Abstract Spatial transcriptomic techniques provide a wealth of information useful in guiding drug development, while three-dimensional (3D) cell cultures have demonstrated power in accelerating drug approvals. However, techniques for robust spatial analysis of 3D cultures are limited. Here, we present a transfection-based method for constructing cellular spheroids through a layer-by-layer approach, in which DNA barcodes encode the spatial positioning of cells. Our technique facilitates multiplex single-cell RNA sequencing, providing spatial maps of gene expression and drug response, while correlative imaging reveals the locations of barcoded cell populations and quantifies local tissue elasticity. We show that model HeLa 3D spheroids display heterogeneous responses to drugs, which may arise through diffusion gradients of the drug, or from differences in metabolism, nutrient supply, and cellular stressors. The ability to create spatially encoded cellular assemblies may help to reveal spatial variation in gene expression within 3D culture models.

Abstract Human milk contains diverse live cells that contribute to infant nutrition, immune protection, and maternal-infant health, yet their cellular biology remains difficult to resolve within this lipid-rich and heterogeneous biofluid. Here, we adapted correlative light, electron, and ion microscopy (CLEIM) for nanoscale imaging and metabolic profiling of human milk cells (HMCs) and integrated these spatial measurements with single-cell transcriptomic analysis. This workflow combines confocal microscopy for cell-type identification, electron microscopy (EM) for ultrastructural mapping, nanoscale secondary ion mass spectrometry (NanoSIMS) for spatial elemental and isotope analysis, and single-cell RNA sequencing (scRNA-seq) for transcriptional interpretation. Using this platform, we generated an ultrastructural atlas of HMCs, including lactocytes, neutrophils, macrophages, dendritic cells, and bacteria-like structures. NanoSIMS revealed sulfur- and iron-enriched neutrophil granules, consistent with antimicrobial functions. Stable isotope tracing further uncovered distinct metabolic specializations among HMCs: 13C-glucose-derived carbon was preferentially incorporated into macrophage lipid droplets, supported by lipid-handling transcriptional features, whereas 15N-amino acid tracing identified metabolically active lactocyte subsets associated with translation and secretory programs. These findings reveal cellular and metabolic heterogeneity in human milk that is not captured by conventional profiling methods. More broadly, this work establishes a spatial multimodal framework for linking cell identity, ultrastructure, metabolic activity, and transcriptional state in complex biological fluids, providing a foundation for future studies of human milk biology and maternal-infant health.

Despite the revolutionary impact of genome engineering tools in medicine, the safe and effective intracellular delivery of CRISPR remains a major obstacle for clinical applications. Here, we implement precision molecular medicine and delivery strategies based on CRISPR/dCas9 systems adapted for epigenetic repression (dCas9-KRAB) to silence oncogenic drivers with high genomic selectivity. As proof-of-principle, we target the EWSR1-FLI1 translocation, which encodes a chimeric and hard-to-drug oncogenic transcription factor driving approximately 85% of the cases of Ewing Sarcoma (EWS)-an aggressive malignancy affecting children and adolescents. We describe the development of a non-viral and programmable polymeric system for the delivery of dCas9-KRAB as ribonucleoprotein (RNP) payloads for selective EWSR1-FLI1 repression. We demonstrate highly efficient intracellular delivery of RNPs loaded in polyamide-amine (PAMAM) polymers functionalized by guanidino groups, resulting in robust silencing of EWSR1-FLI1 both in established cell line xenografts and in patient-derived xenografts (PDXs) of EWS. Moreover, silencing of EWSR1-FLI1 is accompanied by potent anti-tumor effects. To our knowledge, we describe the first non-viral platform for in vivo delivery of dCas9-KRAB/RNPs, which can be adapted for the repression of any oncogene. We further outline dCas9/RNP formulations for future therapeutic applications to treat poor-prognosis cancers driven by hard-to-drug oncogenes.

