Biology subjects

Ren, X.

Publications and source records attributed to Ren, X..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Deep learning-guided design of cell type-specific AAV promoters

Precise cell type targeting is critical for both clinical and experimental applications of adeno-associated viral (AAV) vectors, yet engineering vectors with cell type-specific activity remains a challenge. Here, we compared three strategies leveraging single-cell chromatin accessibility data to design cell type-specific AAV promoters, including a deep learning-based method to generate de novo regulatory sequences. When applied to target retinal ganglion cells or horizontal cells in mouse retina, deep learning-guided design consistently outperformed rational approaches, yielding synthetic promoters with stronger and more specific expression in vivo. Synthetic AAV promoters supported diverse transgenes, enabling the recording and ablation of targeted cells. Promoter activity was also maintained in human retinal organoids, suggesting that deep learning-designed sequences may be suitable for translation. Our findings highlight the potential of deep learning to synthesize cell type-specific AAV promoters and establish a versatile platform for cell type targeting with broad implications for gene therapy and basic research.

synthetic biology↗

bioRxiv2026

Genomic and pan-genomic insight into longan domestication and improvement

Longan (Dimocarpus longan) is a tropical tree in Sapindaceae family with economic importance known for its nutritous fruits. Genetic improvement of longan requires knowledge of reliable molecular markers and functional genes associated with key traits. However, it is largely impeded by lacking a complete genome and pangenome reference encompassing the broad genetic diversity in diverse longan germplasms. Here, we present a telomere-to-telomere (T2T) gap-free genome of longan cultivar Shixia and a graph-based pangenome constructed using newly assembled chromosome-level genomes of 101 accessions. We completely assemble the longan centromere regions primarily composed of Gypsy-CRM retrotransposons. The pan-genome analysis reveals 58,978 non-redundant structural variants (SVs) that exhibit signs of genomic selective sweep during longan domestication and breeding. Additionally, haplotype-resolved genome analysis suggests allele-specific gene expression associated with SV-driven changes in 3D genome architecture. Importantly, the graph-based pan-genome empowers population-scale SV genotyping and genomewide association with various longan traits. SV-GWAS revealed 12 QTLs significantly linked with longan maturity period. Among them, a 758bp insertion is located downstream of the DlDAZ gene encoding a C2H2 zinc-finger transcription factor, and DIDAZ-transgenic tomatoes showed delayed maturity. Together, our T2T genome and pan-genome provide valuable resources to facilitate longan genetic research and precise improvement.

genomics↗

bioRxiv2025

Reconstitution of multistep recruitment of ULK1 to membranes in autophagy

The Human ULK1 autophagy-initiating complex consists of ULK1, FIP200, and the HORMA domain heterodimer ATG13:ATG101. PI3P is essential to recruit ULK1C to membranes for ULK1, but ULK1C subunits do not contain PI3P-binding domains. Here we show that the ATG13:ATG101 dimer forms a complex with the PI3P-binding protein WIPI3, as well as WIPI2. Bound to WIPI2-3, ATG13:ATG101 inserts its Trp-Phe (WF) finger into the membrane. MD simulations show that WIPIs and the WF finger cooperatively stabilizes the complex on membranes. Biochemical reconstitution and cell-based assays show that WIPI3:ATG13 engagement promotes ATG16L1 phosphorylation, autophagy and mitophagy. A kinase domain (KD)-proximal PVP motif in the ULK1 IDR docks onto the ATG13:ATG101 HORMA dimer brings the ULK1 KD close to the membrane. The PVP motif is essential for in vitro ULK1 phosphorylation of ATG16L1 and important for autophagy and mitophagy. These data establish a stepwise pathway for recruitment of the ULK1 KD to the vicinity of the membrane surface.

cell biology↗

bioRxiv2025

High-Concordance Validation of Droplet Digital PCR and Next-Generation Sequencing for EGFR Mutation Detection Across Diverse Biospecimens in a Large-scale NSCLC Cohort Study

