CellMAPS: A no-code model-based customisable multiplex image analysis workflow

BackgroundIdentifying biomarkers that predict patient response to immunotherapy is critical for optimizing treatment strategies and improving clinical outcomes. Despite the success of immunotherapy, a significant proportion of patients do not...

Imaging mass cytometry-derived single cell extraction, transformation and phenotyping to analyze immune tumor microenvironments.

Quantitative analysis can be used in combination with fluorescence microscopy. Although the human eye is able to obtain good qualitative results, when analyzing the spatial organization of telomeres in interphase nuclei, there is a need for quantitative results based on image analysis. We developed a tool for analyzing three-dimensional images of telomeres stained by fluorescence in situ hybridization in interphase nuclei with DNA counterstained with 4',6-diamidino-2-phenylindole. After deconvolution of the image, we segmented individual telomeres. From the location of the telomeres we derived a distribution parameter rhoT, which indicated whether the telomeres were in a disk (rhoT >> 1) or not (rhoT approximately 1). We sorted mouse lymphocyte nuclei and measured rhoT. We also performed a bromodeoxyuridine synchronous cell sorting experiment on live cells and measured rhoT at several instances. Measuring rhoT for nuclei in G0/G1, S, and G2 produced 1.4 +/- 0.1, 1.5 +/- 0.2, and 14 +/- 2, respectively, showing a significant difference between G2 and G0/G1 or S. For the bromodeoxyuridine synchronous cell sorting experiment, we found a cell cycle dependency of rhoT and a correlation between rhoT and an observer. In this study we present a quantitative method to characterize the organization of telomeres using three-dimensional imaging, image processing, and image analysis.

Mutation accumulation in RNAs results in closely located single-nucleotide mutations (SNMs), which is highly associated with the drug resistance of pathogens. Imaging of SNMs in single cells has significance for understanding the heterogeneity of RNAs that are related to drug resistance, but the direct "see" closely located SNMs remains challenging. Herein, we designed an encoded ligation-mediated in situ polymerase chain reaction method (termed enPCR), which enabled the visualization of multiple closely located SNMs in bacterial RNAs. Unlike conventional ligation-based probes that can only discriminate a single SNM, this method can simultaneously image different SNMs at closely located sites with single-cell resolution using modular anchoring probes and encoded PCR primers. We tested the capacity of the method to detect closely located SNMs related to quinolone resistance in the gyrA gene of Salmonella enterica (S. enterica), and found that the simultaneous detection of the closely located SNMs can more precisely indicate the resistance of the S. enterica to quinolone compared to the detection of one SNM. The multiplexing imaging assay for SNMs can serve to reveal the relationship between complex cellular genotypes and phenotypes.

Background: In the past few years, animal models and cultured cells have shown that smooth muscle cells (SMCs) and inflammatory cytokines are important new players in atherosclerotic disease. Validating the roles of these new players in human atherogenesis is crucial for the development of therapeutics targeting them. Multiplex imaging is a powerful tool for mapping cell phenotypes and microenvironment in tissue sections, enabling proof-of-concept studies using biobanked human specimens. However, this technology has seldom been applied to human atherosclerotic lesions. This study is a trial to benchmark the application of the multiplex imaging platform PhenoCycler to biobanked human coronary lesions. We hypothesize that this technology is a powerful tool to prove theories raised by previous studies: SMCs initiate foam cell formation and inflammation is associated with phenotypic switching in SMCs. Methods: We created an 8-plex imaging panel to map lesion cells previously identified as key players in atherogenesis, including foam cells from SMC and macrophage origins, CD68 + SMCs, apoptotic cells, and cells expressing inflammatory cytokines interleukin-1β and tumor necrosis factor α. They were imaged simultaneously on each section of early human lesions (n=9) to test theories related to foam cell formation, phenotypic transition, and inflammatory cytokines. Results: Foam cells of SMC origin greatly outnumbered those of macrophage origin and were enriched in the deep intima, where lipids accumulate in early atherogenesis, distinct from macrophage foam cells in the superficial intima. A higher presence of CD68 + SMCs was observed among lesion SMCs highly expressing interleukin-1β, but not TNF-α. Highly inflamed SMCs exhibited a trend of increased apoptosis compared to macrophages with similar cytokine levels. Conclusions: Our multiplex imaging results confirmed that SMCs form the majority of foam cells in early atherogenesis, and their phenotypic transition to macrophage-like is related to inflammation. Applying multiplex imaging to biobanked human lesions unlocks opportunities to prove that theories based on animal models and cultured cells apply to human atherogenesis.

