Biology subjects

Rose, J.

Publications and source records attributed to Rose, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Ascorbate and TET2 couple balanced hematopoiesis to disease tolerance

Infections stimulate myelopoiesis but this comes at the cost of reduced production of other lineages. It is unclear if this imbalance is protective or damaging, and if factors that promote balanced hematopoiesis can impact infection pathology. Here we show that the ascorbate-TET axis limits excessive myelopoiesis, promotes balanced hematopoiesis, and is essential for surviving malaria. Hematopoietic-specific ascorbate depletion promoted myelopoiesis. The effects of ascorbate were mediated by TET2/TET3 activity in myeloid progenitors. Combined ascorbate and TET2 depletion markedly skewed hematopoiesis to the myeloid lineage and impaired erythropoiesis. Ascorbate is commonly depleted in infections, but the functional significance of this remains unclear. In the absence of ascorbate and TET2, mice infected with Plasmodium parasites could clear the pathogen, but had exacerbated myeloid and impaired erythroid response, anemia, and tissue damage and they succumbed to infection, indicating compromised disease tolerance. Therefore ascorbate and TET2 promote balanced hematopoiesis to impart tolerance to malaria.

immunology↗

bioRxiv2026

Ascorbate is a physiological tumor suppressor metabolite in leukemia

Accumulation of oncometabolites promotes tumorigenesis, but it is not known if there are naturally occurring tumor suppressor metabolites whose deficiency within cancer-initiating cells causes cancer. Many leukemia patients are ascorbate deficient. Here we show that hematopoietic-specific ascorbate depletion in mice is sufficient to cause or promote leukemogenesis. Hematopoietic-specific ascorbate depletion promoted leukemogenesis by acting within leukemia initiating cells. Ascorbate depletion in myeloid leukemia expanded plasmacytoid dendritic cells, an effect mediated by the ascorbate molecular targets TET2 and TET3. Leukemia cells accumulated ascorbate as part of a tumor suppressor response to neoplastic proliferation that limited progression but depleted circulating ascorbate. Ascorbate restoration in mice with established leukemias extended survival. Hematopoietic-specific ascorbate depletion caused leukemia in collaboration with TET2 loss and was sufficient to cause leukemia with high penetrance even in the absence of other engineered mutations. These results identify ascorbate as a physiological tumor suppressor metabolite in blood cancers.

cancer biology↗

bioRxiv2026

Loss of TCA cycle turning promotes stem cell function

The citric acid cycle (TCA cycle) is the common terminal pathway for the oxidation of all nutrients. Citrate oxidation to oxaloacetate produces CO2, and citrate synthase (CS) uses nutrient-derived acetyl groups to regenerate citrate and fuel cycle turning. However, the essentiality of cycle fueling and turning in vivo remains unclear. Here we use hematopoiesis, the most proliferative system in the body, as a model to show that, contrary to common assumptions, TCA cycle turning is dispensable for respiration, survival, and proliferation of stem and progenitor cells in vivo, and its loss promotes stem cell function. Hematopoietic-specific Cs deletion in adult mice blocked citrate cycling without reducing the frequency of hematopoietic stem (HSC) and progenitor cells. HSCs and progenitor cells adapted to TCA cycle loss by markedly increasing nutrient consumption and biosynthesis. Disruption of cycle turning increased HSC regeneration, myeloid progenitor proliferation, and myelopoiesis in vivo. HSCs without a turning TCA cycle outcompeted wild-type HSCs within the same environment. The effect of CS deletion on HSC function was not phenocopied by genetic ablation of cytosolic citrate use and was rescued by ablation of glutamine use in biosynthesis. Therefore, TCA cycle turning restrains nutrient uptake, biosynthesis, cell proliferation, and stem cell function. These results suggest an explanation for the reduction in cycle activity observed in many normal proliferating cells and cancer cells.

biochemistry↗

bioRxiv2026

Neural subspace reorganization reflects value-based decision making

Value-based decision making requires maintaining and comparing multiple option representations associated with different values. Then, the chosen option must be communicated to downstream regions to drive behavior. The neural circuits involved in value-based decisions are increasingly well understood, but less understood is how decisions shape option representations. We recorded from lateral prefrontal cortex (LPFC), a central hub for transforming options into actions, while non-human primates (NHPs) held two sequentially presented option-value pairs (spatial targets and abstract reward cues) in working memory, then chose the option with the higher value. This revealed how decisions dynamically reorganize option representations in LPFC. We found that, once decisions could be made, representations of chosen and unchosen options rotated into orthogonal subspaces and the chosen option representation was expanded. Before decisions, the first- and second-presented options were maintained separately. After decisions, the chosen option was rotated into a subspace with a consistent representation of the prescribed action, regardless of its presentation order. This suggests a mechanism for value-based decisions where the decision drives a neural subspace reorganization that facilitates selective and efficient readout of the chosen action.

