Comprehensive Proteomic Analysis Research Articles

The mouse multi-organ proteome from infancy to adulthood

The early-life organ development and maturation shape the fundamental blueprint for later-life phenotype. However, a multi-organ proteome atlas from infancy to adulthood is currently not available. Herein, we present a comprehensive proteomic analysis of ten mouse organs (brain, heart, lung, liver, kidney, spleen, stomach, intestine, muscle and skin) at three crucial developmental stages (1-, 4- and 8-weeks after birth) acquired using data-independent acquisition mass spectrometry. We detect and quantify 11,533 protein groups across the ten organs and obtain 115 age-related differentially expressed protein groups that are co-expressed in all organs from infancy to adulthood. We find that spliceosome proteins prevalently play crucial regulatory roles in the early-life development of multiple organs, and detect organ-specific expression patterns and sexual dimorphism. This multi-organ proteome atlas provides a fundamental resource for understanding the molecular mechanisms underlying early-life organ development and maturation.

Comprehensive proteomic analysis of HCoV-OC43 virions and virus-modulated extracellular vesicles.

In recent years, coronaviruses have dominated global attention, making it crucial to develop methods to control them and prevent future pandemics. Besides viral proteins, host proteins play a significant role in viral propagation and offer potential therapeutic targets. Targeting host proteins is advantageous because they are less likely to mutate and develop resistance compared to viral proteins, a common issue with many antiviral treatments. In this study, we examined the protein content of the less virulent biosafety level 2 HCoV-OC43 virus as a stand-in for the more virulent SARS-CoV-2. Our findings reveal that several cellular proteins incorporated into the virion regulate viral spread. In addition, we report that the virus extensively modulates the content of extracellular vesicles, enhancing viral dissemination. This underscores the critical interplay between the virus, host proteins, and extracellular vesicles.

Comparative Analysis of mRNA, microRNA of Transcriptome, and Proteomics on CIK Cells Responses to GCRV and Aeromonas hydrophila.

Grass Carp Reovirus (GCRV) and Aeromonas hydrophila (Ah) are the causative agents of haemorrhagic disease in grass carp. This study aimed to investigate the molecular mechanisms and immune responses at the miRNA, mRNA, and protein levels in grass carp kidney cells (CIK) infected by Grass Carp Reovirus (GCRV, NV) and Aeromonas hydrophilus (Bacteria, NB) to gain insight into their pathogenesis. Within 48 h of infection with Grass Carp Reovirus (GCRV), 99 differentially expressed microRNA (DEMs), 2132 differentially expressed genes (DEGs), and 627 differentially expressed proteins (DEPs) were identified by sequencing; a total of 92 DEMs, 3162 DEGs, and 712 DEPs were identified within 48 h of infection with Aeromonas hydrophila. It is worth noting that most of the DEGs in the NV group were primarily involved in cellular processes, while most of the DEGs in the NB group were associated with metabolic pathways based on KEGG enrichment analysis. This study revealed that the mechanism of a grass carp haemorrhage caused by GCRV infection differs from that caused by the Aeromonas hydrophila infection. An important miRNA-mRNA-protein regulatory network was established based on comprehensive transcriptome and proteome analysis. Furthermore, 14 DEGs and 6 DEMs were randomly selected for the verification of RNA/small RNA-seq data by RT-qPCR. Our study not only contributes to the understanding of the pathogenesis of grass carp CIK cells infected with GCRV and Aeromonas hydrophila, but also serves as a significant reference value for other aquatic animal haemorrhagic diseases.

Phytol and α-Bisabolol Synergy Induces Autophagy and Apoptosis in A549 Cells and Additional Molecular Insights through Comprehensive Proteome Analysis via Nano LC-MS/MS.

