Biology subjects

Biswas, A.

Publications and source records attributed to Biswas, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Lateral-eyed tree shrews combine panoramic vision with an expanded oculomotor repertoire

Mammals differ in how they redirect gaze. Primates shift and hold gaze with rapid, precise eye movements that can be decoupled from the head. In lateral-eyed mammals studied to date, gaze is organized around the head: saccades are coupled to head rotation in freely moving animals and typically accompanied by attempted head movements even under head fixation. These observations suggest a trade-off: animals that keep a panoramic field give up the precise, eye-driven control of gaze typical of frontal-eyed animals. But because lateral eyes and head-centered gaze co-occur in every lateral-eyed species studied, it is unknown whether one follows from the other. Here we test this in the tree shrew, an arboreal mammal with laterally placed eyes. Using real-time eye tracking in head-fixed animals, we find that tree shrews saccade frequently under head fixation and that most saccades occur without detectable attempted head movement. Tree shrews make visually evoked saccades, along both the horizontal and vertical axes of the visual field, toward targets. They can also fixate on visual targets. Using widefield calcium imaging, we show that visual cortex over-represents the central visual field. Lastly, we show that extraocular muscles contain morphologically elaborate palisade endings, specialized nerve endings that are a constant feature of frontal-eyed species. Lateral eyes therefore do not preclude eye-driven gaze control. The two are separable dimensions of visual system design, a combination that may suit animals whose locomotion routinely points the head away from where they need to look.

neuroscience↗

bioRxiv2026

The physical state of the nucleus tracks its function across eukaryotes

The nucleus is consistently less dense than the surrounding cytoplasm, a difference proposed to arise from selective nuclear import. The functional significance of low nuclear density, however, remains unknown. Because density is a bulk property of the cell, acute perturbations inevitably have pleiotropic consequences. We therefore turned to the natural experiments offered by the tree of life where nuclei vary in genome size, chromatin organisation, import selectivity and transcriptional state, and asked whether low nuclear density is tied to particular nuclear architectures or functions. Using dinoflagellates, Giardia and ciliates, we show nuclear density is uncoupled from genome size. In ciliates, two nuclei in one cytoplasm reach opposite densities relative to it, governed by nuclear import rather than transcription. We thus confirm nuclear import as a major determinant of nuclear density and reveal nuclei that break the low-density rule.

biophysics↗

bioRxiv2026

Adaptive respiratory bypass restores oxidative metabolism in NADH dehydrogenase-deficient Escherichia coli

Nicotinamide adenine dinucleotide is central to the cellular redox network and continuously cycles between its oxidized (NAD) and reduced (NADH) forms. Membrane-bound NADH dehydrogenases are the primary NADH-oxidizing enzymes and principal entry point for reducing equivalents into the bacterial respiratory chain. While maintaining optimal balance of this redox cofactor is among the most fundamental biochemical constraints on living systems, how cells adapt when the primary NADH oxidation route is lost remains poorly understood. Here we show that Escherichia coli lacking both NADH dehydrogenases initially compensates through costly fermentative lactate secretion, potentially to maintain redox and energy homeostasis, but at the cost of impaired growth and oxidative metabolism. Using adaptive laboratory evolution across four independent lineages, we found that all evolved populations derepressed malate:quinone oxidoreductase by disrupting an ArcA-binding site. This gain-of-function mutation establishes an alternative pathway that funnels electrons directly into the quinone pool, restoring respiratory oxygen consumption to wild-type level. Multi-omics and genome-scale metabolic modeling reveal that this bypass drives broad reorganization of central carbon metabolism, reinstates tricarboxylic acid cycle flux, and shifts cells toward a higher, more oxidative aero-type. These findings uncover a previously unrecognized metabolic buffering capacity that enables bacteria to circumvent loss of canonical respiratory entry points.

microbiology↗

bioRxiv2026

Exosomal miR-23a release coincides with astrocytic calcineurin activation, protects neurons from apoptosis by regulating NOXA in Parkinson's disease models and is enriched in the plasma of patients with Parkinson's disease

