Biology subjects

Deshpande, A.

Publications and source records attributed to Deshpande, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Automation of high-throughput arrayed lentivirus production and titration

Generation of arrayed genome-wide CRISPR libraries in a ready-to-transduce lentiviral format remains laborious, time-consuming, and costly. To address these limitations, the present study developed a fully automated lentivirus production and titration workflow using a Biomek i7 Hybrid automated workstation, integrated with multiple instruments and managed by SAMI EX software. The workflow produced and titrated viruses in 96 and 384-well plate formats, respectively. It employed reverse transfection and triplicate wells per lentivector to reduce variability and yielded an average of three viral particles in transduction unit (TU) per producing HEK293T cell. Titration was performed using U937-mCherry suspension cells, with the percentage of transduced cells converted from U937 (X%) to HEK293T (Y%) values via a linear regression equation (Y% = 4.3X% + 9.3%). The titer calculation was based on the initial seeding cell number, the converted percentage of HEK293T transduced cells, and virus input volume. The titration demonstrated strong reproducibility across LSRFortessa (BD) and Aurora (Cytek) flow cytometers (R2 = 0.9). Among 1,760 unconcentrated virus preparations, median and mean titers reached approximately 1.2 x 106 TU/mL, with over 97% of samples exceeding the high-titer threshold of 2x105 TU/mL, thus demonstrating a robust, scalable, and cost-effective automation platform for high throughput arrayed lentiviral library production and titration.

microbiology↗

bioRxiv2025

Automation of high-throughput arrayed mammalian cell line cultivation

Cell culture automation has traditionally been limited to basic tasks at low throughput, which are insufficient for passaging rapidly proliferating cell lines or for generating stable clonal lines. To address unmet needs, this study implemented a Biomek i7 Hybrid automated workstation, integrated with peripheral instruments and coordinated by SAMI EX software, to enable automated, high throughput mammalian cell culture workflows. The workflows support cell density monitoring, arrayed passaging, sample cherry-picking, plate reformatting, cell density normalization, and cryopreservation in 96-well plates. Integration with the CloneSelect imager allows rapid confluency monitoring and monoclonality assessment (<100 sec per plate). Cell passaging and density normalization require 32 minutes for one plate and 61 minutes for two plates. Workflow consistency was demonstrated across multiple cell lines and biological replicates, with wells showing comparable confluency within three standard deviations, lower coefficient of variation, and substantially narrower interquartile ranges after a single cell passage and density normalization. Four automation pipelines, including monoclonality screening, cell passaging and cherry-picking, density normalization, and cryopreservation, collectively enable clonal line establishment. Depending on scale, one to eight 384-well plates were processed in 69 to 355 minutes, yielding an average of 35 clonal lines per plate suitable for downstream genomic DNA sequence confirmation.

cell biology↗

bioRxiv2025

Nucleosome stability safeguards cell identity, stress resilience and healthy aging

Nucleosomes are the minimal repeating units of chromatin. Their dynamic assembly and disassembly underpins chromatin organization and genome regulation. However, it remains unclear how intrinsic nucleosome stability contributes to higher-level yet fundamental cellular and organismal properties--such as preservation of cell identity, lineage specification, stress resilience and ultimately healthy aging. To address this, we tested the impact of decreased intrinsic nucleosome stability across multiple cell, tissue and organismal models by introducing histone mutants that weaken histone-histone interactions. While nucleosome instability did not broadly alter global chromatin accessibility, DNA damage, cell proliferation or viability, it impaired lineage-specific gene expression programs, altered lineage specification and activated intrinsic inflammatory and stress pathways in a manner reminiscent of aging in mouse tissues and human cells. Consistently, nucleosome instability accelerated the onset of age-associated transcriptional alterations and functional decline in Caenorhabditis elegans and Drosophila melanogaster, and reduced cellular resilience to exogenous perturbations-- including environmental, epigenetic and mitotic stress--in human cells and Saccharomyces cerevisiae. These cross-species findings identify nucleosome stability as an evolutionarily conserved epigenetic safeguard that preserves cell identity and stress resilience and supports organismal function and healthy aging.

genomics↗

bioRxiv2025

The host protein cyclophilin A inhibits HIV-1 nuclear entry by decreasing capsid elasticity