Non-Small Cell Lung Cancer (NSCLC) is a malignancy with a significant prevalence and aggressive nature, posing a considerable challenge in terms of therapeutic interventions. Autophagy and apoptosis, two intricate cellular processes, are integral to NSCLC pathophysiology, each affecting the other through shared signaling pathways. Phytol (Phy) and α-bisabolol (Bis) have shown promise as potential anticancer agents individually, but their combined effects in NSCLC have not been extensively investigated. The present study was to examine the synergistic impact of Phy and Bis on NSCLC cells, particularly in the context of autophagy modulation, and to elucidate the resulting differential protein expression using LCMS/ MS analysis. The A549 cell lines were subjected to the patented effective concentration of Phy and Bis, and subsequently, the viability of the cells was evaluated utilizing the MTT assay. The present study utilized real-time PCR analysis to assess the expression levels of crucial apoptotic genes, specifically Bcl-2, Bax, and Caspase-9, as well as autophagy-related genes, including Beclin-1, SQSTM1, Ulk1, and LC3B. The confirmation of autophagy marker expression (Beclin-1, LC3B) and the autophagy-regulating protein SQSTM1 was achieved through the utilization of Western blot analysis. Differentially expressed proteins were found using LC-MS/MS analysis. The combination of Phy and Bis demonstrated significant inhibition of NSCLC cell growth, indicating their synergistic effect. Real-time PCR analysis revealed a shift towards apoptosis, with downregulation of Bcl-2 and upregulation of Bax and Caspase-9, suggesting a shift towards apoptosis. Genes associated with autophagy regulation, including Beclin-1, SQSTM1 (p62), Ulk1, and LC3B, showed significant upregulation, indicating potential induction of autophagy. Western blot analysis confirmed increased expression of autophagy markers, such as Beclin-1 and LC3B, while the autophagy-regulating protein SQSTM1 exhibited a significant decrease. LC-MS/MS analysis revealed differential expression of 861 proteins, reflecting the modulation of cellular processes. Protein-protein interaction network analysis highlighted key proteins involved in apoptotic and autophagic pathways, including STOML2, YWHAB, POX2, B2M, CDA, CAPN2, TXN, ECHS1, PEBP1, PFN1, CDC42, TUBB1, HSPB1, PXN, FGF2, and BAG3, emphasizing their crucial roles. Additionally, PANTHER pathway analysis uncovered enriched pathways associated with the differentially expressed proteins, revealing their involvement in a diverse range of biological processes, encompassing cell signaling, metabolism, and cellular stress responses. The combined treatment of Phy and Bis exerts a synergistic inhibitory effect on NSCLC cell growth, mediated through the interplay of apoptosis and autophagy. The differential protein expression observed, along with the identified proteins and enriched pathways, provides valuable insights into the underlying molecular mechanisms. These findings offer a foundation for further exploration of the therapeutic potential of Phy and Bis in the management of NSCLC.

Comprehensive Transcriptome and Proteome Analyses Reveal the Drought Responsive Gene Network in Potato Roots.

The root system plays a decisive role in the growth and development of plants. The water requirement of a root system depends strongly on the plant species. Potatoes are an important food and vegetable crop grown worldwide, especially under irrigation in arid and semi-arid regions. However, the expected impact of global warming on potato yields calls for an investigation of genes related to root development and drought resistance signaling pathways in potatoes. In this study, we investigated the molecular mechanisms of different drought-tolerant potato root systems in response to drought stress under controlled water conditions, using potato as a model. We analyzed the transcriptome and proteome of the drought-sensitive potato cultivar Atlantic (Atl) and the drought-tolerant cultivar Qingshu 9 (Q9) under normal irrigation (CK) and weekly drought stress (D). The results showed that a total of 14,113 differentially expressed genes (DEGs) and 5596 differentially expressed proteins (DEPs) were identified in the cultivars. A heat map analysis of DEGs and DEPs showed that the same genes and proteins in Atl and Q9 exhibited different expression patterns under drought stress. Weighted gene correlation network analysis (WGCNA) showed that in Atl, Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG)-enriched pathways were related to pyruvate metabolism and glycolysis, as well as cellular signaling and ion transmembrane transporter protein activity. However, GO terms and KEGG-enriched pathways related to phytohormone signaling and the tricarboxylic acid cycle were predominantly enriched in Q9. The present study provides a unique genetic resource to effectively explore the functional genes and uncover the molecular regulatory mechanism of the potato root system in response to drought stress.

Integrated proteomic, phosphoproteomic, and N-glycoproteomic analyses of small extracellular vesicles from C2C12 myoblasts identify specific PTM patterns in ligand-receptor interactions

Small extracellular vesicles (sEVs) are important mediators of intercellular communication by transferring of functional components (proteins, RNAs, and lipids) to recipient cells. Some PTMs, including phosphorylation and N-glycosylation, have been reported to play important role in EV biology, such as biogenesis, protein sorting and uptake of sEVs. MS-based proteomic technology has been applied to identify proteins and PTM modifications in sEVs. Previous proteomic studies of sEVs from C2C12 myoblasts, an important skeletal muscle cell line, focused on identification of proteins, but no PTM information on sEVs proteins is available.In this study, we systematically analyzed the proteome, phosphoproteome, and N-glycoproteome of sEVs from C2C12 myoblasts with LC–MS/MS. In-depth analyses of the three proteomic datasets revealed that the three proteomes identified different catalogues of proteins, and PTMomic analysis could expand the identification of cargos in sEVs. At the proteomic level, a high percentage of membrane proteins, especially tetraspanins, was identified. The sEVs-derived phosphoproteome had a remarkably high level of tyrosine-phosphorylated sites. The tyrosine-phosphorylated proteins might be involved with EPH-Ephrin signaling pathway. At the level of N-glycoproteomics, several glycoforms, such as complex N-linked glycans and sialic acids on glycans, were enriched in sEVs. Retrieving of the ligand-receptor interaction in sEVs revealed that extracellular matrix (ECM) and cell adhesion molecule (CAM) represented the most abundant ligand-receptor pairs in sEVs. Mapping the PTM information on the ligands and receptors revealed that N-glycosylation mainly occurred on ECM and CAM proteins, while phosphorylation occurred on different categories of receptors and ligands. A comprehensive PTM map of ECM-receptor interaction and their components is also provided.In summary, we conducted a comprehensive proteomic and PTMomic analysis of sEVs of C2C12 myoblasts. Integrated proteomic, phosphoproteomic, and N-glycoproteomic analysis of sEVs might provide some insights about their specific uptake mechanism.

