Antibody-mediated blockade for galectin-3 binding protein in tumor secretome abrogates PDAC metastasis.

The major challenges in pancreatic ductal adenocarcinoma (PDAC) management are local or distant metastasis and limited targeted therapeutics to prevent it. To identify a druggable target in tumor secretome and to explore its therapeutic intervention, we performed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis of tumors obtained from a patient-derived xenograft model of PDAC. Galectin-3 binding protein (Gal-3BP) is identified as a highly secreted protein, and its overexpression is further validated in multiple PDAC tumors and primary cells. Knockdown and exogenous treatment of Gal-3BP showed that it is required for PDAC cell proliferation, migration, and invasion. Mechanistically, we revealed that Gal-3BP enhances galectin-3-mediated epidermal growth factor receptor signaling, leading to increased cMyc and epithelial-mesenchymal transition. To explore the clinical impact of these findings, two antibody clones were developed, and they profoundly abrogated the metastasis of PDAC cells in vivo. Altogether, our data demonstrate that Gal-3BP is an important therapeutic target in PDAC, and we propose its blockade by antibody as a therapeutic option for suppressing PDAC metastasis.

Migrasomal autophagosomes relieve endoplasmic reticulum stress in glioblastoma cells.

BACKGROUND: Glioblastoma (GBM) is more difficult to treat than other intractable adult tumors. The main reason that GBM is so difficult to treat is that it is highly infiltrative. Migrasomes are newly discovered membrane structures observed in migrating cells. Thus, they can be generated from GBM cells that have the ability to migrate along the brain parenchyma. However, the function of migrasomes has not yet been elucidated in GBM cells. RESULTS: Here, we describe the composition and function of migrasomes generated along with GBM cell migration. Proteomic analysis revealed that LC3B-positive autophagosomes were abundant in the migrasomes of GBM cells. An increased number of migrasomes was observed following treatment with chloroquine (CQ) or inhibition of the expression of STX17 and SNAP29, which are involved in autophagosome/lysosome fusion. Furthermore, depletion of ITGA5 or TSPAN4 did not relieve endoplasmic reticulum (ER) stress in cells, resulting in cell death. CONCLUSIONS: Taken together, our study suggests that increasing the number of autophagosomes, through inhibition of autophagosome/lysosome fusion, generates migrasomes that have the capacity to alleviate cellular stress.

Proteomic analysis of breast cancer based on immune subtypes.

BACKGROUND: Immunotherapy is applied to breast cancer to resolve the limitations of survival gain in existing treatment modalities. With immunotherapy, a tumor can be classified into immune-inflamed, excluded and desert based on the distribution of immune cells. We assessed the clinicopathological features, each subtype's prognostic value and differentially expressed proteins between immune subtypes. METHODS: Immune subtyping and proteomic analysis were performed on 56 breast cancer cases with neoadjuvant chemotherapy. The immune subtyping was based on the level of tumor-infiltrating lymphocytes (TILs) and Klintrup criteria. If the level of TILs was ≥ 10%, it was classified as immune-inflamed type without consideration of the Klintrup criteria. In cases of 1-9% TIL, Klintrup criteria 1-3 were classified as the immune-excluded subtype and Klintrup criteria not available (NA) was classified as NA. Cases of 1% TILs and Klintrup 0 were classified as the immune-desert subtype. Mass spectrometry was used to identify differentially expressed proteins in formalin-fixed paraffin-embedded biopsy tissues. RESULTS: Of the 56 cases, 31 (55%) were immune-inflamed, 21 (38%) were immune-excluded, 2 (4%) were immune-desert and 2 (4%) were NA. Welch's t-test revealed two differentially expressed proteins between immune-inflamed and immune-excluded/desert subtypes. Coronin-1A was upregulated in immune-inflamed tumors (adjusted p = 0.008) and α-1-antitrypsin was upregulated in immune-excluded/desert tumors (adjusted p = 0.008). Titin was upregulated in pathologic complete response (pCR) than non-pCR among immune-inflamed tumors (adjusted p = 0.036). CONCLUSIONS: Coronin-1A and α-1-antitrypsin were upregulated in immune-inflamed and immune-excluded/desert subtypes, respectively. Titin's elevated expression in pCR within the immune-inflamed subtype may indicate a favorable prognosis. Further studies involving large representative cohorts are necessary to validate these findings.

