Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology1,2. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance3-7. Furthermore, PSC activation occurs very early during PDAC tumorigenesis8-10, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial-mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell-cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy.

Functional and Clinical Proteomic Exploration of Pancreatic Cancer.

Pancreatic cancer, in most cases being pancreatic ductal adenocarcinoma (PDAC), is one of the most lethal cancers with a median survival time of less than 6 months. Therapeutic options are very limited for patients with PDAC, and surgery is still the most effective treatment, making improvements in early diagnosis critical. One typical characteristic of PDAC is the desmoplastic reaction of its stroma microenvironment, which actively interacts with cancer cells to orchestrate key components in tumorigenesis, metastasis, and chemoresistance. A global exploration of cancer-stroma crosstalk is essential to decipher PDAC biology and design intervention strategies. Over the past decade, the dramatic improvement in proteomics technologies has enabled the profiling of proteins, post-translational modifications (PTMs), and their protein complexes at unprecedented sensitivity and dimensionality. Here, starting with our current understanding of PDAC characteristics, including precursor lesions, progression models, tumor microenvironment, and therapeutic advancements, we describe how proteomics contributes to the functional and clinical exploration of PDAC, providing insights into PDAC carcinogenesis, progression, and chemoresistance. We summarize recent achievements enabled by proteomics to systematically investigate PTMs-mediated intracellular signaling in PDAC, cancer-stroma interactions, and potential therapeutic targets revealed by these functional studies. We also highlight proteomic profiling of clinical tissue and plasma samples to discover and verify useful biomarkers that can aid early detection and molecular classification of patients. In addition, we introduce spatial proteomic technology and its applications in PDAC for deconvolving tumor heterogeneity. Finally, we discuss future prospects of applying new proteomic technologies in comprehensively understanding PDAC heterogeneity and intercellular signaling networks. Importantly, we expect advances in clinical functional proteomics for exploring mechanisms of cancer biology directly by high-sensitivity functional proteomic approaches starting from clinical samples.

Chemical Proteomic Approach for In-Depth Glycosylation Profiling of Plasma Carcinoembryonic Antigen in Cancer Patients.

Carcinoembryonic antigen (CEA) of human plasma is a biomarker of many cancer diseases, and its N-glycosylation accounts for 60% of molecular mass. It is highly desirable to characterize its glycoforms for providing additional dimension of features to increase its performance in prognosis and diagnosis of cancers. However, to systematically characterize its site-specific glycosylation is challenging because of its low abundance. Here, we developed a highly sensitive strategy for in-depth glycosylation profiling of plasma CEA through chemical proteomics combined with multienzymatic digestion. A trifunctional probe was utilized to generate covalent bond of plasma CEA and its antibody upon UV irradiation. As low as 1 ng/ml CEA in plasma could be captured and digested with trypsin and chymotrypsin for intact glycopeptide characterization. Twenty six of 28 potential N-glycosylation sites were well identified, which were the most comprehensive N-glycosylation site characterization of CEA on intact glycopeptide level as far as we known. Importantly, this strategy was applied to the glycosylation analysis of plasma CEA in cancer patients. Differential site-specific glycoforms of plasma CEA were observed in patients with colorectal cancers (CRCs) and lung cancer. The distributions of site-specific glycoforms were different as the progression of CRC, and most site-specific glycoforms were overexpressed in stage II of CRC. Overall, we established a highly sensitive chemical proteomic method to profile site-specific glycosylation of plasma CEA, which should generally applicable to other well-established cancer glycoprotein biomarkers for improving their cancer diagnosis and monitoring performance.

Comprehensive Evaluation and Optimization of the Data-Dependent LC-MS/MS Workflow for Deep Proteome Profiling.

