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Ewing sarcoma (EWS), a rare pediatric bone tumor, poses unique therapeutic challenges due to its distinct microenvironment and limited molecular understanding. To gain a comprehensive molecular and functional view of the tumors in their microenvironment, we performed a deep mass spectrometry-based proteomic analysis of 168 tumors from 72 patients from primary, relapsed, and metastatic tumors. Analysis of more than 10,000 proteins across patients revealed novel insights into cancer prognosis, chemo-resistance, and progression. We found ferroptosis inhibition as a potential mediator of EWS chemo-resistance and identified novel subclasses of EWS that link the tumor immune landscape with DNA damage repair, ubiquitin-related proteins, and patient prognosis. Validation by multiplexed immunofluorescence imaging confirmed the association between patient prognosis and tumor neutrophils, and association of macrophages and T-cells with better prognosis. These results suggest that immuno-oncological treatments might be efficacious for a subset of patients. Altogether, this comprehensive investigation provides valuable insights into the intricate biology of EWS, paving the way for developing novel therapeutic strategies.

Imaging Methods: fluorescence microscopy light microscopy

Organisms: Homo sapiens

Background: Beta-thalassemia is among the most common monogenic disorders, posing a major global health challenge. Editing of genetic modifiers, such as BCL11A erythroid enhancer and HBG promoters, enhances fetal hemoglobin expression and confers major therapeutic potential. Double-strand-break (DSB)-independent genome editing tools, such as base editors (BE), are potentially safer and better suited for multiplexed application than DSB-dependent CRISPR/Cas technology. However, harmful on- and off-target events remain a concern and must be excluded before clinical application, including chromosomal rearrangements invisible to standard detection technologies. Results: Using primary patient-derived CD34+ cells from three donors, we investigate simplex and duplex BE-based disruption of the BCL11A erythroid enhancer and the BCL11A binding site (-115 bp) on the HBG promoter for DNA-level and functional studies at the RNA, protein, and morphological level. Analyses include direct comparison to DSB-based editing, the current clinically applied standard, and CAST-seq to assess recombination events, allowing wider inferences on relative safety. RNA-seq analyses for clones of primary CD34+ cells across all treatments confirm peak HBG induction for duplex BE and comparable effects on apoptotic and immune response signatures. Overall, duplex BE produces robust γ-globin and fetal hemoglobin induction, improves functional correction over simplex editing and results in low incidence of genomic alterations in both target loci. Conclusions: Duplex BE targeting both BCL11A erythroid enhancer and HBG promoter enables functional correction and genome integrity. Our study highlights the efficacy, safety, and therapeutic potential of the present duplex BE approach.

Imaging Methods: fluorescence microscopy

Organisms: Homo sapiens

Upregulation of Epidermal Growth Factor Receptor is evident in most cases of cervical cancer and is usually associated with a poor prognosis. At the same time, therapies directed against EGFR are generally not successful in this type of cancer, which suggests that therapeutic inactivation of EGFR can be easily overcome. In order to evaluate mechanisms by which cervical cancers with alterations in EGFR signaling thrive, we have generated several EGFR mutant clones by CRISPR/Cas9 genome editing. This work details the approach used to generate EGFR mutant cells and describes changes in cell characteristics associated with decreased quantity and altered subcellular distribution of EGFR.

Imaging Methods: confocal microscopy

Organisms: Homo sapiens

The advent of multiplexed protein imaging, also known as spatial proteomics, has enabled the simultaneous detection of over 50 protein markers at the single-cell level. These approaches have revolutionized the study of biological processes within the native tissue context across fields from developmental biology to viral pathogenesis and cancer. With the increased adoption of multiplexed protein imaging to formalin-fixed paraffin-embedded (FFPE) tissues, a robust assessment of various tissue preparation steps on antibody performance is essential to support standardized and robust protocols for maximal performance and reduced batch-to-batch variability. Here we performed a large-scale, multi-site, multi-platform assessment of tissue preparation and staining conditions for multiplexed protein imaging of FFPE tissue samples. 24 antigen retrieval and antibody staining conditions were first tested using a commercial-grade 27-plex immune panel on serial tissue sections imaged with the Akoya PhenoCycler-Fusion. In parallel at two different sites, 12 of these conditions were evaluated on the RareCyte Orion and 6 conditions were tested with automated staining on the Leica BOND RX followed by imaging on the Akoya PhenoCycler-Fusion. The 24 conditions were ranked based on their average coefficient of variation (CV) of marker signal distributions paired with assignment of penalty scores for markers that failed for specific conditions. The top-ranked protocol was 20 minutes heat-induced epitope retrieval (HIER) with a Tris/EDTA (pH 9) buffer and overnight antibody staining at 4°C, followed by 40 and 10 minutes Tris/EDTA HIER with 4°C overnight antibody staining respectively. Conversely, the bottom-ranked protocol was 20 minutes HIER with citraconic anhydride (pH 7.4) and a 1 hour room temperature (25°C) antibody staining. Validation across seven additional academic and industry labs using platforms including imaging mass cytometry, MIBI, Orion, and Lunaphore COMET confirmed the robustness of the 20-minute Tris/EDTA HIER plus overnight 4°C staining (where applicable) across tissue types and technologies. While staining intensity did not correlate with spatial sampling or annotation efficiency, it positively correlated with spatial heterogeneity, suggesting that more robust spatial proteomics data improves sensitivity for diverse cell cluster identification. This work provides a resource for optimizing experimental design, reagent selection, and standardized best practices for generating and evaluating highly multiplexed imaging data.

