Biology subjects

Smith, C.

Publications and source records attributed to Smith, C..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Temperature and Excipient Mediated Modulation of Monoclonal Antibody Interactions Revealed by kD, Rheology, and Raman Spectroscopy

High-concentration monoclonal antibody (mAb) formulations are frequently constrained by elevated viscosity and colloidal instability, stemming from enhanced intermolecular interactions under crowded conditions. This study delineates the thermodynamic and rheological consequences of modulating protein-protein interactions through excipient-mediated and temperature-dependent mechanisms. Using an orthogonal analytical framework comprising diffusion interaction parameter (kD) measurements, high-shear rheometry, and Raman spectroscopic profiling, we interrogated mAb solutions at [~]80 and 160 mg/mL across a physiologically and industrially relevant thermal window (5-45 {degrees}C). In the absence of ionic additives, high kD values ([~]60 mL/g) indicated dominant long-range electrostatic repulsions, resulting in suppressed self-association and lower viscosity. Incorporation of NaCl (0.05% w/v) markedly decreased kD ([~]16-20 mL/g), consistent with Debye screening of surface charges and a shift toward short-range hydrophobic and van der Waals attractions, particularly impactful at elevated protein concentrations and low temperatures. Polysorbate 20 (0.05% v/v) mitigated these interactions via preferential surface adsorption, while sucrose exhibited a dualistic, concentration-dependent influence on viscosity via preferential exclusion and entropic crowding. The combination of NaCl and PS20 yielded the most pronounced rheological suppression, reflecting synergistic attenuation of both long-range repulsion and short-range association. Raman spectral analysis of Amide I/III regions confirmed structural invariance under thermal and shear stress, attributing viscosity modulation to colloidal rather than conformational perturbations. Collectively, these data elucidate the multivariate control of interparticle potentials in mAb solutions and provide a predictive basis for engineering subcutaneous formulations that optimize manufacturability, physical stability, and injectability through strategic manipulation of colloidal interaction landscapes.

biophysics↗

bioRxiv2025

Derepression of transposable elements in mouse prefrontal cortex disrupts social behavior

Here, we present a synthetic biology approach to assess the social behavioral consequences of altered function of the Kruppel-associated box zinc finger protein (KZFP) interacting protein TRIM28 within the prefrontal cortex (PFC) of male and female mice. We reprogrammed natural TRIM28WT by replacing the transcriptionally repressive domain with an enhanced transcriptional activation domain VP64-p65-Rta (TRIM28VPR), or by excising the transcriptional regulatory domain (TRIM28NFD). In vitro validation confirmed that TRIM28WT represses, and TRIM28VPR activates, the expression of a KZFP-regulated luciferase reporter gene. Upon intra-PFC viral-mediated delivery of TRIM28 variants, we observed that inversion of TRIM28 transcriptional control via HSV-TRIM28VPR reduced the salience of novel social interaction for male and female mice while not affecting non-social behaviors. RNA-sequencing revealed HSV-TRIM28VPR promoted transcriptional escape of all classes of TEs, particularly those located within intronic and distal enhancer regions of downregulated immune genes. HSV-TRIM28VPR-driven social deficits were reversible by intra-PFC repletion of interferon cytokines. These novel data point to PFC KZFP-TRIM28 interactions as necessary to stabilize TEs to enable cis-regulation of key immune gene expression and enhance organismal capacity for complex, pro-social behaviors.

neuroscience↗

bioRxiv2025

Patterned illumination enables denser deep-learning based single-molecule localization microscopy

Single-molecule localization microscopy (SMLM) improves resolution beyond diffraction limits by imaging cellular structures at the nanometer scale. Recent advances include using modulation patterns to improve localization precision, and deep learning to accurately process high-density samples with overlapping fluorophore emissions, thus improving imaging speed. A method combining these two advances, SIMCODE, is presented here, allowing high-density modulation-enhanced SMLM. SIMCODE achieved resolution improvements in high-density areas compared to SMLM, deep learning-based SMLM (DECODE), and modulation-enhanced SMLM alone (SIMFLUX). In DNA-PAINT imaging of COS-7 cells, SIMCODE showed improvements in the Fourier Ring Correlation and resolution-scaled Pearson coefficient, with overall improvement increasing as imaging buffer concentration increased five-fold. Modulation-enhanced localization microscopy combined with deep learning thus produced higher quality reconstructions at higher emitter densities (i.e., [~]3x the number of detected spots). This will enable faster imaging, higher labeling densities, and more flexibility in fluorophore choice, which are important for studying dynamic processes and densely labeled structures.