Comprehensive physiology and proteomics analysis revealed the resistance mechanism of rice (Oryza sativa L) to cadmium stress

Cadmium contamination can lead to a decrease in crop yield and quality. However, Cd-tolerant rice can improve rice resistance genes, improve crop tolerance to heavy metals, and protect plants from oxidative damage. In this study, Japonica rice: Chunyou 987 and Indica rice: Chuanzhong you 3607 were used to reveal the molecular response mechanism of Cd-tolerant rice under cadmium concentration of 3 mg/kg through comparative experiments combined with physiology and proteomics. The results showed that compared with indica rice, japonica rice showed more robust resistance to Cd stress and effectively retained many Cd ions in roots. Moreover, it enhanced its enzymatic and non-enzymatic anti-oxidative stress mechanism, which increased the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) by 47.37%, 21.75%, and 55.42%, respectively. The contents of non-enzymatic antioxidant substances ascorbic acid (AsA), glutathione (GSH), cysteine (Cys), proline (PRO), anthocyanins (OPC), and flavonoids were increased by 25.32%, 42.67%, 21.43%, 50.81%, 33.23%, and 72.16%, respectively. Through proteomics analysis, it was found that in response to the damage caused by cadmium stress, Japonica rice makes Photosynthesis functional proteins (psbO and PetH), Photosynthesis antenna proteins (LHCA and ASCAB9), Carbon fixation functional proteins (PEPC and OsAld), Porphyrin metabolism functional proteins (OsRCCR1 and SE5), Glyoxylate and dicarboxylate The expression of metabolism functional proteins (CATC and GLO4.) and Glutathione metabolism functional proteins (APX8 and OsGSTU13) were significantly up-regulated, which stimulated the antioxidant stress mechanism and photosynthetic system, and constructed a robust energy supply system to ensure the normal metabolic activities of life. Strengthening the mechanisms of plant homeostasis. In summary, this study revealed the molecular mechanism of tolerance to Cd stress in japonica rice, and the results of this study will provide a possible way to improve Cd-resistant rice seedlings.

Comparative proteomic analysis of the ovarian fluid and eggs of Siberian sturgeon

BackgroundSturgeon species are living fossils that exhibit unique reproductive characteristics, and elucidation of the molecular processes governing the formation and quality of sturgeon eggs is crucial. However, comprehensive data on the protein composition of sturgeon ovarian fluid (OF) and eggs and their functional significance are lacking. To address this knowledge gap, the aim of the present study was to conduct a comprehensive comparative proteomic analysis of Siberian sturgeon OF and eggs using liquid chromatography–mass spectrometry (LC–MS/MS).ResultsA total of 617 proteins were identified in OF, and 565 proteins were identified in eggs. A total of 772 proteins showed differential abundance. Among the differentially abundant proteins, 365 were more abundant in OFs, while 407 were more abundant in eggs. We identified 339 proteins unique to OFs and 287 proteins specific to eggs, and further investigated the top 10 most abundant proteins in each. The functional annotation of the OF proteins highlighted their predominant association with immune system processes, including the complement and coagulation cascade, neutrophil and leukocyte-mediated immunity, cholesterol metabolism, and regulation of the actin cytoskeleton. Analysis of egg proteins revealed enrichment in metabolic pathways, such as oxidative phosphorylation and fatty acid metabolism, and protein ubiquitination and translation. OF-specific proteins included extracellular matrix and secretory vesicles, and eggs were enriched in proteins localized to mitochondria and ribosome components.ConclusionsThis study presents the first comprehensive characterization of the protein composition of sturgeon OF and eggs and elucidates their distinct functional roles. These findings advance our understanding of sturgeon reproduction, OF-egg signaling and the origin of OF proteins. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium with the dataset identifier PXD044168 to ensure accessibility for further research.