Binding of the host protein cyclophilin A (CypA) to the HIV-1 capsid exerts a variety of effects on infection, including enhancement of reverse transcription, stabilization of the capsid, and promotion of nuclear entry. For several HIV-1 mutants, CypA binding inhibits nuclear entry by an unknown mechanism. We recently demonstrated that HIV-1 cores are elastic and that HIV-1 mutants with inelastic capsids are impaired for nuclear entry and infection of nondividing cells. Here we show that CypA prevents infection of nondividing cells by such mutants and inhibits their entry into the nucleus. CypA binding to mutant cores further reduced their elasticity in vitro, and this effect was reversed by suppressor mutations that restored nuclear entry. We suggest that HIV-1 nuclear entry involves temporal modulation of capsid elasticity by host proteins prior to and during traversal of the nuclear pore.

microbiology↗

bioRxiv2025

Towards universal modeling of transcript isoform expression levels

A holy grail in computational biology is accurate modeling of transcript expression levels using epigenetic features, which would provide a quantitative way to study gene regulation in normal and disease states. Previous studies relied heavily on immortalized cell lines that exhibit properties different from cells in natural tissue environments. Most studies also quantified the expression of each gene by a single expression level, which fails to capture separate expression levels of different transcript isoforms of the same gene. In this study, making use of the latest large-scale dataset of paired transcriptomic and epigenomic data of human samples produced by the International Human Epigenome Consortium (IHEC), we computationally modeled the expression levels of individual transcript isoforms in 324 samples from 29 tissue types. We constructed the models using graph-based methods that integrate both location-specific epigenomic features and multiple types of gene-gene relationships. We found that to infer transcript isoform expression levels in a sample, a model that integrates information from many samples of other tissue types consistently outperforms a model trained on data from this sample itself, providing strong support that it is possible to construct a "universal" model that can accurately infer transcript isoform expression levels across tissue types.

bioinformatics↗

bioRxiv2025

Characterization of full-length and cytoplasmic tail-truncated envelope glycoproteins incorporated into human immunodeficiency virus (HIV-1) virions and virus-like particles

During transport to the surface of infected cells, the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is cleaved to produce the mature functional Env trimer ((gp120/gp41)3). Env cleavage stabilizes the pretriggered Env conformation (PTC), the major target for broadly neutralizing antibodies. Although the mature Env is relatively enriched in virions and virus-like particles (VLPs), conformationally flexible uncleaved Envs typically contaminate preparations of these particles. In non-permissive cells, the long [~]149-residue gp41 cytoplasmic tail (CT) is necessary for Env incorporation into virions. In a minority of HIV-1 strains, the gp41 CT is clipped in virions by the viral protease. Here, we compare Envs with CT truncations and CT alterations that increase or decrease protease clipping in permissive cells. Changes in protease clipping affected amphotericin B sensitivity, but did not alter other viral phenotypes. By contrast, a corresponding CT truncation (L748STOP) increased cell-surface and virion Env levels, cell-cell fusion, and virus infectivity and cytotoxicity. Notably, in diverse HIV-1 strains, the ratio of cleaved/uncleaved Envs in preparations of virions and extracellular vesicles was increased by this CT truncation. ESCRT and ALIX-binding region (EABR) vesicles incorporated significantly more uncleaved CT-truncated Env than HIV-1 VLPs. Env CT deletion/truncation did not qualitatively alter the viral neutralization profile; however, increased antibody concentrations were required to neutralize viruses with the higher levels of cleaved Env that resulted from CT truncation. Specific CT truncations provide a means of enriching the PTC and limiting the incorporation of nonfunctional and conformationally heterogeneous uncleaved Envs into preparations of virions and VLPs. IMPORTANCEThe human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer mediates entry of the virus into host cells. The pretriggered conformation (PTC) of Env is the major target for protective broadly neutralizing antibodies, but the PTC is unstable and therefore difficult to study. The cleavage of the flexible Env precursor stabilizes the PTC. Therefore, the presence of uncleaved Env compromises the purity of the PTC in Env preparations. We found that certain truncations of the Env cytoplasmic tail resulted in improved ratios of cleaved:uncleaved Env in preparations of HIV-1 viruses or virus-like particles. In some contexts, cytoplasmic tail truncation increased the level of Env in virus preparations. Although higher concentrations of antibodies were required to neutralize these viruses, Envs with specific truncations of the cytoplasmic tail retained the PTC. Thus, cytoplasmic tail truncation could assist efforts to purify and characterize the Env PTC on the viral membrane.

