Spatial Profile Research Articles

Spatial transcriptomic landscape unveils immunoglobin-associated senescence as a hallmark of aging.

To systematically characterize the loss of tissue integrity and organ dysfunction resulting from aging, we produced an in-depth spatial transcriptomic profile of nine tissues in male mice during aging. We showed that senescence-sensitive spots (SSSs) colocalized with elevated entropy in organizational structure and that the aggregation of immunoglobulin-expressing cells is a characteristic feature of the microenvironment surrounding SSSs. Immunoglobulin G (IgG) accumulated across the aged tissues in both male and female mice, and a similar phenomenon was observed in human tissues, suggesting the potential of the abnormal elevation of immunoglobulins as an evolutionarily conserved feature in aging. Furthermore, we observed that IgG could induce a pro-senescent state in macrophages and microglia, thereby exacerbating tissue aging, and that targeted reduction of IgG mitigated aging across various tissues in male mice. This study provides a high-resolution spatial depiction of aging and indicates the pivotal role of immunoglobulin-associated senescence during the aging process.

SpaInGNN: Enhanced clustering and integration of spatial transcriptomics based on refined graph neural networks

Recent developments in spatial transcriptomics (ST) technology have markedly enhanced the proposed capacity to comprehensively characterize gene expression patterns within tissue microenvironments while crucially preserving spatial context. However, the identification of spatial domains at the single-cell level remains a significant challenge in elucidating biological processes. To address this, SpaInGNN was developed, a sophisticated graph neural network (GNN) framework that accurately delineates spatial domains by integrating spatial location data, histological information, and gene expression profiles into low-dimensional latent embeddings. Additionally, to fully leverage spatial coordinate data, spatial integration using graph neural network (SpaInGNN) refines the graph constructed for spatial locations by incorporating both tissue image distance and Euclidean distance, following a pre-clustering of gene expression profiles. This refined graph is then embedded using a self-supervised GNN, which minimizes self-reconfiguration loss. By applying SpaInGNN to refined graphs across multiple consecutive tissue slices, this study mitigates the impact of batch effects in data analysis. The proposed method demonstrates substantial improvements in the accuracy of spatial domain recognition, providing a more faithful representation of the tissue organization in both mouse olfactory bulb and human lateral prefrontal cortex samples

Induced collagen type-I secretion by hepatocytes of the melanoma liver metastasis is associated with a reduction in tumour-infiltrating lymphocytes.

Overall patients with melanoma liver metastasis (MLiM) have a dismal prognosis and poor responses to the standard of care treatment. Understanding the role of the tumour microenvironment (TME) is critical for discovering better strategies to overcome intrinsic therapy resistance in MLiM. The aim was to understand the crosstalk signalling pathways between hepatocytes and metastatic melanoma cells in the TME of MLiM. Hepatocytes and melanoma tumour cells of MLiM were assessed using transcriptomic NanoString GeoMx digital spatial profiling (NGDSP) assay. Functional assays were performed using normal hepatocytes and MLiM-derived cell lines. Validation was performed using multiplex immunofluorescence. In NGDSP analysis adjacent normal hepatocytes (ANH) had higher CXCR4 and COL1A1/2 levels than distant normal hepatocytes (DNH), while melanoma cells had higher TNF-α levels. In vitro, MLiM cell lines released TNF-α which upregulated CXCR4 and CXCL12 levels in ANH. CXCL12 activated CXCR4, which triggered AKT and NFκB signalling pathways. Consequently, AKT signalling induced the upregulation of collagen type I. MLiM were significantly encircled by a shield of collagen, whereas other liver metastases showed reduced levels of collagen. Of all the liver metastasis analyzed, the presence of collagen in melanoma liver metastasis was associated with a reduction in tumour-infiltrating lymphocytes. MLiM modified ANH to increase collagen production and created a physical barrier. The collagen barrier was associated with a reduction of immune cell infiltration which could potentially deter MLiM immune surveillance and treatment responses. Spatial analyses of melanoma liver metastasis show that adjacent normal hepatocytes have increased collagen-type I levels. Melanoma liver metastases tumour cells secrete enhanced levels of TNF-α to stimulate CXCR4/CXCL12 upregulation in adjacent normal hepatocytes. Activation of CXCR4 promotes AKT and NF-κB signalling pathways to promote collagen-type I secretion in adjacent normal hepatocytes. Elevated collagen levels were associated with reduced tumour-infiltrating lymphocytes.