BackgroundsNext-generation sequencing (NGS) and droplet digital PCR (ddPCR) are both established methods for detecting EGFR mutations in non-small cell lung cancer (NSCLC). However, comprehensive validation of their concordance in mutation detection and variant allele frequency (VAF) quantification across heterogeneous sample types remains limited. Inconsistent results from different sample types (e.g., cfDNA, FFPE) pose a significant challenge to clinical decisions. This is particularly critical for advanced NSCLC patients, who often rely on liquid biopsy, yet the concordance between liquid and tissue-based testing lacks validation in large-scale studies. Another issue in clinical practice is that many tumor samples are limited in quantity, making it difficult to meet testing requirements. Therefore, it is worth exploring whether pre-capture NGS libraries can serve as substitutes for original DNA. MethodsIn this study, we first developed three assays to detect EGFR L858R, exon 19 deletions (Ex19del) and T790M, respectively using ddPCR platform. Their Limit of Detection (LOD) could reach 0.01% at 100 ng of input DNA. Subsequently, we conducted a large retrospective clinical study to systematically compare the detection performance of ddPCR and NGS across three mutation types using approximately 1,000 EGFR-positive samples, including cell-free DNA (cfDNA), pre-capture NGS libraries of cfDNA (cfDNA-prePCR), FFPE-derived DNA (ffpeDNA), pre-capture NGS libraries of FFPE-derived DNA (ffpeDNA-prePCR), fresh tumor tissue DNA (ttDNA), pre-capture NGS libraries of ttDNA (ttDNA-prePCR), pleural effusion supernatants DNA (peDNA), and pre-capture NGS libraries of peDNA (peDNA-prePCR). They were analyzed for detection concordance and VAF correlation. Especially, we made comparisons of the tumor DNA, including ctDNA and tumor tissue DNA, with their paired pre-capture NGS library. ResultsKey findings demonstrated excellent overall agreement between NGS and ddPCR. The mutation detection concordance rates were 98.72% (overall), with subtype-specific rates of 98.93% (L858R), 99.23% (Ex19del), and 97.14% (T790M). VAF measurements between ddPCR and NGS showed exceptional correlation (Pearsons r = 0.975, P<0.001). Notably, pre-capture NGS libraries showed remarkable VAF concordance with their source materials (0.993 for cfDNA libraries vs cfDNA; 0.998 for tumor tissue libraries vs tumor DNA with EGFR L858R; 0.991 for tumor tissue libraries vs tumor DNA with EGFR Ex19del). ConclusionsNGS and ddPCR demonstrate high concordance in EGFR mutation detection and VAF quantification, supporting their complementary roles in clinical testing. Using pre-capture libraries as an alternative to source samples can avoid repeat biopsies and enables subsequent testing for patients with inadequate FFPE sample quantity. These findings establish an evidence base for integrated diagnostic paradigms leveraging NGSs multiplexing power and ddPCRs sensitivity.

cancer biology↗

bioRxiv2025

CellBin:a generalist framework to process spatial omics data to cell level

Spatial omics has rapidly expanded with increasingly diverse imaging modalities, molecular targets, and chip sizes. However, no general framework currently exists to construct cell level matrices that are robust across platforms and omics types. Here we present CellBin, a universal and scalable frame-work that unifies image stitching, cell segmentation, and spot-to-cell mapping for multiple spatial omics technologies. CellBin integrates a multi-field weighted stitching algorithm for large-area images, a family of U-Net-based models trained across diverse staining modalities, and an optimized computational architecture for high-throughput processing. Across five technological platforms and three omics data types, CellBin achieves robust segmentation and accurate single-cell matrix construction, consistently outperforming seven state-of-the-art methods in F1-score, cell size precision, and annotation accuracy. By providing a generalizable, cross-platform solution, CellBin bridges multiple spatial omics, enabling unified, high-resolution cell level analyses across technologies.

bioinformatics↗

bioRxiv2025

Somatic TYK2 activating mutations in tumor-infiltrating T cells promote anti-cancer immunity