Data-dependent liquid chromatography-tandem mass spectrometry (LC-MS/MS) is widely used in proteomic analyses. A well-performed LC-MS/MS workflow, which involves multiple procedures and interdependent metrics, is a prerequisite for deep proteome profiling. Researchers have previously evaluated LC-MS/MS performance mainly based on the number of identified peptides and proteins. However, this is not a comprehensive approach. This motivates us to develop MSRefine, which aims to evaluate and optimize the performance of the LC-MS/MS workflow for data-dependent acquisition (DDA) proteomics. It extracts 47 kinds of metrics, scores the metrics, and reports visual results, assisting users in evaluating the workflow, locating problems, and providing optimizing strategies. In this study, we compared and analyzed multiple pairs of datasets spanning different samples, methods, and instruments and demonstrated that the comprehensive visual metrics and scores in MSRefine enable us to evaluate the performance of the various experiments and provide optimal strategies for the identification of more peptides and proteins.

Anti-fatigue properties of the ethanol extract of Moringa oleifera leaves in mice.

BACKGROUND: Moringa oleifera (M. oleifera) leaves are rich in nutrients and bioactive ingredients. This study was aimed at evaluating the anti-fatigue effect of the ethanol extract of M. oleifera leaves (MLEE) on mice and its primary mechanism of action using a weight-loaded forced swimming test. In the present study, MLEE was prepared by ultrasound-assisted extraction, and its anti-fatigue effect and antioxidant capacity were evaluated in mice. Mice were administrated MLEE (320 mg kg-1 body weight) for 15 days. RESULTS: MLEE supplementation significantly increased levels of glucose and non-esterified fatty acids (NEFA), while decreasing levels of lactate and blood urea nitrogen in serum (P < 0.05); the levels of glycogen in the liver and muscle were also increased, as was the activity of glycogen synthase and the level of NEFA in muscle (P < 0.05). According to a Western blot analysis, MLEE increased the expression of AMPKα1, JNK, AKT and STAT3 in the muscle of mice. CONCLUSION: Our findings indicate that MLEE has an anti-fatigue effect via the AMPK-linked route, which enables it to control energy metabolism and enhance antioxidant enzyme activity. © 2023 Society of Chemical Industry.

Tumor PKCδ instigates immune exclusion in EGFR-mutated non-small cell lung cancer.

BACKGROUND: The recruitment of a sufficient number of immune cells to induce an inflamed tumor microenvironment (TME) is a prerequisite for effective response to cancer immunotherapy. The immunological phenotypes in the TME of EGFR-mutated lung cancer were characterized as non-inflamed, for which immunotherapy is largely ineffective. METHODS: Global proteomic and phosphoproteomic data from lung cancer tissues were analyzed aiming to map proteins related to non-inflamed TME. The ex vivo and in vivo studies were carried out to evaluate the anti-tumor effect. Proteomics was applied to identify the potential target and signaling pathways. CRISPR-Cas9 was used to knock out target genes. The changes of immune cells were monitored by flow cytometry. The correlation between PKCδ and PD-L1 was verified by clinical samples. RESULTS: We proposed that PKCδ, a gatekeeper of immune homeostasis with kinase activity, is responsible for the un-inflamed phenotype in EGFR-mutated lung tumors. It promotes tumor progression by stimulating extracellular matrix (ECM) and PD-L1 expression which leads to immune exclusion and assists cancer cell escape from T cell surveillance. Ablation of PKCδ enhances the intratumoral penetration of T cells and suppresses the growth of tumors. Furthermore, blocking PKCδ significantly sensitizes the tumor to immune checkpoint blockade (ICB) therapy (αPD-1) in vitro and in vivo model. CONCLUSIONS: These findings revealed that PKCδ is a critical switch to induce inflamed tumors and consequently enhances the efficacy of ICB therapy in EGFR-mutated lung cancer. This opens a new avenue for applying immunotherapy against recalcitrant tumors.

Design of a flexible surface/interlayer for packaging.