Despite the revolutionary impact of genome engineering tools in medicine, the safe and effective intracellular delivery of CRISPR remains a major obstacle for clinical applications. Here, we utilize precision molecular targeting and delivery strategies based on CRISPR/dCas9 systems adapted for epigenetic repression (dCas9-KRAB) to silence oncogenic drivers with high selectivity. As proof-of-principle, we target the EWSR1-FLI1 translocation, which encodes a chimeric and hard-to-drug oncogenic transcription factor driving approximately 85% of the cases of Ewing Sarcoma (EWS)-an aggressive childhood malignancy. We describe the development of a programmable non-viral polymeric system for the delivery of dCas9-KRAB as ribonucleoprotein (RNP) payloads for EWSR1-FLI1 repression. We demonstrate highly efficient intracellular delivery of RNPs loaded in polyamide-amine (PAMAM) polymers functionalized by guanidino groups, resulting in robust silencing of EWSR1-FLI1 both in established cell line xenografts and in EWS-related patient-derived xenografts (PDXs) of EWS. We show that silencing of EWSR1-FLI1 is accompanied by potent anti-tumor effects. Collectively, we characterize an effective non-viral platform for in vivo delivery of dCas9-KRAB/RNPs, which could be adapted for the repression of any oncogene. We further outline dCas9/RNP formulations for future therapeutic applications to treat poor-prognosis cancers driven by hard-to-drug oncogenes.

Polymer-mediated gene delivery is evolving from stochastic design methodologies to precise molecular engineering. Traditional polymers, although effective in nucleic acid complexation, face challenges in terms of structural heterogeneity, unpredictable pharmacokinetics and inefficient endosomal escape. These challenges have driven interest in sequence-defined polymeric systems, which enable atomic-level control over monomer composition, charge distribution and functionality. Sequence-defined polymers provide opportunities to establish robust structure-function relationships, overcome biological barriers and achieve targeted delivery to specific tissues. This Review examines the architectural evolution of polymeric gene carriers and highlights how increasing structural precision correlates with enhanced functional performance. Synthetic methodologies enabling sequence control are analysed, from solid-phase approaches to flow chemistry and supramolecular templating. By integrating polymer science with biological outcomes, we present a strategic framework for addressing persistent challenges in non-viral gene delivery.

Human milk is a complex biofluid containing a diverse array of cells crucial for infant health. Despite their importance, our understanding of these cells remains incomplete due to technical challenges. To fully comprehend human milk cells, high-resolution imaging technologies that can directly measure biological processes are required. We have developed a specialized imaging platform combining light and electron microscopy for human milk cell imaging. To identify different cell types, human milk cells were first stained with several specific cell markers (e.g., EpCAM and MUC1 for lactocytes, CD16 and CD66b for neutrophils, and HLA-DR and CD68 for macrophages) prior to light (confocal) microscopy. Following this, the same cells were processed with osmium staining, resin embedding, and sectioning for electron microscopy, allowing us to observe ultrastructural details. Our imaging workflow has enabled nanoscale visualization of human milk cells, resulting in a first-of-its-kind comprehensive database profiling the organelle-level ultrastructure of different cell types present in human milk. The cells in the human milk are highly heterogenous, featuring a large proportion of lactocytes and lipid droplets, binucleated lactocytes, neutrophil aggregation, neutrophil extracellular traps, dendritic cells/macrophages with bacteria, and immunophagocytosis. This study provides valuable cellular insights contributing to a deeper understanding of human milk biology.

RNA secondary structures, including G-quadruplexes (G4s), have emerged as vital players in protein–RNA interactions. The RNA-binding protein Fused in Sarcoma (FUS), which is strongly implicated in both neurodegenerative disease and cancer, is known to interact with RNA molecules through a variety of GU-rich sequences. However, a definitive consensus motif for FUS–RNA recognition and binding has not yet been determined. Here, we hypothesize that G4 structures, which are inherently G-rich, may play a key role in FUS binding. We examine the role of G4s in FUS–RNA binding by developing an RNA immunoprecipitation sequencing (RIP-seq) protocol under G4-stabilizing and non-stabilizing conditions. We find that G4s regulate the binding of FUS to target RNAs, providing new information on protein–RNA binding motifs, while reinforcing the importance of RNA secondary structures as pivotal regulators of protein interactions. These insights advance our understanding of FUS–RNA binding dynamics and future potential for identifying new therapeutic targets for neurodegenerative disease and other FUS-related pathologies.