microbiology↗

bioRxiv2025

Inhibition of NLRP3 by a CNS-penetrating indazole scaffold

Low-grade inflammation is a hallmark of ageing and a key cause of age-related impairments and diseases1. The NOD-like receptor NLRP3 senses a variety of danger signals and environmental insults, resulting in pro-inflammatory response, inflammasome formation and pyroptosis2,3. Its aberrant activation has been linked to many acute and chronic diseases ranging from atherosclerosis to Alzheimers disease and cancer, making NLRP3 an attractive therapeutic target4,5. Here we report the discovery, characterization, and structure of an indazole-based NLRP3 antagonist, BAL-1516, which potently inhibits inflammasome formation in monocytes and microglia. The cryo-electron microscopy structure of BAL-1516 bound to NLRP3 reveals a previously undescribed compound binding site at a surface groove of the nucleotide-binding domain with contacts to the FISNA and WHD subdomains. The characteristic feature of BAL compound binding is the formation of three hydrogen bonds to the peripheral {beta}-strand of the triple-ATPase; two from the indazoles nitrogen atoms and a third from the compounds linker region. Additional phenyl and thiazole moieties render the compound hydrophobic, allowing excellent blood-brain barrier penetration. The compound binding site is highly specific for NOD-like receptors, and the optimized compound BAL-1516 is able to directly bind mouse NLRP3 despite two conservative residue changes in the binding interface. The BAL compounds represent a first-in-class family of NLRP3 inhibitors, providing a broad design space, including covalent and degradative properties, for the development of NLRP3-directed therapeutics. The innate immune system contains cytosolic proteins that sense cellular stress caused by bacterial, viral and fungal infections or sterile inflammation, to control cellular integrity2. NLRP3 is a well-studied member of the nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) that is involved in the activation of the inflammasome, a multiprotein complex that mediates inflammation6. Upon detection of stress or pathogen-associated signals, NLRP3 triggers the activation of caspase-1, which leads to the production of pro-inflammatory cytokines such as IL-1{beta} and IL-18, driving inflammatory responses and ultimately pyroptotic cell death. In the context of neuroinflammation, NLRP3 plays a significant role in the pathogenesis of various neurodegenerative diseases, including Alzheimers disease, Parkinsons disease, and multiple sclerosis7. Research into targeting NLRP3 signalling with CNS-penetrating molecules holds potential for developing therapeutic strategies to alleviate neuroinflammatory conditions and to slow the progression of neurodegenerative diseases.

immunology↗

bioRxiv2025

Modeling cellular influence delineates functionally relevant cellular neighborhoods in primary and metastatic pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, with liver metastases significantly worsening outcomes. However, distinct features of the tumor microenvironment (TME) between primary and metastatic sites remain poorly defined. Cellular neighborhoods within the TME are recognized as functional units that influence tumor behavior. Conventional spatial methods, which assign equal weights to all cells in a region, fail to capture the nuances of cellular interactions. To address this, we developed Functional Cellular Neighborhood (FunCN) quantification, which integrates both the proportion and proximity of surrounding cells. Applying FunCN to PDAC imaging mass cytometry data, we identified neutrophil-enriched interactions in liver metastases compared to primary tumors, correlating with elevated VISTA expression by tumor cells. Additionally, FunCN clusters around CD8+ T cells in pancreas and liver were associated with higher TIGIT and LAG3, respectively. These findings demonstrate the importance of spatial immune landscapes in PDAC and identify potential therapeutic opportunities.