neuroscience↗

bioRxiv2025

Loss of vitamin C biosynthesis protects from a parasitic infection

The ability to synthesize essential molecules is sometimes lost in evolution. A classic example is ascorbate (Vitamin C), which is synthesized in most animals by L-Gulonolactone Oxidase (GULO), an enzyme lost multiple independent times in animal evolution. This event is thought to be evolutionarily neutral, however, GULO-deficient animals including humans need to obtain ascorbate from their diet and are susceptible to ascorbate deficiency and scurvy. We therefore hypothesized that this disadvantage of GULO loss is offset by physiological benefits. Here we show that ascorbate deficiency protects mice from schistosomiasis, a debilitating parasitic disease which afflicts 250 million people. Schistosoma mansoni worms required host ascorbate to produce eggs in vivo. Consequently, ascorbate-deficient mice were protected from schistosomiasis pathologies and transmission. Intermittent ascorbate deficiency protected Gulo-deficient mice from both scurvy and schistosomiasis mortality. The effects of ascorbate on schistosome reproduction were mediated by ascorbate-dependent histone demethylation which promoted vitellocyte development in female schistosomes. We propose that vitamin deficiencies are not always detrimental but can protect animals from pathogens which need to obtain vitamins from their host.

biochemistry↗

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

Contingency violation in extinction learning

In extinction learning, contextual renewal occurs when an extinguished behavior reemerges after a context change. A key question is how stimuli become integrated as contextual cues. While contingency, the predictive relationship between stimuli and outcomes; is known to be important, its precise contribution remains unclear. Using a ABA renewal design with pigeons in operant chambers, we systematically violated contingency by probabilistically reinforcing responses during extinction. Our results show that partial violations of contingency modulated extinction learning but did not abolish contextual renewal. Instead, pigeons developed meta-learning strategies, adapting their behavior across sessions to optimize reward despite extinction conditions. These findings highlight that context formation is sensitive to contingency levels, but also that animals can flexibly reorganize their learning strategies when contingency is unstable.

animal behavior and cognition↗

bioRxiv2025

Divide and conquer: How avian 'prefrontal' and hippocampal neurons process extinction learning in complementary ways

Context-dependent extinction learning enables organisms to acquire an inhibition of responding to cues that no longer signal reward in specific environmental settings. Both the hippocampus and the prefrontal cortex play key but complementary roles in encoding extinction. To understand what drives the differential contributions of these two structures, we recorded single-unit responses from the pigeon hippocampus (HPC) and the prefrontal nidopallium caudolaterale (NCL), while the animals were engaged in a repeated appetitive ABA extinction learning paradigm. HPC carried more information about experimental phases (acquisition (context A), extinction (context B), and renewal (context A)) than NCL. In addition, hippocampal neurons with mixed selectivity integrated all contextual stimuli and conditioned cues, thereby possibly enabling context integration into phase-dependent extinction events. The mixed selectivity pattern of NCL neurons differed: They encoded all relevant task parameters separately, thereby possibly enabling the weighing and deciding between response alternatives. Repeatedly testing the same animals over many months enabled us to reveal that the activity of NCL neurons increasingly became predictive of the number of responses that animals made during renewal. Thus, NCL neurons possibly assumed a meta-learning ability that enabled a behavioral adaption to the deeper task structure that always followed the sequence of acquisition[->]extinction[->]renewal. Our results make it likely that different processing strategies of hippocampus and prefrontal NCL enable a differential encoding of experimental phase, context, decision-making, and meta-learning during extinction learning.

neuroscience↗

bioRxiv2024

Distinct representational properties of cues and contexts shape fear learning and extinction