Characterization of lymphocyte-rich hepatocellular carcinoma and the prognostic role of tertiary lymphoid structures.

BACKGROUND & AIMS: Lymphocyte-rich hepatocellular carcinoma (LR-HCC) is largely unknown and a rare subtype of HCC with immune-rich stroma. Tertiary lymphoid structures (TLS), frequently observed in LR-HCC, are known to be prognostically significant in various malignancies; however, their significance in HCC remains unevaluated. METHODS: Clinicopathologic data of 191 cases of surgically resected conventional HCC (C-HCC, n = 160) and LR-HCC (n = 31) were retrieved. Immunohistochemistry, multiplex immunofluorescence staining, RNA sequencing and proteomic analysis were conducted. Differences between the subtypes were statistically evaluated. RESULTS: LR-HCC was significantly correlated to larger tumour size, higher Edmondson-Steiner grade, presence of TLS and higher CD3-, CD8- and FOXP3-positive T cell, high PD-1 and PD-L1 expression (p < .001 for all) compared to C-HCC. Patients with LR-HCC exhibited significantly better overall survival (OS) (p = .044) and recurrence-free survival (RFS) (p = .025) than C-HCC. LR-HCC demonstrated TLS signatures with significantly higher proteomic-based immune scores in 14 of 17 types of tumour-infiltrating immune cells. Furthermore, C-HCC with secondary follicles, the most mature form of TLS, exhibited significantly better OS (p = .031) and RFS (p = .033) than those without. Across the global proteome, LR-HCC was well-differentiated from C-HCC and a map of protein-protein interactions between tumour-infiltrating lymphocytes and HCC in tumour microenvironment was completed. CONCLUSION: LR-HCC is clinicopathologically and molecularly distinct and shows better prognosis compared to C-HCC. Also, the presence of secondary follicle can be an important prognostic marker for better prognosis in both LR-HCC and C-HCC.

Dualistic Effects of PRKAR1A as a Potential Anticancer Target in Cancer Cells and Cancer-Derived Stem Cells.

The integration of innovative medical technologies and interdisciplinary collaboration could improve the treatment of cancer, a globally prevalent and often deadly disease. Despite recent advancements, current cancer therapies fail to specifically address recurrence and target cancer stem cells (CSCs), which contribute to relapse. In this study, we utilized three types of cancer cells, from which three types of CSCs were further derived, to conduct a proteomic analysis. Additionally, shared cell surface biomarkers were identified as potential targets for a comprehensive treatment strategy. The selected biomarkers were evaluated through short hairpin RNA treatment, which revealed contrasting functions in cancer cells and CSCs. Knockdown of the identified proteins revealed that they regulate the epithelial-mesenchymal transition (EMT) and stemness via the ERK signaling pathway. Resistance to anticancer agents was consequently reduced, ultimately enhancing the overall anticancer effects of the treatment. Additionally, the significance of these biomarkers in clinical patient outcomes was confirmed using bioinformatics. Our study suggests a novel cancer treatment strategy that addresses the limitations of current anticancer therapies.

Proteomic analysis predicts anti-angiogenic resistance in recurred glioblastoma.