systems biology↗

bioRxiv2025

Unified integration of spatial transcriptomics across platforms

Spatial transcriptomics (ST) has transformed our understanding of tissue architecture and cellular interactions, but integrating ST data across platforms remains challenging due to differences in gene panels, data sparsity, and technical variability. Here, we introduce LO_SCPLOWLOKIC_SCPLOW, a novel framework for integrating imaging-based ST data from diverse platforms without requiring shared gene panels. LO_SCPLOWLOKIC_SCPLOW addresses ST integration through two key alignment tasks: feature alignment across technologies and batch alignment across datasets. Optimal transport-guided feature propagation adjusts data sparsity to match scRNA-seq references through graph-based imputation, enabling single-cell foundation models such as scGPT to generate unified features. Batch alignment then refines scGPT-transformed embeddings, mitigating batch effects while preserving biological variability. Evaluations on mouse brain samples from five different technologies demonstrate that LO_SCPLOWLOKIC_SCPLOW outperforms existing methods and is effective for cross-technology spatial gene program identification and tissue slice alignment. Applying LO_SCPLOWLOKIC_SCPLOW to five ovarian cancer datasets, we identify an integrated gene program indicative of tumor-infiltrating T cells across gene panels. Together, LO_SCPLOWLOKIC_SCPLOW provides a robust foundation for cross-platform ST studies, with the potential to scale to large atlas datasets, enabling deeper insights into cellular organization and tissue environments.

bioinformatics↗

bioRxiv2025

Synergistic RAS-MAPK and AKT Activation in MYC-Driven Tumors via Adjacent PVT1 Rearrangements

MYC-driven (MYC+) cancers are aggressive and often fatal. MYC dysregulation is a key event in these cancers, but overexpression of MYC alone is not always enough to cause cancer. Plasmocytoma Variant Translocation 1 (PVT1), a long non-coding RNA (lncRNA) adjacent to MYC on chromosome 8 is a rearrangement hotspot in many MYC+ cancers. In addition to being co-amplified with MYC, the genomic rearrangement at PVT1 involves translocation, which has had obscure functional consequences. We report that translocation at the PVT1 locus cause asymmetric enrichment of 5-PVT1 and loss of 3-PVT1. Despite being classified as a non-coding RNA, the retained 5 region of PVT1 generates a circular RNA (CircPVT1) that codes for the novel peptide we call Firefox (FFX). FFX augments AKT signaling and synergistically activates MYC and mTORC1 in these cells. Further, the 3 end of PVT1, which is lost during the translocation, codes for a tumor-suppressing micropeptide we named as Honeybadger (HNB). We demonstrate that HNB interacts with KRAS and disrupts the activation of KRAS effectors. Loss of HNB leads to activation of RAS/MAPK signaling pathway, and enhances MYC stability by promoting phosphorylation of MYC at Ser62. These findings identify PVT1 as a critical node that synchronizes MYC, AKT, and RAS-MAPK activities in cancer. Our study thus identifies a key mechanism by which rearrangements at the PVT1 locus activate additional oncogenic pathways that synergize with MYC to exacerbate the aggressiveness of MYC+ cancers. This newfound understanding explains the poor prognosis associated with MYC+ cancers and offers potential therapeutic targets that could be leveraged in treatment strategies for these cancers.

cancer biology↗

bioRxiv2024

Discovery of a Potent and Selective Inhibitor of Human NLRP3 with a Novel Binding Modality and Mechanism of Action

The NLRP3 inflammasome is an intracellular protein complex that causes inflammation via the release of IL-1{beta} and pyroptosis. NLRP3 activation is associated with many age-related inflammatory diseases, and NLRP3 inhibition is a promising therapeutic strategy. We previously performed a DNA encoded library screen to identify novel NLRP3 binding molecules. Herein we describe the characterization of BAL-0028 as a potent and specific inhibitor of NLRP3 signaling. Notably, BAL-0028 is a poor inhibitor of mouse NLRP3 but inhibits human and primate NLRP3 with nanomolar potency. Using cellular and biochemical analyses we demonstrate that BAL-0028 binds to the NLRP3 NACHT domain at a site that is distinct from the MCC950 binding pocket. Using humanized NLRP3 mice we show that a derivative of BAL-0028 inhibits NLRP3 activation in vivo in a peritonitis model. Finally, we demonstrate that BAL-0028 inhibits select hyperactive NLRP3 mutations associated with autoinflammatory diseases more potently than does MCC950. BAL-0028 thus represents a new modality for NLRP3 inhibition in inflammatory diseases. SUMMARYNLRP3 is a target for anti-inflammatory therapies and can be inhibited by the tool compound MCC950. We describe the characterization of a new small molecule inhibitor of NLRP3 BAL-0028 that has a distinct mechanism of action and binding site.

