Large-scale 3-dimensional quantitative imaging of tissues: state-of-the-art and translational implications

Winfree, Seth; Ferkowicz, Michael J.; Dagher, Pierre C.; Kelly, Katherine J.; Eadon, Michael T.; Sutton, Timothy A.; Markel, Troy A.; Yoder, Mervin C.; Dunn, Kenneth W.; El‐Achkar, Tarek M.

doi:10.1016/j.trsl.2017.07.006

Cited by 25 publications

(43 citation statements)

References 71 publications

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“…The UCSF and PREMIERE sites will use miFISH to generate simultaneous gene and protein expression data at the single cell level in 2D space (26). The IU/OSU site will utilize large scale 3D confocal imaging of longitudinal kidney thick sections probing concurrently for 8 different targets using antibodies or fluorescent small molecules, followed by tissue cytometry analysis (27,28). Prior to any staining, this technology uses label-free imaging to determine tissue integrity using endogenous fluorescence and quantify collagen deposition using second harmonic generation.…”

Section: Overview Of Kpmp Technologies: Creating a Molecular And Funcmentioning

confidence: 99%

A Multimodal and Integrated Approach to Interrogate Human Kidney Biopsies with Rigor and Reproducibility: The Kidney Precision Medicine Project

El-Achkar

Eadon

Menon

et al. 2019

Preprint

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Comprehensive and spatially mapped molecular atlases of organs at a cellular level are a critical resource to gain insights into pathogenic mechanisms and therapies tailored to the disease of each patient. Obtaining rigorous and reproducible results from disparate methods and at different sites to interrogate biomolecules at a single cell level or in 3-dimensional (3D) space is a significant challenge that can be a futile exercise if not well controlled. The Kidney Precision Medicine Project (KPMP) is an endeavor to generate 3D molecular atlases of healthy and diseased kidney biopsies using multiple state-of-the-art OMICS and imaging technologies across several institutions. We describe a pipeline for generating a reliable and authentic single cell/region 3D molecular atlas of human adult kidney with emphasis on quality assurance, quality control, validation and harmonization across different OMICS and imaging methods. Our “follow the tissue” approach encompasses sample procurement to data generation, analysis, data sharing, while standardizing, and harmonizing procedures at sample collection, processing, storage and shipping. We provide key features of preanalytical parameters, bioassays, post analyses, reference standards and data depositions. A pilot experiment from a common source tissue processed and analyzed at different institutions was executed to identify potential sources of variation, feasibility of multimodal analyses and unique and redundant features of the macromolecules being characterized by each technology. An important outcome was identification of limitations and strengths of the different technologies, and how composite information can be leveraged for clinical application. A peer review system was established to critically review quality control measures and the reproducibility of data generated by each technology before granting approval to work on clinical biopsy specimens. This unique pipeline establishes a process that economizes the use of valuable biopsy tissue for multi-OMICS and imaging analysis with stringent quality control to ensure rigor and reproducibility of results and which can serve as a model for similar personalized medicine projects.Author ContributionsMTE, RM, BBL, TS, TA, AS, SP, CRA, DD, EAO, SW, GZ, MJ and KD performed the ground work for the quality control group, wrote the KPMP TIS manual of procedures and generated figures. HH, JZ, RS, RM, TS, EAO, MTE, TME, PH, SP, MK, ZL and SJ generated the initial working reference marker list. CEA, TME, JBH, JL, MK and SJ led the Pilot 1 protocol. TME, VD, LB, JG, CEA, ZL, SJ and JBH developed the pathology QC tissue qualification and tissue processing criteria. JBH organized and executed the Pilot tissue collection and distribution. JC designed the SpecTrack system. CP prepared and organized the TIS manual of procedures and performed data organization services. YH led ontology development for QC metadata and knowledge standardization. BS and EA organized data integration efforts and data authentication in the data hub. KS and MS led the OMICS discussion group. SJ led the quality control group. TME and CEA led the tissue processing group. MTE and SJ led the Molecular and Pathology Integration group. MTE and SJ conceived and led the TISAC process. RI, OGT KZ, ZL, PH, BR, PCD, KS, MS, JBH, CEA, LB, JG, TME, MK and SJ conceived the integrated TIS pipeline and QC vision. TME and SJ wrote the initial draft of the paper. All authors contributed to the writing and editing of the manuscript.

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Section: Overview Of Kpmp Technologies: Creating a Molecular And Funcmentioning

confidence: 99%

A Multimodal and Integrated Approach to Interrogate Human Kidney Biopsies with Rigor and Reproducibility: The Kidney Precision Medicine Project

El-Achkar

Eadon

Menon

et al. 2019

Preprint

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“…[12][13][14] With optical sectioning microscopy, querying cell types based on specific markers could also be expanded to 3dimensional (3D) space, increasing the ability to assay cellular structure and tissue architecture. 13,15,16 Multiplex imaging based on cell markers may offer a path to classify cells in tissues, especially with the availability of tissue cytometry software tools that facilitate the analytical process. For example, we recently described the Volumetric Tissue Exploration and Analysis (VTEA) tool which enables the semi-automated classification of labeled cells in 3D image volumes.…”

Section: Introductionmentioning

confidence: 99%

“…For example, we recently described the Volumetric Tissue Exploration and Analysis (VTEA) tool which enables the semi-automated classification of labeled cells in 3D image volumes. 13,15,17 However, multiplexed imaging and tissue cytometry have several challenges that may limit their utility for comprehensive cell classification in situ. These challenges include a potentially lengthy technical workflow, a requirement for image processing expertise to optimize the analysis, and above all, only a finite number of cell-associated markers can be obtained from a single experiment.…”