Imaging Methods: fluorescence microscopy

Organisms: Homo sapiens

Activation of PLK1, a master mitotic kinase, requires phosphorylation of its activation segment on Thr210, within a basic consensus sequence for Aurora kinases. Aurora B-dependent phosphorylation of Thr210 has been reported, but other evidence identified a strict requirement for the Aurora A partner Bora for Thr210 phosphorylation. Here, we investigate the elusive mechanistic basis for this requirement. We show that Aurora A:Bora phosphorylates Thr210 of PLK1 in vitro. On the contrary, T210 was not phosphorylated by isolated Aurora A, additional Aurora A:activator complexes, or Aurora B:INCENP, even when used at high kinase/substrate ratios. A transient interaction of Bora and PLK1, identified by structural modelling and probed mutationally, is uniquely required for Thr210 phosphorylation. Dependency on Bora for Thr210 phosphorylation is eliminated after mutating Lys208, in the Aurora consensus, into arginine. This conservative mutation turns PLK1 into a substrate of nearly all tested active Aurora kinases, including Aurora B. Collectively, these results shine a new light on the specificity of the PLK1 activation mechanism.

Imaging Methods: bright-field microscopy fluorescence microscopy

Organisms: Homo sapiens

Neurons use cell-adhesion molecules (CAMs) to interact with other neurons and the extracellular environment: the combination of CAMs specifies migration patterns, neuronal morphologies, and synaptic connections across diverse neuron types. Yet little is known regarding the intracellular signaling cascade mediating the CAM recognitions at the cell surface across different neuron types. Using mouse genetics and viral labeling, we investigated the neural developmental role of Afadin​, a cytosolic adapter protein that connects multiple CAM families to intracellular F-actin. We introduced the conditional Afadin mouse mutant to an embryonic retinal Cre, Six3Cre. We reported that the mouse mutants lead to the scrambled retinal neuron distribution, including Bipolar Cells (BCs), Amacrine Cells (ACs), and retinal ganglion cells (RGCs), across three cellular layers of the retina. This scrambled pattern was first reported here at neuron-type resolution. Importantly, the mutants do not display deficits for BCs, ACs, or RGCs in terms of neural fate specifications or survival. Additionally, the displayed RGC types still maintain synaptic partners with putative AC types, indicating that other molecular determinants instruct synaptic choices independent of Afadin. Lastly, there is a significant decline in visual function and mis-targeting of RGC axons to incorrect zones of the superior colliculus, one of the major retinorecipient areas. Collectively, our study uncovers a unique cellular role of Afadin in sorting retinal neuron types into proper cellular layers as the structural basis for orderly visual processing.

Imaging Methods: confocal microscopy

Organisms: Mus musculus

Activation of innate immunity at the single-cell level is a heterogenous process, yet the origins of this variability – fundamentally linked to the control of immune responses – remain poorly understood. Here, we combine classical fluctuation tests with genomic and high-content microscopy approaches to investigate the heritability of single-cell gene-expression patterns in the evolutionarily conserved toll-like receptor (TLR) system. Using population-level and single-cell RNA-seq, we show that a subset of TLR4-dependent genes, approximately 7% overall and 15% among upregulated genes, including key cytokines and immune effectors, retained transcriptional memory across more than 25 cell divisions in clonal populations of immortalised murine macrophages. High-content microscopy of selected immune-relevant proteins from these RNA-seq analyses, including the TNF-alfa and IL1-beta cytokines, across thousands of clonal populations for up to ~10 cell divisions, revealed that transcriptional heritability remains strong over multiple generations but is ultimately transient and shaped by environmental and population context. Among these, CD36, a scavenger receptor involved in bacterial recognition, showed strong heritability, and we demonstrated that CD36-positive clones were more susceptible to Listeria monocytogenes infection, directly linking transcriptional heritability to infection outcome. Together, these findings demonstrate widespread, long-term TLR-mediated transcriptional heritability and provide a conceptual framework for understanding how variability in heritable transcriptional traits shapes immune function.