Integrative transcriptomics and proteomics analysis reveal the protection of Astragaloside IV against myocardial fibrosis by regulating senescence

Myocardial fibrosis (MF) is a pivotal pathological process implicated in various cardiovascular diseases, particularly heart failure. Astragaloside IV (AS-IV), a natural compound derived from Astragalus membranaceus, possesses potent cardioprotective properties. However, the precise molecular mechanisms underlying its anti-MF effects, particularly in relation to senescence, remain elusive. Thus, this study aimed to investigate the therapeutic potential and underlying molecular mechanisms of AS-IV in treating ISO-induced MF in mice, employing transcriptomics, proteomics, in vitro, and in vivo experiments. We assessed the positive effects of AS-IV on ISO-induced MF using HE staining, Masson staining, ELISA, immunohistochemical staining, transthoracic echocardiography, transmission electron microscopy, and DHE fluorescence staining. Additionally, we elucidated the regulatory role of AS-IV in MF through comprehensive transcriptomics and proteomics analyses, complemented by Western blotting and RT-qPCR validation of pertinent molecular pathways. Our findings demonstrated that AS-IV treatment markedly attenuated ISO-induced myocardial injury and oxidative stress, concomitantly inhibiting the release of SASPs. Furthermore, integrated transcriptomics and proteomics analyses revealed that the anti-MF mechanism of AS-IV was associated with regulating cellular senescence and the p53 signaling pathway. These results highlight AS-IV exerts its anti-MF effects not only by inhibiting oxidative stress but also by modulating senescence through the p53 signaling pathway.

Identification and validation of the surface proteins FIBG, PDGF-β, and TGF-β on serum extracellular vesicles for non-invasive detection of colorectal cancer: experimental study.

The absence of non-invasive biomarkers for the early diagnosis of colorectal cancer (CRC) has contributed to poor prognosis. Extracellular vesicles (EVs) have emerged as promising candidates for cancer monitoring using liquid biopsy. However, the complexity of EVs isolation procedures and absence of clear targets for detecting serum-derived EVs have hindered the clinical application of EVs in early CRC diagnosis. In the discovery phase, we conducted a comprehensive 4D-DIA proteomic analysis of serum-derived EVs samples from 37 individuals, performing an initial screening of EVs surface proteins. In the technical validation phase, we developed an extraction-free CRC-EVArray microarray to assess the expression of these potential EVs surface proteins in a multicenter study comprising 404 individuals. In the application phase, we evaluated the diagnostic efficacy of the CRC-EVArray model based on machine-learning algorithms. Through 4D-DIA proteomic analysis, we identified 7 potential EVs surface proteins showing significantly differential expression in CRC patients compared to healthy controls. Utilizing our developed high-throughput CRC-EVArray microarray, we further confirmed the differential expression of 3 EVs surface proteins, FIBG, PDGF-β and TGF-β, in a large sample population. Moreover, we established an optimal CRC-EVArray model using the NNET algorithm, demonstrating superior diagnostic efficacy with an AUC of 0.882 in the train set and 0.937 in the test set. Additionally, we predicted the functions and potential origins of these EVs-derived proteins through a series of multi-omics approaches. Our systematic exploration of surface protein expression profiles on serum-derived EVs has identified FIBG, PDGF-β, and TGF-β as novel diagnostic biomarkers for CRC. And the development of CRC-EVArray diagnostic model based on these findings provided an effective tool for the large-scale CRC screening, thus facilitating its translation into clinical practice.

Comprehensive physiology and proteomics analysis revealed the molecular toxicological mechanism of Se stress on indica and japonica rice

Selenium pollution can lead to a decrease in crop yield and quality. However, the toxicological mechanisms of high Se concentrations on crops remain unclear. This study aimed to elucidate the physiological and proteomic molecular responses to Se stress in Oryza sativa. The results showed that under selenium stress, enzymatic activities of catalase, peroxidase, and superoxide dismutase in indica rice decreased by 61%, 28%, and 68%, respectively. The contents of non-enzymatic antioxidant substances ascorbic acid, glutathione, cysteine, proline, anthocyanidin, and flavonoids were decreased by 13%, 39%, 46%, 32%, 20%, and 5%, respectively, which significantly inhibited the antioxidant stress process of plants. At the same time, the results of proteomics analysis showed that rice seedlings, under Se stress, are involved in photosynthesis, photosynthesis-antenna proteins, carbon fixation, porphyrin metabolism, glyoxylate, and dicarboxylate. The differentially expressed proteins in metabolism and glutathione metabolism pathways showed a downward trend. It significantly inhibited the anti-oxidative stress, photosynthesis, and energy cycling process in plant cells, destroyed the homeostasis balance of rice plants, and inhibited the growth and development of rice. This finding reveals the molecular toxicological mechanism of Se stress on rice seedlings and provides a possible way to improve Se-resistant rice seedlings.

Proteomics study the potential targets for Rifampicin-resistant spinal tuberculosis.