When we learn that something is dangerous, a fear memory is formed. However, this memory is not fixed and can be updated through new experiences, such as learning that the threat is no longer present. This process of updating, known as extinction or reversal learning, is highly dependent on the context in which it occurs. How the brain represents cues, contexts, and their changing threat value remains a major question. Here, we used functional magnetic resonance imaging and a novel fear learning paradigm to track the neural representations of stimuli across fear acquisition, reversal, and test phases. We found that initial fear learning creates generalized neural representations for all threatening cues in the brains fear network. During reversal learning, when threat contingencies switched for some of the cues, two distinct representational strategies were observed. On the one hand, we still identified generalized patterns for currently threatening cues, whereas on the other hand, we observed highly stable representations of individual cues (i.e., item-specific) that changed their valence, particularly in the precuneus and prefrontal cortex. Furthermore, we observed that the brain represents contexts more distinctly during reversal learning. Furthermore, additional exploratory analyses showed that the degree of this context specificity in the prefrontal cortex predicted the subsequent return of fear, providing a potential neural mechanism for fear renewal. Our findings reveal that the brain uses a flexible combination of generalized and specific representations to adapt to a changing world, shedding new light on the mechanisms that support cognitive flexibility and the treatment of anxiety disorders via exposure therapy.

neuroscience↗

bioRxiv2024

Breaking Free: Development of Circular AAV Cargos for Targeted Seamless Integration in the Liver

Recent advancements in gene insertion have shifted from DNA repair-dependent mechanisms to more precise approaches, enhancing safety and predictability for editing outcomes. Integrase-mediated programmable genomic integration (I-PGI) utilizes a DNA cargo to insert transgenes in a targeted, unidirectional manner. In vivo, where nuclear delivery of DNA is challenging, adeno-associated virus (AAV) can act as the cargo vector. While I-PGI does not require DNA double-stranded breaks (DSBs) for activity, linear DNA cargo, like AAV, stimulates DNA end joining activity after integration. To mitigate potential risks from DSBs with linear viral cargo, we developed two circular genome types capable of seamless gene insertion in non-dividing cells. We first harnessed the orthogonal property of large serine integrases to produce circle-AAV (cAAV) from linear viral genomes in cells. cAAV demonstrated faithful seamless cargo integration in primary human hepatocytes (PHH) and robust DSB-free insertion structures in vivo. We then investigated the delivery of packaged circular AAV cargo (AAV.AD), which eliminates the need for enzymatic manipulation in the cell. AAV.AD proved to be a viable cargo for I-PGI, exhibiting functional integration in PHH and in vivo, that resulted in seamless insertion structures. Together, these findings provide the first reported evidence of DSB-free programmable genomic integration using integrase and AAV cargo, addressing a previously unrecognized challenge in the field.

bioengineering↗

bioRxiv2024

Engineered Bxb1 variants improve integrase activity and fidelity

Many current genome editing technologies rely on the action of large serine integrases (LSIs) to insert gene-sized DNA sequences into the genome. Bxb1 is the most commonly used LSI for therapeutic efforts, including PASTE, PASSIGE and I-PGI. While Bxb1 demonstrated good activity in vitro in cycling cells, the activity in non-dividing hepatocytes was significantly less efficient. Further, wild-type Bxb1 is known to have detectable off-target activity at cryptic genomic sites, which presents a potential safety risk for therapeutic development. To address these issues, we developed Bxb1 variants that demonstrate increased specificity and potency in vitro and have engineered stabilized Bxb1 variants that increase in vivo activity over 25-fold enabling targeted integration at therapeutically relevant levels.

biochemistry↗

bioRxiv2023

Characterization of SARS-CoV-2 Convalescent Patients' Serological Repertoire Reveals High Prevalence of Iso-RBD Antibodies