biophysics↗

bioRxiv2025

The inflammatory skin disease map (ISD map): an interactive computational resource focused on psoriasis and atopic dermatitis molecular mechanisms

BackgroundInflammatory skin diseases (ISD), including atopic dermatitis (AD) and psoriasis (PsO), emerge from a complex network of inter- and intracellular biochemical interactions under the influence of genetic and environmental factors. The complexity of ISD mechanisms hinders translation of research findings into effective treatments and may explain the low remission rates despite the availability of modern targeted therapies. ObjectiveTo model AD- and PsO-associated mechanisms as networks of context-specific molecular interactions, the so-called ISD map, and to check the usefulness of this map as a graphically guided review of AD and PsO mechanisms and as a mechanistic hypothesis-generating platform. MethodsThe ISD map was built by assembling mechanistically resolved causal interactions obtained from relevant biomedical literature via manual curation. ResultsWe demonstrate that the ISD map (https://imi-biomap.elixir-luxembourg.org/) serves as an interactive, graphical review of AD and PsO molecular mechanisms and as a mechanistic hypothesis-generating platform. By analysing the map structure itself or the map integrated with genetics and functional genomics data, we could generate the following mechanistic hypotheses: (i) AD poor response to dupilumab is associated with a potential upregulation of IFNG, IL22, TSLP, IL-17A and IL25 signalling pathways in keratinocytes and/or single nucleotide polymorphisms (SNPs) in genes encoding regulators of IFNG expression in Th1 cells and (ii) PsO resistance to cytokine-induced apoptosis is associated with SNPs in IFNG signalling genes regulating SOCS1 in keratinocytes. Finally, the IL4/IL13 pathway in the AD submap of the ISD map was converted into a probabilistic Boolean model to simulate the effects of IFNG in sensory perception of itching after treatment with dupilumab. Our findings suggest that inhibiting both IFNG and IL4R may improve the therapeutic management of itching. ConclusionThe ISD map provides a significant interactive, computationally accessible resource of molecular knowledge on AD and PsO that can be used to graphically review known AD and PsO mechanisms and generate mechanistic hypotheses.

bioinformatics↗

bioRxiv2025

Differential Cellular Mechanisms Underlie Language and Executive Decline in Amyotrophic Lateral Sclerosis

Cognitive manifestations, including impairment in language and executive functions, are seen in amyotrophic lateral sclerosis (ALS), but the mechanisms that underlie these deficits remain unclear. To address this, we mapped prefrontal cortex regions from ALS patients by integrating spatial and single-nucleus transcriptomics in a cognitively stratified patient cohort. We uncover that cognitive impairment in ALS is associated with distinct patterns of neuronal dysfunction and glial-vascular dysregulation that vary by region and cognitive subtype. Executive dysfunction is linked to reduced mitochondrial and synaptic activity in neurons localized to the deeper layers of the dorsolateral prefrontal cortex, whereas language-related deficits track with a more diffuse, pan-regional response involving both glial and vascular abnormalities. Our analyses also identify signatures in the prefrontal cortex that span both motor and cognitive phenotypes, including a multicellular gliosis response. The findings reveal that the clinical heterogeneity of ALS is driven by phenotype-specific molecular and cellular interactions in motor and non-motor regions of the brain.

genomics↗

bioRxiv2025

Intersecting impact of CAG repeat and Huntingtin knockout in stem cell-derived cortical neurons

Huntingtons Disease (HD) is caused by a CAG repeat expansion in the gene encoding Huntingtin (HTT). While normal HTT function appears impacted by the mutation, the specific pathways unique to CAG repeat expansion versus loss of normal function are unclear. To understand the impact of the CAG repeat expansion, we evaluated biological signatures of HTT knockout (HTT KO) versus those that occur from the CAG repeat expansion by applying multi-omics, live cell imaging, survival analysis and a novel feature-based pipeline to study cortical neurons (eCNs) derived from an isogenic human embryonic stem cell series (RUES2). HTT KO and the CAG repeat expansion influence developmental trajectories of eCNs, with opposing effects on the growth. Network analyses of differentially expressed genes and proteins associated with enriched epigenetic motifs identified subnetworks common to CAG repeat expansion and HTT KO that include neuronal differentiation, cell cycle regulation, and mechanisms related to transcriptional repression and may represent gain-of-function mechanisms that cannot be explained by HTT loss of function alone. A combination of dominant and loss-of-function mechanisms are likely involved in the aberrant neurodevelopmental and neurodegenerative features of HD that can help inform therapeutic strategies.

