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Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abundant sialoglycoprotein was found enriched in exosomes and it was identified by peptide mass fingerprinting and immunoblot as the galectin-3-binding protein (LGALS3BP). Exosomes were found to contain predominantly complex glycans of the di-, tri-, and tetraantennary type with or without proximal fucose and also high mannose glycans. Diantennary glycans containing bisecting N-acetylglucosamine were also detected. This work provides detailed information about glycoprotein and N-glycan composition of exosomes from ovarian cancer cells, furthermore it opens novel perspectives to further explore the functional role of glycans in the biology of exosomes.

The objectives of this study were to evaluate the formation of lymphvascular niches in lymph nodes of patients with oral squamous cell carcinoma (OSCC), and investigate the roles of lymphangiogenic and angiogenic factors, such as vascular endothelial growth factor (VEGF)-A, VEGF-C, and VEGF-D, expressed in the primary tumors. Forty-four patients with previously untreated clinically late T2 or T3 OSCC of cN0 were evaluated for primary tumors and 166 sentinel lymph nodes (SLNs). Primary tumors were immunohistochemically analyzed for expressions of VEGFs. Densities of lymphatic vessels (LVDpodoplanin) and high endothelial venules (HEVD) in the SLNs were also calculated using antibodies for each marker, podoplanin and MECA-79, respectively. In 25 patients, all lymph nodes were metastasis-negative, whereas, in 19 patients, metastasis was positive for at least one lymph node (either at SLN, non-SLN, or nodal recurrence). From the analyses of 140 SLNs without metastasis, LVDpodoplanin in 50 SLNs of metastasis-positive cases was significantly higher than that in 90 SLNs of metastasis-negative cases (p = 0.0025). HEVD was not associated with lymph node metastasis. The patients with VEGF-A-High or VEGF-D-High tumors had significantly higher LVDpodoplanin than patients with their Low counterparts (p = 0.0233 and p = 0.0209, respectively). In cases with lymph node metastasis, the VEGF-D-expression score was significantly higher than in those without lymph node metastasis (p = 0.0006). These results suggest that lymph node lymphangiogenesis occurs before metastasis in OSCC. VEGF-A and VEGF-D play critical roles in this process. VEGF-D is a potential predictive marker of positive lymph node metastasis in cN0 patients.

The profiling of cancer cell secretomes is considered to be a good strategy for identifying cancer-related biomarkers, but few studies have focused on identifying low-molecular-mass (LMr) proteins (<15 kDa) in cancer cell secretomes. Here, we used tricine–SDS-gel-assisted fractionation and LC–MS/MS to systemically identify LMr proteins in the secretomes of five oral cavity squamous cell carcinoma (OSCC) cell lines. Cross-matching of these results with nine OSCC tissue transcriptome datasets allowed us to identify 33 LMr genes/proteins that were highly upregulated in OSCC tissues and secreted/released from OSCC cells. Immunohistochemistry and quantitative real-time PCR were used to verify the overexpression of two candidates, HMGA2 and MIF, in OSCC tissues. The overexpressions of both proteins were associated with cervical metastasis, perineural invasion, deeper tumor invasion, higher overall stage and a poorer prognosis for post-treatment survival. Functional assays further revealed that both proteins promoted the migration and invasion of OSCC cell lines in vitro . Collectively, our data indicate that the tricine–SDS-gel/LC–MS/MS approach can be used to efficiently identify LMr proteins from OSCC cell secretomes and suggest that HMGA2 and MIF could be potential tissue biomarkers for OSCC.

Most cases of pancreatic cancer are not diagnosed until they are no longer curable with surgery. Therefore, it is critical to develop a sensitive, preferably noninvasive, method for detecting the disease at an earlier stage. In order to identify biomarkers for pancreatic cancer, we devised an in vitro positive/negative selection strategy to identify RNA ligands (aptamers) that could detect structural differences between the secretomes of pancreatic cancer and non-cancerous cells. Using this molecular recognition approach, we identified an aptamer (M9-5) that differentially bound conditioned media from cancerous and non-cancerous human pancreatic cell lines. This aptamer further discriminated between the sera of pancreatic cancer patients and healthy volunteers with high sensitivity and specificity. We utilized biochemical purification methods and mass-spectrometric analysis to identify the M9-5 target as cyclophilin B (CypB). This molecular recognition-based strategy simultaneously identified CypB as a serum biomarker and generated a new reagent to recognize it in body fluids. Moreover, this approach should be generalizable to other diseases and complementary to traditional approaches that focus on differences in expression level between samples. Finally, we suggest that the aptamer we identified has the potential to serve as a tool for the early detection of pancreatic cancer.