P-glycoprotein is implicated in clinical drug resistance; thus, rapid quantitative analysis of its expression and activity is of paramout importance to the design and success of novel therapeutics. The scope for the application of quantitative imaging and image analysis tools in this field is reported here at "proof of concept" level. P-glycoprotein expression was utilized as a model for quantitative immunofluorescence and subsequent spatial intensity distribution analysis (SpIDA). Following expression studies, p-glycoprotein inhibition as a function of verapamil concentration was assessed in two cell lines using live cell imaging of intracellular Calcein retention and a routine monolayer fluorescence assay. Intercellular and sub-cellular distributions in the expression of the p-glycoprotein transporter between parent and MDR1-transfected Madin-Derby Canine Kidney cell lines were examined. We have demonstrated that quantitative imaging can provide dose-response parameters while permitting direct microscopic analysis of intracellular fluorophore distributions in live and fixed samples. Analysis with SpIDA offers the ability to detect heterogeniety in the distribution of labeled species, and in conjunction with live cell imaging and immunofluorescence staining may be applied to the determination of pharmacological parameters or analysis of biopsies providing a rapid prognostic tool.

ConspectusOrganic, soft materials with solution-phase nanoscale structures, such as emulsions, hydrogels, and thermally responsive materials, are inherently difficult to directly image via dry state and cryogenic-transmission electron microscopy (TEM). Therefore, we lack a routine microscopy method with sufficient resolution that can, in tandem with scattering techniques, probe the morphology and dynamics of these and many related systems. These challenges motivate liquid cell (LC) TEM method development, aimed at making the technique generally available and routine. To date, the field has been and continues to be dominantly focused on analyzing solution-phase inorganic materials. These mostly metallic nanoparticles have been studied at electron fluxes that can allow for high-resolution imaging, in the range of hundreds to thousands of e- Å-2 s-1. Despite excellent contrast, in these cases, one often contends with knock-on damage, direct radiolysis, and sensitization of the solvent by virtue of enhanced secondary electron production by the impinging electron beam. With an interest in soft materials, we face both related and distinct challenges, especially in achieving a high-enough contrast within solvated liquid cells. Additionally, we must be aware of artifacts associated with high-flux imaging conditions in terms of direct radiolysis of the solvent and the sensitive materials themselves. Regardless, with care, it has become possible to gain real insight into both static and dynamic organic nanomaterials in solution. This is due, in large part, to key advances that have been made, including improved sample preparation protocols, image capture technologies, and image analysis, which have allowed LCTEM to have utility. To enable solvated soft matter characterization by LCTEM, a generalizable multimodal workflow was developed by leveraging both experimental and theoretical precedents from across the LCTEM field and adjacent works concerned with solution radiolysis and nanoparticle tracking analyses. This workflow consists of (1) modeling electron beam-solvent interactions, (2) studying electron beam-sample interactions via LCTEM coupled with post-mortem analysis, (3) the construction of "damage plots" displaying sample integrity under varied imaging and sample conditions, (4) optimized LCTEM imaging, (5) image processing, and (6) correlative analysis via X-ray or light scattering. In this Account, we present this outlook and the challenges we continue to overcome in the direct imaging of dynamic solvated nanoscale soft materials.

Musculoskeletal disorders such as osteoarthritis, bone fractures, osteoporosis, or ligament injuries are highly prevalent conditions affecting muscles, bones, joints, and adjacent connective tissues. In 2020, they affected over 1.63 billion people worldwide (GBD 2021 Other Musculoskeletal Disorders Collaborators, 2023). Musculoskeletal disorders cause various symptoms such as pain and mobility limitations, and they have a tremendous impact on the quality of life of an individual imposing a large economic burden to a society (Liu et al., 2022;Parto et al., 2023). The underlying pathophysiological processes and clinical manifestations vary depending on the affected tissue and specific disorder.Imaging provides visualization of the anatomy and physiological processes of the body and plays an important role in the diagnostics of musculoskeletal disorders both in clinical practice and research. Conventional radiography and computed tomography (CT) are usually used to assess changes in the mineralized tissues, such as subchondral bones, whereas magnetic resonance imaging (MRI) is best suited for assessing changes in the soft tissues, for example damage in tendons, ligaments, menisci, and cartilage. Novel imaging, image processing, and image analysis techniques have a great potential to enable earlier diagnosis and deeper understanding of musculoskeletal disorders and pathophysiological processes related to them (Kijowski and Fritz 2023) The aim of this Research Topic was to collect articles showcasing applications of novel and innovative imaging, image processing, and image analysis techniques for assessing musculoskeletal disorders and the underlying pathophysiological processes. Although musculoskeletal conditions can affect various joints, and no specific restrictions were placed on this research topic, the articles included in this research topic focused on disorders involving the spine, spinal joints, and associated muscles.

