Comprehensive Proteomic Profiling Research Articles

P28 Data-independent acquisition mass spectrometry improves spatially resolved analysis of the human skin proteome

Abstract Introduction and aims Proteomic analysis of the extracellular matrix (ECM) presents challenges because of the highly crosslinked and low-solubility nature of ECM proteins. Traditional homogenization and protein digestion approaches result in the loss of crucial information regarding protein localization and spatial relationships. To address this, spatially resolved proteomics emerges as a powerful tool for exploring heterogeneity within bulk tissues. This study aims to determine the minimum tissue volume required for comprehensive proteome coverage using data-independent acquisition mass spectrometry (DIA-MS) on skin tissue. The study focused on optimizing spatially resolved proteomic techniques to enhance depth-of-analysis while preserving spatial specificity. Methods Human abdominal skin biopsies were obtained from a single individual and subsequently cryosectioned. Histological assessment was performed through haematoxylin and eosin staining for visualization purposes. Laser-capture microdissection coupled with mass spectrometry facilitated the precise isolation of target regions. Comparative analyses were performed between data-dependent acquisition mass spectrometry (DDA-MS) and DIA-MS, with a particular emphasis on ECM proteins within the dermis. Results Our findings revealed an improvement in proteome coverage with DIA-MS compared with DDA-MS, in addition to clear scaling relationships between the depth-of-analysis and sample concentration. The Results demonstrated the superiority of DIA-MS in achieving robust and comprehensive proteomic profiles, even with minimal tissue volumes. Preliminary findings suggest the capability of DIA-MS in elucidating the complexities of the skin proteome with spatial resolution. Conclusions In conclusion, our study highlights the efficacy of DIA-MS in spatially resolved proteomics on skin tissue. The optimized approach presented here offers a reliable and efficient method for obtaining in-depth proteome information with minimal tissue requirements. These Results form the foundation for ongoing experiments, utilizing DIA-MS to advance spatially resolved proteomic analyses of human skin.

Proteomics profiling of the honeybee parasite Tropilaelaps mercedesae across post-embryonic development

Tropilaelaps mercedesae, an ectoparasitic mite of honeybees, is currently a severe health risk to Apis mellifera colonies in Asia and a potential threat to the global apiculture industry. However, our understanding of the physiological and developmental regulation of this pest remains significantly insufficient. Using ultra-high resolution mass spectrometry, we provide the first comprehensive proteomic profile of T. mercedesae spanning its entire post-embryonic ontogeny, including protonymphs, deutonymphs, mature adults, and reproductive mites. Consequently, a total of 4,422 T. mercedesae proteins were identified, of which 2,189 proteins were significantly differentially expressed (FDR < 0.05) throughout development and maturation. Our proteomic data provide an important resource for understanding the biology of T. mercedesae, and will contribute to further research and effective control of this devastating honeybee pest.

Optimizing Spectronaut Search Parameters to Improve Data Quality with Minimal Proteome Coverage Reductions in DIA Analyses of Heterogeneous Samples.

Data-independent acquisition has seen breakthroughs that enable comprehensive proteome profiling using short gradients. As the proteome coverage continues to increase, the quality of the data generated becomes much more relevant. Using Spectronaut, we show that the default search parameters can be easily optimized to minimize the occurrence of false positives across different samples. Using an immunological infection model system to demonstrate the impact of adjusting search settings, we analyzed Mus musculus macrophages and compared their proteome to macrophages spiked withCandida albicans. This experimental system enabled the identification of "false positives" as Candida albicans peptides and proteins should not be present in the Mus musculus-only samples. We show that adjusting the search parameters reduced "false positive" identifications by 89% at the peptide and protein level, thereby considerably increasing the quality of the data. We also show that these optimized parameters incurred a moderate cost, only reducing the overall number of "true positive" identifications across each biological replicate by <6.7% at both the peptide and protein level. We believe the value of our updated search parameters extends beyond a two-organism analysis and would be of great value to any DIA experiment analyzing heterogeneous populations of cell types or tissues.

Insights into the differential proteome landscape of a newly isolated Paramecium multimicronucleatum in response to cadmium stress

Employing microbial systems for the bioremediation of contaminated waters represent a potential option, however, limited understanding of the underlying mechanisms hampers the implication of microbial-mediated bioremediation. The omics tools offer a promising approach to explore the molecular basis of the bioremediation process. Here, a mass spectrometry-based quantitative proteome profiling approach was conducted to explore the differential protein levels in cadmium-treated Paramecium multimicronucleatum. The Proteome Discoverer software was used to identify and quantify differentially abundant proteins. The proteome profiling generated 7,416 peptide spectral matches, yielding 2824 total peptides, corresponding to 989 proteins. The analysis revealed that 29 proteins exhibited significant (p ≤ 0.05) differential levels, including a higher abundance of 6 proteins and reduced levels of 23 proteins in Cd2+ treated samples. These differentially abundant proteins were associated with stress response, energy metabolism, protein degradation, cell growth, and hormone processing. Briefly, a comprehensive proteome profile in response to cadmium stress of a newly isolated Paramecium has been established that will be useful in future studies identifying critical proteins involved in the bioremediation of metals in ciliates. SignificanceCiliates are considered a good biological indicator of chemical pollution and relatively sensitive to heavy metal contamination. A prominent ciliate, Paramecium is a promising candidate for the bioremediation of polluted water. The proteins related to metal resistance in Paramecium species are still largely unknown and need further exploration. In order to identify and reveal the proteins related to metal resistance in Paramecia, we have reported differential protein abundance in Paramecium multimicronucleatum in response to cadmium stress. The proteins found in our study play essential roles during stress response, hormone processing, protein degradation, energy metabolism, and cell growth. It seems likely that Paramecia are not a simple sponge for metals but they could also transform them into less toxic derivatives or by detoxification by protein binding. This data will be helpful in future studies to identify critical proteins along with their detailed mechanisms involved in the bioremediation and detoxification of metal ions in Paramecium species.

