Immunomagnetic capture and traceless release of native tumor-derived exosomes from human plasma for exploring interaction with recipient cells by aptamer-functionalized nanoflowers.

BACKGROUND: Tumor-derived exosomes (TEXs) play an important role in the development process of cancer, which can transport a large number of carcinogenic molecules to normal cells, and subsequently promote tumor metastasis. However, TEXs that were utilized in most of previous researches were obtained from the cell medium of tumor cell lines, which cannot reflect the physiological state of primary cells in vivo. Isolation of native TEXs from human plasma with intact function is contributed to exploring the interaction between TEXs and recipient cells for understanding their true biological functions. RESULTS: We developed a strategy that involves both capture and release processes to obtain native TEXs from plasma of cancer patients. An MoS2-based immunomagnetic probe (Fe3O4@MoS2-Au-Aptamer, named as FMAA) with the advantages of high surface area, magnetic response and abundant affinity sites was designed and synthesized to capture TEXs through recognizing high-expression tumor-associated antigens of EpCAM. With the assistance of complementary sequences of EpCAM, TEXs were released with non-destruction and no residual labels. According to NTA analysis, 107-108 TEXs were recovered from per mL plasma of breast cancer patients. The interaction between native TEXs and normal epithelial cells confirms TEXs could induce significant activation of autophagy of recipient cells with co-culture for 12 h. Proteomics analysis demonstrated a total of 637 proteins inside epithelial cells had dynamic expression with the stimulation of TEXs and 5 proteins in the pathway of autophagy had elevated expression level. SIGNIFICANCE: This work not only obtains native TEXs from human plasma with non-destruction and no residual labels, but also explores the interaction between TEXs and recipient cells for understanding their true biological functions, which will accelerate the application of TEXs in the field of biomarkers and therapeutic drugs.

Single-cell transcriptome analysis reveals dynamic changes of the preclinical A549 cancer models, and the mechanism of dacomitinib.

The in vitro A549 cells, and A549 xenografts in nude mouse, were two commonly used models for anti-cancer drug discovery. However, the biological and molecular characteristics of these two classic models, and also the dynamic transcriptome changes after dacomitinib exposure remains elusive. We performed single-cell RNA sequencing to define the transcriptome profile at single-cell resolution, and processed tumor samples for bulk RNA and protein analysis to validate the differently expressed genes. Transcriptome profiling revealed that the in vitro A549 cells are heterogeneous. The minimal subpopulation of the in vitro A549 cells, which were characterized by the signature of response to unfolded protein, became the overriding subpopulation of the xenografts. The EGFR non-activating A549 cells were resistant to dacomitinib in vitro, while A549 xenografts were comparatively sensitive as EGFR-activating HCC827 xenografts. Dacomitinib inhibited MAPK signaling pathway, and increased the immune response in the A549 xenografts. A phagocytosis checkpoint stanniocalcin-1 (STC1) was significantly inhibited in dacomitinib-treated xenografts. So here our study gives the first insight of the heterogeneity of the two classic models, and the translational potential of dacomitinib being used into a broader patient population rather than EGFR common activating mutation.

Quantification of GPC1(+) Exosomes Based on MALDI-TOF MS In Situ Signal Amplification for Pancreatic Cancer Discrimination and Evaluation.

Pancreatic cancer (PC) has a high mortality, with a fairly low five-year survival rate, because of its delayed diagnosis. Recently, liquid biopsy, especially based on exosomes, has attracted vast attention, thanks to its low invasiveness. Herein, we constructed a protocol for pancreatic cancer related Glypican 1 (GPC1) exosome quantification, based on in situ mass spectrometry signal amplification, by utilizing mass tag molecules on gold nanoparticles (AuNPs). Exosomes were extracted and purified by size-exclusion chromatography (SEC), captured by TiO2 modified magnetic nanoparticles, and then targeted specifically by anti-GPC1 antibody modified on AuNPs. With matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), the signal of PC biomarker, GPC1, was converted to a mass tag signal and amplified. With addition of a certain amount of internal standard molecules modified on AuNPs, the relative intensity ratio of mass tag to internal standard was proportional to the concentration of GPC1(+) exosomes derived from pancreatic cancer cell lines, PANC-1, with good linearity (R2 = 0.9945) in a wide dynamic range from 7.1 × 10 to 7.1 × 106 particles/µL. This method was further applied to plasma samples from healthy control (HC) and pancreatic cancer patients with different tumor load, and exhibited a great potential in discriminating diagnosed PC patients from HC, and has the monitoring potential in PC progression.

Recent Advances on Small-Molecule Antagonists Targeting TLR7.