Parkinson's disease (PD) is characterized by a progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta and degeneration of their projections in the striatum (STR) of the brain. Astrocytes are recognized as active contributors to PD pathogenesis, yet the role of calcineurin (CN), a Ca/calmodulin-dependent phosphatase which is well characterized in neurons, remains unexplored in astrocytes in the context of PD. Here, we show that CN protein expression is upregulated in the STR of MPTP-treated mice, coinciding with DA neurodegeneration and astrogliosis. In human astrocytoma cells and primary rat astrocytes, the neurotoxin Rotenone induced rapid intracellular Calcium elevation and CN activation. Unexpectedly, this was accompanied by a decrease in intracellular levels of miR-23a, an astrocyte-enriched microRNA previously linked to CN signalling in cardiomyocytes. We found that this decrease reflected active exosomal export: Rotenone increased both exosomal miR-23a levels and total exosome number, while blocking exosome biogenesis with GW4869 restored intracellular miR-23a levels. Functionally, both astrocyte-derived exosomes and miR-23a overexpression protected neuronal cells (SH-SY5Y, PC12) against Rotenone-, 6-OHDA-, and MPP+-induced toxicity. Mechanistically, miR-23a showed direct binding to the 3'UTR of the pro-apoptotic BH3-only protein NOXA, thereby suppressing its expression, subsequently attenuating caspase-3 activation. Finally, exosomal miR-23a levels were significantly elevated in plasma from PD patients compared with age-matched controls, supporting clinical relevance. Together, these findings identify a previously unrecognized astrocyte-to-neuron communication axis during PD pathogenesis, in which astrocytic CN activation accompanied by exosomal miR-23a release represents a neuroprotective stress-responsive pathway with potential biomarker and therapeutic relevance in PD.

neuroscience↗

bioRxiv2026

Integrated in silico identification of fungal-derived dual-target inhibitors for anti-schistosomal drug discovery

Praziquantel, which has limited efficacy against young parasites and reduces susceptibility, is the principal therapy for schistosomiasis, a neglected tropical illness. This work identified fungal-derived compounds with potential dual inhibitory action against Schistosoma mansoni DHODH and cathepsin B1 using an integrated computational method. From 1,831 fungal metabolites, 120 were selected for molecular docking after structure standardisation, drug-likeness evaluation, ADMET, and toxicity screening. Ganoderlactone B (CID_122184973) and Spiroapplanatumine F (CID_132962217) strongly bound to DHODH and CB1, with docking affinities of -10.6 and -10.0 kcal/mol and -9.0 and -8.9, respectively. Docking validation showed good discrimination between active compounds and decoys with area-under-the-curve values of 0.98 for DHODH and 0.96 for CB1. The protein-ligand interaction study showed hydrogen bonding, hydrophobic interactions, and van der Waals contacts at the binding sites. In 100 ns molecular dynamics simulations, protein complexes exhibited modest backbone deviations and maintained compactness, although CID_132962217 showed sustained binding and a constrained conformation. MM-PBSA analysis identified CID_122184973 as the best common ligand for both targets. At the B3LYP/6-31G level, density functional theory calculations showed that CID_139584993 had the lowest HOMO-LUMO energy gap and hardness values of 3.552 and 1.776 eV, respectively, and the highest softness value of 0.282 eV-1, indicating greater electronic reactivity. Electrostatic potential mapping and QSAR predictions supported their interaction and antiparasitic potential. Overall, CID_122184973 and CID_132962217 are promising fungal scaffolds for dual-target anti-schistosomal drug development. Experimental enzyme inhibition, parasite viability, toxicity, and in vivo research are needed to confirm computational findings.

bioinformatics↗

bioRxiv2026

Protein-driven colloid-osmotic pressure controls nuclear size, organization, and function

The size of the cell nucleus is tightly controlled and changes in nuclear size correlate with altered nuclear function during development, cell differentiation and senescence. How nuclear size is regulated and whether changes thereof are functionally relevant remains unclear. Here, we demonstrate that nuclear size is determined by the osmotic pressure exerted by proteins in yeast, human cells and frog egg extracts. The biophysical model we establish solves the long-standing question of how the nuclear-to-cytoplasmic ratio is regulated and maintained. Furthermore, altering protein-driven osmotic balance modulates the physical properties of the nucleus, with a direct effect on chromatin organization and gene expression: Nuclear enlargement causes the dissolution of heterochromatic structures and derepression of subtelomeres and transposons, while simultaneously down-regulating highly expressed housekeeping genes. Importantly, these global transcriptional patterns closely mimic the gene expression changes that occur as yeast, human, and drosophila cells enlarge. Importantly, artificially forcing nuclear compression is sufficient to reverse these size-associated expression changes. Together, our findings provide a quantitative, mechanistic explanation for the coupling between the size of the nucleus and the cell, and they establish nuclear size as a modulator of chromatin organization and gene expression.