Introduction: The escalating global surge in Rifampicin-resistant strains poses a formidable challenge to the worldwide campaign against tuberculosis (TB), particularly in developing countries. The frequent reports of suboptimal treatment outcomes, complications, and the absence of definitive treatment guidelines for Rifampicin-resistant spinal TB (DSTB) contribute significantly to the obstacles in its effective management. Consequently, there is an urgent need for innovative and efficacious drugs to address Rifampicin-resistant spinal tuberculosis, minimizing the duration of therapy sessions. This study aims to investigate potential targets for DSTB through comprehensive proteomic and pharmaco-transcriptomic analyses. Methods: Mass spectrometry-based proteomics analysis was employed to validate potential DSTB-related targets. PPI analysis confirmed by Immunohistochemistry (IHC) and Western blot analysis. Results: The proteomics analysis revealed 373 differentially expressed proteins (DEPs), with 137 upregulated and 236 downregulated proteins. Subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses delved into the DSTB-related pathways associated with these DEPs. In the context of network pharmacology analysis, five key targets-human leukocyte antigen A chain (HLAA), human leukocyte antigen C chain (HLA-C), HLA Class II Histocompatibility Antigen, DRB1 Beta Chain (HLA-DRB1), metalloproteinase 9 (MMP9), and Phospholipase C-like 1 (PLCL1)-were identified as pivotal players in pathways such as "Antigen processing and presentation" and "Phagosome," which are crucially enriched in DSTB. Moreover, pharmaco-transcriptomic analysis can confirm that 58 drug compounds can regulate the expression of the key targets. Discussion: This research confirms the presence of protein alterations during the Rifampicin-resistant process in DSTB patients, offering novel insights into the molecular mechanisms underpinning DSTB. The findings suggest a promising avenue for the development of targeted drugs to enhance the management of Rifampicin-resistant spinal tuberculosis.

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5’-splice site (5’SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5’SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

Comprehensive Proteomics Analysis of Polyhydroxyalkanoate (PHA) Biology in Pseudomonas putida KT2440: The Outer Membrane Lipoprotein OprL is a Newly Identified Phasin

Pseudomonas putida KT2440 is an important bioplastic-producing industrial microorganism capable of synthesizing the polymeric carbon-rich storage material, polyhydroxyalkanoate (PHA). PHA is sequestered in discrete PHA granules, or carbonosomes, and accumulates under conditions of stress, for example, low levels of available nitrogen. The pha locus responsible for PHA metabolism encodes both anabolic and catabolic enzymes, a transcription factor, and carbonosome-localized proteins termed phasins. The functions of phasins are incompletely understood but genetic disruption of their function causes PHA-related phenotypes. To improve our understanding of these proteins, we investigated the PHA pathways of P.putida KT2440 using three types of experiments. First, we profiled cells grown in nitrogen-limited and nitrogen-excess media using global expression proteomics, identifying sets of proteins found to coordinately increase or decrease within clustered pathways. Next, we analyzed the protein composition of isolated carbonosomes, identifying two new putative components. We carried out physical interaction screens focused on PHA-related proteins, generating a protein-protein network comprising 434 connected proteins. Finally, we confirmed that the outer membrane protein OprL (the Pal component of the Pal-Tol system) localizes to the carbonosome and shows a PHA-related phenotype and therefore is a novel phasin. The combined datasets represent a valuable overview of the protein components of the PHA system in P.putida highlighting the complex nature of regulatory interactions responsive to nutrient stress.

Abstract CT033: Avelumab combined with regorafenib in solid tumors with tertiary lymphoid structures: A phase 2 REGOMUNE trial cohort