BCL-2 has been implicated in prostate cancer (PCa) progression and development of castration-resistant disease (CRPC); however, it remains unclear how the BCL-2- and AR-expressing PCa cell populations evolve across the PCa continuum, how AR molecularly regulates BCL-2 and whether BCL-2 represents a common therapeutic target in heterogeneous CRPC. Here we first show the selective induction of BCL-2 by AR pathway inhibitors (ARPIs). Vectra-based quantitative multiplex immunofluorescence (qmIF) and image mass cytometry (IMC) analyses with single-cell resolution in patient PCa and xenograft models reveal markedly increased BCL-2+ (AR+ or AR-) PCa cells in CRPC. Mechanistically, AR represses BCL-2 transcription through several AR binding sites and ARPIs relieve this repression. Therapeutic studies in cells, organoids and xenografts support BCL-2 as a shared vulnerability across diverse CRPC subtypes. A Phase Ib clinical trial (NCT03751436) combining enzalutamide and BCL-2 inhibitor venetoclax demonstrated reduced circulating tumor cells in responding patients. In summary, by integrating high-content single-cell level imaging analyses with mechanistic studies, extensive preclinical therapeutic experiments and a Phase Ib clinical trial, our studies herein elucidate the AR+/-BCL-2+/- PCa cell subpopulation dynamics and credentials BCL-2 as a vital therapeutic target in heterogeneous CRPC.

The interaction between genetic and environmental factors determines the development of type 1 diabetes (T1D). Some viruses are capable of infecting and damaging pancreatic β-cells, whose antiviral response could be modulated by specific viral RNA receptors and sensors such as melanoma differentiation associated gene 5 (MDA5), encoded by the IFIH1 gene. MDA5 has been shown to be involved in pro-inflammatory and immunoregulatory outcomes, thus determining the response of pancreatic islets to viral infections. Although the function of MDA5 has been previously well explored, a detailed immunohistochemical characterization of MDA5 in pancreatic tissues of nondiabetic and T1D donors is still missing. In the present study, we used multiplex immunofluorescence imaging analysis to characterize MDA5 expression and distribution in pancreatic tissues obtained from 22 organ donors (10 nondiabetic autoantibody-negative, 2 nondiabetic autoantibody-positive, 8 recent-onset, and 2 long-standing T1D). In nondiabetic control donors, MDA5 was expressed both in α- and β-cells. The colocalization rate imaging analysis showed that MDA5 was preferentially expressed in α-cells. In T1D donors, we observed an increased colocalization rate of MDA5-glucagon with respect to MDA5-insulin in comparison to nondiabetic controls; such increase was more pronounced in recent-onset with respect to long-standing T1D donors. Of note, an increased colocalization rate of MDA5-glucagon was found in insulin-deficient-islets (IDIs) with respect to insulin-containing-islets (ICIs). Strikingly, we detected the presence of MDA5-positive/hormone-negative endocrine islet-like clusters in T1D donors, presumably due to dedifferentiation or neogenesis phenomena. These clusters were identified exclusively in donors with recent disease onset and not in autoantibody-positive nondiabetic donors or donors with long-standing T1D. In conclusion, we showed that MDA5 is preferentially expressed in α-cells, and its expression is increased in recent-onset T1D donors. Finally, we observed that MDA5 may also characterize the phenotype of dedifferentiated or newly forming islet cells, thus opening to novel roles for MDA5 in pancreatic endocrine cells.

BackgroundMultiplex immunofluorescence (mIF) is a powerful tool for tumor-microenvironment (TME) characterization. However, the adoption of high-plex panels in the clinical setting is limited due to cost and technical complexity. Hematoxylin...

