Biology subjects

Schilling, B.

Publications and source records attributed to Schilling, B..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

γδ17 T cell-stromal networks modulate matrix composition and vascularity in foreign body response

Immune-stromal crosstalk governs tissue fibrosis, which is marked by dysregulated extracellular matrix (ECM) production and aberrant vasculature. Here, we investigate how {gamma}{delta} T cell interactions with stromal cells shape fibrosis in the foreign body response. During the acute reaction, type-1 ({gamma}{delta}IFN{gamma}) and type-17 ({gamma}{delta}17) effector subsets accumulated at the implant. While {gamma}{delta}IFN{gamma} decreased as fibrosis progressed, activated {gamma}{delta}17 persisted as dominant interleukin-17 producers. The {gamma}{delta}17 increased with aging and high-fat diet, both factors associated with chronic inflammation and fibrosis. Co-culture with {gamma}{delta}17 stimulated fibroblast expression of collagen genes and intercellular communication inference linked {gamma}{delta} T cell ligands to activation of ECM remodeling and vascular development programs in fibroblasts and endothelial cells. Finally, genetic deletion of {gamma}{delta} T cells altered expression of ECM components and increased vessel size within the fibrotic matrix. Altogether, our findings implicate {gamma}{delta} T cells in regulating stromal behavior to modulate composition and vascularity of fibrotic tissues.

immunology↗

bioRxiv2025

Multiomic profiling of a unique in-transit melanoma cohort identifies melanoma differentiation as predictor of tumor progression and therapy response

Melanoma patients with in-transit metastasis (ITM), a stage of disease where melanoma has metastasized to sites in between the primary lesion and draining lymph node, vary significantly in their clinical outcomes, but the biology driving differential outcomes in ITM is poorly understood. To elucidate the mechanisms of differential outcomes, we utilized multimodal molecular profiling (WES, RNA-seq, highly multiplexed immunofluorescence, spatial transcriptomics) in 1) evolutionary analysis of longitudinal tumor samples and 2) identifying prognostic tumor intrinsic and microenvironmental features in a unique cohort of patients with unresectable ITM. Among other findings, we observed a persistent dedifferentiated AXL/NGFR clonal lineage pre-existing and following immune checkpoint blockade in in-transit and distant metastases. Concordantly, we found that low pigmentation and high T cell exhaustion signatures were independently associated with distant progression. Our findings highlight tumor cell state and immune dysfunction as key predictors and potential biomarkers of metastatic risk in ITM. STATEMENT OF SIGNIFICANCEWhat drives distant progression in melanoma is unclear. Analyzing tumor and immune features in a rare in-transit melanoma patient cohort, we identify biological signals highlighting how immune and tumor states observable in pre-distant metastasis melanomas shape long-term outcomes, and nominate potential prognostic biomarkers.

cancer biology↗

bioRxiv2025

Tau oligomers modulate synapse fate by eliciting progressive bipartite synapse dysregulation and synapse loss

BackgroundSynapse function is critical for cognition, and synapse loss is highly correlated with cognitive decline in Alzheimers disease and related dementias. Tau oligomers, which accumulate in the brain in Alzheimers disease, can acutely inhibit synaptic plasticity and cause synapse loss. Coordinated presynaptic and postsynaptic function is essential for effective synaptic transmission, and both compartments can be dysregulated by pathogenic tau. However, the series of pathophysiological events triggered by tau oligomers to cause the dysfunction and deterioration of presynaptic terminals and postsynaptic sites remain unclear. MethodsWe developed a proximity labeling tool to map the postsynaptic proteome by fusing PSD-95 with APEX2 (APEX2-PSD-95) which was expressed in human induced pluripotent stem cell (iPSC)-derived neurons. We used APEX2-PSD-95 to map the dynamic changes in the postsynaptic proteome with precise temporal resolution after an acute exposure of human iPSC-derived neurons to recombinant tau oligomers for 30 min. Leveraging immunocytochemistry, electrophysiology and electron microscopy, we further delineated the impact of the acute tau oligomer exposure on presynaptic and postsynaptic compartments over time for up to 14 days. ResultsThe brief exposure of human iPSC-derived neurons to tau oligomers caused a progressive deterioration of synapses, marked by both presynaptic and postsynaptic dysregulation. Postsynaptic proteome mapping revealed an immediate tau oligomer-triggered downregulation of the postsynaptic actin motor proteins Myosin-Va and Myosin-10, which coincided with impaired AMPA receptor (AMPAR) trafficking during synaptic plasticity. This was followed 24 hours later by the upregulation of disease-related proteins, including GSK3{beta}, at postsynaptic sites. The loss of PSD-95-labeled postsynaptic sites at 7 days after tau oligomer exposure preceded the loss of Synapsin-labeled presynaptic terminals at 14 days. The postsynaptic sites that remained exhibited a long-term downregulation of postsynaptic AMPAR levels and sustained synaptic plasticity impairment. Moreover, the remaining presynaptic terminals contained less clusters of vesicles at the presynaptic active zone which was associated with reduced vesicle release probability at synapses. ConclusionOur findings reveal the series of events underlying tau oligomer-induced bipartite synapse deterioration. The progressive decline of synapses involves the emergence of two synapse fates. One synapse fate involves the persistent weakening of both presynaptic and postsynaptic function, and the other results in synaptic loss.