Genetic alterations in tumor cells often lead to the emergence of growth-stimulatory autocrine and paracrine signals, involving overexpression of secreted peptide growth factors, cytokines, and hormones. Increased levels of these soluble proteins may be exploited for cancer diagnosis and management or as points of therapeutic intervention. Here, we combined the use of controlled vocabulary terms and sequence-based algorithms to predict genes encoding secreted proteins from among ≈12,500 sequences represented on oligonucleotide microarrays. Expression of these genes was queried in 150 carcinomas from 10 anatomic sites of origin and compared with 46 normal tissues derived from the corresponding sites of tumor origin and other body tissues and organs. Of 74 different genes identified as overexpressed in cancer tissues, several encode proteins with demonstrated clinical diagnostic application, such as α-fetoprotein in liver carcinoma, and kallikreins 6 and 10 in ovarian cancer, or therapeutic utility, such as gastrin-releasing peptide/bombesin in lung carcinomas. We show that several of the other candidate genes encode proteins with high levels of tumor-associated expression by immunohistochemistry on tissue microarrays and further demonstrate significantly elevated levels of another novel candidate protein, macrophage inhibitory cytokine 1, a distant member of the tranforming growth factor-β superfamily, in the serum of patients with metastatic prostate, breast, and colorectal carcinomas. Our results suggest that the combination of annotation/protein sequence analysis, transcript profiling, immunohistochemistry, and immunoassay is a powerful approach for delineating candidate biomarkers with potential clinical significance and may be broadly applicable to other human diseases.

Ovulation has long been associated with an increased risk in ovarian cancer, yet the underlying molecular mechanisms remain obscure. Two aspects of ovulation have been linked to ovarian cancer pathogenesis. The first is the impact of repetitive tissue injury and repair that occurs with each ovulatory event. The second is the release of follicular fluid that accompanies the follicular rupture and its effect on the ovarian and fallopian tube epithelial cells. Hormones are an important component of follicular fluid, which transiently bathes the ovarian surface and fallopian tube epithelium during ovulation. Much work has been done exploring the role of hormones in fertility, but some, such as estrogen, have also been implicated in the pathogenesis of ovarian and other cancers. Understanding the role of hormones within follicular fluid, as well as how they are altered in disorders which increase ovarian cancer risk, will enhance our ability to assess risk and develop preventative strategies. This review provides an in depth discussion of the logistics of using and studying follicular fluid in ovarian cancer research, and discusses the fluctuations in follicular fluid hormone levels during normal physiological processes versus conditions that increase ovarian cancer risk.

Genetic and transcriptional profiling, as well as surface marker identification of single circulating tumor cells (CTCs) have been demonstrated. However, quantitatively profiling of functional proteins at single CTC resolution has not yet been achieved, owing to the limited purity of the isolated CTC populations and a lack of single-cell proteomic approaches to handle and analyze rare CTCs. Here, we develop an integrated microfluidic system specifically designed for streamlining isolation, purification and single-cell secretomic profiling of CTCs from whole blood. Key to this platform is the use of photocleavable ssDNA-encoded antibody conjugates to enable a highly purified CTC population with <75 ‘contaminated’ blood cells. An enhanced poly-L-lysine barcode pattern is created on the single-cell barcode chip for efficient capture rare CTC cells in microchambers for subsequent secreted protein profiling. This system was extensively evaluated and optimized with EpCAM-positive HCT116 cells seeded into whole blood. Patient blood samples were employed to assess the utility of the system for isolation, purification and single-cell secretion profiling of CTCs. The CTCs present in patient blood samples exhibit highly heterogeneous secretion profile of IL-8 and VEGF. The numbers of secreting CTCs are found not in accordance with CTC enumeration based on immunostaining in the parallel experiments.