Toll-like receptor 7 (TLR7) is a class of pattern recognition receptors (PRRs) recognizing the pathogen-associated elements and damage and as such is a major player in the innate immune system. TLR7 triggers the release of pro-inflammatory cytokines or type-I interferons (IFN), which is essential for immunoregulation. Increasing reports also highlight that the abnormal activation of endosomal TLR7 is implicated in various immune-related diseases, carcinogenesis as well as the proliferation of human immunodeficiency virus (HIV). Hence, the design and development of potent and selective TLR7 antagonists based on small molecules or oligonucleotides may offer new tools for the prevention and management of such diseases. In this review, we offer an updated overview of the main structural features and therapeutic potential of small-molecule antagonists of TLR7. Various heterocyclic scaffolds targeting TLR7 binding sites are presented: pyrazoloquinoxaline, quinazoline, purine, imidazopyridine, pyridone, benzanilide, pyrazolopyrimidine/pyridine, benzoxazole, indazole, indole, and quinoline. Additionally, their structure-activity relationships (SAR) studies associated with biological activities and protein binding modes are introduced.

Integrated Pipeline of Rapid Isolation and Analysis of Human Plasma Exosomes for Cancer Discrimination Based on Deep Learning of MALDI-TOF MS Fingerprints.

Plasma exosomes have shown great potential for liquid biopsy in clinical cancer diagnosis. Herein, we present an integrated strategy for isolating and analyzing exosomes from human plasma rapidly and then discriminating different cancers excellently based on deep learning fingerprints of plasma exosomes. Sequential size-exclusion chromatography (SSEC) was developed efficiently for separating exosomes from human plasma. SSEC isolated plasma exosomes, taking as less as 2 h for a single sample with high purity such that the discard rates of high-density lipoproteins and low/very low-density lipoproteins were 93 and 85%, respectively. Benefitting from the rapid and high-purity isolation, the contents encapsulated in exosomes, covered by plasma proteins, were well profiled by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS). We further analyzed 220 clinical samples, including 79 breast cancer patients, 57 pancreatic cancer patients, and 84 healthy controls. After MS data pre-processing and feature selection, the extracted MS feature peaks were utilized as inputs for constructing a multi-classifier artificial neural network (denoted as Exo-ANN) model. The optimized model avoided overfitting and performed well in both training cohorts and test cohorts. For the samples in the independent test cohort, it realized a diagnosed accuracy of 80.0% with an area under the curve of 0.91 for the whole group. These results suggest that our integrated pipeline may become a generic tool for liquid biopsy based on the analysis of plasma exosomes in clinics.

Simultaneous analysis of cellular glycoproteome and phosphoproteome in cervical carcinoma by one-pot specific enrichment.

Cervical carcinoma is a kind of common gynecological cancer, the physiopathology of which is consanguineously connected with protein glycosylation and phosphorylation. Here, the magnetic binary metal oxide composites with the functionalization of hydrophilic tripeptide is designed and synthesized for simultaneous enrichment of glycopeptide and phosphopeptide in HeLa cells. Multiple kinds of metal sites realize more comprehensive enrichment of phosphopeptides compared with single metal, and ultra-hydrophilic property of glutathione further endows the composites with excellent affinity towards glycopeptides. The composites exhibited low detection limit (0.5 fmol/µL) and high selectivity (digests of ß-casein and BSA, 1:500, m/m) for phosphopeptides, also showed good enrichment efficiency for glycopeptide including detection limit (0.1 fmol/µL) and selectivity (digests of HRP and BSA, 1:50, m/m). Meanwhile, the composites still possess outstanding enrichment capability towards phosphopeptide and glycopeptide after stored for two months or six consecutive times reuse. Eventually, 1177 phosphopeptides and 438 glycopeptides are identified simultaneously from 100 µg HeLa cell digests. As anticipated, potential biomarkers, such as heat shock protein beta-1, DNA topoisomerase 2-alpha, proliferation marker protein Ki-67 and 60 kDa heat shock protein are detected, suggesting its promising application in discovery and screening of cervical carcinoma.

Quantitative proteomic analysis of the effects of dietary deprivation of methionine and cystine on A549 xenograft and A549 xenograft-bearing mouse.

Methionine (Met) and cystine (CySS) are key sulfur donors in cell metabolism and are important nutrients for sustaining tumor growth; however, the molecular effects associated with their deprivation remain to be characterized. Here, we applied a xenograft mouse model to assess the impact of their deprivation on A549 xenografts and the xenograft-bearing animal. Results show that Met and CySS deprivation inhibits A549 growth in vitro, not in vivo. Deprivation was detrimental to the xenograft-bearing mouse, as demonstrated by weight loss and renal dysfunction. Differentially expressed proteins in A549 xenograft and mouse kidneys were characterized using quantitative proteomics. Functional annotation and protein-protein interaction network analysis revealed the enriched signaling pathways, including focal adhesion (Fn1) in the A549 xenograft, and xenobiotic metabolism (Cyp2e1) and glutathione metabolism (Ggt1) in the mouse kidney. Met and CySS deprivation inhibits the migratory and invasive properties of cancer cells, as evidenced by reduced expression of the epithelial to mesenchymal transition marker N-cadherin in A549 cells in vitro. Moreover, IGFBP1 protein expression was inhibited in both A549 xenograft and mouse kidneys. This study provides the first insights into changes within the proteome profile and biological processes upon Met and CySS deprivation in a A549 xenograft mouse model.