neuroscience↗

bioRxiv2025

A multi-omics analysis of human fibroblasts overexpressing an Alu transposon reveals widespread disruptions in aging-associated pathways

During aging and cellular senescence, repetitive elements are frequently transcriptionally derepressed across species and cell types. Among these, the most abundant repeats by copy number in the human genome are Alu retrotransposons. Though Alu elements are often studied for their mutagenic potential, there is increasing appreciation for their contributions to other biological functions, including pro-inflammatory signaling and mitochondrial dysfunction. However, a comprehensive analysis of Alu-driven molecular changes remains to be conducted, and Alus potential contributions to aging features remain incompletely characterized. Here, we show that overexpression of an AluJb transposon in human primary IMR-90 fibroblasts leads to large-scale alterations across the transcriptome, cellular proteome, and secretome. Functional genomics analyses reveal alterations in aging pathways, broadly, and mitochondrial metabolism, proteostasis, cell cycle, and extracellular matrix pathways, more specifically. Our results demonstrate that Alu transcriptional upregulation is sufficient to drive widespread disruptions to cellular homeostasis that mirror aging-associated alterations.

genomics↗

bioRxiv2025

A Fibroblast State Choreographs an Epithelial YAP-dependent Regenerative Program Essential to (Pre)malignancy via ECM-mediated Mechanotransduction

Chronic lung injury generates metaplasia which occasionally, but ominously, progresses to squamous dysplasia and squamous lung cancer. To identify mechanisms through which disrupted tissue homeostasis contributes to malignant initiation and progression, we used in vivo and in vitro heterotypic recombinant models of human bronchial epithelial cells (hBECs) and fibroblasts. We demonstrate that injury-associated TGF-{beta} signaling creates a fibroblast state dependent upon HSP47 upregulation. These fibroblasts accumulated collagen, thus elevating tissue stiffness and activating mechanosignaling that sustained YAP-dependent embryonic-like, pro-malignant activities in adjacent hBECs. This Stress/Tension-Instructive Fibroblast (STIF) state, exhibited by stressed fibroblasts in premalignant and malignant lesions across multiple cancer types, was sufficient to reprogram disease-free hBECs to metaplasia and to drive hBECs with compromised tumor suppressor function to dysplasia, yet could be inhibited and reversed. STIFs suffice to activate epithelial phenotypes reminiscent of oncogene-mediated cell transformation and induce (pre)malignancy via increased force transmission, providing novel targets for prevention. Statement of significanceTissue injury creates a regenerative pro-tumorigenic Stress/Tension-Instructive Fibroblast (STIF) state which is sufficient to activate a YAP-dependent, pre-malignant program to induce or unmask pre-cancerous phenotypes in epithelial cells through mechanotransduction. Inhibition of STIF activity or mechanosignaling prevents metaplasia and progression to dysplasia. HighlightsO_LITissue injury creates a pro-tumorigenic Stress/Tension-Instructive Fibroblast (STIF) state in multiple organs that precedes and persists through cancer C_LIO_LISTIF signaling alone, working through fibroblasts and not epithelial cells, is sufficient to activate embryonic-like plasticity and induce epithelial pre-cancerous metaplastic lesions C_LIO_LISTIFs program (pre)malignant phenotypes in adjacent epithelial cells through mechanosignaling by activating YAP prior to tumor formation C_LIO_LIInhibiting STIFs or mechanosignaling prevents/reverts metaplasia and prevents progression to dysplasia C_LI

cancer biology↗

bioRxiv2025

Age-Dependent Increase and Dysregulation of the Splicing Machinery in Acute Myeloid Leukemia of Older Patients