cell biology↗

bioRxiv2026

Gestational exposure of bisphenol-A limits decidual ECM organization via S100a10-Annexin A2 axis in murine placenta

Environmental exposure to endocrine disrupting chemicals, like bisphenol-A (BPA), can impart detrimental effects on developing feto-placental unit, during pregnancy. Placenta remains a central player maintaining this feto-placental homeostasis for sustenance of a healthy pregnancy. Thus, the bisphenol-A mediated endocrine disruption affects the healthy functioning of placenta by altering key processes, such as tissue remodelling, angiogenesis, and metabolism. However, the underlying mechanism of BPA-altered ECM remodelling remains elusive. Therefore, in this study we investigated the BPA mediated changes in placental tissue remodelling using a bisphenol-A exposed murine model during pregnancy. The results reveal that, the phenotypic changes in feto-placental interface correlates with perturbed placental proteome in response to BPA. Further investigation highlights a S100a10-Annexin A2 axis mediated upregulation of tissue plasminogen activator (tPA), which drives altered extracellular matrix (ECM) degradation in placental decidua. This culminates into functional dysregulation in feto-placental axis, leading to reduced size of fetus and placenta. Therefore, this study provides novel insights of a S100a10-Annexin A2 axis associated mechanism for alteration of ECM remodelling in placental decidua due to BPA exposure, which may lead to toxicity related adverse pregnancy outcome.

developmental biology↗

bioRxiv2026

GO, NO-GO: A SENSITIVE PERIOD FOR DEVELOPING SEX-SPECIFIC VISUALLY-GUIDED PREY PURSUIT-PREDATOR AVOIDANCE TRADE-OFF STRATEGIES

In natural environments, animals face a trade-off between foraging and staying vigilant against predators, a conflict known to shape sensory processing and behavior through natural selection. Even within a species, this trade-off may be resolved differently depending on age, sex, or life history. To identify the neural mechanisms underlying visually-guided pursuit-avoidance trade-offs, we studied how mice responded to a sudden overhead threat while pursuing a moving, prey-like target near the ground. This paradigm let us quantify orienting decisions in adolescent and adult mice of both sexes. We found that adolescence is a key period when male and female mice begin to diverge in how they resolve this conflict. Manipulating the value of the pursued target during adolescence versus adulthood revealed that adolescence is also a sensitive period for shaping adult escape-to-shelter versus continue to approach target behavior. Notably, hunting experience gained specifically during adolescence caused males, but not females, to shift strategy: experienced adolescent males tended to uniquely shift towards a "no-go" (stay-near-Prey) strategy. All the other groups instead favored an active "go" strategy, repeatedly shifting between approaching target and running to shelter. All mice showed this oscillation between approaching target and escaping to a shelter to some degree, but hunting experience during adolescence most significantly shifted this balance between approach and escape for males.

neuroscience↗

bioRxiv2026

Extracting deep learning based morphology segmentation footprint for boar sperm cells

Reliable delineation of the head and tail of swine spermatozoa supports automated assessment of boar semen quality, from morphometric measurement to the quality control of insemination doses. In practice this relies on fluorescent staining, which adds chemistry, cost, and delay to every acquisition and labels only the nucleus. Recent work coupling imaging flow cytometry with machine learning has advanced rapidly, yet the segmentation stage still depends on a stained channel at inference and resolves the head alone. We present a supervised encoder decoder network that segments boar spermatozoa from brightfield images acquired on an Amnis ImageStream Mark II with no stain at inference. Training labels derive from the Hoechst 33342 nuclear channel (Ch7), recorded in registration with brightfield (Ch1); the dye serves only as an annotation source, and the network sees Ch1 alone. The best semantic segmentation model reaches a Dice coefficient of 0.940 on held-out cells. For comparison we evaluate a classical morphological pipeline, four further semantic segmentation models spanning three decoder families and two ImageNet-pretrained backbones, and two zero-shot pipelines built on the Segment Anything Model 2 (SAM 2), prompted either by a dilated box around the predicted head mask or by head and tail boxes emitted by a Gemma 4 Vision Language Model (VLM). The zero-shot route scores 0.637 against Ch7 but labels the tail, which the fluorescence protocol cannot. Cells scoring worst under the supervised model proved to be mostly registration failures rather than segmentation failures, as Ch7 is displaced relative to Ch1. Manual screening for this drift is infeasible at dataset scale, so we propose a flagging system that marks any Dice below 0.792, two standard deviations below the mean, and pairs it with a zero-shot pipeline in which a VLM l and SAM 2 cross-check the flagged cell before human review.