Cancer cells evolve to increase fitness and evade the immune system, but it is not clear if tumor infiltrating lymphocytes (TILs) undergo selection for somatic mutations that augment anti-cancer immunity. Using single molecule whole exome sequencing in TILs, we identified somatic mutations in Tyrosine-protein kinase 2 (TYK2), several of which increased TYK2 phosphorylation, JAK-STAT signaling, and interferon gamma signaling. Among these mutations, TYK2D810V was recurrently observed in patients with diverse cancer types. In vitro, TYK2D810V enhanced T cells effector functions and cytokine production. We generated mice with a germline Tyk2D807V mutation that recapitulated the human TYK2D810Vmutation. These mice were healthy and did not develop autoimmune disorders. However, they possessed enhanced anti-tumor effects in the context of syngeneic and autochthonous cancer models. Adoptive therapy with Tyk2D807V CD8 T cells also decreased cancer growth. Thus, naturally occurring mutations in non-malignant lymphocytes can antagonize cancer and inspire new immunotherapies strategies.

cancer biology↗

bioRxiv2025

Reduced Dependence on Sensorimotor Processing in the Brain is Associated with Higher Math Skills in Adults

Number processing is crucial for math competence, yet the neural underpinnings remain unclear. In this study, we used functional magnetic resonance imaging (fMRI) to 1) identify key brain regions involved in symbolic and embodied number processing, and 2) examine their relations with math abilities in children and adults. We collected fMRI data from 104 adults (Mage = 23.25 years) and 88 4th graders (Mage = 9.75) as they performed number comparison and phonological tasks with numeral (symbolic) and hand (embodied) numerical stimuli. Participants math abilities were assessed using the math subtests of the Woodcock-Johnson Tests of Achievement. Univariate analyses revealed number processing regions in occipital, parietal and temporal cortices including inferior parietal, sensorimotor, supramarginal and insular areas. In adults, less involvement of sensorimotor processing regions during number compared to phonological processing is associated with enhanced math skills. These findings not only elucidate the neural mechanisms underlying number processing and math abilities but also raise questions about the emphasis on embodied number presentation in math learning experiences. Significance StatementChildren and adults show substantial individual differences in math competence, and understanding the underlying neural mechanisms is crucial. By employing functional magnetic resonance imaging (fMRI), we identified key brain regions involved in symbolic and embodied number processing and explored their relations with math skills. Our findings reveal that reduced sensorimotor involvement during number processing correlates with enhanced math performance in adults. This study not only elucidates the neural mechanisms underlying number processing, offering critical insights into the cognitive architecture that supports math abilities, but also raises questions about the emphasis on embodied number representations in math learning experiences.

neuroscience↗

bioRxiv2025

A regulator of amino acid catabolism controls Acinetobacter baumannii gut colonization

Asymptomatic gut colonization increases the risk of clinical infection and transmission by the multidrug-resistant pathogen Acinetobacter baumannii. Ornithine utilization was shown to be critical for A. baumannii competition with the resident microbiota to persist in gut colonization, but the regulatory mechanisms and cues are unknown. Here, we identify a transcriptional regulator, AstR, that specifically activates the expression of the A. baumannii ornithine utilization operon astNOP. Phylogenetic analysis suggests that AstR was co-opted from the Acinetobacter arginine utilization ast(G)CADBE locus and is specialized to regulate ornithine utilization in A. baumannii. Reporter assays showed that astN promoter expression was activated by ornithine but inhibited by glutamate and other preferred amino acids. astN promoter expression was similarly activated by incubation with fecal samples from conventional mice but not germ-free mice, suggesting AstR-dependent activation of the astN promoter responds to intermicrobial competition for amino acids. Finally, AstR was required for A. baumannii to colonize the gut in a mouse model. Together, these results suggest that pathogenic Acinetobacter species evolved AstR to regulate ornithine catabolism, which is required to compete with the microbiota during gut colonization.

microbiology↗

bioRxiv2025

Recapitulating apicobasal tissue polarity in extracellular matrix incorporated airway organoids