While our understanding of SARS-CoV-2 pathogenesis and antibody responses following infection and vaccination has improved tremendously since the outbreak in 2019, the sequence identities and relative abundances of the individual constituent antibody molecules in circulation remain understudied. Using Ig-Seq, we proteomically profiled the serological repertoire specific to the whole ectodomain of SARS-CoV-2 prefusion-stabilized spike (S) as well as to the receptor binding domain (RBD) over a 6-month period in four subjects following SARS-CoV-2 infection before SARS-CoV-2 vaccines were available. In each individual, we identified between 59 and 167 unique IgG clonotypes in serum. To our surprise, we discovered that [~]50% of serum IgG specific for RBD did not recognize prefusion-stabilized S (referred to as iso-RBD antibodies), suggesting that a significant fraction of serum IgG targets epitopes on RBD inaccessible on the prefusion-stabilized conformation of S. On the other hand, the abundance of iso-RBD antibodies in nine individuals who received mRNA-based COVID-19 vaccines encoding prefusion-stabilized S was significantly lower ([~]8%). We expressed a panel of 12 monoclonal antibodies (mAbs) that were abundantly present in serum from two SARS-CoV-2 infected individuals, and their binding specificities to prefusion-stabilized S and RBD were all in agreement with the binding specificities assigned based on the proteomics data, including 1 iso-RBD mAb which bound to RBD but not to prefusion-stabilized S. 2 of 12 mAbs demonstrated neutralizing activity, while other mAbs were non-neutralizing. 11 of 12 mAbs also bound to S (B.1.351), but only 1 maintained binding to S (B.1.1.529). This particular mAb binding to S (B.1.1.529) 1) represented an antibody lineage that comprised 43% of the individuals total S-reactive serum IgG binding titer 6 months post-infection, 2) bound to the S from a related human coronavirus, HKU1, and 3) had a high somatic hypermutation level (10.9%), suggesting that this antibody lineage likely had been elicited previously by pre-pandemic coronavirus and was re-activated following the SARS-CoV-2 infection. All 12 mAbs demonstrated their ability to engage in Fc-mediated effector function activities. Collectively, our study provides a quantitative overview of the serological repertoire following SARS-CoV-2 infection and the significant contribution of iso-RBD antibodies, demonstrating how vaccination strategies involving prefusion-stabilized S may have reduced the elicitation of iso-RBD serum antibodies which are unlikely to contribute to protection.

immunology↗

bioRxiv2023

Concerted epithelial and stromal changes during progression of Barrett's Esophagus to invasive adenocarcinoma exposed by multi-scale, multi-omics analysis

Esophageal adenocarcinoma arises from Barretts esophagus, a precancerous metaplastic replacement of squamous by columnar epithelium in response to chronic inflammation. Multi-omics profiling, integrating single-cell transcriptomics, extracellular matrix proteomics, tissue-mechanics and spatial proteomics of 64 samples from 12 patients paths of progression from squamous epithelium through metaplasia, dysplasia to adenocarcinoma, revealed shared and patient-specific progression characteristics. The classic metaplastic replacement of epithelial cells was paralleled by metaplastic changes in stromal cells, ECM and tissue stiffness. Strikingly, this change in tissue state at metaplasia was already accompanied by appearance of fibroblasts with characteristics of carcinoma-associated fibroblasts and of an NK cell-associated immunosuppressive microenvironment. Thus, Barretts esophagus progresses as a coordinated multi-component system, supporting treatment paradigms that go beyond targeting cancerous cells to incorporating stromal reprogramming. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/544265v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@64c375org.highwire.dtl.DTLVardef@15c6ef2org.highwire.dtl.DTLVardef@177dee0org.highwire.dtl.DTLVardef@c945e7_HPS_FORMAT_FIGEXP M_FIG Graphical AbstractTo obtain a comprehensive picture of the coordinated changes in epithelial, stromal and immune compartments during development of Barretts-associated esophageal adenocarcinoma, patient-matched samples corresponding to various phases of disease progression were collected from 12 patients, each of which had at a given time point lesions at multiple stages progression (matched-normal, metaplasia, dysplasia, and carcinoma). Matched "normal" gastric tissues were also collected. These sample were analyzed by single cell RNA-sequencing (scRNAseq) for single-cell resolution transcriptomics and Copy Number Variant (CNV), by proteomics for extracellular matrix (ECM) proteins, by Atomic Force Microscopy (AFM for tissue stiffness and by CODEX spatial proteomics imaging The integrative multi-omics analysis exposed drastic alterations in cell type composition and shifts in cell states in all three compartments. A large subpopulation of fibroblasts absent in the normal esophagus and characteristic of dysplasia and adenocarcinoma sample, that based on markers would indeed be considered cancer associated fibroblasts (CAF), appeared already in the metaplastic phase. This fibroblast subpopulation had transcriptomes virtually indistinguishable with fibroblasts of the cancer free gastric epithelium in these patients C_FIG

cancer biology↗

bioRxiv2023

Dicarboxylic acid supplementation protects from acute kidney injury via stimulation of renal peroxisomal activity

