Biology subjects

Danuser, G.

Publications and source records attributed to Danuser, G..

At least 19 recordsLinked to original sources

bioRxiv2026

A genetically encoded RhoG FRET biosensor reveals spatially compartmentalized RhoG-Rac1 signaling during cell protrusion

RhoG is a member of the Rho-family of small GTPases, and is closely related to the canonical Rac1 GTPase, implicated in membrane trafficking, dorsal ruffling, macropinocytosis, and cell protrusion, but its activity has been difficult to visualize directly in living cells with high spatial and temporal resolution. Here, we developed and validated a genetically encoded, single-chain Forster resonance energy transfer (FRET) biosensor for RhoG based on a C-terminal full-length RhoG and an intramolecular RhoG-binding domain derived from ELMO1. The biosensor showed a robust dynamic range when comparing constitutively active and inactive RhoG mutants, responded appropriately to regulation by RhoGDI, GAPs, and GEFs, and detected growth factor-stimulated RhoG activation in live cells. Imaging in mouse embryonic fibroblasts revealed dynamic RhoG activation at leading-edge protrusions, dorsal ruffles, and forming pinocytic and macropinocytic structures. To define the signaling relationship between RhoG and its closely related family member Rac1, we combined the RhoG biosensor with a near-infrared Rac1 FRET biosensor for simultaneous live-cell imaging. Morphodynamic mapping showed that both RhoG and Rac1 activities were positively coupled to edge protrusion, with strongest correlations near the leading-edge, but their direct coupling varied with distance from the edge, indicating partial spatial decoupling within protrusive regions. Inhibition of Src-family kinases altered RhoG dynamics, strongly suppressed Rac1 coupling to protrusion, and inverted the normal positive correlation between RhoG and Rac1 activities. Signaling microdomain analysis further showed that Src inhibition selectively prolonged Rac1 microdomain lifetimes without significantly affecting RhoG domains. Together, these results establish a new biosensor for direct visualization of RhoG activity and reveal that RhoG and Rac1 are coordinated but spatially and temporally distinct components of protrusion-associated signaling networks, with Src-family kinases playing a central role in maintaining their normal coupling.

cell biology↗

bioRxiv2026

ARHGAP20 organizes spatial Rap1-RhoA signaling coordination controlling adhesion dynamics during migration

Cell migration requires the precise coordination of signaling pathways that regulate cytoskeletal dynamics and adhesion turnover. Rho GTPase-activating proteins (RhoGAPs) play critical roles in shaping these processes by controlling the spatial and temporal activity of small GTPases. ARHGAP20 is a RhoA-specific GAP, a downstream target of the Ras-related GTPase Rap1, and has been implicated in cancer cell motility, yet its functional role in coordinating migration-associated signaling remains poorly understood. Here, we investigated the role of ARHGAP20 in cell migration and its impact on the coordination between adhesion- and contractility-associated signaling pathways regulated by Rap1A and RhoA respectively. Using loss-of-function approaches in MTLn3 cells, we show that depletion of ARHGAP20 impairs both directed and random migration, leading to reduced cell velocity and displacement, and increased cell adhesion. To explore the underlying signaling mechanisms, we developed a genetically encoded FRET biosensor to monitor Rap1A activity and combined it with a near-infrared RhoA biosensor to simultaneously analyze their spatiotemporal dynamics in living cells. We found that Rap1A and RhoA activities are negatively coordinated during leading-edge dynamics and that ARHGAP20 depletion enhances this local anticorrelation. To further define where ARHGAP20 regulates Rap1A-RhoA signaling, we applied a microdomain-based analytical framework to quantify local signaling clusters. This analysis revealed that ARHGAP20 selectively modulates Rap1A-RhoA coordination outside focal adhesion regions, while leaving signaling correlations and overlap within focal adhesions unchanged. Subcellular localization analyses further reveal that ARHGAP20 is largely excluded from focal adhesions but associates with microtubules, the endoplasmic reticulum, the Golgi apparatus, and multiple Rab-positive vesicular compartments, supporting a trafficking-dependent mechanism for its spatial targeting. Together, our results identify ARHGAP20 as a regulator of cell migration that modulates the coordination between Rap1A and RhoA signaling through intracellular vesicular trafficking, highlighting the GAPs role in organizing the spatial coupling between signaling pathways in adhesion-associated regions required for efficient cell migration.

cell biology↗

bioRxiv2026

Volumetric Cyclic Immunofluorescence for 3D Spatial Profiling of Immune Structures in Human FFPE Tissue