BackgroundHow important are sexual hormones beyond their function in reproductive biology has yet to be understood. In this study, we analyzed the effects of sex steroids on the biology of the embryonic amphibian epidermis, which represents an easily amenable model of non-reproductive mucociliary epithelia (MCE). MCE are integrated systems formed by multiciliated (MC), mucus-secreting (MS) and mitochondrion-rich (MR) cell populations that are shaped by their microenvironment. Therefore, MCE could be considered as ecosystems at the cellular scale, found in a wide array of contexts from mussel gills to mammalian oviduct.ResultsWe showed that the natural estrogen (estradiol, E2) and androgen (testosterone, T) as well as the synthetic estrogen (ethinyl-estradiol, EE2), all induced a significant enhancement of MC cell numbers. The effect of E2, T and EE2 extended to the MS and MR cell populations, to varying degrees. They also modified the expression profile of RNA MCE markers, and induced a range of “non-typical” cellular phenotypes, with mixed identities and aberrant morphologies, as revealed by imaging analysis through biomarker confocal detection and scanning electron microscopy. Finally, these hormones also affected tadpole pigmentation, revealing an effect on the entire cellular ecosystem of the Xenopus embryonic skin.ConclusionsThis study reveals the impact in vivo, at the molecular, cellular, tissue and organism levels, of sex steroids on non-reproductive mucociliary epithelium biogenesis, and validates the use of Xenopus as a relevant model system in this field.

This chapter presents a detailed methodology for monitoring nuclear shape fluctuations and their correlation with chromatin condensation, developed for our analyses of early prophase, but applicable to other contexts. Nuclear shape dynamics play a key role in mitotic progression, and understanding the mechanical and biophysical properties associated with the fast-paced fluctuations may offer insights into key cellular processes like chromatin condensation and nuclear envelope breakdown. By employing live-cell imaging and computational analysis such as segmentation and flickering spectroscopy techniques, this approach leverages high-resolution temporal tracking of nuclear shape changes during cell cycle progression to derive insights into the mechanical forces driving chromatin condensation and nuclear envelope instabilities leading to the nuclear envelope breakdown. The experimental protocol provides a step-by-step guide for synchronizing HeLa cells at a specific cell-cycle transition and manipulating chromatin condensation and cytoskeletal structures through pharmacological perturbations. The data analysis section includes methods for extracting relevant biophysical parameters, such as nuclear effective tension and nuclear invaginations, as well as image-processing analysis, to correlate nuclear deformations with chromatin dynamics. We hope this robust and accessible workflow will serve as a powerful tool for exploring the mechanical coupling between chromatin condensation and nuclear structure across differentconditions, which is crucial for our fundamental understanding of the nuclear function, as well as relevant for diseases resulting in nuclear abnormalities and disrupted cellular functions.

Multiplex imaging platforms have enabled the identificationofthe spatial organization of different types of cells in complex tissueor the tumor microenvironment. Exploring the potential variationsin the spatial co-occurrence or colocalization of different cell typesacross distinct tissue or disease classes can provide significantpathological insights, paving the way for intervention strategies.However, the existing methods in this context either rely on stringentstatistical assumptions or suffer from a lack of generalizability.We present a highly powerful method to study differential spatialco-occurrence of cell types across multiple tissue or disease groups,based on the theories of the Poisson point process and functionalanalysis of variance. Notably, the method accommodates multiple imagesper subject and addresses the problem of missing tissue regions, commonlyencountered due to data-collection complexities. We demonstrate thesuperior statistical power and robustness of the method in comparisonwith existing approaches through realistic simulation studies. Furthermore,we apply the method to three real data sets on different diseasescollected using different imaging platforms. In particular, one ofthese data sets reveals novel insights into the spatial characteristicsof various types of colorectal adenoma.

Advances in multiplexed single-cell immunofluorescence (mIF) and multiplex immunohistochemistry (mIHC) imaging technologies have enabled the analysis of cell-to-cell spatial relationships that promise to revolutionize our understanding of tissue-based diseases and autoimmune disorders. Multiplex images are collected as multichannel TIFF files; then denoised, segmented to identify cells and nuclei, normalized across slides with protein markers to correct for batch effects, and phenotyped; and then tissue composition and spatial context at the cellular level are analyzed. This chapter discusses methods and software infrastructure for image processing and statistical analysis of mIF/mIHC data.