IntroductionLysine succinylation is a post-translational modification associated with the control of several diseases, including acute kidney injury (AKI). It is suggested that hypersuccinylation favors peroxisomal fatty acid oxidation (FAO) instead of mitochondrial. In addition, the medium-chain fatty acids (MCFAs) dodecanedioic acid (DC12) and octanedioic acid (DC8), upon FAO, generate succinyl-CoA, resulting in hypersuccinylation. DC8 is convenient, inexpensive, easily administered, and efficient. We believe this study could be translated in the future to clinical settings, which would highly benefit patients at high risk of AKI. Methods and ResultsTo test the protective roles of MCFAs during AKI, mice were fed with control, 10% DC12, or 10% DC8 diet, then, subjected to either ischemic-AKI, or cisplatin-AKI models. Supplementation was provided until sacrifice. Biochemical, histologic, genetic, and proteomic analysis were performed, the latter involving a lysine-succinylome-based analysis. Both DC8 and DC12 prevented the rise of AKI markers in mice that underwent renal injury. However, DC8 was even more protective against AKI than DC12. Finally, succinylome analysis evidenced that the kidneys of DC8-fed mice showed an extensive succinylation of peroxisomal activity-related proteins, and a decline in mitochondrial FAO, in comparison to control-fed mice. ConclusionDC8 supplementation drives renal protein hypersuccinylation, promoting a shift from mitochondrial to peroxisomal FAO, and protecting against AKI. Significance StatementLysine succinylation of proteins is shown to control several diseases, including acute kidney injury (AKI). Here we show that mice supplemented with the medium-chain fatty acid octanedioic acid successfully presented a high level of succinylation and were protected from both ischemia-reperfusion- and cisplatin-induced AKI. Moreover, our study demonstrates that peroxisomal activity was increased while mitochondrial activity was preserved, suggesting that the metabolism of diet-obtained medium-chain fatty acids by peroxisomes is renoprotective.

pathology↗

bioRxiv2023

Helminth infection impacts hematopoiesis

Helminth infections are common in animals. However, the impact of a helminth infection on the function of hematopoietic stem cells (HSCs) and other hematopoietic cells has not been comprehensively defined. Here we describe the hematopoietic response to infection of mice with Schistosoma mansoni, a parasitic flatworm which causes schistosomiasis. We analyzed the frequency or number of hematopoietic cell types in the bone marrow, spleen, liver, thymus, and blood, and observed multiple hematopoietic changes caused by infection. Schistosome infection impaired bone marrow HSC function after serial transplantation. Functional HSCs were present in the infected liver. Infection blocked bone marrow erythropoiesis and augmented spleen erythropoiesis, observations consistent with the anemia and splenomegaly prevalent in schistosomiasis patients. This work defines the hematopoietic response to schistosomiasis, a debilitating disease afflicting more than 200 million people, and identifies impairments in HSC function and erythropoiesis.

immunology↗

bioRxiv2023

Fructose Induced KHK-C Increases ER Stress and Modulates Hepatic Transcriptome to Drive Liver Disease in Diet-Induced and Genetic Models of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome, and is estimated to affect one billion individuals worldwide. An increased intake of a high-fat diet (HFD) and sugar-sweetened beverages are risk-factors for NAFLD development, but how their combined intake promotes progression to a more severe form of liver injury is unknown. Here we show that fructose metabolism via ketohexokinase (KHK) C isoform increases endoplasmic reticulum (ER) stress in a dose dependent fashion, so when fructose is coupled with a HFD intake it leads to unresolved ER stress. Conversely, a liver-specific knockdown of KHK in C57BL/6J male mice consuming fructose on a HFD is adequate to improve the NAFLD activity score and exert a profound effect on the hepatic transcriptome. Overexpression of KHK-C in cultured hepatocytes is sufficient to induce ER stress in fructose free media. Upregulation of KHK-C is also observed in genetically obesity ob/ob, db/db and lipodystrophic FIRKO male mice, whereas KHK knockdown in these mice improves metabolic function. Additionally, in over 100 inbred strains of male or female mice hepatic KHK expression correlates positively with adiposity, insulin resistance, and liver triglycerides. Similarly, in 241 human subjects and their controls, hepatic Khk expression is upregulated in early, but not late stages of NAFLD. In summary, we describe a novel role of KHK-C in triggering ER stress, which offers a mechanistic understanding of how the combined intake of fructose and a HFD propagates the development of metabolic complications.

molecular biology↗

bioRxiv2022

Elucidation of Amyloid-Beta's Gambit in Oligomerisation:Truncated Aβ fragments of residues Aβ1-23, Aβ1-24 and Aβ1-25 rapidly seed to form SDS-stable, low molecular weight Aβ oligomers that impair synaptic plasticity.