bioinformatics↗

bioRxiv2026

Modulation of noisy gene expression by general sequestration mechanisms

Sequestration of gene products through diverse mechanisms forms a fundamental layer of regulation in intracellular biochemical processes, including post-translational modification, promiscuous binding to genomic decoy sites, and partitioning into membraneless compartments formed through phase separation. Here, we develop a unified stochastic framework to quantify how such sequestration-type processes, when coupled to noisy gene expression, modulate cell-to-cell variation in protein levels. In this model, protein molecules reversibly switch between active (free) and inactive (sequestered) states, whose switching rates are arbitrary functions of the molecular counts. Using exact analytical calculations and the linear noise approximation, we derive expressions for the Fano factor of the active-protein level and identify fluctuation attenuation regimes in terms of the logarithmic sensitivities of the switching rates to protein abundances. We show that inactive-protein-dependent switching, of which genomic decoy binding is a natural example, can preserve Poisson-level fluctuations in the active-protein level under appropriate conditions. Enzymatic inactivation, a type of post-translational modification, emerges as a special case of active-protein-dependent sequestration, where greater responsiveness of the inactivation propensity attenuates active-protein fluctuations. In both decoy binding and enzymatic inactivation, protecting the inactive protein from decay lowers active-protein fluctuations. Finally, a noise-buffering regime associated with intracellular phase separation is recovered when the inactive-to-active switching rate depends inversely on the inactive-protein level. Together, the examples of genomic decoy binding, enzymatic inactivation, and intracellular phase separation suggest that noise buffering observed across diverse intracellular processes is rooted in a broader class of reversible sequestration mechanisms that attenuate protein-level fluctuations.

systems biology↗

bioRxiv2026

Evolutionary constraints limit additional mammalian adaptation of bovine-derived H5N1 influenza viruses in ferrets

The outbreak of clade 2.3.4.4b H5N1 viruses among U.S. dairy cattle has raised concerns that sustained circulation among agricultural mammals could facilitate viral adaptation toward efficient human transmission. However, the evolutionary dynamics governing such adaptation remain poorly understood. Here we investigated the evolution of two bovine-derived H5N1 B3.13 genotype viruses during infection and airborne transmission in ferrets, building on prior characterization of their robust replication and inefficient airborne transmission. Within hosts, viral genetic diversity was limited and viruses were subject to genetic drift and weak purifying selection. Transmission, when it occurred, was characterized by stringent bottlenecks that sharply reduced viral genetic diversity. We found no evidence of mammalian adaptation during infection or transmission. Together, these findings indicate that bovine-derived H5N1 viruses face evolutionary constraints during acute mammalian infection and transmission, limiting movement toward enhanced airborne spread. These constraints may help explain why efficient mammalian replication does not necessarily coincide with efficient transmission. Continued circulation of HA clade 2.3.4.4b viruses nevertheless creates repeated opportunities for rare but consequential evolutionary events, underscoring the importance of sustained surveillance and risk mitigation.

microbiology↗

bioRxiv2026

Identification of a new mycobacterial peptide that controls the activity of the iron-dependent regulator, IdeR.

Mycobacterium tuberculosis (Mtb) must regulate intracellular iron to survive in the host and cause disease. IdeR (Iron-dependent Regulator) is an essential Mtb protein that governs intracellular iron levels by regulating the expression of genes involved in iron metabolism. IdeR expression, however, is iron independent, and the mechanisms that regulate IdeRs function are not fully understood. Here, we report the discovery of a previously unrecognized Mtb peptide (PRI) that regulates IdeR activity. PRI is induced under iron limitation; it binds to IdeR and restricts its activity. In addition, we demonstrate that altering the balance between PRI and IdeR impairs iron homeostasis and intracellular replication of Mtb in macrophages. The findings reveal a new paradigm in mycobacterial iron regulation and open new avenues for targeting iron homeostatic mechanisms in Mtb, which are crucial for virulence and antibiotic resistance.

microbiology↗

bioRxiv2026

PocketBagger: Generalizable pocket druggability prediction via positive-unlabeled learning