cell biology↗

bioRxiv2025

Uncovering the Signatures of Cellular Senescence in the Human Dorsolateral Prefrontal Cortex

Identifying senescent cells poses challenges due to their rarity, heterogeneity, and lack of a definitive marker. We performed Visium spatial transcriptomics (ST) and single nucleus RNA sequencing (snRNA-seq) on non-pathological human tissue to build a transcriptomic atlas of aging and senescence in the dorsolateral prefrontal cortex (dlPFC). We identified markers characteristic of aging dlPFC cortical layers and cell types. We also observed an increase in astrocyte abundance and decrease in somatostatin expressing inhibitory neurons. Overall, the senescence profile in the dlPFC was highly heterogeneous and heavily influenced by cell type identity and cortical layer. Combined unbiased analysis of ST and snRNA-seq datasets revealed gene expression modules encoding for communities of microglia and endothelial cells in the white matter and regional astrocytes programs that were strongly enriched with age and for senescence-related genes. These findings will help facilitate future studies exploring the function of senescent cell subpopulations in the aging brain.

cell biology↗

bioRxiv2025

Transposable Elements are Dysregulated in Brains of Individuals with Major Depressive Disorder

Transposable elements (TEs) are repetitive DNA sequences capable of being transcribed and reintegrated, or transposed, into distinct loci throughout the genome. While thought to be largely transcriptionally silenced in brain, TE transcription is increasingly recognized as dynamic and involved in human health and disease states, including in disorders of the brain. In this study, we annotated TE transcripts in publicly available RNA sequencing (RNAseq) of postmortem human brain tissue to investigate the expression profile of TE transcripts in individuals with Major Depressive Disorder (MDD) compared to healthy controls. Our findings reveal a robust and uniform downregulation of TE transcript expression in the brains of subjects with MDD relative to controls, this occurs most prominently in the orbitofrontal cortex (OFC) brain region, and MDD differentially impacts this TE expression by age and sex. This work points to the aberrant transcription of cortical TEs as a potentially overlooked molecular signature of MDD.

neuroscience↗

bioRxiv2024

A single cell and spatial genomics atlas of human skin fibroblasts in health and disease

Fibroblasts are critical cells that shape the architecture and cellular ecosystems in multiple tissues. Understanding fibroblast heterogeneity and their spatial context in health and disease has enormous clinical relevance. In this study, we constructed a spatially-resolved atlas of human skin fibroblasts from healthy skin and 23 skin disorders. We define 6 major skin fibroblast populations in health and a further three skin disease-specific fibroblast subtypes, and demonstrate the fibroblast composition in different types of skin disease. We characterise a human-specific fibroblastic reticular cell (FRC)-like subtype in the skin perivascular niche and postulate their origin from prenatal skin lymphoid tissue organiser (LTo)-like cells. We also show that inflammatory myofibroblasts (IL11+MMP1+CXCL5+IL7R+) are a conserved fibroblast subtype in inflammatory disorders and cancers across multiple human tissues. We provide a harmonised nomenclature for skin fibroblasts that integrates previous findings from human skin and other tissues.

cell biology↗

bioRxiv2024

Outcome-Driven Microscopy: Closed-Loop Optogenetic Control of Cell Biology

Smart microscopy is transforming biological imaging by integrating real-time analysis with adaptive acquisition to enhance imaging efficiency. Whereas many emerging implementations are event-driven and focus on on-demand data acquisition to reduce phototoxicity, we here present outcome-driven microscopy, which combines smart microscopy with optogenetics to achieve subcellular spatiotemporal control of biology to predefined outcomes. We validate this approach using light-based control of cell migration and nucleocytoplasmic transport, and demonstrate unprecedented spatiotemporal control over cellular behaviour.