immunology↗

bioRxiv2024

Notch signaling stabilizes lengths of motile cilia in multiciliated cells in the lung

Airway multiciliated cells (MCs) maintain respiratory health by clearing mucus and trapped particles through the beating of motile cilia. While it is known that ciliary lengths decrease along the proximal-distal (P-D) axis of the tracheobronchial tree, how this is regulated is unclear. Here, we demonstrate that canonical Notch signaling in MCs plays a critical role in stabilizing ciliary length. Inhibition of Notch signaling in MCs results in ciliary shortening in the trachea, lengthening in the distal airway, and to region-specific alterations in gene expression. We probe how environmental challenges impact MC homeostasis using germ-free and Mycobacterium tuberculosis (M. tb) infection models. While germ-free conditions do not perturb ciliary lengths, M. tb infection leads to lengthening of distal airway cilia, correlating with a downregulation of Notch signaling. These findings reveal that ciliary length and the P-D gradient in the airways are actively regulated, with Notch signaling serving as a stabilizing mechanism.

cell biology↗

bioRxiv2024

Targeting eIF4G1-dependent translation in melanoma

Expression of components of the translation initiation complex (eIF4F) is frequently elevated in cancer, resulting in enhanced synthesis of oncogenic proteins. We thus set out to limit eIF4F pro-oncogenic activity, a notable challenge given its essential role in normal tissues. CRISPR-Cas9-based functional screen using tiling sgRNAs identified the eIF4G1 MA3 domain, a subunit of the eIF4F complex, as a target for developing small molecule inhibitors. Combination of structure-guided in silico modeling and chemical library screening led to the identification of small molecule candidates M19 and its analog M19-6 that binds to the MA3 domain of eIF4G1 and disrupts eIF4F complex. M19-6 treatment reprograms the melanoma translatome, limiting synthesis of factors that promote cell proliferation and neoplastic growth, as well as reducing translation of mRNAs encoding ferroptosis suppressors. Whole genome CRISPR screen indentified ferroptosis activators to augment M19-6 activity, which was confirmed in cultured melanoma cells. M19-6 alleviates melanoma resistance to BRAF and MEK inhibitors, while eliciting anti-tumor and anti-metastatic effects in preclinical mouse models. Among several biomarkers found in M19-6 sensitive cell lines, THBS1 and TGF{beta}I expression were elevated in patients who are non-responders to PD-1 therapy as in patients with metastasis. Our studies identify M19-6 as a therapeutic candidate, offering a novel insights into targeting the eIF4F complex to overcome melanoma resistance to therapy and metastasis. SignificanceWe identify M19-6 as a small molecule that disrupts the eIF4F, translation initiation complex, by targeting the MA3 domain of eIF4G1, resulting in elimination of melanoma cells in culture, overcoming therapy resistance while inhibiting melanoma growth and metastasis in vivo. As M19-6 causes minimal toxicity to melanocytes, it offers a therapeutic modality for melanoma.

cancer biology↗

bioRxiv2024

TAOK2 Drives Opposing Cilia Length Deficits in 16p11.2 Deletion and Duplication Carriers

Copy number variation (CNV) in the 16p11.2 (BP4-BP5) genomic locus is strongly associated with autism. Carriers of 16p11.2 deletion and duplication exhibit several common behavioral and social impairments, yet, show opposing brain structural changes and body mass index. To determine cellular mechanisms that might contribute to these opposing phenotypes, we performed quantitative tandem mass tag (TMT) proteomics on human dorsal forebrain neural progenitor cells (NPCs) differentiated from induced pluripotent stem cells (iPSC) derived from 16p11.2 CNV carriers. Differentially phosphorylated proteins between unaffected individuals and 16p11.2 CNV carriers were significantly enriched for centrosomal and cilia proteins. Deletion patient-derived NPCs show increased primary cilium length compared to unaffected individuals, while stunted cilium growth was observed in 16p11.2 duplication NPCs. Through cellular shRNA and overexpression screens in human iPSC derived NPCs, we determined the contribution of genes within the 16p11.2 locus to cilium length. TAOK2, a serine threonine protein kinase, and PPP4C, a protein phosphatase, were found to regulate primary cilia length in a gene dosage-dependent manner. We found TAOK2 was localized at centrosomes and the base of the primary cilium, and NPCs differentiated from TAOK2 knockout iPSCs had longer cilia. In absence of TAOK2, there was increased pericentrin at the basal body, and aberrant accumulation of IFT88 at the ciliary distal tip. Further, pharmacological inhibition of TAO kinase activity led to increased ciliary length, indicating that TAOK2 negatively controls primary cilium length through its catalytic activity. These results implicate aberrant cilia length in the pathophysiology of 16p11.2 CNV, and establish the role of TAOK2 kinase as a regulator of primary cilium length.