Abstract Background: Mature tertiary lymphoid structures (mTLS) predict improved outcome in patients treated with immune checkpoint inhibitors (ICIs). However, a significant proportion of patients with TLS-positive tumors do not respond to ICI when used as a single agent and may therefore benefit from combination therapy. The multikinase inhibitor, regorafenib, deplete regulatory T cells, an immune population associated with resistance to ICI in TLS-positive tumors. We report the results of the PD-L1 inhibitor avelumab combined with regorafenib in patients with mTLS-positive advanced solid tumors. Methods: ‘Regomune’ is an open-label, multicenter phase II study assessing the combination of avelumab (10mg/kg IV every 14 days) with regorafenib (160 mg daily for 3 weeks in a 4-week cycle) in patients with advanced mTLS-positive solid tumors. The mTLS status was centrally assessed as previously described (Vanhersecke et al Nature Cancer 2021). All patients had confirmed progressive disease at inclusion, based on a central review of imaging. The primary efficacy endpoint was a 6-month non-progression rate using RECIST v1.1 based on blinded central review. 29 assessable patients were deemed necessary, and to meet the primary endpoint, at least 8 patients needed to be progression-free at 6 months. Results: Between January 2021 and July 2022, 132 patients (5 centers) underwent mTLS screening. Of these, 55 (41.7%) were identified as mTLS+, and 38 were included in the study. The top five histological subtypes were MSS colorectal cancer (15.8%), sarcoma (13.1%), oesogastric (10.5%), biliary tract (7.9%), and pancreatic cancer (7.9%). The most frequent grade 3/4 adverse events were palmar-plantar erythrodysesthesia syndrome (23.7%), maculo-papular rash (18.4%), fatigue, and oral mucositis (7.9% each). No treatment-related deaths were reported. Of the 34 patients assessed for efficacy, 16 (47%) showed tumor shrinkage, with 9 achieving a partial response (26.7%) and 7 having stable disease (20.6%). The median duration of response was 6 months. Twelve patients (comprising various tumor types including MSS colorectal cancer, small bowel carcinomas, biliary tract cancer, sarcomas, thyroid and gynecological tumors.) were progression-free at 6 months, marking the study’s primary endpoint achievement. The median progression-free survival and overall survival were 3.6 months and 8.6 months, respectively. Spatial transcriptomics, multiplex immunofluorescence (IF) of tumor tissues, and comprehensive plasma proteomics analysis have collectively uncovered critical biomarkers that significantly determine both sensitivity and resistance to treatment. Conclusions: This is the first histology-agnostic clinical trial employing mTLS as a biomarker for patient selection for treatment with an immune checkpoint inhibitor-based regimen. Durable responses were recorded, even in cases typically resistant to immunotherapy, confirming the predictive value of mTLS. Trial Registration NCT03475953 Citation Format: Antoine Italiano, Sophie Cousin, Carine Bellera, Jean-Philippe Guegan, Jean-Philippe Metges, Antoine Adenis, Rastilav Bahleda, Philippe Cassier, Coralie Cantarel, Michele Kind, Jean Palussiere, Lucile Vanhersecke, Alban Bessede. Avelumab combined with regorafenib in solid tumors with tertiary lymphoid structures: A phase 2 REGOMUNE trial cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT033.

Use of data-independent acquisition mass spectrometry to identify an objective serum indicator of the need for osteoporotic therapeutic intervention

Osteoporosis is characterized by weakened bone microstructure and loss of bone mass. Current diagnostic criteria for osteoporosis are based on the T-score, which is a measure of bone mineral density. However, osteoporotic fragility fractures can occur regardless of the T-score, underscoring the need for additional criteria for the early detection of patients at fracture risk. To identify indicators of reduced bone strength, we performed serum proteomic analysis using data-independent acquisition mass spectrometry with serum samples from two patient groups, one with osteoporosis but no fractures and the other with osteopenia and fragility fractures. Collective evaluation of the results identified six serum proteins that changed to a similar extent in both patient groups compared with controls. Of these, extracellular matrix protein 1 (ECM1), which contributes to bone formation, showed the most significant increase in serum levels in both patient groups. An ELISA-based assay suggested that ECM1 could serve as a serum indicator of the need for therapeutic intervention; however, further prospective studies with a larger sample size are necessary to confirm these results. The present findings may contribute to the provision of early and appropriate therapeutic strategies for patients at risk of osteoporotic fractures. SignificanceThis study aimed to identify objective serum indicators of the need for therapeutic intervention in individuals at risk of osteoporotic fracture. Comprehensive proteome analyses of serum collected from patients with osteoporosis but no fractures, patients with osteopenia and fragility fractures, and controls were performed by data-independent acquisition mass spectrometry. Collective evaluation of the proteome analysis data and ELISA-based assays identified serum ECM1 as a potential objective marker of the risk of fragility fractures in patients with osteoporosis or osteopenia. The findings are an important step toward the development of appropriate bone health management methods to improve well-being and maintain quality of life.

Integrated Transcriptome and Proteome Analysis Reveals the Regulatory Mechanism of Root Growth by Protein Disulfide Isomerase in Arabidopsis.

