Biology subjects

Wong, J.

Publications and source records attributed to Wong, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

LCR-modules: a collection of workflows for cancer genome analysis

The surge of genomic data from advanced sequencing technologies is outpacing current analytical pipelines. We introduce LCR-modules, an open-source suite of bioinformatics tools designed for flexible and automated cancer genome data analysis. LCR-modules enables reproducible analysis of diverse cancer genomics data at scale. The suite comprises 49 Snakemake-based workflows organized into three levels, facilitating tasks from low-level quality control to complex cohort-level analyses. LCR-modules supports various sequencing types and integrates pipelines such as mutation calling, expression quantification, and cohort-level aggregation, ensuring flexibility and reproducibility. LCR-modules represents a significant advancement in genomic data analysis, reducing barriers in reproducibility and scalability and has already been applied to a combination of exomes and genomes from over 10,800 samples.

bioinformatics↗

bioRxiv2025

Bicistronic CAR T-cells Against CD70 & Active Integrin β2 Overcome Antigen Heterogeneity and Preserve Safety in Acute Myeloid Leukemia

The surface antigen landscape of acute myeloid leukemia (AML) displays significant heterogeneity and overlap with healthy hematopoietic cells. This imparts a substantial hurdle to the development of AML-targeting chimeric antigen receptor (CAR) T-cells that can avoid on- target, off-tumor toxicity. Here, we develop a dual-antigen targeting CAR-T against CD70 and the active conformation of integrin {beta}2 (aITGB2), each previously reported as promising AML targets due to minimal off-tumor expression. We show an OR-gated approach for these antigens significantly increases the proportion of AML blasts that can be targeted, in part using a novel ex vivo co-culture method to restore surface protein homeostasis following a freeze-thaw cycle. We test dual-targeting CAR-T constructs with different combinations of costimulatory domains, identifying constructs with superior anti-tumor cytotoxicity in vitro against AML cell line and patient-derived xenograft models. We further show significantly improved in vivo tumor clearance and survival for a dual-targeting CAR in murine models of AML tumor heterogeneity. Finally, we show that this dual-targeting CAR does not increase off-tumor toxicity, especially against hematopoietic stem and progenitor cells. Together, these findings demonstrate a promising clinically-translatable approach for the treatment of AML without the notable toxicity liabilities associated with other leading CAR-T targets for this disease.

immunology↗

bioRxiv2025

Identification and Characterization of Novel Chikungunya Virus Polymerase Inhibitors

Chikungunya virus (CHIKV) and other alphaviruses in the Togaviridae family are positive-sense RNA viruses and major human pathogens, causing millions of infections worldwide. In humans, alphaviruses such as CHIKV, Mayaro and Ross River viruses typically cause arthritogenic disease characterized by debilitating arthralgia, joint inflammation, fever, and rash. Although a vaccine was recently approved for use against CHIKV, no vaccines are licensed against other alphaviruses. No antiviral treatments are available to prevent or treat infections by any alphavirus. To address this unmet need, we used a CHIKV nanoluciferase reporter virus to develop a high-throughput screening assay for novel small-molecule inhibitors. From this campaign, we identified several unique inhibitors of CHIKV replication. Mechanistic characterization of two inhibitors revealed that both target the nsP4 RNA-dependent RNA polymerase, while susceptibility profiling pinpointed unique nsP4 mutations that specifically confer resistance. In silico docking analyses indicated potential binding poses of the inhibitors near the polymerase active site. Collectively, these results define multiple chemotypes for further development and highlight novel molecular targets within nsP4 for CHIKV inhibition. IMPORTANCEChikungunya virus is a mosquito-borne pathogen that has caused millions of human infections worldwide, producing severe fever, rash, and long-lasting joint pain that can persist for months. Related viruses such as Mayaro and Ross River viruses also cause debilitating disease, yet no antiviral drugs are available to treat any infection caused by this family of viruses. In this study, we developed a high-throughput assay that allowed us to rapidly identify compounds capable of blocking chikungunya virus replication. We discovered new hit compounds that inhibit virus growth. In addition, we determined that two of the most promising hit candidates target the viral nsP4 polymerase. By identifying these novel inhibitors and characterizing both their mechanisms of action and resistance profiles, we have established the groundwork for future efforts to develop much needed therapies against chikungunya virus and related pathogens.