Spatial profiling of mismatch repair deficient endometrial tumors identifies patterns associated with recurrence and response to immune checkpoint therapy

Spatial profiling of mismatch repair deficient endometrial tumors identifies patterns associated with recurrence and response to immune checkpoint therapy

PRODIGE - envelope to disk with NOEMA. IV. An infalling gas bridge surrounding two Class 0/I systems in L1448N

The formation of stars has been subject to extensive studies in the past decades on scales from molecular clouds to protoplanetary disks. It is still not fully understood how the surrounding material in a protostellar system, which often shows asymmetric structures with complex kinematic properties, feeds the central protostar(s) and their disk(s). We study the spatial morphology and kinematic properties of the molecular gas surrounding the IRS3A and IRS3B protostellar systems in the L1448N region located in the Perseus molecular cloud. We present 1\,mm Northern Extended Millimeter Array (NOEMA) observations of the large program PROtostars DIsks: Global Evolution (PRODIGE). We analyzed the kinematic properties of molecular lines. Because the spectral profiles are complex, the lines were fit with up to three Gaussian velocity components. The clustering algorithm called density-based spatial clustering of applications with noise ( DBSCAN ) was used to disentangle the velocity components in the underlying physical structure. We discover an extended gas bridge (approx 3000\,au) surrounding both the IRS3A and IRS3B systems in six molecular line tracers (C18O, SO, DCN $CO, HC$_ $N, and CH$_ $ toward the IRS3A system. We find that the observed velocity profile is consistent with analytical streamline models of gravitational infall toward IRS3A. The high-velocity C18O ($2-1$) emission toward IRS3A indicates a protostellar mass of approx 1.2\,$M_ odot$. While high angular resolution continuum data often show IRS3A and IRS3B in isolation, molecular gas observations reveal that these systems are still embedded within a large-scale mass reservoir, whose spatial morphology and velocity profiles are complex. The kinematic properties of the extended gas bridge are consistent with gravitational infall toward the protostar IRS3A.

Intracranial voltage profiles from untangled human deep sources reveal multisource composition and source allocation bias.

Intracranial potentials are used as functional biomarkers of neural networks. As potentials spread away from the source populations, they become mixed in the recordings. In humans, interindividual differences in the gyral architecture of the cortex pose an additional challenge, as functional areas vary in location and extent. We used source separation techniques to disentangle mixing potentials obtained by exploratory deep arrays implanted in epileptic patients of either sex to gain access to the number, location, relative contribution and dynamics of co-active sources. The unique spatial profiles of separated generators made it possible to discern dozens of independent cortical areas for each patient, whose stability maintained even during seizure, enabling the follow up of activity for days and across states. Through matching these profiles to MRI, we associated each with limited portions of sulci and gyri, and determined the local or remote origin of the corresponding sources. We also plotted source-specific 3D coverage across arrays. In average, individual recording sites are contributed to by 3-5 local and distant generators from areas up to several centimeters apart. During seizure, 13-85 % of generators were involved, and a few appeared anew. Significant bias in location assignment using raw potentials is revealed, including numerous false positives when determining the site of origin of a seizure. This is not amended by bipolar montage, which introduce additional errors of its own. In this way, source disentangling reveals the multisource nature and far intracranial spread of potentials in humans, while efficiently addressing patient-specific anatomofunctional cortical divergence.Significance Statement Field potentials are used to better localize zones showing normal and pathological activity. However, as potentials spread throughout the brain volume, they mix with others and make their place of origin uncertain, even when recorded intracranially. We used advanced algorithms to disentangle the activity of each these zones by their unique spatial profiles, which allowed us to determine the 3D outline of normal and epileptic areas and follow their activity for days. Dozens of independent sources per patient can be explored and precisely located. The findings show that standard stereoEEG recordings are contributed by 3-5 populations, which after separation will help to plan clinical intervention to break epileptic networks by more accurately marking epileptic foci and avoiding false positives.

Mapping metastatic breast cancer complexity through single-cell and spatial profiling.

Mapping metastatic breast cancer complexity through single-cell and spatial profiling.

Quality control for single-cell analysis of high-plex tissue profiles using CyLinter.