neuroscience↗

bioRxiv2024

SUMO2 Inhibition Reverses Aberrant Epigenetic Rewiring Driven by Synovial Sarcoma Fusion Oncoproteins and Impairs Sarcomagenesis

Synovial Sarcoma (SySa) is an aggressive soft tissue sarcoma that accounts for 5 - 10% of all soft tissue sarcomas. Current treatment involves radiation and radical surgery including limb amputation, highlighting the urgent need to develop targeted therapies. We reasoned that transcriptional rewiring by the fusion protein SS18-SSX, the sole oncogenic driver in SySa, creates specific vulnerabilities that can be exploited for treatment. To uncover genes that are selectively essential for SySa, we mined The Cancer Dependency Map (DepMap) data to identify genes that specifically impact the fitness of SySa compared to other tumor cell lines. Targeted CRISPR library screening of SySa-selective candidates revealed that the small ubiquitin-like modifier 2 (SUMO2) was one of the strongest dependencies both in vitro as well as in vivo. TAK-981, a clinical-stage small molecule SUMO2 inhibitor potently inhibited growth and colony-forming ability. Strikingly, transcriptomic studies showed that pharmacological SUMO2 inhibition with TAK-981 treatment elicited a profound reversal of a gene expression program orchestrated by SS18-SSX fusions. Of note, genetic or pharmacological SUMO2 inhibition reduced global and chromatin levels of the SS18-SSX fusion protein with a concomitant reduction in histone 2A lysine 119 ubiquitination (H2AK119ub), an epigenetic mark that plays an important role in SySa pathogenesis. Taken together, our studies identify SUMO2 as a novel, selective vulnerability in SySa. Since SUMO2 inhibitors are currently in Phase 1/2 clinical trials for other cancers, our findings present a novel avenue for targeted treatment of synovial sarcoma. SIGNIFICANCEOur study identifies SUMO2 as a selective dependency in synovial sarcoma. We demonstrate that the SUMO2/3 inhibitor TAK-981 impairs sarcomagenesis and reverses the SS18-SSX fusion-driven oncotranscriptome. Our study indicates that SUMO2 inhibition may be an attractive therapeutic option in synovial sarcoma.

cancer biology↗

bioRxiv2024

Spatial multi-omics reveal intratumoral humoral immunity niches associated with tertiary lymphoid structures in pancreatic cancer immunotherapy pathologic responders

Pancreatic adenocarcinoma (PDAC) is a rapidly progressing cancer that responds poorly to immunotherapies. Intratumoral tertiary lymphoid structures (TLS) have been associated with rare long-term PDAC survivors, but the role of TLS in PDAC and their spatial relationships within the context of the broader tumor microenvironment remain unknown. We generated a spatial multi-omics atlas encompassing 26 PDAC tumors from patients treated with combination immunotherapies. Using machine learning-enabled H&E image classification models and unsupervised gene expression matrix factorization methods for spatial transcriptomics, we characterized cellular states within TLS niches spanning across distinct morphologies and immunotherapies. Unsupervised learning generated a TLS-specific spatial gene expression signature that significantly associates with improved survival in PDAC patients. These analyses demonstrate TLS-associated intratumoral B cell maturation in pathological responders, confirmed with spatial proteomics and BCR profiling. Our study also identifies spatial features of pathologic immune responses, revealing TLS maturation colocalizing with IgG/IgA distribution and extracellular matrix remodeling. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/613714v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@d5e1a5org.highwire.dtl.DTLVardef@1d14012org.highwire.dtl.DTLVardef@c57c6corg.highwire.dtl.DTLVardef@16baa56_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIIntegrated multi-modal spatial profiling of human PDAC tumors from neoadjuvant immunotherapy clinical trials reveal diverse spatial niches enriched in TLS. C_LIO_LITLS maturity is influenced by tumor location and the cellular neighborhoods in which TLS immune cells are recruited. C_LIO_LIUnsupervised machine learning of genome-wide signatures on spatial transcriptomics data characterizes the TLS-enriched TME and associates TLS transcriptomes with survival outcomes in PDAC. C_LIO_LIInteractions of spatially variable gene expression patterns showed TLS maturation is coupled with immunoglobulin distribution and ECM remodeling in pathologic responders. C_LIO_LIIntratumoral plasma cell and immunoglobin gene expression spatial dynamics demonstrate trafficking of TLS-driven humoral immunity in the PDAC TME. C_LI SignificanceWe report a spatial multi-omics atlas of PDAC tumors from a series of immunotherapy neoadjuvant clinical trials. Intratumorally, pathologic responders exhibit mature TLS that propagate plasma cells into malignant niches. Our findings offer insights on the role of TLS-associated humoral immunity and stromal remodeling during immunotherapy treatment.