molecular biology↗

bioRxiv2025

Impact of Mutations Affecting 4'-Fluorouridine Susceptibility on Fitness and Treatment Outcomes for Venezuelan Equine Encephalitis Virus

Venezuelan equine encephalitis virus (VEEV) is a prototypical encephalitic alphavirus. Members of the Alphavirus genus are found across the globe, transmitted by arthropod vectors, and cause significant disease burdens in humans and animals. There are currently no FDA-approved antivirals against any member of the Alphavirus genus. While a vaccine exists against chikungunya virus (CHIKV), a member of the arthitogenic alphaviruses, FDA-approved vaccines are not available for other members of this genus, particularly the encephalitic alphaviruses such as VEEV, Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV). 4-Fluorouridine (4-FlU, EIDD-2749) was recently identified as a broad-spectrum antiviral against multiple RNA viruses, including alphaviruses. 4-FlU can potently inhibit VEEV-TC83 replication, with submicromolar potency in cell culture. However, the emergence of antiviral resistance represents a hurdle for antiviral drug development and the implementation of effective treatment strategies. Here, we have identified novel mutations in the VEEV nsP4 RNA-dependent RNA polymerase that reduce susceptibility to 4-FlU, including P187A, Q191L, L289F, and T296I. We rebuilt each mutation in recombinant VEEV-TC83 and characterized the effects of these mutations on fitness and pathogenicity. In addition, we assessed the impact of mutations reducing sensitivity to 4-FlU in a mouse model. Although mutations against 4-FlU arise quickly in vitro, treatment can still alleviate severe disease and lethal encephalitis. Together, these data highlight the promising therapeutic potential of 4-FlU for the treatment of alphavirus encephalitis. ImportanceVEEV is one of several mosquito-spread viruses that can cause serious brain infections in people and animals. Unlike CHIKV, against which an approved vaccine exists, there are no countermeasures to prevent or treat VEEV infections or block its close relatives, EEEV and WEEV. 4-FlU inhibits many different RNA viruses, including VEEV, and is currently being developed to treat multiple viral infections. A major problem with small-molecule antivirals is the appearance of virus populations that are less susceptible to treatment. In this study, we identified mutations in VEEVs RNA-dependent RNA polymerase (nsP4) that confer reduced susceptibility to 4-FlU. We then engineered these mutations into full-length infectious clones and assessed if viruses encoding for these mutations were still pathogenic. In addition, we treated infected mice with 4-FlU and measured how well the compound inhibited virus replication and prevented severe disease, even when mice were infected with viruses harboring mutations that reduced susceptibility to 4-FlU. Although VEEV can develop moderate resistance to 4-FlU in vitro, administration of 4-FlU still reduced severe disease and prevented lethality in the animals infected with viruses that possess mutations that decrease susceptibility to 4-FlU. These results suggest that 4-FlU has strong potential as a future treatment for alphavirus infections like VEEV encephalitis.

microbiology↗

bioRxiv2025

A Mammalian Genomic Signature Shaped by Single Nucleotide Variants Controlling Transcriptome Integrity and Diversity