The airway epithelium is a dynamic barrier that interfaces with the external environment and internal matrix along its apicobasal axis. To recapitulate this tissue arrangement in an organoid format, we present the decellularized ExtraCellular Matrix-incorporated Apical-out Airway Organoid (dECM-AoAO) that integrates basolateral matrix cues through incorporation of human lung dECM microparticles, while maintaining direct apical exposure to the exterior. Compared to the ECM-free AoAO, dECM incorporation effectively diversifies lineage distribution that better recapitulates native epithelial composition. Harnessing dECM-AoAO locomotion powered by its outward-facing ciliary beating, we developed an experimental and computational pipeline for swarm analysis of organoid group motility as a functional readout of ciliary function. Lastly, dECM-AoAO withstood cryopreservation and revival with sustained viability, lineage composition, and ciliary function, enabling future scalability and broad distribution. Together, this work establishes dECM-AoAO as a more physiologically relevant model system for investigating epithelial-ECM crosstalk during airway homeostasis, pathogenesis, and injury responses.

cell biology↗

bioRxiv2025

YY1 relieves p300 autoinhibition to promote histone acetylation in advanced prostate cancer, enhancing the oncogenic signaling of Androgen Receptor Splice Variant 7

Genetic and epigenetic aberrations often act in concert to establish oncogenic transcriptomic programs in aggressive cancers. For example, the development of castration-resistant prostate cancer (CRPC), an advanced prostate cancer form, is closely associated with over-expression and/or hyper-activation of transcription factors (TFs) such as Androgen Receptor (AR) and Yin Yang 1 (YY1), as well as p300, a prominent histone acetyltransferase. How exactly these cancer-related lesions are coordinated to generate a malignant cell state remains elusive. Here, we demonstrate that YY1, which is frequently over-expressed in advanced prostate cancers, allosterically stimulates the acetyltransferase activity of p300 in cis, leading to the globally elevated acetylation of histone H3 lysine 18 and 27 (H3K18ac and H3K27ac). Mechanistically, YY1s N-terminal activation domain (AD) directly interacts with p300s TAZ2 domain, relieving the autoinhibition of p300 to facilitate substrate acetylation. Our integrated genome-wide mapping and transcriptomic studies reveal significant co-localization of genomic binding sites of YY1, androgen receptor splice variant 7 (AR-V7, a constitutively active form of AR) and p300 in CRPC cells, where the YY1-mediated p300 activation and resultant histone acetylation increases promote the oncogenic gene-expression programs downstream of YY1 and AR/AR-V7. Both in vitro and in vivo functional assays demonstrate a critical requirement of the above signaling for the advanced disease progression and drug resistance seen in CRPC. Altogether, this study uncovers that YY1 acts to alleviate p300s autoinhibition at target genes co-bound by oncogenic TFs (YY1 and/or AR/AR-V7) in CRPC, thereby sustaining tumorigenicity. Additionally, the blockade of YY1-mediated gene activation resensitizes CRPC to treatment to the clinic anti-AR agent (enzalutamide), which provides a rationale for overcoming the therapeutic resistance often seen in advanced prostate cancers.

cancer biology↗

bioRxiv2025

Resorbable Poly-(D,L)-Lactide Anchorage of Nanoparticulate Mineralized Collagen Materials Maximizes In Vivo Skull Regeneration