Section: Introductionmentioning

confidence: 99%

“…These challenges include a potentially lengthy technical workflow, a requirement for image processing expertise to optimize the analysis, and above all, only a finite number of cell-associated markers can be obtained from a single experiment. 15 The scarcity of markers is the most limiting, as each time a new marker is discovered or needed, new experiments on additional tissue sections are required. For sparse tissue such as a kidney biopsy, this could lead to tissue exhaustion.…”

Section: Introductionmentioning

confidence: 99%

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In situclassification of cell types in human kidney tissue using 3D nuclear staining

Woloshuk

Khochare

Almulhim

et al. 2020

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To understand the physiology and pathology of disease, capturing the heterogeneity of cell types within their tissue environment is fundamental. In such an endeavor, the human kidney presents a formidable challenge because its complex organizational structure is tightly linked to key physiological functions. Advances in imaging-based cell classification may be limited by the need to incorporate specific markers that can link classification to function. Multiplex imaging can mitigate these limitations, but requires cumulative incorporation of markers, which may lead to tissue exhaustion. Furthermore, the application of such strategies in large scale 3dimensional (3D) imaging is challenging. Here, we propose that 3D nuclear signatures from a DNA stain, DAPI, which could be incorporated in most experimental imaging, can be used for classifying cells in intact human kidney tissue. We developed an unsupervised approach that uses 3D tissue cytometry to generate a large training dataset of nuclei images (NephNuc), where each nucleus is associated with a cell type label. We then devised various supervised machine learning approaches for kidney cell classification and demonstrated that a deep learning approach outperforms classical machine learning or shape-based classifiers.Specifically, a custom 3D convolutional neural network (NephNet3D) trained on nuclei image volumes achieved a balanced accuracy of 80.26%. Importantly, integrating NephNet3D classification with tissue cytometry allowed in situ visualization of cell type classifications in kidney tissue. In conclusion, we present a tissue cytometry and deep learning approach for in situ classification of cell types in human kidney tissue using only a DNA stain. This methodology is generalizable to other tissues and has potential advantages on tissue economy and non-exhaustive classification of different cell types.

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“…Significant advancement in molecular interrogation of tissue specimens presents a unique opportunity to uncover important pathways that are differentially activated in these two forms of kidney stone disease. These techniques such as transcriptomics, proteomics and large-scale quantitative 3-dimensional imaging can provide an unbiased and comprehensive approach towards discovery [6][7][8][9][10] . We present here a detailed study contrasting CaOx and brushite clinical stone phenotypes using these orthogonal omics approaches.…”

Section: Introductionmentioning

confidence: 99%

A precision medicine approach uncovers a unique signature of neutrophils in patients with brushite kidney stones

Makki

Winfree

Lingeman

et al. 2019

Preprint

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Kidney stone disease is very common and causes significant morbidity. The main goal of this study was to uncover stone-type specific molecular signatures using a precision medicine approach that combines unbiased transcriptomics, proteomics and innovative 3D imaging strategies. We found that increased neutrophil infiltration and NETosis are unrecognized new factors specifically associated with brushite stone formation. These discoveries underscore the unequivocal presence of inflammation in human stone disease, and the divergence of cellular and molecular pathways in distinct forms of nephrolithiasis. Abstract Background:We have previously found that papillary histopathology differs greatly between calcium oxalate and brushite stone formers (SF); the latter have much more papillary mineral deposition, tubular cell injury and tissue fibrosis. Methods:In this study, we applied unbiased orthogonal "omics" approaches on biopsied renal papillae and extracted stones from patients with brushite or calcium oxalate (CaOx) stones. Our goal was to discover stone type-specific molecular signatures to advance our understanding of the underlying pathogenesis.Results: Brushite SF did not differ from CaOx SF with respect to metabolic risk factors for stones, but did exhibit increased tubule plugging in their papillae. Brushite SF had upregulation of inflammatory pathways in papillary tissue, and increased neutrophil markers in stone matrix compared to those with CaOx stones. Large-scale 3D tissue cytometry on renal papillary biopsies showed an increase in the number and density of neutrophils in the papillae of brushite vs. CaOx patients, thereby linking the observed inflammatory signatures to the neutrophils in the tissue. To explain how neutrophil proteins appear in the stone matrix, we measured neutrophil extracellular trap (NET) formation, NETosis, and found it significantly increased in the papillae of brushite compared to CaOx patients. Conclusions:We show that increased neutrophil infiltration and NETosis is an unrecognized factor that differentiates brushite and CaOx SF, and may explain the markedly increased scarring and inflammation seen in the papillae of brushite patients. Given the increasing prevalence of brushite stones, the role of neutrophil activation in brushite stone formation requires further study.

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Large-scale 3-dimensional quantitative imaging of tissues: state-of-the-art and translational implications

Cited by 25 publications

References 71 publications

A Multimodal and Integrated Approach to Interrogate Human Kidney Biopsies with Rigor and Reproducibility: The Kidney Precision Medicine Project

A Multimodal and Integrated Approach to Interrogate Human Kidney Biopsies with Rigor and Reproducibility: The Kidney Precision Medicine Project

In situclassification of cell types in human kidney tissue using 3D nuclear staining

A precision medicine approach uncovers a unique signature of neutrophils in patients with brushite kidney stones

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