Abstract SummaryReliable structure-based prediction of small-molecule druggability is hindered by a fundamental labeling problem. Experimentally confirmed liganded sites (positives) are observable, but credible "undruggable" pockets (negatives) are almost impossible to define. Standard supervised machine learning consequently relies on arbitrary definitions of undruggable, leading to bias and false negatives. Here we introduce PocketBagger, a positive-unlabeled (PU) learning framework for pocket druggability prediction trained exclusively on experimentally determined Protein Data Bank1 (PDB) structures. PocketBagger uses PU bagging to learn key features associated with reliable druggable pockets and considers all remaining pockets in the structurally characterized proteome as unlabeled. We demonstrate the capability of PocketBagger through the training of a simple Random Forest classifier and demonstrate its power in recall (0.804), even when challenged with increasingly difficult generalizability assessments and entire protein-family hold outs. We benchmark and demonstrate the added value of PU learning by comparing PocketBagger to a leading deep-learning predictor. However, PocketBagger is intended to be used as a framework for any model architecture. Along with the code, the data generated by PocketBagger are deployed in canSAR.ai, providing scalable, generalizable pocket druggability predictions to the drug discovery community.

bioinformatics↗

bioRxiv2026

Open-eyed meditation suppresses functional connectivity in EEG across a broad frequency range

Functional connectivity (FC) is a statistical measure that reflects the degree of phase consistency between two signals and provides insights about potential interactions between two brain regions. Previous studies have reported conflicting results on the effect of meditation on FC, with some showing enhancement while others reporting suppression of FC. However, even though meditation increases power over a broad frequency range between 15-200 Hz and beyond, most FC studies have reported changes over fixed and narrow frequency bands below 50 Hz. Further, meditation-induced changes in power spectral density (PSD) and FC have never been compared with changes with other factors such as age, gender and stimulus. We recorded electroencephalogram (EEG) from open-eyed meditators (N=35) and their gender-and age-matched controls (N=36) and found that meditation was associated with a state decrease in FC across a broad frequency range (15-200 Hz), while PSD showed both trait and state enhancement. Furthermore, visual gratings, which are known to enhance narrow-band gamma power, led to reduced gamma FC in both meditators and controls. We also compared the effect of aging and gender on a different dataset of healthy middle-aged (N=78) and elderly (N=89) participants and found differences in distinct frequency bands that were limited to a narrow range. We also found that often-used average referencing heavily distorted the FC and gave uninterpretable results. Overall, our results suggest distinct neural mechanisms underlying healthy aging, vision, and meditation and further recommend caution while using average referencing to study phase-based metrics. Significance statementMeditation research has reported inconsistent effects on functional connectivity (FC), partly because most studies examined only narrow low-frequency bands despite meditation altering brain activity across a much broader frequency band. This study demonstrates that meditation produces a broadband state reduction in FC across 15-200 Hz, while simultaneously enhancing power. In contrast, healthy aging, gender, and visual stimulation showed frequency-specific effects confined to alpha (8-12 Hz) and high-beta (20-36 Hz) bands, highlighting meditations unique large-scale neural signature. The study also shows that average referencing can severely distort phase-based FC estimates, leading to misleading interpretations. These findings clarify conflicting literature, distinguish meditation from other neural modulators, and provide important methodological guidance for EEG connectivity research.

neuroscience↗

bioRxiv2026

Glutamylated vimentin proteoform identified by a synthetic binder links to epithelial-mesenchymal plasticity

Cytoskeletal protein expression and filament dynamics change significantly during cell state transitions. However, post-translational modifications of cytoskeletal proteins during these transitions have rarely been described. Here, using a synthetic glutamylation-binder (SB2B49) selected against a bi-glutamylated peptide epitope, we identify a distinct pool of glutamylated vimentin filaments. We demonstrate that vimentin glutamylation is enzymatically added by tubulin tyrosine ligase-like (TTLLs) and removed by cytosolic carboxypeptidases (CCPs). Mass spectrometry and mutagenesis reveal that glutamylation occurs on specific vimentin residues. We find that glutamylated vimentin levels are dynamically modulated during epithelial-mesenchymal plasticity. During collective migration in scratch-wound assays, glutamylated vimentin filaments are transiently depleted at the wound edge, a process controlled by canonical glutamylation writer-erasers. Our findings reveal a new layer of vimentin regulation via glutamylation and establish the glutamylation-binder as a valuable tool for exploring the diversity of vimentin proteoforms and glutamylation modifications in both physiological and pathological contexts.