BackgroundA clinical demand exists for bone biomaterials that mirror tissue-specific extracellular matrix (ECM) properties and regulate progenitor cell fate. Nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffolds have demonstrated to promote skull regeneration in vivo without addition of growth factors or exogenous progenitor cells, offering a materials-only solution for cranial defect reconstructions. Further enhancement of the safety and regenerative potential of MC-GAG is however necessary for clinical translation. Here, we investigated the regenerative effect of MC-GAG scaffold fixation with a resorbable poly(D,L-lactide) (PDLLA) implant for cerebral protection. MethodsFourteen-millimeter cranial defects were created in New Zealand white rabbits, divided into four groups: 1) defect only, 2) defect with PDLLA, 3) MC-GAG, and 4) MC-GAG with PDLLA. Initial bone healing assessment was conducted at 3 and 9 months using microcomputed tomography (microCT), histology, reference point indentation, and scanning electron microscopy. Long-term effects were evaluated through in vivo microCT imaging at 3, 6, and 9 months, with biomechanical testing of explanted skulls at 9 months. ResultsAt 3 months, MC-GAG significantly enhanced mineralization compared to empty and PDLLA-treated defects, with even greater mineralization observed for MC-GAG-PDLLA. Histologically, MC-GAG demonstrated trabecular mineralization, while MC-GAG-PDLLA had a structural organization resembling native bone. Biomechanical assessment showed enhanced toughness and stiffness for both MC-GAG and MC-GAG-PDLLA. At 6 months, MC-GAG-PDLLA displayed the highest bone mass, and at 9 months, both MC-GAG-containing scaffolds demonstrated superior bone regeneration compared to controls. By 9 months, PDLLA implants were completely resorbed and MC-GAG-PDLLA exhibited mechanical properties surpassing those of all other groups. ConclusionsFixation of MC-GAG scaffolds with PDLLA implants improved mineralization and bone regeneration compared to either material alone for cranial defect reconstruction. Clinical Relevance StatementCranioplasties are essential for cerebral protection and neurological restoration, but current materials have limitations, necessitating alternative bone biomaterials for improved outcomes.

bioengineering↗

bioRxiv2025

Engineering lung-sensing T cells using synthetic receptors targeting RAGE

Our goal was to leverage synthetic biology approaches to engineer lung-sensing T cells that trigger synthetic transcriptional programs only when in the lung. First, we identified a lung-specific cell surface protein, receptor for advanced glycation endproducts (RAGE), expressed at high levels exclusively in the lung. Then, we engineered chimeric antigen receptors (CAR) and synthetic notch receptors (SynNotch) to bind this protein. We showed that anti-RAGE CAR-T cells traffic to and proliferate in the lung exclusively. Anti-RAGE SynNotch receptors activate transcription of a fluorescent reporter only when co-cultured with RAGE+ cell lines or primary lung cells. Finally, we tested an anti-RAGE SynNotch to anti-CD19 CAR circuit in vivo in mice implanted with lung and flank tumors and found that this approach cleared CD19-expressing lung tumors without affecting CD19-expressing flank tumors. Thus, we demonstrate that RAGE is a lung-specific target and that T cells expressing anti-RAGE receptors can sense and activate lung-specific therapeutic transcriptional programs. This approach could be extended to allow cell-based therapies that target and deliver genetically encoded payloads to treat lung diseases while avoiding systemic toxicity.

synthetic biology↗

bioRxiv2025

Clonal Expansion of Alveolar Fibroblast Progeny Drives Pulmonary Fibrosis

Pulmonary fibrosis has been called a fibroproliferative disease but the functional importance of proliferating fibroblasts to pulmonary fibrosis has not been systematically examined. In response to alveolar injury, resting alveolar fibroblasts differentiate into fibrotic fibroblasts that express high levels of collagens. However, what role, if any, proliferation plays in the accumulation of fibrotic fibroblasts remains unclear. Through EdU incorporation, genetic lineage tracing, and single cell RNA sequencing, we resolve the proliferation dynamics of lung fibroblasts during post-injury fibrogenesis. Our data show substantial DNA replication in progeny of alveolar fibroblasts in two models of pulmonary fibrosis. By genetically labeling individual cells, we observe clonal expansion of alveolar fibroblast descendants principally in regions of fibrotic remodeling. The transcriptome of proliferating fibroblasts closely resembles that of fibrotic fibroblasts, suggesting that fibroblasts can first differentiate into fibrotic fibroblasts and then proliferate. Genetic ablation of proliferating fibroblasts and selective inhibition of cytokinesis in alveolar fibroblast descendants significantly mitigates pulmonary fibrosis and rescues lung function. Furthermore, fibroblasts in precision-cut lung slices from human fibrotic lungs exhibit higher proliferation rates than those in non-diseased lungs. This work establishes fibroblast proliferation as a critical driver of pulmonary fibrosis and suggests that specifically targeting fibroblast proliferation could be a new therapeutic strategy for fibrotic diseases.