In Alzheimers disease (AD), Amyloid-beta (A{beta}) oligomers are considered an appealing therapeutic- and diagnostic target. However, to date, the molecular mechanisms associated with the pathological accumulation or structure of A{beta} oligomers remains an enigma to the scientific community. Here we demonstrate the strong seeding properties of unique A{beta} fragment signatures and show that the truncated A{beta} peptides of residues A{beta}1-23, A{beta}1-24 and A{beta}1-25, rapidly seed to form small, SDS-PAGE stable assemblies of [~]5kDa to [~]14kDa molecular mass range. Mass spectrometry analysis of SDS-PAGE fractionated and gel extracted oligomers revealed that the truncated A{beta} isoforms of residues 1-23 to 1-25 form stable entities with low molecular weight (LMW) oligomers, which strongly resemble the regularly reported A{beta} entities of putative dimeric or trimeric assemblies found in human post-mortem AD and Tg mouse brain extracts. Furthermore, electrophysiological recordings in the mouse hippocampus indicate that LMW A{beta} assemblies formed by fragments A{beta}1-23 to A{beta}1-25 significantly impair long-term-potentiation (LTP) in the absence of full-length A{beta}1-42. Extensive antibody screening highlights the important observation, that the LMW A{beta} assemblies formed by these truncated A{beta} peptides escape immuno-detection using conventional, conformation specific antibodies but, more importantly, the clinical antibody aducanumab. Our novel findings suggest that there are new A{beta} target "loopholes" which can be exploited for the development of therapeutic antibodies with binding properties against stable target hotspots present in A{beta} oligomers. We provide here a first example of a new class of monoclonal antibody with unique binding properties against LMW A{beta} oligomers, in the absence of binding to large fibrillar A{beta} assemblies, or dense amyloid plaques. Our research supports a novel, unparalleled approach for targeting early, pathological A{beta} species during the insidious phase of AD and prior to the appearance of large oligomeric or protofibrilar assemblies.

neuroscience↗

bioRxiv2022

Data-Independent Acquisition and Quantification of Extracellular Matrix from Human Lung in Chronic Inflammation-Associated Carcinomas

Early events associated with chronic inflammation and cancer involve significant remodeling of the extracellular matrix (ECM), which greatly affects its composition and functional properties. Using lung squamous cell carcinoma (LSCC), a chronic inflammation-associated cancer (CIAC), we optimized a robust proteomic pipeline to discover potential biomarker signatures and protein changes specifically in the stroma. We combined ECM enrichment from fresh human tissues, data-independent acquisition strategies, and stringent statistical processing to analyze Tumor and matched adjacent histologically normal ( Matched Normal) tissues from patients with LSCC. Overall, 1,802 protein groups were quantified with at least two unique peptides, and 56% of those proteins were annotated as extracellular. Confirming dramatic ECM remodeling during CIAC progression, 529 proteins were significantly altered in the Tumor compared to Matched Normal tissues. The signature was typified by a coordinated loss of basement membrane proteins and small leucine-rich proteins. The dramatic increase in the stromal levels of SERPINH1/heat shock protein 47, that was discovered using our ECM proteomic pipeline, was validated by immunohistochemistry (IHC) of Tumor and Matched Normal tissues, obtained from an independent cohort of LSCC patients. This integrated workflow provided novel insights into ECM remodeling during CIAC progression, and identified potential biomarker signatures and future therapeutic targets. Statement of significance of the studyThe extracellular matrix (ECM) is a complex scaffolding network composed of glycoproteins, proteoglycans and collagens, which binds soluble factors and, most importantly, significantly impacts cell fate and function. Alterations of ECM homeostasis create a microenvironment promoting tumor formation and progression, therefore deciphering molecular details of aberrant ECM remodeling is essential. Here, we present a multi-laboratory and refined proteomic workflow, featuring i) the prospective collection of tumor and matched histologically normal tissues from patients with lung squamous cell carcinoma, ii) the enrichment for ECM proteins, and iii) subsequent label-free data-independent acquisition (DIA)-based quantification. DIA is a powerful strategy to comprehensively profile and quantify all detectable precursor ions contained in the biological samples, with high quantification accuracy and reproducibility. When combined with very stringent statistical cutoffs, this unbiased strategy succeeded in capturing robust and highly confident proteins changes associated with cancer, despite biological variability between individuals. This label-free quantification workflow provided the flexibility required for ongoing prospective studies. Discussions with clinicians, surgeons, pathologists, and cancer biologists represent an opportunity to interrogate the DIA digitalized maps of the samples for newly formulated questions and hypotheses, thus gaining insights into the continuum of the disease and opening the path to novel ECM-targeted therapies.

cancer biology↗