BACKGROUND: Recurrence is common in glioblastoma multiforme (GBM) because of the infiltrative, residual cells in the tumor margin. Standard therapy for GBM consists of surgical resection followed by chemotherapy and radiotherapy, but the median survival of GBM patients remains poor (~ 1.5 years). For recurrent GBM, anti-angiogenic treatment is one of the common treatment approaches. However, current anti-angiogenic treatment modalities are not satisfactory because of the resistance to anti-angiogenic agents in some patients. Therefore, we sought to identify novel prognostic biomarkers that can predict the therapeutic response to anti-angiogenic agents in patients with recurrent glioblastoma. METHODS: We selected patients with recurrent GBM who were treated with anti-angiogenic agents and classified them into responders and non-responders to anti-angiogenic therapy. Then, we performed proteomic analysis using liquid-chromatography mass spectrometry (LC-MS) with formalin-fixed paraffin-embedded (FFPE) tissues obtained from surgical specimens. We conducted a gene-ontology (GO) analysis based on protein abundance in the responder and non-responder groups. Based on the LC-MS and GO analysis results, we identified potential predictive biomarkers for anti-angiogenic therapy and validated them in recurrent glioblastoma patients. RESULTS: In the mass spectrometry-based approach, 4957 unique proteins were quantified with high confidence across clinical parameters. Unsupervised clustering analysis highlighted distinct proteomic patterns (n = 269 proteins) between responders and non-responders. The GO term enrichment analysis revealed a cluster of genes related to immune cell-related pathways (e.g., TMEM173, FADD, CD99) in the responder group, whereas the non-responder group had a high expression of genes related to nuclear replisome (POLD) and damaged DNA binding (ERCC2). Immunohistochemistry of these biomarkers showed that the expression levels of TMEM173 and FADD were significantly associated with the overall survival and progression-free survival of patients with recurrent GBM. CONCLUSIONS: The candidate biomarkers identified in our protein analysis may be useful for predicting the clinical response to anti-angiogenic agents in patients with recurred GBM.

Radiation-induced C-reactive protein triggers apoptosis of vascular smooth muscle cells through ROS interfering with the STAT3/Ref-1 complex.

Damage to normal tissue can occur over a long period after cancer radiotherapy. Free radical by radiation can initiate or accelerate chronic inflammation, which can lead to atherosclerosis. However, the underlying mechanisms remain unclear. Vascular smooth muscle cells (VSMCs) proliferate in response to JAK/STAT3 signalling. C-reactive protein (CRP) can induce VSMCs apoptosis via triggering NADPH oxidase (NOX). Apoptotic VSMCs promote instability and inflammation of atherosclerotic lesions. Herein, we identified a VSMCs that switched from proliferation to apoptosis through was enhanced by radiation-induced CRP. NOX inhibition using lentiviral sh-p22phox prevented apoptosis upon radiation-induced CRP. CRP overexpression reduced the amount of STAT3/Ref-1 complex, decreased JAK/STAT phosphorylation and formed a new complex of Ref-1/CRP in VSMC. Apoptosis of VSMCs was further increased by CRP co-overexpressed with Ref-1. Functional inhibition of NOX or p53 also prevented apoptotic activity of the CRP-Ref-1 complex. Immunofluorescence showed co-localization of CRP, Ref-1 and p53 with α-actin-positive VSMC in human atherosclerotic plaques. In conclusion, radiation-induced CRP increased the VSMCs apoptosis through Ref-1, which dissociated the STAT3/Ref-1 complex, interfered with JAK/STAT3 activity, and interacted with CRP-Ref-1, thus resulting in transcription-independent cell death via p53. Targeting CRP as a vascular side effect of radiotherapy could be exploited to improve curability.

Lipid signatures reflect the function of the murine primary placentation†.

The placenta regulates maternal-fetal communication, and its defect leads to significant pregnancy complications. The maternal and embryonic circulations are primitively connected in early placentation, but the function of the placenta during this developmentally essential period is relatively unknown. We thus performed a comparative proteomic analysis of the placenta before and after primary placentation and found that the metabolism and transport of lipids were characteristically activated in this period. The placental fatty acid (FA) carriers in specific placental compartments were upregulated according to gestational age, and metabolomic analysis also showed that the placental transport of FAs increased in a time-dependent manner. Further analysis of two mutant mice models with embryonic lethality revealed that lipid-related signatures could reflect the functional state of the placenta. Our findings highlight the importance of the nutrient transport function of the primary placenta in the early gestational period and the role of lipids in embryonic development. SUMMARY SENTENCE: The placenta is activated characteristically in terms of lipid transport during primary placentation, and the lipid-related signatures closely reflect the functional state of the placenta.