BackgroundMany functional features of mammalian genomic sequences remain poorly defined, especially how sequence motifs and genetic variants within non-coding regions (NCRs) regulate transcriptome integrity and diversity. We have shown that G-tracts unusually positioned between the polypyrimidine tract and 3' AG repress usage of the AG and are enriched at cryptic splice sites in cancer cells but their broader role across the extensive NCRs of mammalian genomes is unknown. ResultsHere, we identify a widely evolved genomic signature, G-tract-AG motifs consisting of guanine tracts closely upstream of AG dinucleotides, which is significantly associated with single-nucleotide variants (SNVs) identified in genome-wide association studies, particularly within NCRs. Approximately 9,000 such G-tracts within human genes are disrupted by variants of the cis-splicing quantitative trait loci in the Genotype-Tissue Expression project. Functionally, G-tracts repress splicing at the adjacent 3' AG, primarily by stalling the second transesterification step. Disruption of G-tracts by SNVs relieves this repression, enabling splicing and generating novel transcript isoforms. These G-tract-disrupting SNVs are in cis across the majority of protein-coding genes and are among thousands of rare variants causing genetic diseases. ConclusionsG-tract-AG signatures are widespread bipartite motifs with dual functions: G-tracts repress AG usage to safeguard transcriptome integrity, while SNV-induced disruption releases AGs for splicing to promote transcriptome diversity. Our findings provide mechanistic insights into the regulation of transcriptome integrity and diversity by a mammalian genomic signature, particularly for NCR SNVs associated with diverse traits and a new framework for their functional annotation.

genetics↗

bioRxiv2025

Mechanistic insight into the regulation of virulence factor type 1 pili in pathogenic E. coli by rhomboid protease GlpG

With the rise in antimicrobial resistance, understanding the virulence factors utilized by pathogenic E. coli is essential for the development of alternative therapeutics. While previous work has shown that disruption of the E. coli rhomboid protease gene glpG leads to defects in bacterial colonization, here we provide mechanistic insight into the loss of fitness. We show GlpG is essential for the assembly of type 1 pili, a virulence factor required for the colonization of eukaryotic cells. Since pili are critical for biofilm formation and bacterial persistence, the absence of GlpG proteolytic activity reduces the production of biofilm. Working towards new potential antimicrobial targets for treating infections, we show that biofilm formation is hampered by GlpG inhibition. Our data demonstrates that GlpG plays a key role in protein quality control of type 1 pili and alters the paradigm for GlpG proteolysis, previously implicated in the cleavage of only membrane embedded substrates.

biochemistry↗

bioRxiv2025

Evaluation of Klebsiella pneumoniae antigen MrkA reveals expression-limited therapeutic potential

Klebsiella pneumoniae (KP) poses a major global health concern, particularly as hospital-acquired strains become increasingly antibiotic resistant. MrkA, the major type 3 fimbrial (T3F) subunit, is a recognised prospective vaccine and monoclonal antibody (mAb) target. We assessed T3F structural (mrkABCDF) and regulatory (mrkHIJ) gene prevalence and allelic variation across 1649 KP genomes, using 100 diverse isolates for detailed phenotypic characterisation. Despite high conservation MrkA expression itself is variable with genotype failing to predict phenotype. We constructed isogenic KP T3F mutants with absent and supraphysiological MrkA expression to investigate functional consequences and therapeutic responses during infection. In a severe pneumonia model blood dissemination inversely correlated with T3F expression. In vivo antigen profiling revealed only [~]20% of wild-type lung bacteria express MrkA, dropping to [~]5% in blood, with low MrkA expression defining bacteraemic KP irrespective of infection route. Critically, anti-MrkA mAb efficacy was contingent on antigen abundance, with therapeutic benefit restricted to supraphysiological MrkA expression. These findings reveal substantial in vivo MrkA expression variability with critical onward implications for vaccine and mAb development.

microbiology↗

bioRxiv2025

Fine needle aspiration biopsy of breast specimens effectively harvests cells for patient-derived organoids modeling breast ductal carcinoma in situ