Rintatolimod in Advanced Pancreatic Cancer Enhances Antitumor Immunity through Dendritic Cell-Mediated T-Cell Responses.

Amid the need for new approaches to improve survival in pancreatic ductal adenocarcinoma (PDAC), immune-based therapies have garnered interest. Rintatolimod, a Toll-like receptor 3 (TLR-3) agonist, is a potential candidate due to its dual impact on restraining PDAC cell functions and boosting the antitumor immune response. This study investigates the effect of TLR-3 activation through rintatolimod on the peripheral immune landscape of patients with advanced PDAC. Paired blood samples of 30 patients with advanced PDAC, collected at baseline and after 12 rintatolimod intravenous infusions, underwent comprehensive transcriptomic NanoString and proteomic flow cytometry profiling. The impact of rintatolimod and immunologic factors on survival outcomes was assessed through univariate Cox proportional hazards models. Rintatolimod treatment enhances peripheral immune activity at the transcriptomic and proteomic levels, particularly involving type 1 conventional dendritic cells (cDC1) and T cells. Post-rintatolimod, the increased peripheral abundance of BTLA+ XCR1+ cDC1s and CD4+SELL+ T cells correlated with improved clinical outcomes. Patients with stable disease exhibited pronouncedDCand T-cell activation gene overexpression. Notably, the expression of immune checkpoints PD-L1 and PD-L2 decreased post-rintatolimod across all patients. However, those with progressive disease showed increased expression of genes encoding IDO1 and PD-1. This study presents compelling evidence of the immune-stimulatory properties linked to TLR-3 activation through rintatolimod. Rintatolimod may break immunologic tolerance by enhancing antitumor immunity through DC-mediated Th-cell responses. Furthermore, our findings lay the groundwork for investigating the potential synergy between TLR-3 activation and immune checkpoint inhibitor therapy to improve therapeutic outcomes. See related commentary by Martínez-Riaño et al., p. 3355.

Comprehensive profiling of the human fecal proteome from IBD patients with DIA-MS enables evaluation of disease-relevant proteins.

Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), is characterized by chronic gastrointestinal inflammation. A high unmet need exists for noninvasive biomarkers in IBD to monitor changes in disease activityand guide treatment decisions. Stool is an easily accessed, disease proximal matrix in IBD, however the composition of the IBD fecal proteome remains poorly characterized. A data-independent acquisition LC-MS/MS approach was used to profile the human fecal proteome in two independent cohorts (Cohort 1: healthy n=5, UC n=5, CD n=5, Cohort 2: healthy n=20, UC n=10, and CD n=10) to identify noninvasive biomarkers reflective of disease activity. 688 human proteins were quantified, with 523 measured in both cohorts. In UC stool 96 proteins were differentially abundant and in CD stool 126 proteins were differentially abundant compared to healthy stool (absolute log2 fold change>1, p-value<0.05). Many of these fecal proteins are associated with infiltrating immune cells and ulceration/rectal bleeding, which are hallmarks of IBD pathobiology. Mapping the identified fecal proteins to a whole blood single-cell RNA sequencing data set revealed the involvement of various immune cell subsets to the IBD fecal proteome. Findings from this study not only confirmed the presence of established fecal biomarkers for IBD, such as calprotectin and lactoferrin, but also revealed new fecal proteins from multiple pathways known to be dysregulated in IBD. These novel proteins could serve as potential noninvasive biomarkers to monitor specific aspects of IBD disease activity which could expedite clinical development of novel therapeutic targets.

Abstract 1425: Epigenetic modulation synergizes with a novel TCR β chain directed antibody-fusion molecule to suppress checkpoint-refractory tumors irrespective of MHC I status

