Effects of HLEC on the secreted proteins of epithelial ovarian cancer cells prone to metastasize to lymph nodes

Objective： To study explores the effect of HLEC on the secreted proteins of epithelial ovarian cancer （EOC） cells （SKOV3-PM4） with directional highly lymphatic metastasis. Methods： Supernatants of four groups of cultured cells, namely, SKOV3 （A）, SKOV3＋HLEC （B）, SKOV3-PM4 （C）, SKOV3-PM4＋HLEC （D）, were collected, and their proteins were detected by antibody arrays and iTRAOcZD-LC-MALDI- TOF/TOF/MS. Significantly differential proteins were further analyzed via bioinformatics and validated in human serums and cell media via ELISA. Results： Results of antibody arrays and mass spectrometry demonstrated that GRN and VEGFA were upregulated in group C （compared with group A）, whereas IGFBP7 and SPARC were downregulated in group D （compared with group C）. Comprehensive bioinformatics analysis results showed that IGFBP7 and VEGFA were closely linked to each other. Further validation with serums showed statistical significance in VEGFA and IGFBP7 levels among groups of patients with ovarian cancers, benign tumors, and control groups. Two proteins were upegulated in the first group. VEGFA in the control group was downregulated. For IGFBP, upregulation in the control group and down-regulation in the first group were also observed. Conclusion： The HLEC microenvironment is closely associated with directional metastasis to lymph nodes and with differential proteins including cell stromal proteins and adhesion factors. The upregulation of VEGFA and GRN and the downregulation of SPARC and IGFBP7 are closely associated with directional metastasis to lymph nodes in EOC cells.

Preparation of BCEC-Astrocyte Co-culturing Membrane Plate Insert

To prepare a hand-made micropore membrane culture plate insert forco-culture. Methods The plate insert was made using plastic centrifuge tube and micropore membrane.After seeding brain capillary endothelial cells (BCECs) on it (under the effect ofastrocyte-conditioned medium), the plate insert was assessed by analysis of trans-endothelialelectrical resistance (TEER). Results The plate insert has a stability of at least 15 d underculture condition. TEER increased significantly under co-culture condition from (66.1 +- 13.3)Ωcm^2 to (182.2 +- 6.7) Ωcm^2. Conclusion This micropore membrane culture plate insert can beeasily made, on which BCEC culture can be successfully performed. Moreover, it is adjustable andrecyclable. It follows that the plate insert is a useful tool for co-culture and the relatedresearch fields.