Tumors are complex assemblies of cellular and acellular structures patterned on spatial scales from microns to centimeters. Study of these assemblies has advanced dramatically with the introduction of high-plex spatial profiling. Image-based profiling methods reveal the intensities and spatial distributions of 20-100 proteins at subcellular resolution in 103-107 cells per specimen. Despite extensive work on methods for extracting single-cell data from these images, all tissue images contain artifacts such as folds, debris, antibody aggregates, optical aberrations and image processing errors that arise from imperfections in specimen preparation, data acquisition, image assembly and feature extraction. Here we show that these artifacts dramatically impact single-cell data analysis, obscuring meaningful biological interpretation. We describe an interactive quality control software tool, CyLinter, that identifies and removes data associated with imaging artifacts. CyLinter greatly improves single-cell analysis, especially for archival specimens sectioned many years before data collection, such as those from clinical trials.

Shaping the spatial intensity profiles of ultra-short lasers for enhanced selective thin film processing in the fabrication of silicon membrane filters

Shaping the spatial intensity profiles of ultra-short lasers for enhanced selective thin film processing in the fabrication of silicon membrane filters

Establishment of Theoretically Guided Understanding for Magneto‐Optical Interactions through Ferromagnetic Fluids in Uniform, Static Magnetic Field

Prior magneto‐optical transmission models through ferrofluids are limited by oversimplifications which hinder accuracy and generalizability. To overcome this challenge, this study models the magnetic field effect on the magneto‐optical transmission through hematite ferrofluid using the method of invariant embedding. Optical coherence tomography (OCT) is used to extract the changes in the refractive index, enabling a discovery of a magnetodielectric effect for the hematite ferrofluid. To establish a basis for control and understanding, the influence of the magnetic field on the spatial transmission profile at different wavelengths, optical propagation lengths, and the effect of variation of each size parameter on the optical transmission have been investigated. Further parametric studies allowed finding out an analogy to nonuniform, cascade, volume holographic diffraction gratings, which is crucial for a wide range of optical and bioimaging applications. Compared to the experimental results, the presented model achieves 99% accuracy over the wavelength range (300–1100) nm under uniform static magnetic field (0–6.5) mT. Besides, the conspicuous lead of evidentiary understanding of magneto‐optical interactions with magnetic fluids, the structural milestones of the model can be further utilized to model similar challenging constructs in complex media. The innumerable applications of study extend to involve communication engineering, biomedical, optical, and security applications.

Fast magnetohydrodynamic oscillations of a coronal loop embedded in a potential coronal arcade

Observations indicate variable widths exhibited by fan coronal loops and flare loops that tend to widen towards the apex. Short-period, quasi-periodic pulsations in solar flares are often interpreted in terms of the fast-sausage oscillations of flare loops and the collective vertical vibrations of arcade loops are attributed with the vertical kink mode. Both phenomena are used as a seismological tool to estimate the physical parameters in the corona. We performed an analytical study of fast sausage and kink oscillations in coronal loops, given the effects of loop curvature, expansion, and Alfv \'e n speed variation. We modelled a coronal loop as a dense expanding curved magnetic slab embedded within a potential coronal arcade, using a zero-beta plasma limit. We obtained the dispersion relation that governs fast waves in the model and studied it both numerically and analytically. The effects of loop expansion and variable Alfv \'e n speed reduce the cut-off frequency and increase the cut-off wavenumbers for fast sausage and kink waves. Moreover, the principal vertical kink mode has a cut-off and strongly attenuates in the leaky regime. The frequency increase is found to be minor for the global sausage mode both in the trapped and leaky regimes, with a frequency shift within a few percent. We found that in our model, where the Alfv \'e n speed increases from the footpoints to the loop top, the spatial profile of the longitudinal fundamental is broadened and the antinodes of the first overtone are shifted towards the footpoints. Using the classical expression for the cut-off wavenumber of the global sausage mode in a straight waveguide results in an underestimation of the density contrast constraint in flare loops. Instead, the suggested formula accounting for variations in loop widths provides more accurate results. The frequency of the global sausage mode can be correctly determined with the straight slab model.

Membrane Interactions of GET1 and GET2 Facilitate Fiber Cell Initiation through the Guided Entry of the TA Protein Pathway in Cotton.