Urine ß-2-glycoprotein 1 as a biomarker for diagnosis of systemic lupus erythematosus.

OBJECTIVE: The need for a biomarker with robust sensitivity and specificity in diagnosing systemic lupus erythematosus (SLE) remains unmet. Compared with blood samples, urine samples are more easily collected; thus, we aimed to identify such a biomarker based on urinary proteomics which could distinguish patients with SLE from healthy controls (HCs). METHODS: Urine samples were collected from 76 SLE patients who visited rheumatology clinic in 2019 at Asan medical center and from 25 HCs. Urine proteins were analyzed using sequential windowed acquisition of all theoretical fragment ion spectra-mass spectrometry, and the candidate marker was confirmed by enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristic curve analysis was used to determine the diagnostic value of the candidate biomarker. RESULTS: Of 1157 proteins quantified, 153 were differentially expressed in urine samples from HCs. Among them were previously known markers including α-1-acid glycoprotein 1, α-2-HS-glycoprotein, ceruloplasmin, and prostaglandin-H2 D-isomerase. Moreover, the amount of ß-2 glycoprotein (APOH) was increased in the urine of patients with SLE. The ELISA results also showed the level of urine APOH was higher in patients with SLE than in HCs and patients with rheumatoid arthritis. Moreover, the level was not different between SLE patients with and without nephritis. The urine APOH had an area under the curve value of 0.946 at a cut-off value of 228.53 ng/mg (sensitivity 91.5%, specificity 92.0%) for the diagnosis of SLE. CONCLUSION: The results indicate that the urine APOH level can be an appropriate screening tool in a clinical setting when SLE is suspected.

Long-Lasting Growth Hormone Regulated by the Ubiquitin-Proteasome System.

To increase the half-life of growth hormones, we proposed its long-lasting regulation through the ubiquitin-proteasome system (UPS). We identified lysine residues (K67, K141, and K166) that are involved in the ubiquitination of human growth hormone (hGH) using ubiquitination site prediction programs to validate the ubiquitination sites, and then substituted these lysine residues with arginine residues. We identified the most effective substituent (K141R) to prevent ubiquitination and named it AUT-hGH. hGH was expressed and purified in the form of hGH-His, and ubiquitination was first verified at sites containing K141 in the blood stream. Through the study, we propose that AUT-hGH with an increased half-life could be used as a long-lasting hGH in the blood stream.

Tissue-specific roles of GCN2 in aging and autosomal dominant retinitis pigmentosa.

The organisms have the capacity to sense and adapt to their surroundings for their life in a dynamic environment. In response to amino acid starvation, cells activate a rectifying physiological program, termed the integrated stress response (ISR), to restore cellular homeostasis. General controlled non-repressed (GCN2) kinase is a master regulator of the ISR and modulates protein synthesis in response to amino acid starvation. We previously established the GCN2/ATF4/4E-BP pathway in development and aging. Here, we investigated the tissue-specific roles of GCN2 upon dietary restriction of amino acid in a Drosophila model. The knockdown of GCN2 in the gut and fat body, an energy sensing organ in Drosophila, abolished the beneficial effect of GCN2 in lifespan extension upon dietary restriction of amino acids. Proteome analysis in an autosomal dominant retinitis pigmentosa (ADRP) model showed that dietary restriction of amino acids regulates the synthesis of proteins in several pathways, including mitochondrial translation, mitochondrial gene expression, and regulation of biological quality, and that gcn2-mutant flies have reduced levels of these mitochondria-associated proteins, which may contribute to retinal degeneration in ADRP. These results indicate that the tissue-specific regulation of GCN2 contributes to normal physiology and ADRP progression.

Impact of proactive high-throughput functional assay data on BRCA1 variant interpretation in 3684 patients with breast or ovarian cancer.