cell biology↗

bioRxiv2024

Brisk: Exact resource-efficient dictionary for k-mers

The rapid advancements in DNA sequencing technology have led to an unprecedented increase in the generation of genomic datasets, with modern sequencers now capable of producing up to ten terabases per run. However, the effective indexing and analysis of this vast amount of data pose significant challenges to the scientific community. K-mer indexing has proven crucial in managing extensive datasets across a wide range of applications, including alignment, compression, dataset comparison, error correction, assembly, and quantification. As a result, developing efficient and scalable k-mer indexing methods has become an increasingly important area of research. Despite the progress made, current state-of-the-art indexing structures are predominantly static, necessitating resource-intensive index reconstruction when integrating new data. Recently, the need for dynamic indexing structures has been recognized. However, many proposed solutions are only pseudo-dynamic, requiring substantial updates to justify the costs of adding new datasets. In practice, applications often rely on standard hash tables to associate data with their k-mers, leading to high k-mer encoding rates exceeding 64 bits per k-mer. In this work, we introduce Brisk, a drop-in replacement for most k-mer dictionary applications. This novel hashmap-like data structure provides high throughput while significantly reducing memory usage compared to existing dynamic associative indexes, particularly for large k-mer sizes. Brisk achieves this by leveraging hierarchical minimizer indexing and memory-efficient super-k-mer representation. We also introduce novel techniques for efficiently probing k-mers within a set of super-k-mers and managing duplicated minimizers. We believe that the methodologies developed in this work represent a significant advancement in the creation of efficient and scalable k-mer dictionaries, greatly facilitating their routine use in genomic data analysis.

bioinformatics↗

bioRxiv2024

Haploinsufficiency of lysosomal enzyme genes in Alzheimers disease

There is growing evidence suggesting that the lysosome or lysosome dysfunction is associated with Alzheimers disease (AD). Pathway analysis of post mortem brain-derived proteomic data from AD patients shows that the lysosomal system is perturbed relative to similarly aged unaffected controls. However, it is unclear if these changes contributed to the pathogenesis or are a response to the disease. Consistent with the hypothesis that lysosome dysfunction contributes to AD pathogenesis, whole genome sequencing data indicate that heterozygous pathogenic mutations and predicted protein-damaging variants in multiple lysosomal enzyme genes are enriched in AD patients compared to matched controls. Heterozygous loss-of-function mutations in the palmitoyl protein thioesterase-1 (PPT1), -L-iduronidase (IDUA), {beta}-glucuronidase (GUSB), N-acetylglucosaminidase (NAGLU), and galactocerebrosidase (GALC) genes have a gene-dosage effect on A{beta}40 levels in brain interstitial fluid in C57BL/6 mice and significantly increase A{beta} plaque formation in the 5xFAD mouse model of AD, thus providing in vivo validation of the human genetic data. A more detailed analysis of PPT1 heterozygosity in 18-month-old mice revealed changes in -, {beta}-, and {gamma}-secretases that favor an amyloidogenic pathway. Proteomic changes in brain tissue from aged PPT1 heterozygous sheep are consistent with both the mouse data and the potential activation of AD pathways. Finally, CNS-directed, AAV-mediated gene therapy significantly decreased A{beta} plaques, increased life span, and improved behavioral performance in 5xFAD/PPT1+/- mice. Collectively, these data strongly suggest that heterozygosity of multiple lysosomal enzyme genes represent risk factors for AD and may identify precise therapeutic targets for a subset of genetically-defined AD patients. Significance StatementLysosomes play a role in the degradation of aggregation-prone proteins such as amyloid {beta} (A{beta}). Homozygous lysosomal enzyme gene defects result in fatal pediatric lysosomal storage diseases and, historically, carriers were considered normal. However, a human genetic analysis identified deleterious heterozygous variants in multiple lysosomal enzyme genes that are enriched in Alzheimers disease (AD) patients. Those findings were validated in vivo by demonstrating that heterozygous loss-of-function (LoF) mutations in five different lysosomal enzyme genes affect A{beta} processing and exacerbate A{beta} plaque formation. CNS-directed gene therapy ameliorated the effects of a heterozygous LoF mutation in one of those genes in a mouse model of AD. These findings provide insights into the role of lysosomes in AD and have important therapeutic implications.

neuroscience↗

bioRxiv2024

GenomicGapID: Leveraging Spatial Distribution of Conserved Genomic Sites for Broad-Spectrum Microbial Identification