BackgroundPatient-derived organoids (PDOs) generated from benign breast tissue and breast carcinomas have successfully recapitulated their respective in vivo counterparts. PDOs model tumorigenesis and allow for screening of novel therapeutics personalized to individual patients. However, acquiring cells to generate PDOs is cumbersome. We demonstrate the feasibility of fine needle aspiration biopsy (FNAB) for harvesting cells for PDOs modeling ductal carcinoma in situ (DCIS). MethodsSurgical specimens from patients with biopsy-proven DCIS were used for this study. Core needle biopsy (CNB) was performed on fresh specimens in the operating room, and tissue was mechanically dissociated before culture in basement membrane extract (BME) and organoid medium to generate PDOs. FNAB was performed in the gross room on fresh specimens, and the remaining aspirate was similarly submitted for PDO culture. ResultsPDOs were successfully generated in 15/18 specimens obtained by CNB and 7/11 specimens obtained by FNAB. The average time to initial organoid growth was 4 days for FNAB specimens compared to 19.3 days for CNB specimens. Tumor cells were seen on 7/11 FNAB smears and 16/18 CNB touch preps. Immunofluorescence staining confirmed the presence of both luminal and myoepithelial cells in derived PDOs. ConclusionsFNAB effectively obtains cells for PDOs modeling DCIS. CNB after mincing yielded PDOs with a high success rate, but they were slow to establish. Notably, the time to organoid growth was significantly shorter for FNAB specimens. Thus, FNAB offers an efficient alternative for breast PDO culture and can reduce the time and resources spent on generating PDO cultures.

cell biology↗

bioRxiv2025

Commensal taxa in gut microbiota limit antibiotic resistance during extended oral antibiotic use

Certain bacterial infections, such as those involving prosthetics, can require antimicrobial therapy over months to years, potentially increasing the burden of antimicrobial resistance. Here we longitudinally track the antimicrobial resistome in mice during continuous antibiotic dosing over 21 months. The burden of antibiotic resistance genes (ARGs) initially increases, but, surprisingly, declines in later months, approaching levels observed in untreated animals. ARG burden is regulated by taxonomy and declines as ARG-harboring taxa that initially bloom are replaced by commensals. Furthermore, we find that the dynamics of antibiotic-induced ARG burden are influenced by age-related differences in microbial taxonomy and can be removed by fecal microbiota transplantation. We show that commensals may regulate the resistome by limiting the growth of ARG-harboring taxa, thereby providing antimicrobial expansion resistance.

microbiology↗

bioRxiv2025

High precision fluorescence tomography-guided system for pre-clinical radiation research: system design and validation

PurposeCone-beam computed tomography (CBCT), commonly used for image guidance in pre-clinical studies, is limited in soft tissue localization and lacks the functional information needed for image-guided radiation studies and treatment assessment. To address these limitations, we developed 3D fluorescence tomography (FT) for high precision functional image-guided research and integrated it with small animal irradiators. MethodsThe FT system, integrated with a commercial bioluminescence-guided platform in a standalone configuration, enabling compact multi-projection and multi-spectral imaging. A dual-axis galvo mirror scanner was used for laser spots scanning to excite internal fluorophore. A transportable mouse bed allows animals imaged in the optical system and transferred to irradiators for CBCT imaging and FT-guided irradiation. Spatial geometry-based methods were developed to map the laser spots and fluorescence images onto the animal numerical mesh surface, generated from the CBCT image, to serve as input data for FT reconstruction. A self-calibration method using Born-ratio data, fluorescence normalized to the excitation image, was adopted for reconstruction. Mouse phantom embedded with QD800 reporter and orthotopic glioblastoma (GBM) model injected with IRDye 800CW labelled U87-EGFR cells were used to verify the FT-guided system in target localization. ResultsThe laser spot mapping accuracy are within 0.7 mm maximum deviation. Our system can reconstruct the target QD800 as deep as 17 mm in the mouse phantom with single projection fluorescence imaging at localization accuracy < 1 mm deviation. Using the Born ratio approach, we effectively eliminated excitation light leakage and autofluorescence, enabling our FT system to localize IRDye 800CW-labeled EGFR-overexpressing GBM cells in the mouse brain with approximately 1 mm accuracy. ConclusionWe developed a compact FT system integrated with a small animal irradiator, achieving sub-millimeter localization accuracy in phantom and in vivo models. Its precision, flexibility, and compatibility position it as a powerful tool for advancing functional imaging-guided pre-clinical radiation research.

bioengineering↗

bioRxiv2025

DiffMethylTools: a toolbox of the detection, annotation and visualization of differential DNA methylation