Acute myeloid leukemia (AML) is characterized by age-related resistance to therapy and poor outcomes. Initial remission rates vary from 30-80% with current therapies, but relapses are common. The 5-year survival is 30% overall but less than 10% for those 60 years of age or older. Resistance to current therapies is the central clinical challenge. This study investigated how age influences the AML proteome in patients with increasing age. We analyzed bone marrow or leukapheresis samples from 14 AML patients, including 9 older ([≥] 66 years) and 5 younger ([≤] 58 years) individuals, using proteomic technologies (Data-Independent Acquisitions, DIA-MS). We identified 4,471 protein groups, of which 889 exhibited significant changes and regulation between the two age groups. Proteins upregulated in older patients were predominantly associated with DNA transcription and translation, while downregulated proteins were linked to metabolic, catabolic, and extracellular matrix or structural processes. Significant changes in older samples affected the splicing machinery. Significant alterations in several of these factors, suggests either dysregulated or elevated splicing activity and reduced transcription initiation factor activity in older AML patients. Our study provides novel insights into molecular changes during AML arising in the older adult, highlighting the importance of age-specific therapeutic strategies. Statement of Significance of the StudyThis study is significant as it investigates a devastating and highly aggressive cancer, acute myeloid leukemia (AML), which often presents in patients with very low 5-year survival rates (32%), however which is even lower in patients over 60 years old (10%). This study really focuses on the complications and severe outcome in older patients which is not well understood. This is particularly relevant as older AML patients often do not respond to the available treatments. Understanding the pathways and protein profiles that are dysregulated in older AML patients compared to younger AML patients may in the future help to develop more customized therapeutic interventions or allow for some precision treatments. AML patients were stratified into cohort groups by age (older vs younger) and the proteomic profiling highlighted the particularly strong dysregulation of the splicing machinery in the older AML patients. While alternative splicing is often observed in cancers, here the emphasis is pointed to the fact that the dysregulation of the spliceosome and other pathways are greatly exacerbated in the older AML patients, likely leading to even worse outcomes and poor response to treatments in the older patients.

cancer biology↗

bioRxiv2025

Targeting Langerhans cells using a modular mannosylated nucleic acid-based vaccine platform

The skin is a diverse reservoir of immune cells with strong potential for immunotherapeutic delivery. Langerhans cells (LCs) in the epidermis are antigen-presenting cells, accessible for vaccination using carbohydrate-conjugated therapeutics targeting their endocytic lectin receptor, Langerin. As carbohydrate-lectin binding is highly dependent on valency, scaffolds that enable control over ligand spacing and stoichiometry are instrumental in enhancing receptor binding and selectivity. Here we utilized a self-assembled nucleic acid-based Holliday Junction scaffold, fully modified for nuclease protection and with a well-defined carbohydrate arrangement to optimize drug delivery to LCs. In vitro screening with Langerin-expressing cells revealed that mannosylated Holliday Junctions showed the strongest binding. This was confirmed in human epidermal cell suspensions, demonstrating specificity and valency-driven interactions. Topical administration of mannosylated scaffolds on skin explants enabled effective targeting of epidermal LCs. Finally, in an antigen-presentation assay, in vitro differentiated LCs loaded with mannosylated and peptide-conjugated scaffolds significantly enhanced T cell activation. Overall, our study presents a promising nucleic acid-based platform for precise LC targeting and drug delivery, with broad potential for skin-directed immunotherapies.