Bacterial detection and identification methods can be broadly classified as either untargeted with expansive taxonomic coverage or targeted with narrow taxonomic focus. Untargeted approaches, such as culture and sequencing, are often time-consuming and/or costly, whereas targeted methods, such as PCR, can offer faster and more cost-effective results but require a priori knowledge of the likely pathogen to select the appropriate assay. GenomicGapID, a novel approach that leverages the spatial distribution of conserved genetic regions across microbial genomes, represents a significant advancement in the field of microbial identification. This technique has the potential to provide the taxonomic breadth of culture and sequencing, while maintaining the speed, simplicity, and cost-effectiveness of PCR. By leveraging the conservation and relative positioning of highly conserved coding regions across different species, GenomicGapID enables the development of universal primer sets that amplify the non-conserved gaps between these regions. This creates a unique electrophoretic signature that facilitates rapid and accurate target agnostic microbial identification. In this study, we apply the principles of GenomicGapID to the critical task of identifying clinical pathogens. We focus on expanding the coverage of a previously developed universal bacterial identification system, which initially targeted the 16s-23s internal transcribed spacer (ITS) region and was capable of discerning 45 pathogens. To enhance this system, we assembled a comprehensive database of 189 clinically relevant bacterial species. We then identified conserved primer binding sites that produce unique amplicon size signatures for each species. While we found that the use of amplicon size signatures alone would require an impractical number of universal primer sets, we demonstrate that this challenge can be effectively mitigated through concurrent melt analysis. Ultimately, we show that just three universal primer sets, guided by the GenomicGapID framework, are sufficient to cover 189 clinical bacterial pathogens, representing a majority of microbes identified in positive cultures in a clinical microbiology setting, with experimental validation of a subset of these pathogens. This study not only enhances the existing universal bacterial identification system but also establishes GenomicGapID as a versatile and powerful tool in microbial diagnostics and beyond with the potential to open new areas of investigation in genomics, with significant implications for molecular biology, clinical practice, and public health.

microbiology↗

bioRxiv2024

Identification of a new Plasmodium falciparum E2 ubiquitin conjugating enzyme

The ubiquitin-proteasome system (UPS) is essential for Plasmodium falciparum survival and represents a potential target for antimalarial therapies. We utilised a ubiquitin-activity based probe (Ub-Dha) to capture active components of the ubiquitin conjugating machinery during asexual blood-stage development. Several E2 ubiquitin-conjugating enzymes, the E1 activating enzyme, and the HECT E3 ligase PfHEUL were identified and validated through in vitro ubiquitination assays. We also demonstrate selective functional interactions between PfHEUL and a subset of both human and P. falciparum E2s. Additionally, the Ub-Dha probe captured an uncharacterized protein, PF3D7_0811400 (C0H4U0) with no known homology to ubiquitin-pathway enzymes in other organisms. Through structural and biochemical analysis, we validate it as a novel E2 enzyme, capable of binding ubiquitin in a cysteine-specific manner. These findings contribute to our understanding of the P. falciparum UPS, identifying promising novel drug targets and highlighting the evolutionary uniqueness of the Ub-proteasome system in this parasite.

cell biology↗

bioRxiv2024

Chromatin remodeler BRG1 recruits huntingtin to repair DNA double-strand breaks in neurons

Persistent DNA double-strand breaks (DSBs) are enigmatically implicated in neurodegenerative diseases including Huntingtons disease (HD), the inherited late-onset disorder caused by CAG repeat elongations in Huntingtin (HTT). Here we combine biochemistry, computation and molecular cell biology to unveil a mechanism whereby HTT coordinates a Transcription-Coupled Non-Homologous End-Joining (TC-NHEJ) complex. HTT joins TC-NHEJ proteins PNKP, Ku70/80, and XRCC4 with chromatin remodeler Brahma-related Gene 1 (BRG1) to resolve transcription-associated DSBs in brain. HTT recruitment to DSBs in transcriptionally active gene- rich regions is BRG1-dependent while efficient TC-NHEJ protein recruitment is HTT-dependent. Notably, mHTT compromises TC-NHEJ interactions and repair activity, promoting DSB accumulation in HD tissues. Importantly, HTT or PNKP overexpression restores TC-NHEJ in a Drosophila HD model dramatically improving genome integrity, motor defects, and lifespan. Collective results uncover HTT stimulation of DSB repair by organizing a TC-NHEJ complex that is impaired by mHTT thereby implicating dysregulation of transcription-coupled DSB repair in mHTT pathophysiology. Highlights* BRG1 recruits HTT and NHEJ components to transcriptionally active DSBs. * HTT joins BRG1 and PNKP to efficiently repair transcription related DSBs in brain. * Mutant HTT impairs the functional integrity of TC-NHEJ complex for DSB repair. * HTT expression improves DSB repair, genome integrity and phenotypes in HD flies.

neuroscience↗

bioRxiv2024

A rapid, inexpensive, culture-free, universal bacterial identification system using internal transcribed spacer targeting primers: a proof-of-principle study