The clinical utility of BRCA1/2 genotyping was recently extended from the selection of subjects at high risk for hereditary breast and ovary cancer to the identification of candidates for poly (ADP-ribose) polymerase (PARP) inhibitor treatment. This underscores the importance of accurate interpretation of BRCA1/2 genetic variants and of reducing the number of variants of uncertain significance (VUSs). Two recent studies by Findlay et al. and Starita et al. introduced high-throughput functional assays, and proactively analyzed variants in specific regions regardless of whether they had been previously observed. We retrospectively reviewed all BRCA1 and BRCA2 germline genetic test reports from patients with breast or ovarian cancer examined at Asan Medical Center (Seoul, Korea) between September 2011 and December 2018. Variants were assigned pathogenic or benign strong evidence codes according to the functional classification and were reclassified according to the ACMG/AMP 2015 guidelines. Among 3684 patients with available BRCA1 and BRCA2 germline genetic test reports, 429 unique variants (181 from BRCA1) were identified. Of 34 BRCA1 variants intersecting with the data reported by Findlay et al., three missense single-nucleotide variants from four patients (0.11%, 4/3684) were reclassified from VUSs to likely pathogenic variants. Four variants scored as functional were reclassified into benign or likely benign variants. Three variants that overlapped with the data reported by Starita et al. could not be reclassified. In conclusion, proactive high-throughput functional study data are useful for the reclassification of clinically observed VUSs. Integrating additional evidence, including functional assay results, may help reduce the number of VUSs.

Differential Urinary Proteome Analysis for Predicting Prognosis in Type 2 Diabetes Patients with and without Renal Dysfunction.

Renal dysfunction, a major complication of type 2 diabetes, can be predicted from estimated glomerular filtration rate (eGFR) and protein markers such as albumin concentration. Urinary protein biomarkers may be used to monitor or predict patient status. Urine samples were selected from patients enrolled in the retrospective diabetic kidney disease (DKD) study, including 35 with good and 19 with poor prognosis. After removal of albumin and immunoglobulin, the remaining proteins were reduced, alkylated, digested, and analyzed qualitatively and quantitatively with a nano LC-MS platform. Each protein was identified, and its concentration normalized to that of creatinine. A prognostic model of DKD was formulated based on the adjusted quantities of each protein in the two groups. Of 1296 proteins identified in the 54 urine samples, 66 were differentially abundant in the two groups (area under the curve (AUC): p-value < 0.05), but none showed significantly better performance than albumin. To improve the predictive power by multivariate analysis, five proteins (ACP2, CTSA, GM2A, MUC1, and SPARCL1) were selected as significant by an AUC-based random forest method. The application of two classifiers-support vector machine and random forest-showed that the multivariate model performed better than univariate analysis of mucin-1 (AUC: 0.935 vs. 0.791) and albumin (AUC: 1.0 vs. 0.722). The urinary proteome can reflect kidney function directly and can predict the prognosis of patients with chronic kidney dysfunction. Classification based on five urinary proteins may better predict the prognosis of DKD patients than urinary albumin concentration or eGFR.

Targeting ß-catenin overcomes MEK inhibition resistance in colon cancer with KRAS and PIK3CA mutations.

BACKGROUND: Mitogen-activated protein kinases (MEK 1/2) are central components of the RAS signalling pathway and are attractive targets for cancer therapy. These agents continue to be investigated in KRAS mutant colon cancer but are met with significant resistance. Clinical investigations have demonstrated that these strategies are not well tolerated by patients. METHODS: We investigated a biomarker of response for MEK inhibition in KRAS mutant colon cancers by LC-MS/MS analysis. We tested the MEK inhibitor in PIK3CA wild(wt) and mutant(mt) colon cancer cells. In addition, we tested the combinational effects of MEK and TNKS inhibitor in vitro and in vivo. RESULTS: We identified ß-catenin, a key mediator of the WNT pathway, in response to MEK inhibitor. MEK inhibition led to a decrease in ß-catenin in PIK3CA wt colon cancer cells but not in mt. Tumour regression was promoted by combination of MEK inhibition and NVP-TNS656, which targets the WNT pathway. Furthermore, inhibition of MEK promoted tumour regression in colon cancer patient-derived xenograft models expressing PIK3CA wt. CONCLUSIONS: We propose that inhibition of the WNT pathway, particularly ß-catenin, may bypass resistance to MEK inhibition in human PIK3CA mt colon cancer. Therefore, we suggest that ß-catenin is a potential predictive marker of MEK inhibitor resistance.