molecular biology↗

bioRxiv2025

Intervertebral disc impairments in a mouse model of Alzheimer's Disease

Chronic low back pain, frequently associated with intervertebral disc (IVD) degeneration, is highly prevalent in individuals with Alzheimers disease (AD), and the pain intensity is highly correlated with the degree of cognitive impairment. While the incidences of both afflictions increase dramatically in the elderly population, it is unknown whether AD exacerbates the health of the IVD. Utilizing one-year-old male and female 5xFAD mice that constitutively express human APP and PSEN1 transgenes with five AD-linked mutations, we measured the lumbar IVDs extracellular matrix composition, the three-dimensional structure, histopathological degeneration, and mechanical behavior. The collagen, glycosaminoglycans, and advanced glycation end-products content of the IVD were not appreciably different between the 5xFAD animals and their wild-type littermates. Likewise, the 5xFAD IVDs were not histopathologically degenerated. However, the IVD volume, measured by contrast-enhanced microCT, was larger in the 5xFAD animals. Furthermore, dynamic microcompression revealed that 5xFAD IVDs exhibited higher loss tangent, indicating altered tissue damping and fluid-flow dynamics within the disc. These results suggest that although the IVDs of mice with AD are not more degenerated, they may be more susceptible to damage accumulation due to the elevated absorption of energy. Elderly individuals with AD may thus be more prone to IVD injuries that lead to eventual degeneration and spinal pain. Future work will focus on defining the molecular mechanisms and the consequences of these mechanical and structural changes in the IVD and their consequences to low back pain in individuals with AD.

bioengineering↗

bioRxiv2025

Multiplexed Targeted Spatial Mass Spectrometry Imaging Assays to monitor lipids and NAD+ metabolites in CD38 knockout mice exhibiting improved metabolism.

Mass spectrometry imaging (MSI) is a rapidly advancing technology that provides mapping of the spatial molecular landscape of tissues for a variety of analytes. Matrix-assisted laser desorption/ionization (MALDI)-MSI is commonly employed, however, confident in situ identification and accurate quantification of analytes remain challenging. We present a novel imaging methodology combining trapped ion mobility spectrometry (TIMS)-based parallel accumulation-serial fragmentation (PASEF) with MALDI ionization for targeted imaging parallel reaction monitoring (iprm-PASEF). We investigated the spatial distribution of lipids and metabolites in liver tissues from male wild-type and CD38 knockout mice (CD38-/-). CD38, an enzyme involved in nicotinamide adenine dinucleotide (NAD) metabolism, significantly influences liver metabolic function and contributes to age-related NAD decline. Although CD38 deletion previously was linked to improved metabolic phenotypes, the underlying spatial metabolic mechanisms are poorly understood. The spatial iprm-PASEF workflow enabled confident identification and differentiation of lipid isomers at the MS2 fragment ion level and confirmed increased NAD+ and decreased adenosine diphosphate ribose (ADPR), a by-product of NAD+ hydrolysis, in CD38-/- livers. This approach provided confident, specific, and robust MS2-based identification and quantification of fragment ions in spatial MSI experiments. Additionally, the innovative iprm-PASEF opens unprecedented opportunities for spatial metabolomics and lipidomics, offering spatially resolved insights into molecular mechanisms.

cell biology↗

bioRxiv2025

Odd-chain dicarboxylic acid feeding recapitulates the biochemical phenotype of glutaric aciduria type-1 in mice

Glutaric aciduria type-1 (GA1) is an inherited mitochondrial neurometabolic disorder with a poorly understood pathogenesis and unmet medical needs. GA1 can be diagnosed via its hallmark biochemical signature consisting of glutaric aciduria, 3-hydroxyglutaric aciduria, and increased plasma glutarylcarnitine. These glutaryl-CoA-derived metabolites are thought to originate solely in the mitochondria. Here, we demonstrate that wild-type mice fed an 11-carbon odd-chain dicarboxylic acid (undecanedioic acid, DC11) recreates the biochemical phenotype of GA1. Odd-chain dicarboxylic acids like DC11 are not present in food but can arise from several endogenous processes, such as lipid peroxidation and fatty acid {omega}-oxidation. DC11 is chain-shortened in peroxisomes to glutaryl (DC5)-CoA, which then gives rise to the GA1-like pattern of DC5 metabolites in urine, tissues, and blood. Glutaric acid released from peroxisomes during DC11 chain-shortening can enter mitochondria, be activated to CoA by the enzyme succinyl-CoA:glutarate-CoA transferase (SUGCT), and become substrate for glutaryl-CoA dehydrogenase (GCDH), the enzyme that is mutated in GA1. Our data provide proof-of-concept that the generation of dicarboxylic acids by {omega}-oxidation, which is stimulated during the same catabolic states known to trigger acute encephalopathy in GA1, may exacerbate disease by increasing the glutaryl-CoA substrate load in mitochondria.

biochemistry↗

bioRxiv2025

Proteomic Analysis of Breast Cancer Subtypes Identifies Stromal Contributions that Dictate Aggressive Malignant Behavior