Abstract Background: Epigenetic silencing of MHC-I, and poor T cell infiltration and function in the tumor microenvironment (TME) are prevalent in solid malignancies and associate with lack of response to immune checkpoint blockade (ICB). Novel combination therapies may overcome these barriers, thereby providing clinical benefit across solid tumors. STAR0602 is a first-in-class bifunctional therapeutic molecule, that simultaneously engages a nonclonal mode of T cell receptor (TCR) activation with costimulation to promote activation and expansion of αβ T cell subsets expressing distinct variable β (Vβ) TCR chains. Here, we examined the tumor-suppressive ability and mode of action of a murine surrogate of STAR0602 (mSTAR1302) in combination with the class I HDAC inhibitor Entinostat, an epigenetic modulator that enhances tumor immune cell recognition, in multiple tumor models, including ICB-refractory. Methods: mSTAR1302 and/or Entinostat were administered to mice bearing ICB-responsive breast EMT6 wild-type (wt) and ICB-refractory EMT6 β2-microglobulin (B2m)-deleted and CT26 (Kras G12Dmut colon) tumors. Anti-tumor efficacy, survival, and protective memory were monitored. Immune cell depletion studies were conducted. Comprehensive proteomic and immune profiling of tumor, tumor-draining lymph node (tdLN), and spleen cell populations was conducted. Antigen-specific IFNg T cell responses were evaluated by ELISpot. Results: Combination therapy demonstrated significant enhancement in tumor suppression and prolonged survival in CT26 (MHC-I+), EMT6 wt (MHC-I+) and EMT6 β2m-deleted (MHC-Inull) tumors relative to single agent therapy, as well as enhanced tumor-specific protective memory responses. Tumor suppression was independent of CD4+ T cell immunity and associated with sustained activation and expansion of Vβ13 CD8+ T cells. Conclusions: Ongoing mechanistic studies indicate combination therapy to elicit complimentary mechanisms of tumor suppression beyond CD8+ T cells. Ongoing proteomic and single-cell transcriptomic investigation of MHC-I competent and null tumors will provide a deeper insight into the mode of action of this combination therapy. Collectively, these data support the use of STAR0602 in combination with Entinostat for the treatment of solid malignancies, including for patients harboring ICB-refractory tumors devoid of MHC class I. Citation Format: Katherine E. Lothstein, Asma S. Khelifa, Masaya Miyamoto, Christine M. Minnar, Lisa Poppe, Nicholas Roller, Andrew Bayliffe, Ke Liu, Jacques Moisan, Madan Katragadda, Zhen Su, Jeffrey Schlom, Sofia R. Gameiro. Epigenetic modulation synergizes with a novel TCR β chain directed antibody-fusion molecule to suppress checkpoint-refractory tumors irrespective of MHC I status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1425.

Abstract 1847: Human protein atlas: A resource for target expression across 19 tissues

Abstract Proteins are the primary targets and off-targets of therapeutics such as small molecule and biologics. Thus, a thorough understanding of their expression profiles across various human tissues in health and disease is essential through all stages of drug development. However, generating such a comprehensive proteome profile for each tissue poses a major analytical challenge due to the large dynamic range of protein abundance within a tissue and the enormous complexity introduced by distinct proteoforms. These proteoforms may arise from genetic variations (e.g. mutations), posttranscriptional events (e.g. isoforms from alternative splicing) or posttranslational modifications, resulting in more than a million protein species from 20,000 protein-coding genes. Thanks to advancements in sensitivity and speed in the recent years, mass spectrometry (MS)-based proteomics can now be routinely applied to quantify ten thousand of proteins in biological samples. Taking advantage of MS being an unbiased and matrix-agnostic method, we pivoted the generation of a high quality and uniform protein expression atlas of 19 healthy human tissues and 3 cancerous tissues. We constructed the atlas in two parts aiming to address the following two aspects respectively: 1) a deep library providing evidence for protein/proteoform presence in each of the 22 tissues; 2) quantitative profiles for cross-comparison in all tissues. For the first part, each tissue sample was subjected to an extensive fractionation process followed by MS analysis of each of the fractions. In total, we identified 20’025 protein groups mapped to 17’715 protein-coding genes, providing protein-level evidence for &amp;gt; 90% of all protein-coding genes annotated in the neXtProt repository. Among all identified proteins, 10’755 were detectable across all tissues. Notably, we identified 37 proteins mapping to putative genes. In part two of the atlas, quantitative expression profiles were generated for 15’772 protein groups using TrueDiscovery DIA-MS across the 22 tissues. These data allow us to investigate ubiquitous vs. tissue-restricted protein expression of various protein classes such as surface receptors or kinases. Moreover, we performed correlation analysis between protein expression and mRNA abundance across 19 tissues, demonstrating that for a significant portion of the proteome, mRNA level-information is not an adequate proxy for protein expression. Collectively, this MS-based resource alleviates dependency on antibody-based reagents. We envision that the tissue atlas can be expanded to cover various disease indications, providing valuable insights for target discovery and assessment of target/off-target tissue distribution. Citation Format: Sandra Schaer, Marco Tognetti, Roland Bruderer, Polina Shichkova, Yuehan Feng, Lukas Reiter. Human protein atlas: A resource for target expression across 19 tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1847.