Impact resistance and thermal insulation are important factors to be considered in the fields of encapsulation and drug transportation. In this study, a classic circular sleeve structure is designed by integrating the multi-level surface topography of the sleeve and a hollow sandwich in the wall, which effectively improves the energy absorption efficiency and thermal insulation effect. With the increase of the levels of surface structure, the stiffness of the whole structure and the stress on the topmost structure decreases, which is conducive to protecting the structure. In addition, the thermal conduction efficiency can be limited and the heat preservation ability would be improved as the reduction of the contacting area of packages with internal objects is attributed to such specific topography. Moreover, the synergistic effect of the hollow sandwich further enhances the advantages of mechanics and heat insulation. Based on the findings of this study, this novel design has potential applications in fields such as thermal insulation, packaging, and pharmaceuticals.

A fully integrated sample preparation strategy for highly sensitive intact glycoproteomics.

A fully integrated sample preparation technology, termed Intact GlycoSISPROT, was developed for the highly sensitive analysis of site-specific glycopeptides. Through integrating all glycoproteomic sample preparation steps into a single spintip, Intact GlycoSISPROT provided a tool for site-specific glycosylation analysis with low micrograms to even nanograms of protein sample.

Photocatalysis engineered hydrophilic reactors on hydrophobic paper for the visual and colorimetric assay of alkaline phosphatase activity.

Taking advantage of the intrinsic photocatalysis of TiO2, hydrophilic reactor arrays were lithographically patterned on a hydrophobic paper via a simple UV irradiation. As a proof-of-concept, alkaline phosphatase (ALP) was used as the model analyte for colorimetric analysis. As ALP can induce hydrolysis of pyrophosphate-Zn(II) framework, the released Zn2+ ions are subsequently coordinated with red-colored zincon to form blue-colored zincon-Zn(II) chelate complex, and these color differences were applied for further colorimetric assay. The sensing platform showed response to ALP ranging from 20 ~ 800 U L-1 with a detection limit of 3 U L-1, and the recoveries of ALP in serum samples were in the range 95.7 ~ 104.5% with relative standard deviations from 2.10 to 3.84%. Additionally, the distinct wettability features of the proposed sensing platform effectively prevent lateral fluid spread out of hydrophilic reactors, thus allowing not only the use of minimum amount of analyte but it has also a high potential for simultaneous quantification of multiple samples.

Stable and EGF-Induced Temporal Interactome Profiling of CBL and CBLB Highlights Their Signaling Complex Diversity.

The epidermal growth factor receptor (EGFR) signal modulates cell proliferation, migration, and survival. Aberrant activation of EGFR constitutes the major cause of various cancers. Receptor ubiquitination and degradation mediated by CBL proteins play negative regulatory roles and control the intensity and duration of the signaling. With the construction of stable cell lines inducibly expressing FLAG-tagged CBL or CBLB, we identified 102 and 82 stable interacting proteins of CBL and CBLB, respectively, through the affinity purification followed by mass spectrometry (AP-MS) approach. Time-resolved profiling at six different time points combined with functional annotations of the temporal interactomes provides insights into the dynamic assembly of signal proteins upon EGFR signaling activation. Comparison between the interactomes of CBL and CBLB indicates their redundant but also complementary functions. Importantly, we validated the stable association of EPS15L1 and ITSN2 and temporal association of TNK2 to both CBL and CBLB through biochemical assays. Collectively, these results offer a useful resource for CBL and CBLB interactomes and highlight their prominent and diverse roles in the EGFR signaling network.

Riboflavin deficiency alters cholesterol homeostasis partly by reducing apolipoprotein B100 synthesis in HepG2 cells.