Techniques for bacterial detection and identification can be characterized as either untargeted and taxonomically broad or targeted and taxonomically narrow. Untargeted techniques (e.g., culture and sequencing) are time-consuming and/or expensive while targeted techniques (polymerase chain reaction; PCR) can be faster and less expensive but require a strong pre-test suspicion of the potential organism to choose the correct test. We have developed a universal bacterial identification system that is as taxonomically broad as culture and amplicon sequencing but as fast, easy, and affordable as PCR. The platform utilizes a unique universal polymerase chain reaction (PCR) primer set that targets the internal transcribed spacer (ITS) regions between conserved bacterial genes, creating a distinguishable electrophoretic pattern for each bacterial species. Bioinformatic simulation demonstrates that at least 45 commonly isolated pathogenic species can be uniquely identified from a single set of PCR primers using this approach. We experimentally confirmed these predictions on seven representative human pathogens, including gram-negatives and gram-positives, aerobes and anaerobes, and spore formers. Without a priori knowledge of the organism, this system can rapidly identify the unique pattern generated by multiple species in a single reaction. Using quantitative PCR, the system can also determine the corresponding concentration of the organism in question. We also show that the primers are resilient to human DNA contamination at physiologic concentrations, eliminating the need for complex and time intensive extraction methods. Proof-of-principle testing on actual clinical specimens demonstrate that this assay can identify more than twice the species as current multiplex PCR assays (e.g., BioFire) using only one universal primer pair in a single PCR reaction, providing results in <3 hours for <$20 without bioinformatic turnaround time. SIGNIFICANCE STATEMENTThis paper describes a novel bacterial identification system that is as taxonomically broad as culture and amplicon sequencing but as fast, easy, and affordable as PCR. The assay covers more than twice the species as the leading multiplex PCR assays but uses only one universal primer pair in a single PCR reaction. It is resilient to human genomic contamination precluding the need for timely or costly methods to clear human DNA. This approach represents a major advancement in the decades-long struggle to rapidly and accurately identify bacteria.

bioengineering↗

bioRxiv2024

Human CCR4-NOT is a global regulator of gene expression and is a novel silencer of retrotransposon activation

CCR4-NOT regulates multiple steps in gene regulation and has been well studied in budding yeast, but much less is known about the human complex. Auxin-induced degradation was used to rapidly deplete the scaffold subunit CNOT1, and CNOT4, to characterize the functions of human CCR4-NOT in gene regulation. Depleting CNOT1 increased RNA levels and caused a widespread decrease in RNA decay. In contrast, CNOT4 depletion only modestly changed steady-state RNA levels and, surprisingly, led to a global acceleration in mRNA decay. Further, depleting either subunit resulted in a global increase in RNA synthesis. In contrast to most of the genome, the transcription of KRAB-Zinc-Finger-protein (KZNFs) genes, especially those on chromosome 19, was repressed. KZNFs are transcriptional repressors of retrotransposable elements (rTEs), and consistent with the decreased KZNFs expression, rTEs, mainly Long Interspersed Nuclear Elements (LINEs), were activated. These data establish CCR4-NOT as a global regulator of gene expression and a novel silencer of rTEs.

molecular biology↗

bioRxiv2024

BRAIN-METASTATIC MELANOMA MODELS UNCOVER IMMUNE AND MOLECULAR CORRELATES OF RESPONSE TO IMMUNOTHERAPY

Despite the promising results of immune checkpoint blockade (ICB) therapy, outcomes for patients with brain metastasis (BrM) remain poor. Identifying resistance mechanisms has been hindered by limited access to patient samples and relevant preclinical models. Here, we developed two mouse melanoma BrM models that recapitulate the disparate responses to ICB seen in patients. We demonstrate that these models capture the cellular and molecular complexity of human disease and reveal key factors shaping the tumor microenvironment and influencing ICB response. BR1-responsive tumor cells express inflammatory programs that polarize microglia into reactive states, eliciting robust T cell recruitment. In contrast, BR3-resistant melanoma cells are enriched in neurological programs and exploit tolerance mechanisms to maintain microglia homeostasis and limit T cell infiltration. In humans, BR1 and BR3 expression signatures correlate positively or negatively with T cell infiltration and BrM patient outcomes, respectively. Our study provides clinically relevant models and uncovers mechanistic insights into BrM ICB responses, offering potential biomarkers and therapeutic targets to improve therapy efficacy.

immunology↗