Abstract 1842: Global proteomics reveals integrin signaling as a driver of infiltration in glioblastoma

Abstract Background: Glioblastoma (GBM), a notably aggressive brain cancer, presents significant treatment challenges due to its propensity for deep, diffuse infiltration into healthy brain tissue. This trait enables GBM to resist and survive following surgery and conventional adjuvant therapies. Emerging evidence suggests that signaling between GBM stem cells (GSCs) may be instrumental in this invasive behavior. Our study aimed to unravel the intricacies of these intercellular communication networks within glioblastoma by employing comprehensive proteomic profiling across a diverse array of patient-derived GSC lines. Methods: We conducted our study using a sex-stratified cohort comprising 17 patient-derived GSC lines. Proteomic profiling encompassed analyses of both GSC lysates and conditioned media under various oxygen conditions (normoxia at 20% O2, hypoxia at 1% and 0.2% O2). Additionally, we incorporated the glioma cerebral organoid (GLICO) system, facilitating co-cultures of GSCs with human cerebral organoids. For in-depth proteomic analysis, we utilized advanced TMT-based liquid chromatography-tandem mass spectrometry (LC-MS/MS; Q-Exactive, Thermo Fisher). Results: Our findings corroborate that GSCs adhere to the tripartite model of proliferation, invasion, and hypoxic response. A salient discovery was the consistent enrichment of integrin signaling pathways in GSCs under hypoxic conditions. Specifically, we observed a significant association between upregulation in integrin signaling (BIOCARTA_INTEGRIN_PATHWAY) and elevated KRAS activity (SWEET_KRAS_TARGETS_UP: r=0.43, p&amp;lt;0.01), as well as hypoxic responses (WINTER_HYPOXIA_UP: r=0.76, p&amp;lt;0.0001) in GSCs. Notably, in the GLICO model, both integrin signaling and KRAS pathways were markedly upregulated in the presence of GSCs compared to cerebral organoids alone (p&amp;lt;0.05). Conclusions: This research highlights the pivotal role of integrin signaling in mediating the invasive attributes of glioblastoma. The data underscore the importance of further exploring key modulators within the integrin signaling pathway, presenting a promising avenue for the discovery of novel therapeutic targets in glioblastoma treatment. Citation Format: Lennart J. van Winden, Rifat Shahriar Sajid, Okty Abbasi Borhani, Evelyn Rose Kamski-Hennekam, Nakita Gopal, Alberto Leon, Phedias Diamandis. Global proteomics reveals integrin signaling as a driver of infiltration in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1842.

Proteomic Analysis of Cell Wall Proteins with Various Linkages in Fusarium graminearum.

The fungal cell wall, primarily comprising a glucan-chitin matrix and cell wall proteins (CWPs), serves as a key mediator for fungal interactions with the environment and plays a pivotal role in virulence. In this study, we employed a comprehensive proteomics approach to analyze the CWPs in the plant pathogenic fungus Fusarium graminearum. Our methodology successfully extracted and identified 1373 CWPs, highlighting their complex linkages, including noncovalent bonds, disulfide bridges, alkali-sensitive linkages, and glycosylphosphatidylinositol (GPI) anchors. A significant subset of these proteins, enriched in Gene Ontology terms, suggest multifunctional roles of CWPs. Through the integration of transcriptomic and proteomic data, we observed differential expression patterns of CWPs across developmental stages. Specifically, we focused on two genes, Fca7 and Cpd1, which were upregulated in planta, and confirmed their localization predominantly outside the plasma membrane, primarily in the cell wall and periplasmic space. The disruption of FCA7 reduced virulence on wheat, aligning with previous findings and underscoring its significance. Overall, our findings offer a comprehensive proteomic profile of CWPs in F. graminearum, laying the groundwork for a deeper understanding of their roles in the development and interactions with host plants.

Proteomic workflows for deep phenotypic profiling of 3D organotypic liver models.

Organotypic human tissue models constitute promising systems to facilitate drug discovery and development. They allow to maintain native cellular phenotypes and functions, which enables long-term pharmacokinetic and toxicity studies, as well as phenotypic screening. To trace relevant phenotypic changes back to specific targets or signaling pathways, comprehensive proteomic profiling is the gold-standard. A multitude of proteomic workflows have been applied on 3D tissue models to quantify their molecular phenotypes; however, their impact on analytical results and biological conclusions in this context has not been evaluated. The performance of twelve mass spectrometry-based global proteomic workflows that differed in the amount of cellular input, lysis protocols and quantification methods was compared for the analysis of primary human liver spheroids. Results differed majorly between protocols in the total number and subcellular compartment bias of identified proteins, which is particularly relevant for the reliable quantification of transporters and drug metabolizing enzymes. Using a model of metabolic dysfunction-associated steatotic liver disease, we furthermore show that critical disease pathways are robustly identified using a standardized high throughput-compatible workflow based on thermal lysis, even using only individual spheroids (1500 cells) as input. The results increase the applicability of proteomic profiling to phenotypic screens in organotypic microtissues and provide a scalable platform for deep phenotyping from limited biological material.

Multiplatform tear proteomic profiling reveals novel non-invasive biomarkers for diabetic retinopathy.