Riboflavin deficiency led to lower blood cholesterol level and higher content of hepatic cholesterol in rats and the mechanisms are not clarified yet. We hypothesized that riboflavin deficiency might alter cholesterol homeostasis via apolipoprotein B100, one of the important proteins in cholesterol transport. To test this hypothesis, HepG2 cells were cultured in riboflavin-deficient media for 4 days to develop riboflavin deficiency. Compared to riboflavin-sufficient cells, the mRNA (0. 37 ± 0.04 vs 1.03 ± 0.29 relative expression level, n = 3) and protein expressions of apolipoprotein B100 (intracellular: 173.7 ± 14.4 vs 254.8 ± 47.2 µg/mg protein; extracellular: 93.8 ± 31.1 vs 161.6 ± 23.9 µg/mg protein; n = 3) were significantly reduced in riboflavin-deficient cells (P < 0.05). Endoplasmic reticulum oxidoreductin 1 and protein disulfide isomerase, two enzymes involved in the oxidative folding of apolipoprotein B100, were also lower remarkably in expression at both mRNA and protein levels. Meanwhile, intracellular cholesterol was increased (256.3 ± 17.1 µM/g protein vs 181.4 ± 23.9 µM/g protein, n = 4) and extracellular cholesterol decreased (110.0 ± 23.2 µM/g protein vs 166.2 ± 34.6 µM/g protein, n = 4) significantly in riboflavin-deficient cells (P < 0.05). Very low-density lipoprotein was also diminished (29.0 ± 6.1 µM/g protein vs 67.0 ± 11.0 µM/g protein, n = 4) in the culture media (P < 0.05). These findings suggest that riboflavin deficiency alters cholesterol homeostasis partly by reducing apolipoprotein B100 synthesis in HepG2 cells.

Study on the clinical effects of targeted therapy on elderly patients with gastric cancer.

To explore the clinical effects of targeted drug therapy on elderly patients with gastric cancer. Totally 200 metastatic gastric cancer patients who came to our hospital from January 2017 to January 2020 were selected and randomized into four groups, with 50 patients in each group. Bevacizumab (Group I), apatinib (Group II), and recombinant human endostatin (Group III) adopted respectively. While the control group received no targeted drug. Clinical data and clinical effect was collected and compared. After the therapy, the vascular endothelial growth factor (VEGF), soluble vascular endothelial growth factor receptor-2 (sVEGFR2) and human epithelial growth factor receptor-2 (HER2) positive detection of Group I, Group II, and Group III were better than the control group (P<0.05). In addition, the therapeutic effects of Group I, Group II, and Group III were higher and the incidence of adverse reactions was lower than the control group (P<0.05). Targeted drugs have obvious clinical effects in gastric cancer. It can effectively inhibit tumor growth and reduce the occurrence of complications, which is worthy of extensive clinical application and promotion.

AutoProteome Chip System for Fully Automated and Integrated Proteomics Sample Preparation and Peptide Fractionation.

With recent advances in LC-MS systems, current MS-based proteomics has an increasing need for automated, high-throughput sample preparation with neglectable sample loss. In this study, we developed a microfluidic system for fully automated proteomics sample preparation. All of the required proteomics sample preparation steps for both protein digestion and peptide fractionation are fully integrated into a disposable plastic chip device (named AutoProteome Chip). The AutoProteome Chip packed with mixed-mode ion exchange beads and C18 membrane in tandem could be fabricated with very low cost and high stability in organic reagents. Benefiting from its low backpressure, the AutoProteome Chip could be precisely driven by gas pressure, which could be easily multiplexed. As low as 2 ng of standard protein BSA could be trapped into the AutoProteome chip and processed within 2 h. Fully automated processing of 10 µg of protein extracts of HEK 293T cells achieved more than 97% of digestion efficiency with missed cleavage less than 2 and comparable performance with conventional approaches. More than 4700 proteins could be readily identified within 80 min of LC-MS analysis with good label-free quantification performance (Pearson correlation coefficient >0.99). Furthermore, deep proteome profiling by integrated high-pH RP fractionation in the same AutoProteome Chip resulted in more than 7500 proteins being identified from only 20 µg of protein extracts of HEK 293T cells and comparable reprodicibility as single-shot analysis. The AutoProteome Chip system provided a valuable prototype for developing a fully automated proteome analysis workflow and for proteomic applications with high demand for processing throughput, reproducibility, and sensitivity.