Breast cancer manifests as multiple subtypes with distinct patient outcomes and treatment strategies. Here, we optimized proteomic analysis of Formalin-Fixed Paraffin-Embedded (FFPE) specimens from patients diagnosed with five breast cancer subtypes, luminal A, luminal B, Her2, triple negative (TNBC) and metaplastic breast cancers (MBC), and from disease-free individuals undergoing reduction mammoplasty (RM). We identified and quantified [~]6,000 protein groups (with >2 peptides per protein) with significant changes in over 26% of proteins comparing each cancer subtype with control RM. Stringent statistical filters allowed us to deeply mine 576 significant conserved protein changes shared by all subtypes and protein changes unique to each subtype. The most aggressive subtype, MBC, revealed exacerbated stromal stress responses, as illustrated by a collagenolytic extracellular matrix (ECM) and immune participation biased towards neutrophils and eosinophils. Immunostaining of breast tissue sections confirmed differences across subtypes, in particular, a strong upregulation of SERPINH1, neutrophil-specific myeloperoxidase and eosinophil cationic protein in MBC. In summary, we present deep proteomic, digitalized protein abundance profiles, generated from FFPE breast cancer tissues, that revealed significant changes in ECM and cellular proteins. Statement of Significance of the StudyThis study is significant as it discovered deep proteomic signatures for the highly aggressive and malignant metaplastic breast cancer (MCB) which is now considered a fifth subtype based upon its remarkable intra-tumoral heterogeneity that illustrates its unique cell plasticity. To efficiently analyze formalin-fixed paraffin-embedded (FFPE) breast tissues from patients with different breast cancer subtypes and disease-free individuals, we optimized a novel workflow in which we combined paraffinization and Folch extraction. We identified and confidently quantified [~]6,000 protein groups. We were able to find robust changes in extracellular matrix (ECM) with cancer, even though no ECM enrichments were performed. Interestingly, despite the relatively small human cohort size (42 patients), distinct protein signatures emerged throughout all cancer subtypes - common and unique - with remarkable statistical significance for many cancer-relevant proteins and pathways. This Pilot study indicates the hypothesis that an altered stroma can dictate epithelial tumor cell fate. We also observed that MBC was characterized by an especially immunosuppressed tumor environment. We do note the limitation of the relatively small cohort size of our study, and in the future additional patient cohorts will be needed to further validate our findings.

cancer biology↗

bioRxiv2024

Inhibition of the metalloprotease ADAM19 as a Novel Senomorphic Strategy to Ameliorate gut permeability and senescence markers by modulating senescence-associated secretory phenotype (SASP)

Accumulation of DNA damage can accelerate aging through cellular senescence. Previously, we established a Drosophila model to investigate the effects of radiation-induced DNA damage on the intestine. In this model, we examined irradiation-responsive senescence in the fly intestine. Through an unbiased genome-wide association study (GWAS) utilizing 156 strains from the Drosophila Genetic Reference Panel (DGRP), we identified meltrin {beta} (the drosophila orthologue of mammalian ADAM19) as a potential modulator of the senescence-associated secretory phenotype (SASP). Knockdown of meltrin {beta} resulted in reduced gut permeability, DNA damage, and expression of the senescence marker {beta}-galactosidase (SA-{beta}-gal) in the fly gut following irradiation. Additionally, inhibition of ADAM19 in mice using batimastat-94 reduced gut permeability and inflammation in the gut. Our findings extend to human primary fibroblasts, where ADAM19 knockdown or pharmacological inhibition decreased expression of specific SASP factors and SA-{beta}-gal. Furthermore, proteomics analysis of the secretory factor of senescent cells revealed a significant decrease in SASP factors associated with the ADAM19 cleavage site. These data suggest that ADAM19 inhibition could represent a novel senomorphic strategy.

molecular biology↗

bioRxiv2024

Substrate Stiffness Dictates Unique Doxorubicin-induced Senescence-associated Secretory Phenotypes and Transcriptomic Signatures in Human Pulmonary Fibroblasts