To investigate a comprehensive proteomic profile of the tear fluid in patients with diabetic retinopathy (DR) and further define non-invasive biomarkers. A cross-sectional, multicentre study that includes 46 patients with DR, 28 patients with diabetes mellitus (DM), and 30 healthy controls (HC). Tear samples were collected with Schirmer strips. As for the discovery set, data-independent acquisition mass spectrometry was used to characterize the tear proteomic profile. Differentially expressed proteins between groups were identified, with gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis further developed. Classifying performance of biomarkers for distinguishing DR from DM was compared by the combination of three machine-learning algorithms. The selected biomarker panel was tested in the validation cohort using parallel reaction monitoring mass spectrometry. Among 3364 proteins quantified, 235 and 88 differentially expressed proteins were identified for DR when compared to HC and DM, respectively, which were fundamentally related to retina homeostasis, inflammation and immunity, oxidative stress, angiogenesis and coagulation, metabolism, and cellular adhesion processes. The biomarker panel consisting of NAD-dependent protein deacetylase sirtuin-2 (SIR2), amine oxidase [flavin-containing] B (AOFB), and U8 snoRNA-decapping enzyme (NUD16) exhibited the best diagnostic performance in discriminating DR from DM, with AUCs of 0.933 and 0.881 in the discovery and validation set, respectively. Tear protein dysregulation is comprehensively revealed to be associated with DR onset. The combination of tear SIR2, AOFB, and NUD16 can be a novel potential approach for non-invasive detection or pre-screening of DR. Chinese Clinical Trial Registry Identifier: ChiCTR2100054263. https://www.chictr.org.cn/showproj.html?proj=143177 . Date of registration: 2021/12/12.

Kinase inhibitor pulldown assay (KiP) for clinical proteomics

Protein kinases are frequently dysregulated and/or mutated in cancer and represent essential targets for therapy. Accurate quantification is essential. For breast cancer treatment, the identification and quantification of the protein kinase ERBB2 is critical for therapeutic decisions. While immunohistochemistry (IHC) is the current clinical diagnostic approach, it is only semiquantitative. Mass spectrometry-based proteomics offers quantitative assays that, unlike IHC, can be used to accurately evaluate hundreds of kinases simultaneously. The enrichment of less abundant kinase targets for quantification, along with depletion of interfering proteins, improves sensitivity and thus promotes more effective downstream analyses. Multiple kinase inhibitors were therefore deployed as a capture matrix for kinase inhibitor pulldown (KiP) assays designed to profile the human protein kinome as broadly as possible. Optimized assays were initially evaluated in 16 patient derived xenograft models (PDX) where KiP identified multiple differentially expressed and biologically relevant kinases. From these analyses, an optimized single-shot parallel reaction monitoring (PRM) method was developed to improve quantitative fidelity. The PRM KiP approach was then reapplied to low quantities of proteins typical of yields from core needle biopsies of human cancers. The initial prototype targeting 100 kinases recapitulated intrinsic subtyping of PDX models obtained from comprehensive proteomic and transcriptomic profiling. Luminal and HER2 enriched OCT-frozen patient biopsies subsequently analyzed through KiP-PRM also clustered by subtype. Finally, stable isotope labeled peptide standards were developed to define a prototype clinical method. Data are available via ProteomeXchange with identifiers PXD044655 and PXD046169.

Abstract A042: Cancer associated fibroblasts drive transcriptional changes in tumor cells from classical to basal phenotype and promote epithelial-to-mesenchymal transition in human pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a heterogeneous tumor comprised of epithelial tumor, endothelial, immune, and importantly, cancer associated fibroblasts (CAFs) cells. CAFs drive a complex tumor microenvironment (TME) through mechanisms of intratumoral interactions that are incompletely understood. It’s crucial to account for the complex role of CAFs in modern ex-vivo tumor systems as regulators of both promoting and restraining tumor growth, providing growth factor support, reprogramming immune cells, and altering the TME. Using our patient-derived organoid (PDO) and CAF coculture, we identify novel intercellular interactions between CAF and PDO that promote tumor cell proliferation, impair response to therapy, and alter transcriptional phenotype. Methods: We interrogate mechanisms of cellular crosstalk in the PDAC TME using a novel three-dimensional, patient-matched coculture of PDO and CAFs, established from patients undergoing surgical resection. Molecular characterization included bulk RNA sequencing-based transcriptomics, comprehensive proteome profiling for 107 secreted proteins with curated validation by ELISA, cellular phenotyping by multiparameter flow cytometry, and qPCR for validation of curated gene lists. Results: Bulk RNAseq of FACS sorted CAF – PDO cocultures from 12 patients demonstrate Moffitt classification transcriptional changes in 42% of samples compared to PDO monoculture, suggesting CAFs drive basal tumor phenotype. To investigate protein level changes, we compared the secreted proteome of PDOs that transitioned to a basal phenotype after coculture to PDOs that did not undergo transition. Proteome analysis from coculture supernatant revealed that HGF, GDF15, TFF3, and VEGF-A are significantly increased in samples that undergo classical to basal transition. Pathway analysis comparing transcriptional data from classical and basal PDOs identifies significant upregulation of pathways associated with epithelial-to-mesenchymal transition (EMT), inflammation, and NF-kB in basal PDOs. Further, coculture leads to decreased expression of E-cadherin and increased expression of N-cadherin on the surface of PDOs while CAF markers remain unchanged. This interaction is accompanied by an increase in PDO proliferation in coculture, suggesting that CAF presence enhances tumorgenicity and changes epithelial tumor cell fitness. Ongoing experiments will investigate tumor cell-CAF interactions spatially using matched patient tissue to complement the in vitro coculture system. Conclusions: Interactions between cancer cells and CAFs drive overall tumor biology via a complex TME and contribute to poor PDAC patient outcomes. Using our patient matched PDO - CAF coculture, we demonstrate that a CAF competent TME drives PDO plasticity towards a more basal transcriptional phenotype and enhanced EMT. We introduce an elegant approach combining in vitro coculture experiments and high dimensional assays to investigate the complex biology of the TME and inform the mechanisms driving cancer biology in individual patients with PDAC. Citation Format: Samantha Guinn, Joseph Tandurella, Jae W. Lee, Daniel J. Zabransky, Mili Ramani, Jignasha Patel, Elana J. Fertig, Elizabeth M. Jaffee, Richard A. Burkhart, Jacquelyn W. Zimmerman. Cancer associated fibroblasts drive transcriptional changes in tumor cells from classical to basal phenotype and promote epithelial-to-mesenchymal transition in human pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A042.