The guided entry of TA proteins (GET) pathway, which is responsible for the post-translational targeting and insertion of the tail-anchored (TA) protein into the endoplasmic reticulum (ER), plays an important role in physiological processes such as protein sorting, vesicle trafficking, cell apoptosis, and enzymatic reactions in which the GET1/2 complex is indispensable. However, a comprehensive study of the GET1 and GET2 genes and the GET pathway in cotton has not yet been carried out. Here, 12 GET1 and 21 GET2 genes were identified in nine representative plant species, and the phylogenetic relationships, gene structures, protein motifs, cis-regulatory elements (CREs), and temporal and spatial expression profiles were analyzed thoroughly. Our study indicated that GhGET1s and GhGET2s might be localized on ER membranes. According to expression profiling and CREs analysis, GhGET2-A02 was identified as a promising candidate for fiber cell development, interacting with two GhGET1s in the membrane, with a binding bias toward GhGET1-A06. Silencing of GhGET1-A06 or GhGET2-A02 reduced fiber initiation and elongation. In summary, our research provides important evidence for understanding the gene families and functions of GET1 and GET2 in cotton and provides clues for molecular breeding of high-quality cotton fiber varieties.

Loss of Surfactant Protein A Alters Perinatal Lung Morphology and Susceptibility to Hyperoxia-Induced Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia (BPD) is a condition of poor alveolar formation that causes chronic breathing impairment in infants born prematurely. Preterm lungs lack surfactant and are vulnerable to oxidative injuries driving the development of BPD. Our recent studies reported that surfactant protein A (SP-A) genetic variants influence susceptibility to neonatal lung disease. SP-A modulates activation of alveolar macrophages and parturition onset in late gestation. We asked whether a lack of SP-A alters alveolarization in a mouse model of hyperoxia-induced BPD. SP-A-deficient and control newborn mice were exposed to either clinically relevant 60% O2 hyperoxia or normoxia for 5–7 days. Alveolar formation was then assessed by mean linear intercept (MLI) and radial alveolar count (RAC) measurements in lung tissue sections. We report that the combination of SP-A deficiency and hyperoxia reduces alveolar growth compared to WT mice. The morphometric analysis of normoxic SP-A-deficient lungs showed lower RAC compared to controls, indicating reduced alveolar number. In the presence of hyperoxia, MLI was higher in SP-A-deficient lungs compared to controls. Differences were statistically significant for female pups. Spatial proteomic profiling of lung tissue sections showed that hyperoxia caused a 4-fold increase in the DNA damage marker γH2Ax in macrophages of SP-A-deficient lungs compared to normoxia. Our short report suggests an important role for SP-A in perinatal lung development and the protection of lung macrophages from oxidant injury. These studies warrant future investigation to discern the temporal interaction of SP-A, gender, oxidant injury, and lung macrophages in perinatal alveolar formation and development of BPD.

Double-slot micro-ring resonators with trapezoidal subwavelength grating as ultra-sensitive biochemical sensors

Silicon-based refractive index sensors are of significance in the detection of gases, biological substances and chemical compounds. Among these, optical microcavities can confine the optical field to the micrometre-scale region, and possess the advantages of high Q factor, small size and easy integration. In this paper, a trapezoidal subwavelength grating (SWG) is introduced into a slot micro-ring resonator, and the mode splitting is employed to enrich the supported standing wave modes and optimize the spatial profiles of the resonant modes. The modes’ Q factor is improved and the high sensitivity and low detection limit is achieved. The optimal trapezoidal subwavelength grating double slot micro-ring resonator (T-SWGDSMRR) structure is obtained by designing the structural parameters and analyzing their effects on the sensing performance parameters and spectral characteristics. The T-SWGDSMRR, designed for detecting the glucose solution, demonstrated a low detection limit of 3.3×10−5 RIU and an ultra-high Q factor of up to 100825, accompanied by a refractive index sensitivity of 424 nm/RIU. Finally, a cascaded double micro-ring sensor is proposed using the vernier effect, through cascading the T-SWGDSMRR with a referential ring, the sensitivity is enhanced to 12828 nm/RIU, and the limit of detection is 3.12×10−6 RIU.

Glial 'omics in ischemia: Acute stroke and chronic cerebral small vessel disease.