Chelidonine selectively inhibits the growth of gefitinib-resistant non-small cell lung cancer cells through the EGFR-AMPK pathway.

Tyrosine kinase inhibitors (TKIs) have been widely used for the clinical treatment of patients with non-small cell lung cancer (NSCLC) harboring mutations in the EGFR. Unfortunately, due to the secondary mutation in EGFR, eventual drug-resistance is inevitable. Therefore, to overcome the resistance, new agent is urgently required. Chelidonine, extracted from the roots of Chelidonium majus, was proved to effectively suppress the growth of NSCLC cells with EGFR double mutation. Proteomics analysis indicated that mitochondrial respiratory chain was significantly inhibited by chelidonine, and inhibitor of AMPK effectively blocked the apoptosis induced by chelidonine. Molecular dynamics simulations indicated that chelidonine could directly bind to EGFR and showed a much higher binding affinity to EGFRL858R/T790M than EGFRWT, which demonstrated that chelidonine could selectively inhibit the phosphorylation of EGFR in cells with EGFR double-mutation. In vivo study revealed that chelidonine has a similar inhibitory effect like second generation TKI Afatinib. In conclusion, targeting EGFR and inhibition of mitochondrial function is a promising anti-cancer therapeutic strategy for inhibiting NSCLC with EGFR mutation and TKI resistance.

Multiomic analysis of a dried single-drop plasma sample using an integrated mass spectrometry approach.

An easy-to-use and fast approach was developed for integrated proteomic and metabolic profiling in a dried single-drop plasma sample. Plasma collection, room temperature storage, and sample preparation for both proteins and metabolites were seamlessly integrated in one spintip device. MS-based multiomic profiling using the same nano LC-MS system identified more than 150 proteins and 160 metabolites from the 1 µL plasma sample in 6 hours. Further combination with micro-flow LC and targeted MS made it a promising approach for the fast profiling of molecular biomarkers with high sensitivity and accuracy.

A Fully Integrated Spintip-Based Approach for Sensitive and Quantitative Profiling of Region-Resolved in Vivo Brain Glycoproteome.

Region- and cell type-resolved global proteome and specific post-translational modifications (PTMs) profiling of tissues has drawn great attention recently for interpreting the heterogeneous multicellular microenvironment of various in vivo systems. Due to access to low microgram of proteins and low abundance of glycoproteins, spatially resolved glycoproteome analysis of in vivo tissue sections remains challenging. Several glycoproteomics sample preparation strategies were established for processing microgram-level of protein samples, but these strategies were not either fully integrated or directly compatible with tissue samples when considering protein extraction in strong lysis buffers. Moreover, these approaches mainly focused on identification of glycosylation sites, but pay less attention to quantification, all of which limit their applications. Here we designed a fully integrated spintip-based glycoproteomic approach (FISGlyco) which achieves all the steps of glycoprotein enrichment, digestion, deglycosylation, and desalting in single spintip device. Sample loss is significantly reduced, and the total processing time is reduced to 4 h, while detection sensitivity and label-free quantification precision is greatly improved. 607 N-glycosylation sites were successfully identified and quantified from only 5 µg of mouse brain proteins. By seamlessly combining with laser capture microdissection (LCM), the first region-resolved N-glycoproteome profiling of four mouse brain regions, including isocortex, hippocampus, thalamus, and hypothalamus, was achieved, with 1875, 1794, 1801, and 1417 N-glycosites identified, respectively. Our approach could be a generic approach for region and even cell type specific glycoproteome analysis of in vivo tissue sections.

Estimated daily quercetin intake and association with the prevalence of type 2 diabetes mellitus in Chinese adults.

