The disadvantage of 10% EDTA decalcification is a long time-consuming. It needs to identify a quick and straightforward decalcification method when the preparation of lymphedema models using mouse tail which was a sample of bone wrapped in other tissues. In the present study, mouse tail samples were decalcified in 10% EDTA at 25, 37, and 42°C, respectively, with continuous shaking (150 rpm/min). The histologic integrity of samples was evaluated by hematoxylin and eosin staining, and the preservation of antigenicity was tested by either immunohistochemistry or immunofluorescence. The decalcification was distinctly accelerated by temperature. Results of hematoxylin and eosin staining were similar among different temperature groups. Immunohistochemistry and immunofluorescence staining revealed almost no signals in samples decalcified at 42°C for 1 week. Clear signals were detected when samples were decalcified at 37 and 25°C.

CONTEXT:The potential relationship between exosomal microRNAs (miRNAs) and clinical symptoms in breast cancer patients and the expression levels of exosomal miRNAs in patients undergoing surgery and chemotherapy are still unclear. AIMS:The aim of this study was to explore the correlation among exosomal miRNAs and clinical features and treatment in breast cancer patients. MATERIALS AND METHODS:First, exosomes were isolated from the serum of patients with breast cancer and healthy controls. Next, the features of exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. Then, we detected the expression of the top-ranked miRNAs (miR-3662, miR-16-1, miR-146a, miR-1290, and miR-29c) in sixty breast cancer patients and twenty healthy controls using quantitative real-time polymerase chain reaction. STATISTICAL ANALYSIS USED:The differential expression was measured by the Mann-Whitney U-test. RESULTS:The relative expression of miR-3662, miR-146a, and miR-1290 in serum exosomes was significantly higher in patients than healthy controls. Moreover, significant differences were found in the lymph node metastasis and clinical stage of breast cancer as the miRNA levels changed, but their expression levels in exosomes and sera were not correlated. In addition, exosomal miR-3662, miR-146a, and miR-1290 were shown to be valuable biomarkers to monitor patient condition in the course of surgery and chemotherapy. CONCLUSIONS:Exosomal miR-3662, miR-146a, and miR-1290 may have promising predictive value and could be utilized as biomarkers for diagnosis and preventative strategy development.

Background: Colorectal cancer (CRC) with lymphatic invasion is one of the critical prognostic factors in lymph node metastasis. Lymphangiogencsis has a significant effect on lymphatic metastasis and tumor progression. However, the significance of intratumoral and peritumoral lymphangiogenesis has been controversial in CRC. The aim of this study is to investigate the different role of introtumoral and peritumoral lymphangiogenesis in CRC progression and prognosis. Methods: Lymphangiogenesis of 120 CRC specimens, as measured by lymphatic vessel density (LVD), was examined by immunostaining for podoplanin, a lymphatic vessel-specific marker. The mean number of lymphatic vessels of three hotspots was measured in intratumoral and peritumoral areas as intratumoral LVD (LVDit) and peritumoral LVD (LVDpt), respectively. The association of LVDit and LVDpt with the clinicopathological findings and prognosis was investigated. Results: Compared to the peritumoral lymphatics, the intratumoral lymphatics were small, collapsed and irregular. The mean LVDpt was higher than the mean LVDit (P<0.001). LVDit was positively correlated with tumor size (P=0.009), tumor histologic grade (P=0.023), and overall survival (P=0.036). LVDpt was correlated with lymph node metastasis (P<0.001), tumor stage (P=0.004), and overall survival (P=0.016). Conclusions: LVDpt plays a prominent role in lymph node metastasis, whereas LVDit is more closely correlated with tumor growth and histopathological differentiation. Both LVDpt and LVDit contribute to CRC progression and prognosis.

PURPOSE:To evaluate a new strategy for profiling proteomic changes in colorectal cancer (CRC). METHODES:We used laser capture microdissection (LCM) to obtain cells from 20 CRC and paired normal mucosal tissues. The differential proteins between the microdissected tumor cells and normal mucosa epithelia were analyzed by acetylation stable isotopic labeling coupled with L linear ion trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ-FT MS). Western blotting was used to assess the differential expression of proteins. We used bioinformatics tools for cluster and ingenuity pathway analysis of the differential proteins. RESULTS:In total, 798 confident proteins were quantified and 137 proteins were differentially expressed by at least twofold, including 67 that were upregulated and 70 that were downregulated in cancer. Two differential proteins, solute carrier family 12 member 2 (SLC12A2) and Ras-related protein Rab-10, were validated by Western blotting, and the results were consistent with acetylation stable isotopic labeling analysis. According to gene ontology analysis, CRC-related differential proteins covered a wide range of subcellular locations and were involved in many biological processes. According to ingenuity pathway analysis of the differential proteins, the most relevant canonical pathway associated with CRC was the 14-3-3-mediated signaling pathway, and seven reliable functional networks including cellular growth and proliferation, amino acid metabolism, inflammatory response, embryonic development, carbohydrate metabolism, cellular assembly and organization, and cell morphology were obtained. CONCLUSIONS:Combination of LCM, acetylation stable isotopic labeling analysis and LTQ-FT MS is effective for profiling proteomic changes in CRC cells.