Protein disulfide isomerase (PDI, EC 5.3.4.1) is a thiol-disulfide oxidoreductase that plays a crucial role in catalyzing the oxidation and rearrangement of disulfides in substrate proteins. In plants, PDI is primarily involved in regulating seed germination and development, facilitating the oxidative folding of storage proteins in the endosperm, and also contributing to the formation of pollen. However, the role of PDI in root growth has not been previously studied. This research investigated the impact of PDI gene deficiency in plants by using 16F16 [2-(2-Chloroacetyl)-2,3,4,9-tetrahydro-1-methyl-1H-pyrido[3,4-b]indole-1-carboxylic acid methyl ester], a small-molecule inhibitor of PDI, to remove functional redundancy. The results showed that the growth of Arabidopsis roots was significantly inhibited when treated with 16F16. To further investigate the effects of 16F16 treatment, we conducted expression profiling of treated roots using RNA sequencing and a Tandem Mass Tag (TMT)-based quantitative proteomics approach at both the transcriptomic and proteomic levels. Our analysis revealed 994 differentially expressed genes (DEGs) at the transcript level, which were predominantly enriched in pathways associated with "phenylpropane biosynthesis", "plant hormone signal transduction", "plant-pathogen interaction" and "starch and sucrose metabolism" pathways. Additionally, we identified 120 differentially expressed proteins (DEPs) at the protein level. These proteins were mainly enriched in pathways such as "phenylpropanoid biosynthesis", "photosynthesis", "biosynthesis of various plant secondary metabolites", and "biosynthesis of secondary metabolites" pathways. The comprehensive transcriptome and proteome analyses revealed a regulatory network for root shortening in Arabidopsis seedlings under 16F16 treatment, mainly involving phenylpropane biosynthesis and plant hormone signal transduction pathways. This study enhances our understanding of the significant role of PDIs in Arabidopsis root growth and provides insights into the regulatory mechanisms of root shortening following 16F16 treatment.

Abstract 3772: Tissue-niche-based and cell-type-selective in-depth proteomics

Abstract Introduction: The development, physiology function, and pathogenesis of the tissues are intricately orchestrated by the spatial organization of diverse cell populations. Proteins are the fundamental components within individual cells that control every function. Therefore, by integrating both information on cellular location and molecular profiling, spatial proteomics has emerged as an indispensable field for understanding mechanisms governing tissue homeostasis, disease progression, and therapeutic responses. Here, I introduce a spatial proteomics method designed to target selective cell phenotypes within definite tissue niches for in-depth spatial biology analysis. Method: In this approach, we developed a sequential pipeline highlighted with immunofluorescence staining, microscopic photobleaching, fluorescence cell sorting, and liquid chromatography-mass spectrometry (LC-MS) analysis. The process begins with staining 400-μm-thick tissue macrosections with fluorescence-conjugated antibodies to visualize specific cell types of interest. We employed two different fluorescence-conjugated antibodies of the same clone (e.g., Alexa488-anti-CD11c and Alexa647-anti-CD11c antibodies) for the staining on every targeted cell (e.g., CD11c+ immune cells). We then introduced “photobleaching barcodes” using a confocal microscope to photobleach/exhaust either one of the fluorescence signals stained on cells by exposing it to the matched laser. This allows us to create differentiable fluorescence barcodes that label cells at distinct regions, incorporating cell spatial information into our fluorescence detection. After tissue dissociation, barcoded cells were sorted into different groups which were also classified by their original locations in the macrosection. Finally, protein extraction and LC-MS analysis were conducted on the collected cells to enable comprehensive spatial proteomic analysis. Results: The initial application in investigating dendritic cell (DC) subsets in mouse spleen during lipopolysaccharide (LPS)-induced inflammation reveals significant proteome differences among three splenic DC subsets, categorized by their locations in or outside spleen T-cell zones as well as control DCs. Our result aligns with previously published proteome data in splenic DCs, underscoring the feasibility and reliability of our technology. Currently, we are in the process of evaluating breast cancer cell heterogeneity in human biopsy specimens using our approach to profile the tumor-immune microenvironment. Conclusion: Spatial proteomic findings from both applications are poised to discover potential drug targets that benefit treatment efficacy for inflammatory diseases and breast cancer. This technology features deep protein profiling for cell-type-specific spatial proteomics and is designed for broad applications across diverse tissue specimens in various diseases. Citation Format: Yi-Chien Wu, Elie Abi Khalil, Samuel Weng, Steve Lee. Tissue-niche-based and cell-type-selective in-depth proteomics [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 3772.

Abstract 4082: Anchored IL-12 synergizes with an epigenetic modulator to promote immune remodeling and overcome anti-PD1-refractory murine tumors