Cells are subjected to dynamic mechanical environments which impart forces and induce cellular responses. In age-related conditions like pulmonary fibrosis, there is both an increase in tissue stiffness and an accumulation of senescent cells. While senescent cells produce a senescence-associated secretory phenotype (SASP), the impact of physical stimuli on both cellular senescence and the SASP is not well understood. Here, we show that mechanical tension, modeled using cell culture substrate rigidity, influences senescent cell markers like SA-{beta}-gal and secretory phenotypes. Comparing human primary pulmonary fibroblasts (IMR-90) cultured on physiological (2 kPa), fibrotic (50 kPa), and plastic (approximately 3 GPa) substrates, followed by senescence induction using doxorubicin, we identified unique high-stiffness-driven secretory protein profiles using mass spectrometry and transcriptomic signatures, both showing an enrichment in collagen proteins. Consistently, clusters of p21+ cells are seen in fibrotic regions of bleomycin induced pulmonary fibrosis in mice. Computational meta-analysis of single-cell RNA sequencing datasets from human interstitial lung disease confirmed these stiffness SASP genes are highly expressed in disease fibroblasts and strongly correlate with mechanotransduction and senescence-related pathways. Thus, mechanical forces shape cell senescence and their secretory phenotypes.

cell biology↗

bioRxiv2024

Loss of long-chain acyl-CoA dehydrogenase protects against acute kidney injury

Proximal tubular epithelial cells (PTECs) are particularly vulnerable to acute kidney injury (AKI). While fatty acids are the preferred energy source for PTECs via fatty acid oxidation (FAO), FAO-mediated H2O2 production in mitochondria has been shown to be a major source of oxidative stress. We have previously shown that a mitochondrial flavoprotein, long-chain acyl-CoA dehydrogenase (LCAD), which catalyzes a key step in mitochondrial FAO, directly produces H2O2 in vitro. Further we have established that loss of a lysine deacylase, Sirtuin 5 (Sirt5-/-), induces hypersuccinylation and inhibition of mitochondrial FAO genes to stimulate peroxisomal FAO and to protect against AKI. However, the role of LCAD has yet to be determined. Mass spectrometry data acquisition revealed that LCAD is hypersuccinylated in Sirt5-/- kidneys after AKI. Following two distinct models of AKI, cisplatin treatment or renal ischemia/reperfusion (IRI), LCAD knockout mice (LCAD-/-) demonstrated renoprotection against AKI. Specifically, LCAD-/- kidneys displayed mitigated renal tubular injury, decreased oxidative stress, preserved mitochondrial function, enhanced peroxisomal FAO, and decreased ferroptotic cell death. LCAD deficiency confers protection against two distinct models of AKI. This suggests a therapeutically attractive mechanism whereby preserved mitochondrial respiration as well as enhanced peroxisomal FAO by loss of LCAD mediates renoprotection against AKI.

pathology↗

bioRxiv2024

A comprehensive multi-omics signature of doxorubicin-induced cellular senescence in the postmenopausal human ovary.

A major aging hallmark is the accumulation of cellular senescence burden. Over time senescent cells contribute to tissue deterioration through chronic inflammation and fibrosis driven by the Senescence-Associated Secretory Phenotype (SASP). The human ovary is one of the first organs to age, and prominent age-related fibroinflammation within the ovarian microenvironment is consistent with the presence of senescent cells, but these cells have not been characterized in the human ovary. We thus established a doxorubicin-induced model of cellular senescence to establish a "senotype" (gene/protein signature of a senescence cell state) for ovarian senescent cells. Explants of human postmenopausal ovarian cortex and medulla were treated with doxorubicin for 24 hours followed by culture for up to 10 days in a doxorubicin-free medium. Tissue viability was confirmed by histology, lack of apoptosis, and continued glucose consumption by explants. Single nuclei sequencing and proteomics revealed an unbiased signature of ovarian senescence. We identified distinct senescence profiles for the cortex and medulla, driven predominantly by epithelial and stromal cells. Proteomics uncovered subregional differences in addition to 120 proteins common to the cortex and medulla SASP. Integration of transcriptomic and proteomic analyses revealed 26 shared markers, defining a senotype of doxorubicin-induced senescence unique to the postmenopausal ovary. A subset of these proteins: Lumican, SOD2, MYH9, and Periostin were mapped onto native tissue to reveal compartment-specific localization. This senotype will help determine the role of cellular senescence in ovarian aging, inform biomarker development to identify, and therapeutic applications to slow or reverse ovarian aging, senescence, and cancer.

molecular biology↗

bioRxiv2024

SILAC-based quantification reveals modulation of the immunopeptidome in BRAF and MEK inhibitor sensitive and resistant tumor cells