cell biology↗

bioRxiv2026

Bisphenol-A mediated ubiquitinome alteration triggers PPAR-alpha ubiquitination, affecting trophoblast cell migration

Pregnant women are frequently exposed to various endocrine-disrupting chemicals (EDCs), such as bisphenol A (BPA), causing harm to both the developing placenta and fetus. BPA can promote placental dysfunction by altering key cellular processes such as differentiation, invasion, and migration in trophoblast cells. These cellular processes are also tightly managed by the ubiquitin proteasomal system via maintenance of the ubiquitinated protein pool. However, the BPA-mediated dysregulation of this ubiquitin proteasomal homeostasis is poorly understood. Therefore, we identified 19 deubiquitinases (DUBs) and a dynamic ubiquitinome profile of extravillous trophoblast cells (HTR8/SVneo), which reduced trophoblast cell migration post-BPA exposure. Further investigation using an integrated substrate-ligase-deubiquitinase network shows that BPA binding to PPAR-alpha or indirect regulation of its E3 Ligase MuRF1 and DUB USP5 via BPA resulted in hyper-ubiquitination of PPAR-alpha, triggering its nuclear localization. In the nucleus, the ubiquitinated PPAR-alpha can deregulate its migration-associated target gene expression, causing a reduction in the migration of HTR8/SVneo cells. This physiological alteration of extravillous trophoblast cells (EVTs) through BPA can disrupt placental homeostasis. Hence, we assumed that BPA-induced cellular alteration in EVTs can promote placental defects, which might contribute to adverse pregnancy outcomes.

biochemistry↗

bioRxiv2026

Ferroptosis links α-synuclein pathology across brain and skeletal muscle in Parkinsons disease

Parkinsons disease (PD) is increasingly recognized as a multisystem disorder, yet the mechanisms linking neurodegeneration with muscle dysfunction remain largely unknown. In this study, using an A53T -synuclein (Syn) transgenic mouse model, we demonstrate coordinated pathological changes across the brain-muscle organs characterized by systemic inflammation, iron accumulation, oxidative stress, and ferroptosis-associated lipid peroxidation. Our quantitative proteomics data revealed dysregulated iron metabolism and ferroptosis in the brain and skeletal muscle. Biochemical validation confirmed increased expression of Transferrin receptor 1 (TFRC), elevated lipid peroxidation, and suppression of antioxidant defenses, including SLC7A11 and GPX4, indicating enhanced ferroptotic susceptibility. Cell-surface proteomics and biophysical assays further revealed that pathological Syn directly interacts with TFRC, promoting iron accumulation and ferroptosis-associated oxidative damage in neuronal and muscle cells. Together, our findings identify ferroptosis as a shared pathological mechanism across the brain and muscle, mediated by the Syn-TFRC interaction, thus linking neurodegeneration and peripheral muscle pathology in PD.

neuroscience↗

bioRxiv2026

Distinct clonal dynamics and interactions within the microenvironment near tumor stroma interfaces in rare histologic variants of bladder cancer

Rare histologic variants of bladder carcinoma, such as squamous cell and neuroendocrine carcinoma, generally have a worse prognosis compared to pure urothelial carcinoma (PUC), but the underlying molecular determinants are not well understood. We developed a novel computational genomics framework to characterize the dynamics of tumor-stroma-immune interactions at tumor borders and interiors from spatial transcriptomics data. We profiled bladder carcinoma samples of different histological variants on the Visium platform. Differences in clonal phylogeny and spatial heterogeneity of major subclones between the samples suggested disparate clonal spatiotemporal dynamics and interaction with stromal and immune compartments - which was notably prominent at the tumor-stroma interface. Our framework captured immune heterogeneity in the tumor microenvironment, including variations in the presence and architecture of tertiary lymphoid structures. Our analyses further indicated that there are substantial histology-specific differences in cell type composition, clonal spatial heterogeneity, inter-cellular signaling, and cellular processes. These variations collectively suggest divergent mechanisms of microenvironment remodeling across bladder cancer histologies. Cell-free DNA profiling from liquid biopsy captured tumor and microenvironment signatures from tumor boundaries and interiors, potentially allowing for tracking clonal dominance non-invasively. Our method tracks the trajectory of neoplastic disease in bladder cancer samples while identifying aggressive features.

genomics↗