micro-scale vortices and inertial focusing, specifically for the high-purity extraction of CTCs from blood samples. First, we systematically varied parameters including channel dimensions and flow rates to arrive at an optimal device for maximum trapping efficiency and purity. Second, we validated the final device for capture of cancer cell lines in blood, considering several factors, including the effect of blood dilution, red blood cell lysis and cell deformability, while demonstrating cell viability and independence on EpCAM expression. Finally, as a proof-of-concept, CTCs were successfully extracted and enumerated from the blood of patients with breast (N = 4, 25-51 CTCs per 7.5 mL) and lung cancer (N = 8, 23-317 CTCs per 7.5 mL). Importantly, samples were highly pure with limited leukocyte contamination (purity 57-94%). This Vortex approach offers significant advantages over existing technologies, especially in terms of processing time (20 min for 7.5 mL of whole blood), sample concentration (collecting cells in a small volume down to 300 μL), applicability to various cancer types, cell integrity and purity. We anticipate that its simplicity will aid widespread adoption by clinicians and biologists who desire to not only enumerate CTCs, but also uncover new CTC biology, such as unique gene mutations, vesicle secretion and roles in metastatic processes.
During mammalian embryogenesis, de novo hematopoiesis occurs transiently in multiple anatomical sites including the yolk sac, dorsal aorta, and heart tube. A long-unanswered question is whether these ...local transient hematopoietic mechanisms are essential for embryonic growth. Here, we show that endocardial hematopoiesis is critical for cardiac valve remodeling as a source of tissue macrophages. Colony formation assay from explanted heart tubes and genetic lineage tracing with the endocardial specific Nfatc1-Cre mouse revealed that hemogenic endocardium is a de novo source of tissue macrophages in the endocardial cushion, the primordium of the cardiac valves. Surface marker characterization, gene expression profiling, and ex vivo phagocytosis assay revealed that the endocardially derived cardiac tissue macrophages play a phagocytic and antigen presenting role. Indeed, genetic ablation of endocardially derived macrophages caused severe valve malformation. Together, these data suggest that transient hemogenic activity in the endocardium is indispensable for the valvular tissue remodeling in the heart.
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•Hemogenic endocardium is a source of cardiac macrophages in cardiac valves•Endocardial macrophages are more phagocytic than macrophages from other source(s)•Genetic ablation of endocardial macrophages induces defects in valve formation•Endocardially derived macrophages are indispensable for normal valve remodeling
Shigeta et al. found that a subset of macrophages in the heart valves originates locally from the fetal endocardium. This local type of macrophages is more phagocytic than other macrophages and critical for the valve remodeling during cardiogenesis.
Metastatic non-small cell lung cancer (NSCLC) is a highly fatal and immunogenic malignancy. Although the immune system is known to recognize these tumor cells, one mechanism by which NSCLC can evade ...the immune system is via overexpression of programmed cell death ligand 1 (PD-L1). Recent clinical trials of PD-1 and PD-L1 inhibitors have returned promising clinical responses. Important for personalizing therapy, patients with higher intensity staining for PD-L1 on tumor biopsies responded better. Thus, there has been interest in using PD-L1 tumor expression as a criterion for patient selection. Currently available methods of screening involve invasive tumor biopsy, followed by histological grading of PD-L1 levels. Biopsies have a high risk of complications, and only allow sampling from limited tumor sections, which may not reflect overall tumor heterogeneity. Circulating tumor cell (CTC) PD-L1 levels could aid in screening patients, and could supplement tissue PD-L1 biopsy results by testing PD-L1 expression from disseminated tumor sites. Towards establishing CTCs as a screening tool, we developed a protocol to isolate CTCs at high purity and immunostain for PD-L1. Monitoring of PD-L1 expression on CTCs could be an additional biomarker for precision medicine that may help in determining response to immunotherapies.
The discovery of immune checkpoint inhibition (ICI) sparked a revolution in the era of targeted anticancer therapy. However, although monoclonal antibodies targeting the cytotoxic T‐lymphocyte ...antigen‐4 and programmed death‐1 axes have improved survival in patients with advanced cancers, these immunotherapies are associated with a wide spectrum of dermatological immune‐related adverse events (irAEs), ranging from mild to life‐threatening. Several publications have addressed the clinical and histopathological classification of these skin‐directed irAEs, their impact on anti‐tumour immunity and survival, and the critical role of supportive oncological dermatology in their management. In this paper, we review the current understanding of the mechanistic drivers of immune‐related skin toxicities with a focus on inflammatory, immunobullous and melanocyte/pigment‐related reactions. We detail the specific immune‐based mechanisms that may underlie different cutaneous reactions. We also discuss potential mechanisms as they relate to extracutaneous irAEs and the lessons learned from these, the potential overlap with cutaneous irAEs, techniques to study differences in immune‐related vs. de novo skin reactions, and how treatment of these AEs impacts cancer treatment, patient quality of life and overall survival. An improved understanding of the mechanistic basis of cutaneous irAEs will allow clinicians to develop and use blood‐based biomarkers that could help ultimately predict onset and/or severity of these irAEs, and to implement rational mechanistic‐based treatment strategies that are targeted to the irAEs while potentially avoiding reducing the anti‐tumour effect of ICIs.
Immune checkpoint inhibitor therapies have revolutionized the field of cancer therapy, but are associated with cutaneous immune‐related adverse events (irAEs), ranging from mild to life‐threatening. We review the mechanisms involved in the pathogenesis of cutaneous irAEs, with an emphasis on inflammatory/immunobullous reactions and pigment changes. We also discuss experimental methods to study the immunopathogenesis of cutaneous irAEs, and lessons learned from the study of extracutaneous irAEs.
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Poorly organized tumour vasculature often results in areas of limited nutrient supply and hypoxia. Despite our understanding of solid tumour responses to hypoxia, how nutrient deprivation regionally ...affects tumour growth and therapeutic response is poorly understood. Here, we show that the core region of solid tumours displayed glutamine deficiency compared with other amino acids. Low glutamine in tumour core regions led to dramatic histone hypermethylation due to decreased α-ketoglutarate levels, a key cofactor for the Jumonji-domain-containing histone demethylases. Using patient-derived (V600E)BRAF melanoma cells, we found that low-glutamine-induced histone hypermethylation resulted in cancer cell dedifferentiation and resistance to BRAF inhibitor treatment, which was largely mediated by methylation on H3K27, as knockdown of the H3K27-specific demethylase KDM6B and the methyltransferase EZH2 respectively reproduced and attenuated the low-glutamine effects in vitro and in vivo. Thus, intratumoral regional variation in the nutritional microenvironment contributes to tumour heterogeneity and therapeutic response.
The cellular mechanisms driving cardiac tissue formation remain poorly understood, largely due to the structural and functional complexity of the heart. It is unclear whether newly generated myocytes ...originate from cardiac stem/progenitor cells or from pre-existing cardiomyocytes that re-enter the cell cycle. Here, we identify the source of new cardiomyocytes during mouse development and after injury. Our findings suggest that cardiac progenitors maintain proliferative potential and are the main source of cardiomyocytes during development; however, the onset of αMHC expression leads to reduced cycling capacity. Single-cell RNA sequencing reveals a proliferative, "progenitor-like" population abundant in early embryonic stages that decreases to minimal levels postnatally. Furthermore, cardiac injury by ligation of the left anterior descending artery was found to activate cardiomyocyte proliferation in neonatal but not adult mice. Our data suggest that clonal dominance of differentiating progenitors mediates cardiac development, while a distinct subpopulation of cardiomyocytes may have the potential for limited proliferation during late embryonic development and shortly after birth.
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