Vascular injury and pathologies underlie common diseases including ischemic stroke and cerebral small vessel disease (CSVD). Prior work has identified a key role for glial cells, including microglia, in the multifaceted and temporally evolving neuroimmune response to both stroke and CSVD. Transcriptional profiling has led to important advances including identification of distinct gene expression signatures in ischemia-exposed, flow cytometrically sorted microglia and more recently single cell RNA sequencing-identified microglial subpopulations or clusters. There is a reassuring degree of overlap in the results from these two distinct methodologies with both identifying a proliferative and a separate type I interferon responsive microglial element. Similar patterns were later seen using multimodal and spatial transcriptomal profiling in ischemia-exposed microglia and astrocytes. Methodological advances including enrichment of specific neuroanatomic/functional regions (such as the neurovascular unit) prior to single cell RNA sequencing has led to identification of novel cellular subtypes and generation of new credible hypotheses as to cellular function based on the enhanced cell sub-type specific gene expression patterns. A ribosomal tagging strategy focusing on the cellular translatome analyses carried out in the acute phases post stroke has revealed distinct inflammation-regulating roles for microglia and astrocytes in this setting. Early spatial transcriptomics experiments using cerebral ischemia models have identified regionally distinct microglial cell clusters in ischemic core versus penumbra. There is great potential for combination of these methods for multi-omics approaches to further elucidate glial responses in the context of both acute ischemic stroke and chronic CSVD.

Spatial transcriptomics analysis identifies therapeutic targets in diffuse high-grade gliomas.

Diffuse high-grade gliomas are the most common malignant adult neuroepithelial tumors in humans and a leading cause of cancer-related death worldwide. The advancement of high throughput transcriptome sequencing technology enables rapid and comprehensive acquisition of transcriptome data from target cells or tissues. This technology aids researchers in understanding and identifying critical therapeutic targets for the prognosis and treatment of diffuse high-grade glioma. Spatial transcriptomics was conducted on two cases of isocitrate dehydrogenase (IDH) wild-type diffuse high-grade glioma (Glio-IDH-wt) and two cases of IDH-mutant diffuse high-grade glioma (Glio-IDH-mut). Gene set enrichment analysis and clustering analysis were employed to pinpoint differentially expressed genes (DEGs) involved in the progression of diffuse high-grade gliomas. The spatial distribution of DEGs in the spatially defined regions of human glioma tissues was overlaid in the t-distributed stochastic neighbor embedding (t-SNE) plots. We identified a total of 10,693 DEGs, with 5,677 upregulated and 5,016 downregulated, in spatially defined regions of diffuse high-grade gliomas. Specifically, SPP1, IGFBP2, CALD1, and TMSB4X exhibited high expression in carcinoma regions of both Glio-IDH-wt and Glio-IDH-mut, and 3 upregulated DEGs (SMOC1, APOE, and HIPK2) and 4 upregulated DEGs (PPP1CB, UBA52, S100A6, and CTSB) were only identified in tumor regions of Glio-IDH-wt and Glio-IDH-mut, respectively. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) enrichment analyses revealed that upregulated DEGs were closely related to PI3K/Akt signaling pathway, virus infection, and cytokine-cytokine receptor interaction. Importantly, the expression of these DEGs was validated using GEPIA databases. Furthermore, the study identified spatial expression patterns of key regulatory genes, including those involved in protein post-translational modification and RNA binding protein-encoding genes, with spatially defined regions of diffuse high-grade glioma. Spatial transcriptome analysis is one of the breakthroughs in the field of medical biotechnology as this can map the analytes such as RNA information in their physical location in tissue sections. Our findings illuminate previously unexplored spatial expression profiles of key biomarkers in diffuse high-grade glioma, offering novel insight for the development of therapeutic strategies in glioma.