Mapping how parenchymal, immune, and stromal cells interact with extended structures such as blood vessels and nerves is challenging using conventional thin-section tissue imaging. Volumetric methods such as light-sheet fluorescence microscopy (LSFM) are ideal in these cases and LSFM is widely used in animal models such as fish and mice. This is enabled by well-established clearing methods, genetically encoded reporters, and vascular dyes. However, LSFM is rarely utilized for human tissue, which is primarily available as formaldehyde-fixed paraffin embedded (FFPE) specimens. Here, we present an approach to volumetric cyclic immunofluorescence (v-CyCIF) that enables 40-plex or greater imaging of immune and other cell types in human specimens up to 1 mm thick. We demonstrate the use of v-CyCIF to study neuroimmune interactions and lymphoid structures in normal and cancer tissues. Following LSFM imaging, specimens can be re-embedded and sectioned to ~50 {micro}m thick for high-resolution confocal imaging of punctate structures and cell-cell junctions. v-CyCIF therefore provides a flexible framework for multi-scale 3D profiling of clinical specimens across imaging formats and resolutions.

cancer biology↗

bioRxiv2026

RhoG, Rac1 and Cdc42 cooperation in cell protrusion revealed bymultiplexed optogenetics and biosensor imaging

The small GTPase Rac1 controls cell protrusion for a wide variety of critical cell functions. Its regulation by upstream guanine exchange factors (GEFs) has been the focus of multiple studies, but regulation by the GTPase RhoG remains poorly understood. RhoG is known to activate the ELMO/DOCK180 GEF complex, which in turn interacts with Rac1. It is unclear which aspects of protrusion are controlled by RhoG, and which of RhoGs effects on protrusion are mediated by Rac1. To address these questions, we developed biosensors and optogenetic tools to activate one GTPase while observing another, and to simultaneously visualize the activity of two GTPases. New tools included a photoactivable RhoG, a RhoG biosensor, and red shifted biosensors of RhoG and Rac1. RhoG and Rac1 activation events in protrusions were spatio-temporally correlated with one another and with protrusion velocity. Causal inference indicated that RhoG indeed unidirectionally activated Rac1. Photoactivation of RhoG and Rac1 indicated that specific aspects of protrusion behavior were controlled by RhoG, and only some via Rac1. Further dissection of RhoG to Rac1 signaling through simultaneous GTPase activation and biosensor visualization showed that PA-RhoG activates Rac1 predominantly through DOCK180 and that PA-RhoG can activate Cdc42 independently of Rac1.

cell biology↗

bioRxiv2026

Allosteric Biosensors Unravel GTPase-Effector Feedback

Fluorescent biosensors that report protein conformation in vivo have been invaluable for understanding how the spatio-temporal dynamics of signaling controls cells. However, for GTPases these biosensors report the activated conformation using reagents that block the binding of downstream proteins, generating dominant negative effects and altering normal cell physiology. We present here a generalizable design to make GTPase biosensors (AlloRac1 and AlloCdc42), in which a circularly permuted fluorescent protein is inserted into a conserved loop allosterically connected to the effector binding site, generating activity-dependent fluorescence without blocking ligand interactions. The Rac1 biosensor showed that effector interactions led to increased Rac1 activation, indicating an auto-regulatory positive feedback made visible by the new biosensor design. This feedback regulated the kinetics and localization of Rac1 activity, including Rac1 activity gradients that controlled motility. Feedback was generated through Rac1 interaction with the effector Pak1, which led to further activation of Rac1 by the guanine exchange factor {beta}-Pix. The new biosensor approach enables quantitative imaging of previously obscure spatio-temporal dynamics in GTPase regulation.

cell biology↗

bioRxiv2026

Modulation of Oncogenic KRAS Signaling by Branched Actin-driven Cell Membrane Protrusions