Abstract Background Most patients with solid malignancies harbor innate or acquired resistance to immune checkpoint blockade (ICB), prompting the need for novel therapeutic strategies. Interleukin-12 (IL-12) is a promising cytokine for cancer therapy due to its ability to bridge innate and adaptive immunity. However, a narrow therapeutic index limits the use of systemic IL-12 therapy. Here, we investigated the tumor-suppressive effects and mode of action of intra-tumorally delivered murine Interleukin-12 anchored to aluminum hydroxide (referred as mANK-101) in combination with the class I HDAC inhibitor entinostat, in various ICB-refractory murine tumor models, including CT26 (colorectal) and MOC-1 (HPV16neg). We hypothesized that combining Entinostat with an anchored form of IL-12 could overcome systemic toxicity while maintaining anti-tumor activity. Methods Entinostat and intra-tumoral mANK-101 were administered to mice bearing well-established αPD-1-refractory CT26 (colorectal) and MOC-1 (HPV16neg) tumors. Antitumor activity, survival, and protective memory upon tumor rechallenge were evaluated. Comprehensive proteomic and immune cell analysis was performed in MOC-1 tumors, tumor-draining lymph node (tdLN), and periphery. Tumor-specific T cell responses were examined. Results We demonstrate that intra-tumoral mANK-101 synergizes with entinostat to suppress multiple αPD1-refractory tumors, resulting in significant tumor eradication (62-88%), survival benefit (P < 0.0001), and protective memory, including CT26 (colon, Kras G12Dmut) and MOC-1 (oral, HPV16neg). Analysis of MOC-1 tumor-bearing mice demonstrated these effects to be associated with peripheric activation of CD8+ and NK lymphocytes, augmented polyfunctional IFNγ+/TNFα+-producing CD8+ T cells, CD8+ T cell effector memory, and tumor-specific T cell responses. Significant decrease in CD4+ Tregs and increased CD8/Treg ratio were also observed. Ongoing functional studies, proteomic and immune cell analysis at the tumor site, tdLN, and periphery, including single cell transcriptomics and epigenetic studies, will allow for a deeper understanding of the synergistic effect of mANK-101 with the epigenetic modulator Entinostat. Conclusions Collectively, these findings form a rationale for the clinical combination of intralesional delivery of ANK-101 with entinostat for patients with ICB-refractory malignancies, including colorectal and HPV16neg head and neck cancers. Citation Format: Asma S. Khelifa, Katherine L. Lothstein, Masaya Miyamoto, Rob Tighe, Sailaja Battula, Howard L. Kaufman, Jeffrey Schlom, Sofia R. Gameiro. Anchored IL-12 synergizes with an epigenetic modulator to promote immune remodeling and overcome anti-PD1-refractory murine tumors [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 4082.

Abstract 74: Exploring the expression of cancer-intrinsic PD1 in osteosarcoma cells: A comprehensive proteomic analysis of its functional landscape

Abstract Cancer treatment was revolutionized by immune checkpoint inhibitors (ICIs) targeting the PD1/PDL1 axis - a brake to the immune system. Despite remarkable success, some patients do not respond to therapy or rapidly deteriorate due to an unclear mechanism. As reported for several types of tumors, PD1 receptor is not solely expressed on immune cells but also on cancer cells. Depending on the tumor type, it may act either as a suppressor or a tumor promoter. The limited response to ICIs was also reported in osteosarcoma (OSA), the most common type of bone cancer, mostly affecting children and young adults. With no significant improvement in therapy over the past 30 years, there is an urgent need for therapeutic advancement. This study aimed to determine if PD1 protein is expressed in OSA and whether it has a functional role, potentially affecting the response to ICIs. Strikingly, our results revealed both surface and intracellular PD1 expression in U2OS osteosarcoma cells, confirmed by Western Blot, flow cytometry and immunofluorescence analyses. siRNA-mediated PDCD1 gene silencing significantly increased cell migration and viability in scratch and viability assays. LC-MS-based global proteomics showed that PDCD1 knockdown markedly affects U2OS cells’ proteomic landscape compared to the control. To dissect PD1 receptor downstream signaling effects, we subjected the identified proteins to GO and GSEA large-scale data interpretation tools. The results strongly suggested that PDCD1 silencing leads to the enrichment of proteins involved in cell growth, migration and motility, corresponding to the cellular effects we initially observed. Importantly, the analysis clearly indicated positive regulation of MAPK cascade in cells with perturbed PD1 expression, aligning with PD1 mediated MAPK regulation in T cells. To reveal the mechanism underlying PD1 signaling, we focused on proteins with significantly changed expression in response to the knockdown. Using the STRING database, we created their interacting network, revealing that numerous proteins are closely related, and the majority of them are phosphoproteins, indicating significance in cellular signaling. Remarkably, many of the STRING-analyzed proteins belong to intersecting pathways, such as AKT1S1 and LAMTOR proteins, the critical components for mTORC1 signaling, or selected members of the Src family kinases along with their negative regulator CSK. In summary, we report previously undiscovered functional expression of PD1 in OSA cells, providing insights into the landscape of PD1 downstream signaling. Our studies indicate the role of PD1 in regulating cell growth, proliferation, and motility, suggesting osteosarcoma-expressed PD1 as a tumor suppressor. Further studies are urgently needed to investigate its therapeutic significance and explore if we could make ICIs more effective to treat osteosarcoma. Citation Format: Katarzyna Joanna Dziubek, Jakub Faktor, Ted Hupp, Maciej Parys, Sachin Kote. Exploring the expression of cancer-intrinsic PD1 in osteosarcoma cells: A comprehensive proteomic analysis of its functional landscape [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 74.