Background To demonstrate the use of surface-enhanced Raman spectroscopy (SERS) to determine sialic acid (SA) levels in saliva using silver nanoparticles as substrates, in adnexal mass patients scheduled for surgical intervention to remove invasive masses, with the aim to compare SA levels in benign tumor vs ovarian cancer patients. Methods Quantification of SA levels was accomplished by measuring their SERS and calibrating with analytical reagent SA. The mean SA concentration in saliva from 37 benign adnexal mass resulted smaller (5.1 mg/dL) than the mean concentration in 15 Ovarium cancer patients (23 mg/dL). The cancer condition was determined by biopsy of the removed adnexal mass. The CA-125 biomarker was also measured. The predictive potential of both biomarkers is discussed, together with the malignity risk index (MRI). Results Our results showed a sensitivity/specificity of 80%/100% with a cutoff to distinguish between benign/cancer cases of SA 15.5 mg/dL, as established from a ROC analysis. Our results suggest that SA may be a more useful biomarker than CA-125 to detect ovarian cancer. Conclusions Our results suggest that the SA levels measured from saliva may be as good predictors as the MRI index for the presence of ovarian cancer in sensitivity/negative predictive value and outperforms it in specificity/positive predictive value.

Purpose Thyrotropin-secreting pituitary adenomas (TSHomas) represent a rare subtype of pituitary tumors. Neurosurgery (NCH) is still considered the first-line therapy. In this study we aimed to investigate the outcome of different treatment modalities, including first line somatostatin analogs (SSA) treatment, with a specific focus on neurosurgery-related complications. Methods We retrospectively evaluated thirteen patients diagnosed for TSHomas (9 M; age range 27–61). Ten patients had a magnetic resonance evidence of macroadenoma, three with slight visual field impairment. In the majority of patients, thyroid ultrasonography showed the presence of goiter and/or increased gland vascularization. Median TSH value at diagnosis was 3.29 mU/L (normal ranges 0.2–4.2 mIU/L), with median fT4 2.52 ng/dL (0.9–1.7 ng/dL). Results Three patients (two microadenoma) were primarily treated with NCH and achieved disease remission, whereas ten patients (nine macroadenomas) were initially treated with SSA. Despite the optimal biochemical response observed during medical treatment in most patients (mean TSH decrease −72 %), only two stayed on medical therapy alone, achieving stable biochemical control at the end of the follow-up. The remaining patients (n = 7) underwent NCH later on during their clinical history, followed by radiotherapy or adjuvant SSA treatment in two cases. Noteworthy, five of them developed hypopituitarism. All patients reached a biochemical control, after a multimodal therapeutic approach. Conclusions Neurosurgery ultimately led to complete disease remission or to biochemical control in majority of patients, whereas resulting in a considerable percentage of post-operative complications (mainly hypopituitarism, 50 %). In the light of the optimal results unanimously reported for medical treatment with SSA, our experience suggests that a careful evaluation of risk/benefit ratio should be taken into consideration when directing the treatment approach in patients with TSHoma.

Primary aldosteronism due to unilateral aldosterone-producing adenoma (APA) is a surgically curable form of hypertension. Bilateral APA can also be surgically curable in theory but few successful cases can be found in the literature. It has been reported that even using successful adrenal venous sampling (AVS) via bilateral adrenal central veins, it is extremely difficult to differentiate bilateral APA from bilateral idiopathic hyperaldosteronism (IHA) harbouring computed tomography (CT)-detectable bilateral adrenocortical nodules. We report a case of bilateral APA diagnosed by segmental AVS (S-AVS) and blood sampling via intra-adrenal first-degree tributary veins to localize the sites of intra-adrenal hormone production. A 36-year-old man with marked long-standing hypertension was referred to us with a clinical diagnosis of bilateral APA. He had typical clinical and laboratory profiles of marked hypertension, hypokalaemia, elevated plasma aldosterone concentration (PAC) of 45.1 ng dl −1 and aldosterone renin activity ratio of 90.2 (ng dl −1 per ng ml −1 h −1 ), which was still high after 50 mg-captopril loading. CT revealed bilateral adrenocortical tumours of 10 and 12 mm in diameter on the right and left sides, respectively. S-AVS confirmed excess aldosterone secretion from a tumour segment vein and suppressed secretion from a non-tumour segment vein bilaterally, leading to the diagnosis of bilateral APA. The patient underwent simultaneous bilateral sparing adrenalectomy. Histopathological analysis of the resected adrenals together with decreased blood pressure and PAC of 5.2 ng dl −1 confirmed the removal of bilateral APA. S-AVS was reliable to differentiate bilateral APA from IHA by direct evaluation of intra-adrenal hormone production.