DNA methylation is a fundamental epigenetic mechanism, and its significant changes (i.e., differential methylation) regulate gene expression, cell-type specification and disease progression without altering the underlying DNA sequence. Differential methylation was usually detected via existing statistical tools by comparing two groups of methyomes (i.e. whole-genome methylation profiles) and has wide applications of various downstream investigations for human disease studies. However, few toolboxes were available to efficiently streamline methylation investigation by integrating robust detection, annotation and visualization of differential methylation. Also, differential methylation detected via tools has poor reproducibility and no tools were tested on the increasing volume of long read methylomes. To address these issues, we introduced DiffMethylTools, an end-to-end solution to eliminate analytical and computational difficulties for differential methylation dissection. Comparison of detection performance on six datasets including three long-read methylomes demonstrated that DiffMethylTools achieved overall better performance of detecting differential methylation than existing tools like MethylKit, DSS, MethylSig, and bsseq. Besides, DiffMethylTools supported versatile input formats for seamless transition from upstream methylation detection tools, and offered diverse annotations and visualizations to facilitate downstream investigations. DiffMethylTools therefore offered a robust, interpretable, and user-friendly solution for differential methylation investigation, benefiting the dissection of methylations roles in human disease studies.

bioinformatics↗

bioRxiv2025

Stable coexistence of Citrobacter rodentium with a lytic bacteriophage during in vivo murine infection

Bacteriophages are ubiquitously present in bacterial communities, yet phage-bacteria interactions in complex environments like the gut remain poorly understood. While antibiotic resistance is driving a renewed interest in phage therapy, most studies have been conducted in in vitro systems, offering limited insight into the complexity of such dynamics in physiological contexts. Here, we use Citrobacter rodentium (CR), a natural mouse-restricted enteric pathogen and well-established model for human enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) infections, to investigate phage-pathogen interactions in vivo. We isolate and characterise Eifel2, a novel lytic phage infecting CR, and generate anti-phage specific antibodies that enable the visualisation of phage infections in vitro. In a murine model of CR infection, oral administration of Eifel2 led to robust phage replication in the gut without reducing the bacterial burden or infection-associated inflammation, confirming the establishment of a stable coexistence in the gut. Despite the emergence of a sub-population of phage-resistant CR mutants in vivo, they did not undergo clonal expansion, indicating that additional selective pressures impaired their widespread dissemination in the gut. Together, our findings demonstrate that imaging approaches can capture key infection stages in vitro, while in vivo models are essential for capturing the complexity of phage-bacteria interactions. This work highlights the importance of studying phage therapy in host-pathogen contexts that include a normal microbiota and a suitable host environment, where dynamic co-existence rather than eradication may define therapeutic outcomes. ImportanceBacteriophages, or phages, are viruses which can either kill or persist inside bacteria. Current interests in phage biology are in part ignited by the fact that they could be used to treat infections caused by antibiotic-resistant bacteria. However, most of our understanding of phage-bacterial interactions comes from in vitro models and/or in vivo gut models relying on altering the endogenous microbiota. Here we report the finding of a novel phage, Eifel2, which specifically targets Citrobacter rodentium (CR), the mouse equivalent of human diarrhoeagenic E. coli pathogens. Despite effectively killing CR in vitro, CR and Eifel2 develop a coexistence relationship in mice with an intact microbiota. While CR phage-resistant mutants emerge, host and microbial factors constrain their expansion. This work highlights the importance of studying phage therapy in host-pathogen contexts that include the complete microbiota, where therapeutic outcomes may rely on dynamic co-existence and containment rather than eradication.