For over three decades, we have known that oncogenic RAS alters the actin cytoskeleton organization and cell surface morphology1,2. RAS activates the GTPase RAC1, which triggers the growth of branched actin networks to promote cell membrane protrusions3,4. In melanoma, the hyperactive RAC1 mutant, Rac1P29S, was recently shown to drive extended lamellipodia, which then empower cell proliferation through sequestration and localized inhibition of the merlin tumor suppressor5. This discovery illustrates cell morphological programs not only as outputs but also as regulators of human oncogenic signals. Hence, we wondered whether the pronounced branched actin-driven membrane protrusions (BAMPs) downstream of oncogenic RAS are not mere outputs of RAS signaling but rather an active component in mediating the oncogenic penetrance of RAS mutants. We used volumetric light sheet microscopy and biochemical approaches to investigate the role of BAMPs in regulating the molecular signaling of oncogenic KRAS in pancreatic and lung cancer models. We found that elevated BAMP formation regulated the interaction of oncogenic KRAS with downstream effectors, specifically with the RAC1 GEF TIAM1. This implies that BAMPs amplify their own upstream regulators in a positive feedback. This meritorious cycle upregulates cyclin D1 expression by inactivating the merlin tumor suppressor, independently of the mitogen activated protein kinase pathway (MAPK). In the absence of BAMPs, cells carrying oncogenic KRAS mutations are unable to attain their full penetrance in proliferation. Overall, this work unveils the long-overlooked role of branched actin-driven cell morphology in the functionalization of KRAS mutants as potent oncogenes.

cancer biology↗

bioRxiv2026

Reconstructing Actin Dynamics of the Leading Edge from Observational Data

Mesenchymal cell migration starts with the protrusion of the cells leading edge, enabled by the branching growth of the actin network. Two crucial molecular players in protrusion are actin filaments and the Arp2/3 protein complex, the branching agent. Traditionally, protrusion models are intuited from several perturbative experiments. Recent multiplex microscopy imaging data promise to drastically change this paradigm by providing measurements of naturally fluctuating densities of key proteins at the leading edge of unperturbed cells. We report the first attempt to reconstruct the mechanistic protrusion model from such data. We analyze fluctuations of F-actin and Arp2/3 densities and cell edge velocity using phase space and regression analysis to reconstruct linearized stochastic actin-Arp2/3-velocity coupled dynamics. We then build a nonlinear partial differential equation model of these dynamics based on previous knowledge of this system and parsimony. The resulting model recovers the rates of reactions and mechanical and transport processes in the lamellipodium from a single non-perturbative experiment. The model posits that the only essential nonlinearities in the lamellipodial dynamics stem from the retrograde flow of F-actin. The model suggests that the protrusion is dominated by a damped oscillatory cycle resulting from a combination of positive feedback from Arp2/3 to protrusion velocity and negative feedback from F-actin to the velocity. Significance statementTraditionally, models of intracellular mechanochemistry are intuited from perturbative experiments. Multiplex microscopy imaging changes this by providing measurements of naturally fluctuating densities of key proteins in unperturbed cells. We use such data to reconstruct a mechanistic model of cell leading edge. We build equations for actin system and edge velocity using data, previous knowledge and parsimony. The resulting model recovers the rates of reactions and mechanical and transport processes at the leading edge. The model suggests that the protrusion is dominated by a damped oscillatory cycle resulting from a combination of positive feedback from Arp2/3 to protrusion velocity and negative feedback from F-actin to the velocity.

biophysics↗

bioRxiv2025

Resolving phenotyping discordance with SPACEMAP, an integrated machine learning framework

Multiplex imaging technologies have revolutionized our ability to study cellular behavior within the tissue microenvironment. Translating this complex data into meaningful biological insights requires a unified analytical framework. To address this, we developed SPACEMAP (Spatial Phenotyping And Classification with Enhanced Multiplex Analysis Pipeline), a comprehensive Python and Qupath-based platform for multiplex imaging analysis. SPACEMAP integrates image registration, segmentation, artifact removal, tissue and zone classification, spatial feature extraction, and a consolidated phenotyping approach into a single system. A core feature of SPACEMAP is its high-fidelity phenotyping. To evaluate classification performance, we benchmarked our method RESOLVE, against three established approaches, Leiden clustering, Self-Organizing Maps, and SCIMAP revealing substantial disagreement among them. SPACEMAP overcomes this through two complementary workflows: a machine learning model trained on expert-labeled cells, and a consensus classifier that integrates high-confidence cells across methods. Here, we validated SPACEMAP on in-house colorectal cancer samples and a public dataset, demonstrating its robustness.

bioinformatics↗

bioRxiv2025

Phosphoinositide lipids have bidirectional and spatially distinct roles in filopodial dynamics