Proteomics Analysis of Knee Subchondral Bone Identifies Differentially Expressed Proteins Associated with Osteoarthritis.

Osteoarthritis (OA) is a prevalent debilitating whole-joint disorder. Currently, a growing number of proteomic studies have been performed to evaluate molecular biomarkers in several tissues from OA patients; however, little is known about the protein profiles in subchondral bone of OA. In this study, proteomic analysis was performed on subchondral bone from patients with OA to identify differentially expressed proteins (DEPs). Bioinformatics tools were used to further investigate these DEPs. Thereafter, DEPs were validated in the samples from patients with OA, as well as in bilateral ovariectomy-induced OA (OVX-OA) rats using immunohistochemistry. A comprehensive subchondral bone proteome profile of patients with OA was constructed. Additionally, biological information analysis showed that a majority of DEPs participated in the dysregulation of the complement and coagulation cascades. The validation experiments suggested that SerpinA5, the protein involved in the complement and coagulation cascades, was significantly increased in severely damaged subchondral bone of patients with OA compared to the control group. Furthermore, the increase of SerpinA5 in OVX-OA rats compared to control rats was also confirmed. Our results indicated that the dysregulation of coagulation and complement pathways plays a role in the progression of OA, and it provides a promising therapeutic target of OA.

A comprehensive proteomic profiling of urinary exosomes and the identification of early non-invasive biomarker in patients with coronary artery disease

Urinary small extracellular vesicles or exosomes (uEVs) source could be an emerging trove of biomarkers in coronary artery disease (CAD). It is a chronic inflammatory disease having a long asymptomatic phase of fatty-fibrous development in arteries leading to angina, myocardial infarction, and death. Our study was aimed at identifying differential protein expression profiling of uEVs in CAD. We collected urine samples of CAD patients (n = 41) age 18–65 years and gender matched healthy controls (n = 41). We isolated uEVs using differential ultracentrifugation. Further, uEV samples were characterized by western blotting exosome markers (Flotillin, TSG, CD63, and CD9), nano tracking analysis, and transmission and scanning electron microscopy. A total of 508 proteins were identified by iTRAQ-based mass spectrometry. We observed protein expression levels of AZGP1, SEMG1/2, ORM1, IGL, SERPINA5, HSPG2, prosaposin, gelsolin, and CD59 were upregulated, and UMOD, KNG1, AMBP, prothrombin, and TF were downregulated. Protein-protein interactions, gene ontology and pathway analysis were performed to functionally annotate identified uEVs proteins. A novel uEVs differential protein signature is shown. On validating UMOD protein by ELISA in two clinically different CAD, stable-CAD patients had lower levels than healthy controls whereas recent myocardial infarction patients had lowest. Our findings suggest UMOD importance as early diagnostic biomarker. SignificanceCoronary artery disease is a chronic inflammatory disease caused by gradual deposition of cholesterol and fat along with other proteins to develop plaque inside arteries. This further leads to blockage of artery, heart attack and death. There are no identifiable early biomarkers to diagnose this. For the first time, we have identified the differentially expressed proteins isolated from non-invasive uEV of CAD patients compared to healthy controls by using MS Orbitrap and iTRAQ labelling of peptides. We have identified decreased levels of UMOD protein in CAD. These findings have been confirmed by ELISA. Furthermore, the levels of UMOD were observed as more highly decreased in recent myocardial infarction CAD patients, indicating the importance of this protein as an early diagnostic biomarker. Conclusively, our study represents a non-invasive urinary EVs trove of differentially expressed proteins in CAD. This will form a groundwork for understanding the pathophysiology of CAD and will help in future translational research utilizing uEVs.