NEDD4L limits cAMP signaling through ubiquitination of CREB-regulated transcription coactivator 3.

The transcription factor cAMP-responsive element-binding protein (CREB) is involved in a variety of physiologic processes. Although its activity appears to be largely correlated with its phosphorylation status, cAMP-mediated dephosphorylation and the subsequent nuclear migration of the CREB-regulated transcription factors (CRTCs) are required to stimulate CREB transcriptional activity. Among the 3 identified mammalian homologs of CRTCs, CRTC3 has been shown to be expressed predominantly in adipose tissues in response to catecholamine signals that regulate lipid metabolism. Here, we show that prolonged cAMP signaling down-regulates CRTC3 in a proteasome-dependent manner and that neural precursor cell-expressed developmentally down-regulated gene 4-like (NEDD4L), a specific ubiquitin ligase for CRTC3, is responsible for this process. By recognizing the PY motif of CRTC3, NEDD4L interacts with CRTC3 and promotes its polyubiquitination. Interaction between NEDD4L and CRTC3 is further boosted by cAMP signaling, and this enhanced interaction appears to be dependent on the cAMP-mediated phosphorylation of NEDD4L at the Ser448 site. Furthermore, we show that food withdrawal stimulates NEDD4L phosphorylation in mice, which then show a decrease of adipose tissue CRTC3 protein levels. Together, these results suggest that NEDD4L plays a key role in the feedback regulation of cAMP signaling by limiting CRTC3 protein levels.-Kim, Y.-H., Yoo, H., Hong, A.-R., Kwon, M., Kang, S.-W., Kim, K., Song, Y. NEDD4L limits cAMP signaling through ubiquitination of CREB-regulated transcription coactivator 3.

Top-Down Proteomics Enables Comparative Analysis of Brain Proteoforms Between Mouse Strains.

Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains.

Tumour growth rate of follicular thyroid carcinoma is not different from that of follicular adenoma.

OBJECTIVE: Distinguishing malignancy from benign thyroid nodule has always been challenging, especially in follicular lesions. Thyroid nodules with small size and indeterminate cytology do not lead to immediate surgery. We tried to evaluate whether tumour size and tumour growth rate can distinguish follicular thyroid carcinoma (FTC) from follicular adenoma (FA). DESIGN AND PATIENTS: This retrospective study included patients with pathologically proven FTCs (n = 50) and FAs (n = 110) who underwent preoperative serial neck ultrasonography (US) at least 3 times: it comprises 30% of all follicular tumours (32% FAs and 25% FTCs). The growth rates of follicular tumours on serial US were measured using at least 3 consecutive examinations during a median follow-up of 4.1 years (range, 0.7-13.3 years) by experienced radiologists. RESULTS: The FA and FTC groups showed no significant difference in clinicopathological characteristics, including age, proportion of large nodules (>4 cm) and preoperative cytology. The maximum diameter of thyroid nodule was gradually increased in both groups with statistical significance (P < .001 and P < .001, respectively). No significant differences in change of maximum diameter of thyroid nodule (P = .132) and tumour volume (P = .208) were found between the FA and FTC groups during the follow-up. The median time to a significant tumour growth from baseline was not different between the FA and FTC groups (1.4 years and 1.7 years, respectively, P = .556). When we divided the patients into four groups (rapid, moderate, slow and no growth) according to the growth velocity of the thyroid tumours, no significant difference in growth velocity was found among the groups. CONCLUSIONS: The tumour size and growth rate of the thyroid nodule itself could not predict malignancy. Diagnostic approaches that use molecular markers would be more important than clinical features for the decision of diagnostic surgery for patients with follicular tumours.