Brain metal homeostasis is essential for healthy neurological function, and disturbed brain metal homeostasis has deleterious consequences for neurodevelopment or cognitive outcome following injury or during disease. Specific regions of the brain (e.g. the hippocampus and subregions within) are known to be enriched with transition metals (i.e. ions of iron, copper, and zinc). Neither the physiological need for localized enrichment, nor the mechanisms driving the enrichment, however, are well understood. In this study we have applied a multimodal template, incorporating elemental mapping using X-ray fluorescence microscopy with spatial transcriptomics, to help reveal a molecular basis for metallomic heterogeneity across key subregions of the hippocampus. Our results reveal that significant differences in iron, zinc, and copper enrichment are associated with regional enrichment of specific transcripts related to metal transport, metal storage, and metal regulatory proteins. In addition to providing novel biological insight into the neurometallomic profile of the hippocampus, this study also provides an important template for others to integrate transcriptomics into multimodal workflows investigating the neurometallome.

Secondary structure is a principal determinant of lncRNA function, predominantly regarding scaffold formation and interfaces with target molecules. Noncanonical secondary structures that form in nucleic acids have known roles in regulating gene expression and include G-quadruplexes (G4s), intercalated motifs (iMs), and R-loops (RLs). In this paper, we used the computational tools G4-iM Grinder and QmRLFS-finder to predict the formation of each of these structures throughout the lncRNA transcriptome in comparison to protein-coding transcripts. The importance of the predicted structures in lncRNAs in biological contexts was assessed by combining our results with publicly available lncRNA tissue expression data followed by pathway analysis. The formation of predicted G4 (pG4) and iM (piM) structures in select lncRNA sequences was confirmed in vitro using biophysical experiments under near-physiological conditions. We find that the majority of the tested pG4s form highly stable G4 structures, and identify many previously unreported G4s in biologically important lncRNAs. In contrast, none of the piM sequences are able to form iM structures, consistent with the idea that RNA is unable to form stable iMs. Unexpectedly, these C-rich sequences instead form Z-RNA structures, which have not been previously observed in regions containing cytosine repeats and represent an interesting and underexplored target for protein-RNA interactions. Our results highlight the prevalence and potential structure-associated functions of noncanonical secondary structures in lncRNAs, and show G4 and Z-RNA structure formation in many lncRNA sequences for the first time, furthering the understanding of the structure-function relationship in lncRNAs.

Central nervous system (CNS) injuries and neurodegenerative diseases have markedly poor prognoses and can result in permanent dysfunction due to the general inability of CNS neurons to regenerate. Differentiation of transplanted stem cells has emerged as a therapeutic avenue to regenerate tissue architecture in damaged areas. Electrical stimulation is a promising approach for directing the differentiation outcomes and pattern of outgrowth of transplanted stem cells, however traditional inorganic bio-electrodes can induce adverse effects such as inflammation. This study demonstrates the implementation of two organic thin films, a polymer/reduced graphene oxide nanocomposite (P(rGO)) and PEDOT:PSS, that have favorable properties for implementation as conductive materials for electrical stimulation, as well as an inorganic indium tin oxide (ITO) conductive film. Transcriptomic analysis reveals that electrical stimulation improves neuronal differentiation of SH-SY5Y cells on all three films, with the greatest effect for P(rGO). Unique material- and electrical stimuli-mediated effects are observed, associated with differentiation, cell-substrate adhesion, and translation. The work demonstrates that P(rGO) and PEDOT:PSS are highly promising organic materials for the development of biocompatible, conductive scaffolds that will enhance electrically-aided stem cell therapeutics for CNS injuries and neurodegenerative diseases.

Antisense oligonucleotides (ASOs) are a well-established therapeutic modality based on RNA interference, but low cellular uptake, limited ability to direct ASO trafficking, and a range of intracellular barriers to successful activity compromise both gene silencing outcomes and clinical translations. Herein, we demonstrate that polymers can increase ASO internalisation via intracellular trafficking pathways that are distinct from lipid-based delivery reagents. For the first time, we spatially define internalisation and dissociation stages in the polymer-mediated cytosolic delivery of ASOs using Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS), which enables visualisation of ASO localisation at the organelle level. We find that polymer-ASO complexes are imported into cells, from which free ASO enters the cytosol following complex dissociation. This information enables a better understanding of the intracellular trafficking pathways of nucleic acid therapeutics and may be exploited for therapeutic delivery to enhance the effectiveness of nucleic acid therapeutics in the future.