THE CELL “SURFACEOME”: A STRATEGY TO TARGET SENESCENT CELLS IN VIVO

Abstract Senescent cell accumulation drives aging and related pathologies including metabolic disorders and neurodegeneration, and their selective elimination is a promising therapeutic approach to treat multiple diseases of aging in humans. However, developing drugs to eliminate senescent cells requires specific biomarkers to selectively identify and target these cells in vivo. In this study, we have performed a comprehensive and quantitative proteomic profiling of surface proteomes (surfaceomes) of senescent cells to identify potential senescence biomarker candidates in human tissues. We induced senescence using ionizing radiation in a variety of cell types including lung fibroblasts, monocytes, vascular smooth muscle cells, aortic endothelial cells and preadipocytes. Senescence was validated using a panel of canonical markers including reduced proliferation, increased senescence-associated β-galactosidase activity, and increased expression of p21, p16, IL6, and others. The cell surface proteome was enriched using an optimized surface biotinylation approach (Glyco-cell surface capture) followed by data-independent acquisition (DIA) LC-MS/MS analysis on a Q-Exactive HF Orbitrap mass spectrometer. Our analysis revealed novel cell surface targets that were further validated by flow cytometry and immunohistochemistry. These results provide clinically useful information for establishing biomarkers and therapeutic targets to aid in the translation of senescence-targeted therapies to treat age-associated diseases.

IL-10 Levels Differentially Regulate IFNg Signaling of CART19 and CART22

Introduction: CD19-directed CART (CART19) are highly effective in the treatment of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). However, some pts relapse with CD19 negative disease after CART19, which has led to the development of CD22-directed CART (CART22). While CART22 has produced 70% CR in CD19 negative ALL, this is lower than CART19 and there is less evidence for long-term disease control compared to CART19. Interestingly, toxicity patterns are different with CART22 pts experiencing a later and less severe CRS even at high disease burden. To better understand these differences in efficacy and toxicity we conducted comprehensive serum proteomic profiling on serial samples from pts treated with CART-19 or -22. Methods: Serial serum samples from pts enrolled on two clinical trials of the murine CART19 CTL019 (NCT01626495 &amp; NCT02906371) and CART22 (NCT02650414) were examined. Pts were categorized as having severe (Grade 3 or 4) or minimal CRS (Grade 1 or 2). All statistical analyses were performed using R (v4.2.2) and PRISM (v9). Differential expression analyses were performed between CART19 and CART22 pts. Significance was defined as an absolute fold change &amp;gt; 2, and a false discovery rate (FDR) &amp;lt; 0.05. All in vitro experiments were performed in triplicate. Two-tailed t tests were calculated. Results: 43 pts (CART19 N=26 and CART22 N=17) with 223 unique timepoints were included in this analysis. All CART22 pts had minimal CRS; 13 CART19 pts had minimal and 13 had severe CRS. Both cohorts had high disease burden prior to infusion (CART19 mean = 29.9%, CART22 mean=44.3%, p=0.19). All pts who received CART22 had previously received CD19 directed immunotherapy (CART19 N=16, blinatumomab N=1). At the pre-infusion timepoint, 12 proteins were significantly different between pts in the CART19 and CART22 cohorts. The protein most significantly differentially expressed in patients who subsequently underwent CART22 treatment was the anti-inflammatory cytokine IL-10. IL-10 levels were consistently elevated in patients who received CART22, surpassing levels at all observed timepoints, even when compared with patients treated with CART19 who developed severe CRS (See Fig 1). We therefore hypothesized the IL-10 levels may be impacting the efficacy and CRS associated with CART22. We next tested the impact of IL-10 on IFNγ as a surrogate marker of CRS in a series of in vitro experiments. CART-19 or -22 cells from the same donor were co-cultured with NALM-6 cells in the presence and absence of IL-10. Without IL-10 present, CART22 cells produced higher levels of IFNγ than CART19 cells (8959 vs 6395 pg/mL, p=0.005). Inversely, with IL-10 present, CART19 cells produced more IFNγ than CART22 cells (21006 vs 15157 pg/mL, p=0.05). We evaluated the length of immune synapse (IS) formation, the time between CART cell interaction and blast killing, using live-cell microscopy, and found that CART22 cells had a trend towards a prolonged IS formation relative to CART19 cells (median time to detachment 9.1 min vs. 3.5 min, p=ns). Finally, given the differences in time of onset of CRS following CART19 vs CART22, we looked at levels of IFNγ and its canonical responder CXCL9 between pts with minimal CRS only. As expected, we found a later peak of IFNγ in CART22 pts compared to CART19 pts (Figure 2). Interestingly, despite similar peaks in IFNγ, CXCL9 levels were higher in CART22 pts, implying dysregulation of the IFNγ/CXCL9 axes in CART22 but not CART19 pts. Conclusions: Comparing comprehensive serum proteomic profiling between CART19 and CART22 pts we make several novel observations. First, IL-10 levels are significantly higher in CART22 pts at pre-infusion, suggesting that previous immunotherapy has caused an upregulation of this anti-inflammatory cytokine. Second, we show that IL-10 levels have a differential impact on IFNγ levels in CART19 vs CART22 cells. This may impact both CRS associated with CART cells and efficacy and may contribute to observed differences in IFNγ and CXCL9 levels seen between the two patient populations. These findings are significant for the clinical translation of CART22 and other immunotherapies, as previous immunotherapy may lead to sustained alterations in the cytokine milieu. Furthermore, IL-10 is a targetable cytokine that could be modulated in future studies to improve efficacy of CART22.