Neural circuits are assembled by filopodia, dynamic actin-rich protrusions that connect environment sensing with structural changes. Using rapid timelapse imaging and acute pharmacological perturbation in Xenopus laevis retinal ganglion cells, we reveal that phosphoinositide lipid conversions impose spatially distinct control over filopodial dynamics. The class I phosphoinositide 3-kinase (PI3K) inhibitor alpelisib and Oculocerebrorenal syndrome of Lowe protein/Inositol polyphosphate 5-phosphatase inhibitor YU142670 markedly reduced filopodial tip motility. Class II PI3K inhibitor PITCOIN4 and YU142670 curtailed base dynamics, indicating location-specific lipid requirements. Despite no detectable change in PI(4,5)P2, both alpelisib and YU142670 depleted PI(3,4)P2 at filopodial tips. Their co-application was non-additive, with partial alleviation of YU142670-induced stalling by alpelisib, consistent with networked control of lipid conversion and actin remodelling. Live imaging with a TAPP1-3xcPH probe combined with quantitative cross-correlation and Granger causality analysis showed that PI(3,4)P2 at filopodial tips both generates tip extension and responds to forward filopodial movement. Disrupting actin polymerisation rapidly erased tip PI(3,4)P2 prior to filopodial stalling. These findings define a vulnerable node with implications for neurodevelopmental miswiring.

cell biology↗

bioRxiv2025

Robust statistical assessment of Oncogenotype to Organotropism translation in xenografted zebrafish

Organotropism results from the functional versatility of metastatic cancer cells to survive and proliferate in diverse microenvironments. This adaptivity can originate in clonal variation of the spreading tumor and is often empowered by epigenetic and molecular reprogramming of cell regulatory circuits. Related to organotropic colonization of metastatic sites are environmentally-sensitive, differential responses of cancer cells to therapeutic attack. Accordingly, understanding the organotropic profile of a cancer and probing the underlying driver mechanisms are of high clinical importance. However, determining systematically the organotropism of one cancer versus the organotropism of another cancer, potentially with the granularity of comparing the same cancer type between patients or tracking the evolution of a cancer in a single patient for the purpose of personalized treatment, has remained very challenging. It requires a host organism that allows observation of the spreading pattern over relatively short experimental times. Moreover, organotropic patterns often tend to be statistically weak and superimposed by experimental variation. Thus, an assay for organotropism must give access to statistical powers that can separate meaningful heterogeneity, i.e., heterogeneity that determines organotropism, from meaningless heterogeneity, i.e., heterogeneity that causes experimental noise. Here we describe an experimental workflow that leverages the physiological properties of zebrafish larvae for an imaging-based assessment of organotropic patterns over a time-frame of 3 days. The workflow incorporates computer vision pipelines to automatically integrate the stochastic spreading behavior of a particular cancer xenograft in tens to hundreds of larvae allowing subtle trends in the colonization of particular organs to emerge above random cell depositions throughout the host organism. We validate our approach with positive control experiments comparing the spreading patterns of a metastatic sarcoma against non-transformed fibroblasts and the spreading patterns of two melanoma cell lines with previously established differences in metastatic propensity. We then show that integration of the spreading pattern of xenografts in 40 - 50 larvae is necessary and sufficient to generate a Fish Metastatic Atlas page that is representative of the organotropism of a particular oncogenotype and experimental condition. Finally, we apply the power of this assay to determine the function of the EWSR1::FLI1 fusion oncogene and its transcriptional target SOX6 as plasticity factors that enhance the adaptive capacity of metastatic Ewing sarcoma.

cancer biology↗

bioRxiv2025

Autonomous learning of pathologists' cancer grading rules

Deep learning reveals that tissue morphology contains rich pathophysiological information beyond human understanding. However, approaches to convert these spatially distributed signals into subcellular insights informing disease mechanisms are lacking. We introduce Delta-Marches, an interpretability-first approach that nominates distinguishing morphological features rather than explaining existing models' decisions. Delta-Marches simulates idealized morphological changes between classes by coupling latent-space traversals with generative AI. Comparing each image to its class-shifted counterpart allows feature extractors to infer the most affected aspects, reducing sample-to-sample variability and resolving transformations at subcellular resolution. Prototyped in renal carcinoma grading, Delta-Marches generates realistic grade transitions and pinpoints tumor-cell nuclear phenotypes as key determinants. It also reveals reduced vasculature with increasing grade, a known pattern absent from standard rubrics. Applied to dysplastic progression in colorectal tissue, it recovers glandular remodeling and goblet-cell loss, generalizing across tissue types and scales. These results show Delta-Marches parses complex image phenotypes and catalyzes hypothesis generation.

bioinformatics↗

bioRxiv2024

Caveolin-1 regulates context-dependent signaling and survival in Ewing Sarcoma.