The immunopeptidome is constantly monitored by T cells to detect foreign or aberrant HLA peptides. It is highly dynamic and reflects the current cellular state, enabling the immune system to recognize abnormal cellular conditions, such as those present in cancer cells. To precisely determine how changes in cellular processes, such as those induced by drug treatment, affect the immunopeptidome, quantitative immunopeptidomics approaches are essential. To meet this need, we developed a pulsed SILAC-based method for quantitative immunopeptidomics. Metabolic labeling with lysine, arginine, and leucine enabled isotopic labeling of nearly all HLA peptides across all allotypes (> 90% on average). We established a data analysis workflow that integrates the de novo sequencing-based tool Peptide-PRISM for comprehensive HLA peptide identification with MaxQuant for accurate quantification. We employed this strategy to explore the modulation of the immunopeptidome upon MAPK pathway inhibition (MAPKi) and to investigate alterations associated with early cellular responses to inhibitor treatment and acquired resistance to MAPKi. Our analyses demonstrated significant changes in the immunopeptidome early during MAPKi treatment and in the resistant state. Moreover, we identified putative tumor-specific cryptic HLA peptides linked to these processes that might represent exploitable targets for cancer immunotherapy. SynopsisWe have developed a mass spectrometric method that allowed us to investigate the effects of common MAPK inhibitors on the immunopeptidome of melanoma cells. This approach has led to the discovery of new potential targets for cancer immunotherapy.

immunology↗

bioRxiv2024

Mass Spectrometry Imaging of the Subchondral Bone in Osteoarthritis Reveals Tissue Remodeling of Extracellular Matrix Proteins that Precede Cartilage Loss

Osteoarthritis (OA) is a degenerative condition of the skeletal extracellular matrix (ECM) marked by the loss of articular cartilage and changes to subchondral bone homeostasis. Treatments for OA beyond full joint replacement are lacking primarily due to gaps in molecular knowledge of the biological drivers of disease. Mass Spectrometry Imaging (MSI) enables molecular spatial mapping of the proteomic landscape of tissues. Histologic sections of human tibial plateaus from knees of human OA patients and cadaveric controls were treated with collagenase III to target ECM proteins prior to MS Imaging of bone and cartilage proteins using a timsTOF fleX mass spectrometer. Spatial MSI data of the knee were processed and automatically segmented identifying distinct areas of knee joint damage. ECM peptide markers were compared between i) the medial halves of OA patient joints and the medial side of non-OA (cadaveric) joints, and ii) between the same medial OA tissues and their corresponding, less OA impacted, lateral joint halves. Distinct peptide signatures distinguished OA medial tissues from the cadaveric medial and OA lateral tissues (AUROC >0.85). Overall, 31 peptide candidates from ECM proteins, including Collagen alpha-1(I), Collagen alpha-1(III), and surprisingly, Collagen alpha-1(VI) and Collagen alpha-3(VI), exhibited significantly elevated abundance in diseased tissues. Highly specific hydroxyproline-containing collagen peptides, mainly from collagen type I, dominated OA subchondral bone directly under regions of lost cartilage. The identification of specific protein markers for subchondral bone remodeling in OA advances our molecular understanding of disease progression in OA and provides potential new biomarkers for OA detection and disease grading.

cell biology↗

bioRxiv2024

Senescent cell heterogeneity and responses to senolytic treatment are related to cell cycle status during cell growth arrest.

Cellular senescence has been strongly linked to aging and age-related diseases. It is well established that the phenotype of senescent cells is highly heterogeneous and influenced by their cell type and senescence-inducing stimulus. Recent single-cell RNA-sequencing studies identified heterogeneity within senescent cell populations. However, proof of functional differences between such subpopulations is lacking. To identify functionally distinct senescent cell subpopulations, we employed high-content image analysis to measure senescence marker expression in primary human endothelial cells and fibroblasts. We found that G2-arrested senescent cells feature higher senescence marker expression than G1-arrested senescent cells. To investigate functional differences, we compared IL-6 secretion and response to ABT263 senolytic treatment in G1 and G2 senescent cells. We determined that G2-arrested senescent cells secrete more IL-6 and are more sensitive to ABT263 than G1-arrested cells. We hypothesize that cell cycle dependent DNA content is a key contributor to the heterogeneity within senescent cell populations. This study demonstrates the existence of functionally distinct senescent subpopulations even in culture. This data provides the first evidence of selective cell response to senolytic treatment among senescent cell subpopulations. Overall, this study emphasizes the importance of considering the senescent cell heterogeneity in the development of future senolytic therapies.

cell biology↗