PURPOSE: Quercetin is one of potential antidiabetic substances because of its powerful antioxidant and anti-inflammatory actions. The purpose of this study is to estimate daily quercetin intake and assess the relationship between dietary quercetin intake and the prevalence of type 2 diabetes mellitus (T2DM) in a Chinese population. METHODS: Dietary intake was investigated by a validated 100-item food frequency questionnaire. Daily intakes of quercetin and nutrients were calculated accordingly. T2DM was diagnosed based on the criteria of the American Diabetes Association. Adjusted logistic regression models were used to analyze the relationship between the quartiles of quercetin intake and the prevalence of T2DM. RESULTS: The prevalences of T2DM were 8.35% in men and 4.68% in women. The main food sources of quercetin were apple, orange, and green tea. Daily intake of quercetin was 20.9 ± 2.32 mg/day (mean ± SD). After adjusting for potentially confounding factors, the odds ratios (95% CI) for T2DM across the ascending quartiles of quercetin intake were: 1.00 (reference), 0.75 (0.60-0.95), 0.76 (0.59-0.99), and 0.63 (0.51-0.94). CONCLUSIONS: The results of the present study showed that quercetin intake was inversely related to the prevalence of T2DM in the Chinese population, suggesting a protective effect of quercetin in the development of T2DM.

Non-lipopeptide fungi-derived peptide antibiotics developed since 2000.

The 2,5-diketopiperazines (DKPs) are the smallest cyclopeptides and their basic structure includes a six-membered piperazine nucleus. Typical peptides lack a special functional group in the oligopeptide nucleus. Both are produced by at least 35 representative genera of fungi, and possess huge potential as pharmaceutical drugs and biocontrol agents. To date, only cyclosporin A has been developed into a commercial product. This review summarises 186 fungi-derived compounds reported since 2000. Antibiotic (antibacterial, antifungal, synergistic antifungal, antiviral, antimycobacterial, antimalarial, antileishmanial, insecticidal, antitrypanosomal, nematicidal and antimicroalgal) activities are discussed for 107 of them, including 66 DKPs (14 epipolythiodioxopiperazines, 20 polysulphide bridge-free thiodiketopiperazines, and 32 sulphur-free prenylated indole DKPs), 15 highly N-methylated, and 26 non-highly N-methylated typical peptides. Structure-activity relationships, mechanisms of action, and research methods are covered in detail. Additionally, biosynthases of tardioxopiperazines and neoechinulins are highlighted. These compounds have attracted considerable interest within the pharmaceutical and agrochemical industries.

Integrated and Quantitative Proteomic Approach for Charting Temporal and Endogenous Protein Complexes.

Proteins often assemble into multiprotein complexes for carrying out their biological functions. Affinity purification combined with mass spectrometry (AP-MS) is a method of choice for unbiasedly charting protein complexes. Typically, genetically tagged bait protein and associated proteins are immunoprecipitated from cell lysate and subjected to in-gel or on-bead digestion for MS analysis. However, the sample preparation procedures are often time-consuming and skipping reduction and alkylation steps results in incomplete digestion. Here, by seamlessly combining AP with the simple and integrated spintip-based proteomics technology (SISPROT), we developed an integrated AP-MS workflow for simultaneously processing more than 10 AP samples from cells cultured in six-well plates in 2 h. Moreover, we developed a quantitation-based data analysis workflow for differentiating potential interacting proteins from nonspecific interferences. The AP-SISPROT ensures high digestion efficiency especially for large transmembrane proteins such as EGFR and high quantification precision for profiling temporal interaction network of key EGFR signaling protein GRB2 across four time points of EGF treatment. More importantly, the integration feature allows minimum sample lose and helps the development of an ideal AP-MS workflow for studying endogenous protein complexes by the CRISPR Cas9 technology for the first time. By generating endogenously expressed bait protein fused with affinity tag, protein complexes associated with endogenous Integrin-linked kinase (ILK) was identified with much higher selectivity as compared with overexpressed and tagged ILK. The AP-SISPROT technology and its combination with CRISPR Cas9 technology should be generally applicable for studying protein complexes in a more efficient and physiologically relevant manner.