Bacterial functional responses to environmental variability: a case study in three distinct mountain lakes within a single watershed

The utilization of carbon substrates (CSs) is crucial for the functioning of heterotrophic bacterial communities and aquatic ecosystems. This study used the Biolog EcoplateTM technique to explore spatial and temporal community-level physiological profiles (CLPPs) of bacteria in three eutrophic-dystrophic mountain lakes in Bulgaria. Bacterial metabolic activity varied among the lakes, being highest in the lake dominated by phytoplankton and lowest in the lake dominated by macrophyte primary producers. CLPP highlighted specific bacterial metabolic preferences to certain carbon guilds (CGs), showing a higher affinity for carbohydrates and polymers and lower preferences for amino acids and amines. The overall metabolic dissimilarity among the lakes was approximately 32%, primarily driven by the CGs of amino acids and amines. The most preferred CSs included the carbohydrates D-Mannitol and N-Acetyl-D-glucosamine, and the carboxylic acid D-Galacturonic acid. Bacterial functional diversity was high (H′: 3.18 − 3.33) due to the high evenness of CS utilization. Multidimensional analyses revealed significant influences of lake-specific characteristics, sampling month, and the interaction between these factors on CLPP patterns. Nutrients and water temperature were identified as the most influential environmental factors affecting bacterial communities. This study enhances our understanding of bacterial functional responses to changing environments, demonstrating the effectiveness of the Biolog EcoplateTM technique in providing insights into these changes.

Rapid and Robust Workflows Using Different Ionization, Computation, and Visualization Approaches for Spatial Metabolome Profiling of Microbial Natural Products in Pseudoalteromonas

Rapid and Robust Workflows Using Different Ionization, Computation, and Visualization Approaches for Spatial Metabolome Profiling of Microbial Natural Products in <i>Pseudoalteromonas</i>

Amyloid-β (Aβ) immunotherapy induced microhemorrhages are linked to vascular inflammation and cerebrovascular damage in a mouse model of Alzheimer’s disease

BackgroundAnti-amyloid-β (Aβ) immunotherapy trials have revealed amyloid-related imaging abnormalities (ARIA) as the most prevalent and serious adverse events linked to pathological changes in cerebral vasculature. Recent studies underscore the critical involvement of perivascular macrophages and the infiltration of peripheral immune cells in regulating cerebrovascular damage. Specifically, Aβ antibodies engaged at cerebral amyloid angiopathy (CAA) deposits trigger perivascular macrophage activation and the upregulation of genes associated with vascular permeability. Nevertheless, further research is needed to understand the immediate downstream consequences of macrophage activation, potentially exacerbating CAA-related vascular permeability and microhemorrhages linked to Aβ immunotherapy.MethodsThis study investigates immune responses induced by amyloid-targeting antibodies and CAA-induced microhemorrhages using RNA in situ hybridization, histology and digital spatial profiling in an Alzheimer's disease (AD) mouse model of microhemorrhage.ResultsIn the present study, we have demonstrated that bapineuzumab murine surrogate (3D6) induces profound vascular damage, leading to smooth muscle cell loss and blood–brain barrier (BBB) breakdown. In addition, digital spatial profiling (DSP) reveals that distinct immune responses contribute to vascular damage with peripheral immune responses and perivascular macrophage activation linked to smooth muscle cell loss and vascular fibrosis, respectively. Finally, RNA in situ hybridization identifies two distinct subsets of Trem2+ macrophages representing tissue-resident and monocyte-derived macrophages around vascular amyloid deposits. Overall, these findings highlight multifaceted roles of immune activation and vascular damage in driving the development of microhemorrhage.ConclusionsIn summary, our study has established a significant link between CAA-Aβ antibody immune complex formation, immune activation and vascular damage leading to smooth muscle cell loss. However, the full implications of this cascade on the development of microhemorrhages requires further exploration. Additional investigations are warranted to unravel the precise molecular mechanisms leading to microhemorrhage, the interplay of diverse immune populations and the functional roles played by various Trem2+ macrophage populations in response to Aβ immunotherapy.

Rigor and Reproducibility of Digital Spatial Profiling on Clinically Sourced Human Tissues.

Spatial transcriptomic profiling enables precise quantification of gene expression with simultaneous localization of expression profiles onto tissue structures. This new technology promises to improve our understanding of the disease mechanisms. Therefore, there is intense interest in applying these methods in clinical trials or as laboratory developed tests to aid in diagnosis of disease. Before these technologies can be more broadly deployed in clinical research and diagnostics, it is necessary to thoroughly understand their performance in real world conditions. In this study, we vet technical reproducibility, data normalization methods and assay sensitivity focusing predominantly on one widely used spatial transcriptomic methodology, digital spatial profiling. Using clinically sourced human tissue specimens, we find that digital spatial profiling exhibits high rigor and reproducibility. Our approach lays the foundation for incorporation of digital spatial profiling methods into clinical workflows.