Imaging Methods: fluorescence microscopy

Organisms: Mus musculus

Optical multifrequency time-harmonic elastography (OMTHE) was used for rapid mechanical characterization of extra-cellular matrix-derived collagen networks at micrometer resolution. OMTHE was optimized for point-wise shear wave excitation in small sample volumes and compared to tabletop magnetic resonance elastography (ttMRE) and optical intensity changes. Dynamic stiffening due to the fluid-gel transition during collagen polymerization and chemical crosslinking using glutaraldehyde was tracked by shear waves speed (SWS) at vibration frequencies between 3 and 10 kHz and frame rates up to 4 kHz. During collagen polymerization, after an initial lag phase, SWS increased on average 6 ± 3 min earlier than optical density, suggesting that a load-bearing percolating fiber network was established before fibril thickening enhanced light scattering. In contrast, chemical crosslinking showed a lag-free, diffusion-driven SWS increase from 1.7 ± 0.4 m/s to 2.5 ± 0.5 m/s, matching the relative SWS change from ground-truth ttMRE. In conclusion, OMTHE provides a unique research tool that quantifies biomechanical property changes in small biological samples with spatiotemporal resolutions of micrometers and seconds.

Imaging Methods: bright-field microscopy

We present cloneXplorer, a fully automated live cell analysis and clone picking platform that uses time lapse monitoring of cell proliferation, cytokine secretion, and surface marker expression in ~100,000 single cell co-cultures to identify and isolate desired cells for clonal expansion or sequence analysis. We employ this platform in a cell line development workflow, in which single cells are selected from a heterogeneous source population based on cell proliferation rate and a fluorescent reporter, and are successfully expanded into monoclonal cell lines in >95% of attempts. Next, we devise a TCR epitope matching workflow by engineering Jurkat cells to express NFAT-GFP, CD8, and a TCR that recognizes a specific antigen. When Jurkat cells are co-cultured with a K562 library of 100 antigen presenting cells (APC), we show that the molecules recovered from microwells with positive GFP expression have the correct antigen in ~80% of attempts, determined by sequencing. To monitor immune activation in mouse and human primary samples, we implement a fluorescent sandwich assay to detect IFN-γ secretion in individual co-cultures. Finally, we combine these capabilities in a proof-of-concept demonstration, which uses IFN-γ secretion and the presence of CD8 surface markers as hierarchical gates to isolate and expand single T cells, and we verify their antigen-specificity by tetramer staining. Together, these results showcase potential applications of the cloneXplorer platform in cell line development and immune discovery applications.

Imaging Methods: fluorescence microscopy

Organisms: Homo sapiens Mus musculus

This study integrates temporal image analysis, automated watering and weighing, and Bayesian growth modeling to dissect how 47 diverse maize inbred lines respond to drought, heat, and their combination. The results demonstrate genotype-specific effects, and that the combined stress is more closely related to heat than drought stress responses. A follow-up experiment also demonstrates that early stress impacts days to anthesis and plant area long after the stress has concluded. These findings underscore the need to evaluate maize diversity under multifactorial stress regimes to accurately identify resilient genotypes for environment-adaptive breeding.

Organisms: Zea mays

The main source of circulating erythropoietin (Epo) in the adult are kidney Norn cells, a recently identified interstitial cell type capable of becoming renal Epo-producing (REP) cells following a local decrease in tissue oxygenation. REP cells are restricted to small clusters in the cortico-medullary border region, suggesting that their microenvironment is relevant for cell differentiation and/or proper regulation of Epo production. Possibly for the same reason, REP cells cease to produce Epo in injured kidneys, which is rapidly reverted by stabilizers of the hypoxia-inducible factor (HIF). To shed new light on the mechanisms governing Epo production, we combined spatial transcriptomics, mRNA-FISH and sequential immunofluorescence, enabling the characterization of the direct neighbourhood of active REP cells. While in the hypoxic mouse kidney REP cells were closest to proximal tubule (PT) segments (S) S1 to S2/3 and endothelial cells, Epo was reinduced by HIF-stabilizers in injured kidneys in the vicinity of damaged PT cells that expressed high levels of injury markers. In contrast, the Norn and endothelial cell profiles remained normal. The REP cell microenvironment switched from pathways involved in energy metabolism under hypoxic conditions to inflammatory and fibrotic pathways under injury conditions. In summary, these data demonstrate that in the diseased kidney HIF-stabilizers reinduce Epo expression in REP cells with a metabolically inactive PT neighbourhood, consistent with a causal role of tubular cells during the loss of Epo expression.

Imaging Methods: fluorescence microscopy

Organisms: Mus musculus

PLA (Proximity Ligation Assay) and Immunoflourescence(IF) Images

Imaging Methods: fluorescence microscopy

Organisms: Homo sapiens