Toll-like receptor (TLR) agonists are being developed as anti-cancer therapeutics due to their potent immunostimulatory properties. However, clinical trials testing TLR agonists as monotherapy have often failed to demonstrate significant improvement over standard of care. We hypothesized that the anti-cancer efficacy of TLR agonist immunotherapy could be improved by combinatorial approaches. To prevent increased toxicity, often seen with systemic combination therapies, we developed a hydrogel to deliver TLR agonist combinations at low doses, locally, during cancer debulking surgery. Using tumor models of WEHI 164 and bilateral M3-9-M sarcoma and CT26 colon carcinoma, we assessed the efficacy of pairwise combinations of poly(I:C), R848, and CpG in controlling local and distant tumor growth. We show that combination of the TLR3 agonist poly(I:C) and TLR7/8 agonist R848 drives anti-tumor immunity against local and distant tumors. In addition, combination of local poly(I:C) and R848 sensitized tumors to systemic immune checkpoint blockade, improving tumor control. Mechanistically, we demonstrate that local therapy with poly(I:C) and R848 recruits inflammatory monocytes to the tumor draining lymph nodes early in the anti-tumor response. Finally, we provide proof of concept for intraoperative delivery of poly(I:C) and R848 together via a surgically applicable biodegradable hydrogel.

DNA i-motif structures are formed in the nuclei of human cells and are believed to provide critical genomic regulation. While the existence, abundance, and distribution of i-motif structures in human cells has been demonstrated and studied by immunofluorescent staining, and more recently NMR and CUT&Tag, the abundance and distribution of such structures in human genomic DNA have remained unclear. Here we utilise high-affinity i-motif immunoprecipitation followed by sequencing to map i-motifs in the purified genomic DNA of human MCF7, U2OS and HEK293T cells. Validated by biolayer interferometry and circular dichroism spectroscopy, our approach aimed to identify DNA sequences capable of i-motif formation on a genome-wide scale, revealing that such sequences are widely distributed throughout the human genome and are common in genes upregulated in G0/G1 cell cycle phases. Our findings provide experimental evidence for the widespread formation of i-motif structures in human genomic DNA and a foundational resource for future studies of their genomic, structural, and molecular roles.

Epithelial-mesenchymal transition (EMT) is a reversible and dynamic biological process in which epithelial cells acquire mesenchymal characteristics including enhanced stemness and migratory ability. EMT can facilitate cancer metastasis and is a known driver of cellular resistance to common chemotherapeutic drugs, such as docetaxel. Current chemotherapeutic practices such as docetaxel treatment can promote EMT and increase the chance of tumor recurrence and resistance, calling for new approaches in cancer treatment. Here we show that prolonged docetaxel treatment at a sub-IC50 concentration inhibits EMT in immortalized human mammary epithelial (HMLE) cells. Using immunofluorescence, flow cytometry, and bulk transcriptomic sequencing to assess EMT progression, we analyzed a range of cellular markers of EMT in docetaxel-treated cells and observed an upregulation of epithelial markers and downregulation of mesenchymal markers in the presence of docetaxel. This finding suggests that docetaxel may have clinical applications not only as a cytotoxic drug but also as an inhibitor of EMT-driven metastasis and multidrug resistance depending on the concentration of its use.

Spatial transcriptomics has been widely used to capture gene expression profiles, realised as a two-dimensional (2D) projection of RNA captured from tissue sections. Wree-dimensional (3D) cultures such as spheroids and organoids are highly promising alternatives to oversimplified and homogeneous 2D cell culture models, but existing spatial transcriptomic platforms do not currently have sufficient resolution for robust analysis of 3D cultures. We present a transfection-based method for fluorescent DNA barcoding of cell populations, and the subsequent construction of spheroidal cellular architectures using barcoded cells in a layer-by-layer approach. For the first time, changes in gene expression throughout this 3D culture architecture are interrogated using multiplex single-cell RNA sequencing in which DNA barcodes are used to encode the spatial positioning of cells. We show that transfection with fluorophore-conjugated barcode oligonucleotides enables both imaging and sequencing at single-cell resolution, providing spatial maps of gene expression and drug response. Additionally, we show that fluorophore-conjugated DNA barcodes support correlative imaging studies such as mechano-microscopy to capture information about spatially-varying mechanical heterogeneity in 3D cultures. We ability to create customised, encoded cellular assemblies is a general approach that can resolve spatial differences in gene expression in 3D cell culture models.