cell biology↗

bioRxiv2025

Glycosylation-enabled Site-specific Growth Factor Engineering for Biomaterial Functionalization

In native tissue environments, growth factors (GFs) are often physically associated with the extracellular matrix (ECM) framework. Despite the enormous potential of the chemoselective click chemistry for GF functionalization of biomaterials to recapitulate such GF-ECM association, its application is limited by the lack of a universal strategy for reliable production of clickable GFs. Here we present a novel platform technology that leverages intrinsic post-translational protein glycosylation to enable site-specific metabolic engineering of GFs with azido tags for subsequent ECM hydrogel conjugation. Using Vascular Endothelial Growth Factor as a model, we demonstrated efficient, glycosylation-dependent azido incorporation during its recombinant expression with preserved bioactivity. We further expanded the utility of this strategy to non-glycosylated proteins through engineered N-linked glycosylation via the incorporation of a signal peptide that directs newly synthesized proteins to the secretory pathway where glycosylation takes place along with a sequon for glycan attachment. The resulting GF with site-specific azido incorporation can be effectively immobilized within dibenzocyclooctyne-bearing ECM hydrogel via the copper-free click chemistry, exhibiting sustained GF retention and delivering augmented angiogenic responses. Our approach thereby offers an unprecedented opportunity to streamline recombinant protein engineering for biomaterial functionalization in tissue engineering and regenerative medicine applications.

bioengineering↗

bioRxiv2025

Mechanistic basis for protection against fatty liver disease by CIDEB loss-of-function mutations

Background & AimsSomatic and germline CIDEB mutations are associated with protection from chronic liver diseases. The mechanistic basis and whether CIDEB suppression would be an effective therapy against fatty liver disease remain unclear. Methods21 CIDEB somatic mutations were introduced into cells to assess functionality. In vivo screening was used to trace Cideb mutant clones in mice fed normal chow, western (WD), and choline-deficient, L-amino acid-defined, high-fat (CDA-HFD) diets. Constitutive and conditional Cideb knockout mice were generated to study Cideb in liver disease. Isotope tracing was used to evaluate fatty acid oxidation and de novo lipogenesis. Transcriptomics, lipidomics, and metabolic analyses were utilized to explore molecular mechanisms. Double knockout models (Cideb/Atgl and Cideb/Ppara) tested mechanisms underlying Cideb loss. ResultsMost CIDEB mutations showed that they impair function, and lineage-tracing showed that loss-of-function clones were positively selected with some, but not all fatty liver inducing diets. Cideb KO mice were protected from WD, CDA-HFD, and alcohol diets, but had the greatest impact on CDA-HFD induced liver disease. Hepatocyte-specific Cideb deletion could ameliorate disease after MASLD establishment, modeling the impact of therapeutic siRNAs. Cideb loss protected livers via increased {beta}-oxidation, specifically through ATGL and PPARa activation. ConclusionsCideb deletion is more protective in some types of fatty liver disease. {beta}-oxidation is an important component of the Cideb protective mechanism. CIDEB inhibition represents a promising approach, and somatic mutations in CIDEB might predict the patient populations that might benefit the most.

physiology↗

bioRxiv2025

Phase transition pathways encode distinct physicochemical properties of biomolecular condensates