microbiology↗

bioRxiv2025

Denoising 7T Structural MRI with Conditional Generative Diffusion Models

Purpose7T MRI offers ultra-high resolution and improved sensitivity for iron deposition in neurodegenerative disorders, but commonly used acquisitions are long and hence challenging, especially for elderly subjects. Efficiently denoising a short acquisition to achieve the image quality of a longer acquisition would be of translational benefit. Materials and MethodsWe introduce a conditional diffusion model derived from generative AI (a 7T Conditional Diffusion Model, 7TCDM) that was trained on native single-acquisition 2D reconstructions and referenced multi-repetition images to guide the denoising process and improve SNR and contrast. 7TCDM model was tested on 2D T2-weighted gradient-echo imaging from 19 participants, including healthy controls and individuals with mild cognitive impairment or Alzheimers disease (AD). 7TCDMs performance was assessed using Mean Squared Error (MSE), Peak Signal-to-Noise Ratio (PSNR), Structural Similarity Index Measure (SSIM), and comprehensive reader studies. ResultsReferencing the multi-repetition ground truth, 7TCDM improved the single-acquisition original image by 29.1% in MSE, 5.8% in PSNR, and 9.4% in SSIM, and outperformed convolutional neural network-based models in all metrics. Expert rater evaluations confirmed superior image quality, with significantly enhanced detail and contrast preservation in regions such as the hippocampi, white matter lesions, and small cortical veins. The model also demonstrated robust performance in both the concurrently acquired and publicly available 3D multi-echo gradient echo acquisitions, which the model was not trained on. ConclusionsThe 7T Conditional Diffusion Model provides high-quality denoised images from shorter scans, increasing the feasibility of scanning patients in shorter times while preserving essential anatomical and pathological details.

neuroscience↗

bioRxiv2025

Oncogenic virus hijacks SOX18 pioneer function to enhance viral persistence

Kaposis sarcoma herpesvirus (KSHV) establishes lifelong oncogenic infection in lymphatic endothelial cells (LECs) by ensuring episomal maintenance of its genome via the viral protein LANA. Efficient viral genome maintenance typically involves host DNA replication and episome tethering, but the extent of cell-type-specific regulation remains unclear. Here, we identify that KSHV hijacks the pioneering function of the endothelial-specific transcription factor SOX18 to facilitate persistence of viral episomes. Upon infection, LANA co-opts SOX18 to recruit the SWI/SNF chromatin-remodeling complex via its ATPase subunit BRG1, enhancing chromatin accessibility and enabling efficient viral genome persistence. Disruption of SOX18 or BRG1--genetically or pharmacologically--leads to reduced episome load and attenuated hallmarks of virus infection. This work highlights how viruses can harness lineage-specific transcriptional regulators to establish persistent nuclear retention of their episome into the host genome.

molecular biology↗

bioRxiv2025

Assessing the Effectiveness of 3D-Printed Ceramic Structures for Coral Restoration: Growth, Survivorship, and Biodiversity Using Visual Surveys and eDNA

Coral reef degradation has spurred the development of artificial structures to mitigate losses in coral cover. These structures serve as substrates for coral transplantation, with the expectation that growing corals will attract reef-associated taxa -- while the substrates ability to directly support biodiversity is often neglected. We evaluated a novel 3D-printed modular tile made of porous terra cotta, designed with complex surface structures to enhance micro- and cryptic biodiversity, through a restoration project in Hong Kong. Over four years, we monitored 378 outplanted coral fragments using diver assessments and photography, while biodiversity changes were assessed through visual surveys and eDNA metabarcoding. Coral survivorship was high, with 88% of transplants surviving by the studys end. The restoration site exhibited greater fish and macroinvertebrate abundance compared to a nearby unrestored area. eDNA analyses revealed 23.5% higher eukaryote ASV richness at the restoration site than the unrestored site and a 13.3% increase relative to a natural reference coral community. This study highlights the tiles dual functionality: (1) supporting coral growth and (2) enhancing cryptic biodiversity, an aspect often neglected in traditional reef restoration efforts. Our findings underscore the potential of 3D-printed ceramic structures to improve both coral restoration outcomes and broader reef ecosystem recovery.

ecology↗

bioRxiv2025

Targeted Irradiation and STAT3 Inhibition Reprogram the AML Microenvironment and Extend Survival: Toward Translational Immunoradiotherapy