cancer biology↗

bioRxiv2024

Natural killer cells associate with epithelial cells in the pancreatic ductal adenocarcinoma tumor microenvironment

Pancreatic ductal adenocarcinoma (PDAC) exhibits dense fibrosis and immune exclusion. While fibrosis has been studied globally and at the region-of-interest level, its impact on stromal-ductal architecture and immune cell localization remains unknown. Here, we establish cancer-associated fibroblast (CAF)-stratified ductal spatial architecture as a fundamental determinant of immune exclusion in PDAC. Focusing on malignant PDAC epithelial ductal regions, the critical interface where immune cells must access tumor epithelium, we demonstrate that periductal fibroblast organization dictates leukocyte proximity. Through integrative analysis of treatment-naive patient samples from three independent cohorts - including imaging mass cytometry, multiplex immunohistochemistry, and single-cell RNA sequencing - we uncovered that activated, pro-inflammatory leukocytes preferentially localized near malignant ducts in regions with low fibroblast density. Stratifying epithelial-ductal regions by CAF abundance revealed a graded constraint: increasing fibroblast content corresponded to reduced leukocyte-epithelial proximity and elevated collagen I deposition. Despite their exclusion in high-CAF ducts, leukocytes in low-CAF ducts retained functional competence. Mechanistically, ligand-receptor inference implicated collagen-CD44 signaling as an adhesion axis anchoring immune cells within fibroblast-rich zones, with CD44 blockade enhancing natural killer cell invasion and motility in fibrotic spheroid models. Thus, by establishing ductal regions as critical spatial units of immune exclusion, these findings provide a framework for dissecting stromal-immune interactions and reveal targetable "stromal checkpoints" that can be leveraged to overcome CAF-driven barriers to leukocyte motility and infiltration in PDAC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/593868v3_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@17df874org.highwire.dtl.DTLVardef@142c809org.highwire.dtl.DTLVardef@15af4a7org.highwire.dtl.DTLVardef@74183a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Virtual patient analysis identifies strategies to improve the performance of predictive biomarkers for PD-1 blockade

Patients with metastatic triple-negative breast cancer (TNBC) show variable responses to PD-1 inhibition. Efficient patient selection by predictive biomarkers would be desirable, but is hindered by the limited performance of existing biomarkers. Here, we leveraged in-silico patient cohorts generated using a quantitative systems pharmacology model of metastatic TNBC, informed by transcriptomic and clinical data, to explore potential ways to improve patient selection. We tested 90 biomarker candidates, including various cellular and molecular species, by a cutoff-based biomarker testing algorithm combined with machine learning-based feature selection. Combinations of pre-treatment biomarkers improved the specificity compared to single biomarkers at the cost of reduced sensitivity. On the other hand, early on-treatment biomarkers, such as the relative change in tumor diameter from baseline measured at two weeks after treatment initiation, achieved remarkably higher sensitivity and specificity. Further, blood-based biomarkers had a comparable ability to tumor- or lymph node-based biomarkers in identifying a subset of responders, potentially suggesting a less invasive way for patient selection.

systems biology↗