The same intrinsically disordered proteins (IDPs) can form biomolecular condensates through distinct thermodynamic phase transition processes, such as temperature-dependent and osmosis-dependent pathways. These distinct thermodynamic driving forces should, in principle, induce phase transition by modulating different features of the solvent environments, so that different driving forces should correspond with distinct sequence grammars for phase transition. However, whether the molecular driving force is pathway-dependent and how these different pathways can define the properties and functions of condensates are largely unknown. Here, by employing a diverse set of solid-state and solution-state NMR techniques, we uncover that different phase transition pathways of the monomer unit of an IDP define the types of molecular interactions driving phase transition and stabilizing dense phases. By establishing a complete chemical shift profile of the IDP unit, we identified a unique interaction mode that specifically initiates the upper critical solution temperature transition process, an anion-dependent cation-pi interaction, in which Asp acts as a bistable molecular switch regulating a stepwise Arg-Tyr interaction in a critical temperature-dependent manner. We further show that the pathway-dependent molecular interactions encode condensates formed by the same IDP to exhibit different physical and electrochemical properties, which in turn enable distinct functions of condensates. Our study shows that besides the defined sequence grammar of a given IDP, the molecular driving forces under specific transition processes are different and can determine the structure and properties of condensates, which emphasizes an overlooked role of transition pathway on encoding the functions of condensates.

biophysics↗

bioRxiv2025

Chemoselective Characterization of New Extracellular Matrix Deposition in Bioengineered Tumor Tissues

The extracellular matrix (ECM), present in nearly all tissues, provides extensive support to resident cells through structural, biomechanical, and biochemical means, and in return the ECM undergoes constant remodeling from interacting cells to adapt to the evolving tissue states. Bioengineered 3D tissues, commonly known as cell-ECM composites, are robust model systems to recapitulate and investigate native pathophysiology. Key to this engineered morphogenesis process are the intricate cell-ECM interactions reflected by how cells respond to and thereby modulate their surrounding microenvironments through their ongoing ECM secretome. However, investigating ECM-regulated new ECM production has been challenging due to the proteomic background from the pre-existing biomaterial ECM. To address this hindrance, here we present a chemoselective strategy to label, enrich, and characterize newly synthesized ECM (newsECM) proteins produced by resident cells, allowing distinction from the pre-existing ECM background. Applying our analytical pipeline to bioengineered tumor tissues, either built upon decellularized ECM (dECM-tumors) or as ECM-free tumor spheroids (tumoroids), we observed distinct ECM synthesis patterns that were linked to their extracellular environments. Tumor cells responded to the dECM presence with elevated ECM remodeling activities, mediated by augmented digestion of pre-existing ECM coupled with upregulated synthesis of tumor-associated ECM. Our findings highlight the sensitivity of newsECM profiling to capture remodeling events that are otherwise under-represented by bulk proteomics and underscore the significance of dECM support for enabling native-like tumor cell behaviors. We anticipate the described newsECM analytical pipeline to be broadly applicable to other tissue-engineered systems to probe ECM-regulated ECM synthesis and remodeling, both fundamental aspects of cell-ECM crosstalk in engineered tissue morphogenesis.

bioengineering↗

bioRxiv2025

AggreBots: configuring CiliaBots through guided, modular tissue aggregation

Ciliated biobots, or CiliaBots, are a class of engineered multicellular tissues that are capable of self-actuated motility propelled by the motile cilia located on their exterior surface. Correlations have been observed between CiliaBot motility patterns and their morphology and cilia distribution. However, precise control of these structural parameters to generate desired motility patterns predictably remains lacking. Here, we developed a novel Aggregated CiliaBot (AggreBot) platform capable of producing designer motility patterns through spatially controlled aggregation of epithelial spheroids made from human airway cells (referred to as CiliaBot Building Blocks or CBBs), yielding AggreBots with configurable geometry and distribution of active cilia. Guided multi-CBB aggregation led to the production of rod-, triangle-, and diamond-shaped AggreBots, which consistently effected greater motility than traditional single-spheroid CiliaBots. Furthermore, CBBs were found to maintain internal boundaries post-aggregation through the combined action of pathways controlling cellular fluidity and tissue polarity. This boundary fidelity, combined with the use of CBBs with immotile cilia due to mutations in the CCDC39 gene, allowed for the generation of hybrid AggreBots with precision control over the coverage and distribution of active cilia, further empowering control of motility patterns. Our results demonstrate the potential of AggreBots as self-propelling biological tissues through the establishment of morphological "levers" by which alterations to tissue motility can be theoretically planned and experimentally verified.

bioengineering↗