Plasticity is a hallmark function of cancer cells, yet the mechanisms that enable dynamic switching between survival states remain incompletely understood. Here, we identify Caveolin-1, a membrane-domain scaffolding protein, as a context-dependent regulator of survival signaling in Ewing sarcoma (EwS). Single-cell analyses reveal a distinct subpopulation of EwS cells marked by high CD99 and elevated Caveolin-1 expression. These CD99High cells exhibit unique morphology, transcriptional programs, and markedly enhanced survival both under chemotherapeutic challenge and in vivo. Importantly, CD99High and CD99Low states are reversible, providing EwS cells with a flexible route to survival-oriented plasticity. Mechanistically, we show that Caveolin-1 in CD99High cells orchestrates PI3K/AKT survival signaling by modulating the spatial organization of PI3K activity on the plasma membrane. We propose that the CD99High state establishes a Caveolin-1-driven signaling architecture that supports survival through mechanisms distinct from those used by CD99Low cells. These findings uncover a dynamic state transition in EwS cells and position Caveolin-1 as a key driver of context-specific survival signaling.

cancer biology↗

bioRxiv2024

Combinatorial responsiveness of single chemosensory neurons to external stimulation of mouse explants revealed by DynamicNeuronTracker

Calcium fluorescence imaging enables us to investigate how individual neurons of live animals encode sensory input or drive specific behaviors. Extracting and interpreting large-scale neuronal activity from imaging data are crucial steps in harnessing this information. A significant challenge arises from uncorrectable tissue deformation, which disrupts the effectiveness of existing neuron segmentation methods. Here, we propose an open-source software, DynamicNeuronTracker (DyNT), which generates dynamic neuron masks for deforming and/or incompletely registered 3D calcium imaging data using patch-matching iterations. We demonstrate that DyNT accurately tracks densely populated neurons, whereas a widely used static segmentation method often produces erroneous masks. DyNT also includes automated statistical analyses for interpreting neuronal responses to multiple sequential stimuli. We applied DyNT to analyze the responses of pheromone-sensing neurons in mice to controlled stimulation. We found that four bile acids and four sulfated steroids activated 15 subpopulations of sensory neurons with distinct combinatorial response profiles, revealing a strong bias toward detecting sulfated estrogen and pregnanolone. TeaserDynamicNeuronTracker extracts neuronal activity from deforming 3D calcium imaging datasets using patch-matching iterations.

neuroscience↗

bioRxiv2024

Anchorage-independent cell proliferation promoted by fascin's F-actin bundling

The actin filament (F-actin) bundling protein fascin-1 is highly enriched in many metastatic cancers. Fascins contribution to metastasis have been ascribed to its enhancement of cell migration and invasion. However, mouse genetic studies clearly point to functions also in tumorigenesis, yet without mechanistic underpinnings. Here, we show that fascin expression promotes the formation of a non-canonical signaling complex that enables anchorage-independent proliferation. This complex shares similarities to focal adhesions and we refer to them as pseudo-adhesion signaling scaffolds (PASS). PASS are enriched with tyrosine phosphorylated proteins and require fascins F-actin-bundling activity for its assembly. PASS serve as hubs for the Rac1/PAK/JNK proliferation signaling axis, driven by PASS-associated Rac-specific GEFs. Experimental disruption of either fascin or RacGEF function abrogates sustained proliferation of aggressive cancers in vitro and in vivo. These results add a new molecular element to the growing arsenal of metabolic and oncogenic signaling programs regulated by the cytoskeleton architecture.

cell biology↗

bioRxiv2024

Imaging of cellular dynamics in vitro and in situ: from a whole organism to sub-cellular imaging with self-driving, multi-scale microscopy

Many biological processes span multiple time and length scales, including developmental processes and cancer metastasis. While light-sheet fluorescence microscopy (LSFM) has become a fast and efficient method for imaging of organisms, cells and sub-cellular dynamics, simultaneous observations across these scales have remained challenging. Moreover, continuous high-resolution imaging inside living organisms has mostly been limited to few hours as regions of interest quickly move out of view due to sample movement and growth. Here, we present a self-driving, multi-resolution light-sheet microscope platform controlled by a custom Python-based software, to simultaneous observe and quantify sub-cellular dynamics and entire organisms in vitro and in vivo over hours of imaging. We apply the platform to the study of developmental processes, cancer invasion and metastasis, and we provide quantitative multi-scale analysis of immune-cancer cell interactions in zebrafish xenografts.