Demonstrating the feasibility of large-scale development of standardized assays to quantify human proteins.

Multiple reaction monitoring (MRM) mass spectrometry has been successfully applied to monitor targeted proteins in biological specimens, raising the possibility that assays could be configured to measure all human proteins. We report the results of a pilot study designed to test the feasibility of a large-scale, international effort for MRM assay generation. We have configured, validated across three laboratories and made publicly available as a resource to the community 645 novel MRM assays representing 319 proteins expressed in human breast cancer. Assays were multiplexed in groups of >150 peptides and deployed to quantify endogenous analytes in a panel of breast cancer-related cell lines. The median assay precision was 5.4%, with high interlaboratory correlation (R(2) > 0.96). Peptide measurements in breast cancer cell lines were able to discriminate among molecular subtypes and identify genome-driven changes in the cancer proteome. These results establish the feasibility of a large-scale effort to develop an MRM assay resource.

Priming with ceramide-1 phosphate promotes the therapeutic effect of mesenchymal stem/stromal cells on pulmonary artery hypertension.

Some molecules enriched in damaged organs can contribute to tissue repair by stimulating the mobilization of stem cells. These so-called "priming" factors include bioactive lipids, complement components, and cationic peptides. However, their therapeutic significance remains to be determined. Here, we show that priming of mesenchymal stromal/stem cells (MSCs) with ceramide-1 phosphate (C1P), a bioactive lipid, enhances their therapeutic efficacy in pulmonary artery hypertension (PAH). Human bone marrow (BM)-derived MSCs treated with 100 or 200 µM C1P showed improved migration activity in Transwell assays compared with non-primed MSCs and concomitantly activated MAPK(p42/44) and AKT signaling cascades. Although C1P priming had little effect on cell surface marker phenotypes and the multipotency of MSCs, it potentiated their proliferative, colony-forming unit-fibroblast, and anti-inflammatory activities. In a monocrotaline-induced PAH animal model, a single administration of human MSCs primed with C1P significantly attenuated the PAH-related increase in right ventricular systolic pressure, right ventricular hypertrophy, and thickness of α-smooth muscle actin-positive cells around the vessel wall. Thus, this study shows that C1P priming increases the effects of MSC therapy by enhancing the migratory, self-renewal, and anti-inflammatory activity of MSCs and that MSC therapy optimized with priming protocols might be a promising option for the treatment of PAH patients.

Optical Aptamer Probes of Fluorescent Imaging to Rapid Monitoring of Circulating Tumor Cell.

Fluorescence detecting of exogenous EpCAM (epithelial cell adhesion molecule) or muc1 (mucin1) expression correlated to cancer metastasis using nanoparticles provides pivotal information on CTC (circulating tumor cell) occurrence in a noninvasive tool. In this study, we study a new skill to detect extracellular EpCAM/muc1 using quantum dot-based aptamer beacon (QD-EpCAM/muc1 ALB (aptamer linker beacon). The QD-EpCAM/muc1 ALB was designed using QDs (quantum dots) and probe. The EpCAM/muc1-targeting aptamer contains a Ep-CAM/muc1 binding sequence and BHQ1 (black hole quencher 1) or BHQ2 (black hole quencher2). In the absence of target EpCAM/muc1, the QD-EpCAM/muc1 ALB forms a partial duplex loop-like aptamer beacon and remained in quenched state because the BHQ1/2 quenches the fluorescence signal-on of the QD-EpCAM/muc1 ALB. The binding of EpCAM/muc1 of CTC to the EpCAM/muc1 binding aptamer sequence of the EpCAM/muc1-targeting oligonucleotide triggered the dissociation of the BHQ1/2 quencher and subsequent signal-on of a green/red fluorescence signal. Furthermore, acute inflammation was stimulated by trigger such as caerulein in vivo, which resulted in increased fluorescent signal of the cy5.5-EpCAM/muc1 ALB during cancer metastasis due to exogenous expression of EpCAM/muc1 in Panc02-implanted mouse model.