Recurrences frequently occur following surgical removal of primary tumors. In many cancers, adjuvant therapies have limited efficacy. Surgery provides access to the tumor microenvironment, creating an opportunity for local therapy, in particular immunotherapy, which can induce local and systemic anti-cancer effects. Here, we develop a surgically optimized biodegradable hyaluronic acid-based hydrogel for sustained intraoperative delivery of Toll-like receptor 3 agonist poly(I:C) and demonstrate that it significantly reduces tumor recurrence after surgery in multiple mouse models. Mechanistically, poly(I:C) induces a transient interferon alpha (IFNα) response, reshaping the tumor/wound microenvironment by attracting inflammatory monocytes and depleting regulatory T cells. We demonstrate that a pre-existing IFN signature predicts response to the poly(I:C) hydrogel, which sensitizes tumors to immune checkpoint therapy. The safety, immunogenicity, and surgical feasibility are confirmed in a veterinary trial in canine soft tissue tumors. The surgically optimized poly(I:C)-loaded hydrogel provides a safe and effective approach to prevent cancer recurrence.

DNA G-quadruplexes (G4s) have been identified as important biological targets for transcriptional, translational, and epigenetic regulation. The stabilisation of G4s with small molecule ligands has emerged as a technique to regulate gene expression and as a potential therapeutic approach for human diseases. Here, we demonstrate that ligand stabilisation of G4s causes altered chromatin accessibility dependent on the targeting specificity of the molecule. In particular, stabilisation of a target G4 using the highly specific GTC365 ligand resulted in differential accessibility of 61 genomic regions, while the broad-targeting G4 ligand, GQC-05, stabilised many G4s and induced a global shift towards increased accessibility of gene promoter regions. Interestingly, while we observed distinct effects of each ligand on RNA expression levels and the induction of DNA double-stranded breaks, both ligands modified DNA damage response pathways. Our work represents the dual possibility of G4-stabilising ligands for specific or global chromatin modulation via unique targeting characteristics.

Cancer is a heterogenous disease, with multiple cellular subpopulations present within a single tumour mass that differ genetically and morphologically, and thus respond differently to chemotherapeutics. Epithelial-to-Mesenchymal transition (EMT) has been shown to play a role in tumour heterogeneity. Single-cell sequencing is critical to identify cell-type-specific transcriptomic differences with multiplexing methods increasing experimental scope with reduced cost. Cell hashing with barcoded antibodies is commonly used to multiplex samples but is limited to samples expressing target antigens. Antigen-independent methods of barcoding cells, such as barcoded lipid-anchors, have gained traction but present substantial populations that cannot be unambiguously demultiplexed. Herein we report a multiplexed single-cell transfection-enabled cell hashing sequencing (scTECH-seq) platform, which uses antigen-independent endocytic uptake to barcode cells, resulting in efficient, uniform barcoding with high cell recovery. We apply this methodology to identify distinct metastable cell states in human mammary cells undergoing EMT and show that stabilisation of G-quadruplex DNA has the potential to inhibit EMT.

Understanding the chemical events following trauma to the central nervous system could assist in identifying causative mechanisms and potential interventions to protect neural tissue. Here, we apply a partial optic nerve transection model of injury in rats and use synchrotron X-ray fluorescence microscopy (XFM) to perform elemental mapping of metals (K, Ca, Fe, Cu, Zn) and other related elements (P, S, Cl) in white matter tracts. The partial optic nerve injury model and spatial precision of microscopy allow us to obtain previously unattained resolution in mapping elemental changes in response to a primary injury and subsequent secondary effects. We observed significant elevation of Cu levels at multiple time points following the injury, both at the primary injury site and in neural tissue near the injury site vulnerable to secondary damage, as well as significant changes in Cl, K, P, S, and Ca. Our results suggest widespread metal dyshomeostasis in response to central nervous system trauma and that altered Cu homeostasis may be a specific secondary event in response to white matter injury. The findings highlight metal homeostasis as a potential point of intervention in limiting damage following nervous system injury.