bioengineering↗

bioRxiv2024

Spatial proteomics of ER tubules reveals CLMN, an ER-actin tether at focal adhesions that promotes cell migration

The endoplasmic reticulum (ER) is structurally and functionally diverse, yet how its functions are organized within morphological subdomains is incompletely understood. Utilizing TurboID-based proximity labeling and CRISPR knock-in technologies, here we map the proteomic landscape of the human ER and nuclear envelope. Spatial proteomics reveals enrichments of proteins into ER tubules, sheets, and nuclear envelope. We uncover an ER-enriched actin-binding protein, Calmin (CLMN), and define it as an ER-actin tether that localizes to focal adhesions adjacent to ER tubules. CLMN depletion perturbs focal adhesion disassembly, actin dynamics, and cell movement. Mechanistically, CLMN-depleted cells also exhibit defects in calcium signaling near ER-actin interfaces, suggesting CLMN promotes calcium signaling near adhesions to facilitate their disassembly. Collectively, we map the sub-organelle proteome landscape of the ER, identify CLMN as an ER-actin tether, and describe a non-canonical mechanism by which ER tubules engage actin to regulate cell migration.

cell biology↗

bioRxiv2024

RhoA activation promotes glucose uptake to elevate proliferation in MAPK inhibitor resistant melanoma cells.

Cutaneous melanomas harboring a B-RafV600E mutation are treated with immune check point inhibitors or kinase inhibitor combination therapies relying on MAPK inhibitors (MAPKi) Dabrafenib and Trametinib (Curti and Faries, 2021). However, cells become resistant to treatments over the timespan of a few months. Resistance to MAPKi has been associated with adoption of an aggressive amoeboid phenotype characterized by elevated RhoA signaling, enhanced contractility and thick cortical filamentous actin (F-actin) structures (Kim et al., 2016; Misek et al., 2020). Targeting active RhoA through Rho-kinase (ROCK) inhibitors, either alone or in combination with immunotherapies, reverts MAPKi-resistance (Misek et al., 2020; Orgaz et al., 2020). Yet, the mechanisms for this behavior remain largely unknown. Given our recent findings of cytoskeletons role in cancer cell proliferation (Mohan et al., 2019), survival (Weems et al., 2023), and metabolism (Park et al., 2020), we explored possibilities by which RhoA-driven changes in cytoskeleton structure may confer resistance. We confirmed elevated activation of RhoA in a panel of MAPKi-resistant melanoma cell lines, leading to a marked increase in the presence of contractile F-actin bundles. Moreover, these cells had increased glucose uptake and glycolysis, a phenotype disrupted by pharmacological perturbation of ROCK. However, glycolysis was unaffected by disruption of F-actin bundles, indicating that glycolytic stimulation in MAPKi-resistant melanoma is independent of F-actin organization. Instead, our findings highlight a mechanism in which elevated RhoA signaling activates ROCK, leading to the activation of insulin receptor substrate 1 (IRS1) and P85 of the PI3K pathway, which promotes cell surface expression of GLUT1 and elevated glucose uptake. Application of ROCK inhibitor GSK269962A results in reduced glucose uptake and glycolysis, thus impeding cell proliferation. Our study adds a mechanism to the proposed use of ROCK inhibitors for long-term treatments on MAPKi-resistant melanomas.

cancer biology↗

bioRxiv2023

Multiplexed 3D Analysis of Cell Plasticity and Immune Niches in Melanoma

Diseases like cancer involve alterations in in cell proportions, states, and local interactions as well as complex changes in 3D tissue architecture. However, disease diagnosis and most multiplexed spatial profiling studies rely on inspecting thin (4-5 micron) tissue specimens. Here, we use confocal microscopy and cyclic immunofluorescence (3D CyCIF) to show that few if any cells are intact in these thin sections; this reduces the accuracy of cell phenotyping and interaction analysis. In contrast, high-plex 3D CyCIF imaging of intact cells in thick tissue sections enables accurate quantification of marker proteins and detailed analysis of intracellular structures and organelles. Precise imaging of cell membranes also makes it possible to detect juxtacrine signalling among interacting tumour and immune cells and reveals the formation of spatially-restricted cytokine niches. Thus, 3D CyCIF provides insights into cell states and morphologies in preserved human tissues at a level of detail previously limited to cultured cells.

cancer biology↗