Proteomic characterization of epithelial ovarian cancer delineates molecular signatures and therapeutic targets in distinct histological subtypes

Clear cell carcinoma (CCC), endometrioid carcinoma (EC), and serous carcinoma (SC) are the major histological subtypes of epithelial ovarian cancer (EOC), whose differences in carcinogenesis are still unclear. Here, we undertake comprehensive proteomic profiling of 80 CCC, 79 EC, 80 SC, and 30 control samples. Our analysis reveals the prognostic or diagnostic value of dysregulated proteins and phosphorylation sites in important pathways. Moreover, protein co-expression network not only provides comprehensive view of biological features of each histological subtype, but also indicates potential prognostic biomarkers and progression landmarks. Notably, EOC have strong inter-tumor heterogeneity, with significantly different clinical characteristics, proteomic patterns and signaling pathway disorders in CCC, EC, and SC. Finally, we infer MPP7 protein as potential therapeutic target for SC, whose biological functions are confirmed in SC cells. Our proteomic cohort provides valuable resources for understanding molecular mechanisms and developing treatment strategies of distinct histological subtypes.

Global Proteomics Identifies CDK11A/B As Integral to Myeloma Proliferation and High-Risk Disease

Category Multiple Myeloma and Plasma Cell Dyscrasias: Basic and Translational Introduction Although there have been many treatment advances for multiple myeloma (MM) that have led to increases in overall survival (OS) and progression free survival (PFS) for up to 20 years, the subset of patients with high-risk disease continue to have poor clinical outcomes, with median OS of less than 3 years. Our studies have focused on improving gene expression profiling (GEP) and IMWG risk classification of newly diagnosed MM (NDMM) by proteomics. We have developed a 60-protein risk score (PEP60) that captures a group of high-risk NDMM cases misclassified as low-risk by GEP70, a powerful 70-gene mRNA risk score. We discovered that protein and mRNA have low heterogeneity in MM (R=0.37). Strikingly, CDK11A/B was ranked top 14 th for inferior survival (OS) at the protein level but did not make it into the top 1000 most significant genes at the mRNA level, which was supported by low correlation (R=0.25). CDK11A/B has been previously identified as a druggable target in MM (Tiedemann et al. Cancer Res. 2012; 72(3): 757-768) and our study is the first to confirm its significance at the protein level in a large global proteomics study of patient samples (n = 167). Altogether, this study highlights the importance of discovering high-risk targets at the protein level and identifies CDK11A/B as a high-risk protein that would have been missed in transcript-only analysis. Materials and Methods Total protein from primary bone marrow samples of CD138+ purified plasma cells was subjected to mass spectrometric analysis (n = 167 NDMM). A chromatogram library was assembled with Data Independent acquisition (DIA) spectra and precursor spectra were acquired after each DIA duty cycle, as previously described (Searle et al. Nat Commun 2018, 9:5128). Following data acquisition, data were searched using an empirically corrected library and a quantitative analysis was performed to obtain a comprehensive proteomic profile. Proteins were identified and quantified using EncyclopeDIA and visualized with Scaffold DIA using 1% false discovery thresholds at both the protein and peptide level. Expression was normalized by Variance Stabilizing Normalization (VSN). Heterogeneity between protein and mRNA (microarray) expression were compared across genes using Pearson correlation. Using the maxstat package in R, log-rank tests on 4,760 proteins were conducted and 60 high-risk proteins were selected (FDR&amp;lt;0.1%) to develop a prognostic protein risk score (PEP60). Kaplan-Meier curves revealing overall survival (OS) were constructed using the survival package in R. Results: A geometric mean ratio of 34 upregulated proteins divided by 26 downregulated proteins (PEP60), sorted from low to high, show that patient deaths increase in Heatmap with increase in PEP60 score. Kaplan-Meier estimates of OS PEP60 and OS GEP70 in low-risk myeloma and high-risk myeloma showed increased inferior survival for PEP60 OS (P&amp;lt;0.001; HR = 4.49) compared to GEP70 OS (P&amp;lt;0.001; HR = 3.64). Kaplan-Meier estimates of OS for PEP60high/GEP70high (black), PEP60high/GEP70low (red), PEP60low/GEP70high (brown), and PEP60low/GEP70low (blue) showed significant inferior survival for the PEP60high/GEP70high group (P&amp;lt;0.001, HR = 9.07) compared to PEP60high/GEP70low (P&amp;lt;0.001, HR = 3.70) and PEP60low/GEP70high (P&amp;lt;0.001, HR = 3.03), demonstrating that PEP60 and GEP70 capture separate high-risk patients when only one is high ( Fig 1). Correlation of CDK11A/B mRNA (microarray) and protein show low correlation (R=0.25).Kaplan-Meier estimates of OS for CDK11A/B were highly significant for protein (P&amp;lt;0.001; HR = 3.00) ( Fig 2) but not as significant for mRNA (P=0.02; HR = 1.86). Conclusion Protein and mRNA correlation of paired samples from 167 NDMM patients reveals a wide heterogeneity between expression at the mRNA and protein levels. Global proteomics Identified a subset of patients that have inferior survival that were previously missed in our high-risk model using mRNA expression (GEP70). CDK11A/B was identified as highly significant for inferior survival (OS) at the protein level but not at the mRNA level. This study shows the importance of proteomics for capturing high-risk proteins and identifies CDK11 A/B as a high-risk, druggable target in patients samples.