PurposeDespite advances in acute myeloid leukemia (AML) therapy, relapses remain challenging. While AML is radiation-sensitive, total body irradiation (TBI) causes organ toxicities and activates tolerogenic/proangiogenic STAT3 signaling. CSI-2, a myeloid cell-targeted STAT3 inhibitor, promotes anti-leukemic immune responses but has limited efficacy against high disease burden. We investigated whether image-guided targeted marrow irradiation (TMI), which focuses radiation on leukemia sites while sparing critical organs, could synergize with CSI-2 to improve leukemia clearance and establish durable immunity. MethodsMice were intravenously engrafted with CMM-AML cells reaching 20-30% in bone marrow (BM) infiltration (moderate-to-high disease burden) before receiving IV-injections of CSI-2 (5mg/kg) with or without TMI. Fluorescently labeled CSI-2 biodistribution was assessed using flow cytometry and quantitative multiphoton microscopy. Survival was monitored for 3-4 months before evaluating BM composition using flow cytometry and immunohistochemistry. ResultsTMI significantly improved vascular permeability and scavenger receptor/TLR9-dependent uptake of CSI-2 by AML cells and leukemia-associated myeloid cells. Combined TMI/CSI-2 treatment more effectively reduced high leukemia burden than CSI-2 alone, achieving >80% survival at 120 days with increased CD8+ cytotoxic and CD4+ helper T cell infiltration. TMI/CSI-2-treated mice were protected from AML rechallenge suggesting that they developed protective immune memory. In an aggressive MLL-AF9 AML model, TMI/CSI-2 combination significantly extended survival compared to either monotherapy. ConclusionTMI/CSI-2 strategy represents a novel organ-sparing immunoradiotherapy that synergistically enhances leukemia clearance while promoting long-term protective immunity. These findings warrant further investigation of this strategy for high-burden or relapsed AML and provide the foundation for clinical translation.

immunology↗

bioRxiv2025

Unraveling Tyrosine-Kinase Inhibitor Resistance in NSCLC Cells via Same-cell Measurement of RNA, Protein, and Morphological Responses

Non-small cell lung cancer (NSCLC) frequently develops resistance to tyrosine kinase inhibitors (TKIs), limiting the long-term success of targeted therapies. A deeper understanding of resistance mechanisms at the molecular and cellular levels may enable the development of more effective treatment strategies. Here, we applied the Teton detection assay on the AVITI24 platform to measure concurrently RNA, protein, and cellular morphology in NSCLC cell lines treated with the TKIs gefitinib and osimertinib. This single-cell, multiomic analysis revealed distinct expression and morphological profiles between drug-sensitive and resistant cells, including differences in MAPK-related pathway activity. Stratifying responses at the single-cell level uncovered subtle responses not detectable in bulk measurements. We identified CDK4/6 activity as a route of cell survival under TKI treatment and demonstrated that co-treatment with the CDK4/6 inhibitor palbociclib enhanced TKI efficacy. The ability to measure multiomics and cellular morphology in the same cells opens new avenues for future studies aimed at improving personalized treatment strategies in NSCLC and overcoming the obstacles posed by drug resistance.

genomics↗

bioRxiv2025

High-Throughput Multiomics Profiling of Model Systems Using the AVITI24 Platform

We present a multiomics platform comprising Teton, a detection assay system, and AVITI24, a dual-flowcell instrument that performs both cellular imaging and sequencing readout. Teton integrates a compartmentalized flowcell for cell culture with methods to measure morphology, RNA, and protein at subcellular resolution. The platform quantifies morphological features through cell painting of 6 cellular components, RNA expression of up to 350 transcripts via sequencing of oligonucleotides hybridized to mRNA, and protein expression of up to 200 targets using antibody-linked oligonucleotide sequencing. The flow cell accommodates >1 million cells in a 10 cm squared open-well format or can be subdivided into 12 or 48 wells to support experiments with multiple conditions or time points. We describe and validate the detection methods of the platform and showcase its capabilities by co-culturing three cancer cell lines and elucidating the cellular pathways triggered by various drug treatments as a function of time. Using multiple time points enables us to capture the dynamics of cellular processes including receptor activation and signaling cascades. The results demonstrate how different cancer cells evade TNF-induced apoptosis by activating compensatory signaling programs that maintain survival despite pro-apoptotic cues. Our model system replicates previously published results and highlights the versatility of the platform in enabling rapid, high-throughput analysis of complex cellular responses in varied biological contexts.

genomics↗