Paclitaxel Treatment Alters Integrin Manifestation of Tailor-Made KLK-Expressing Cell Aggregates Ovarian malignancy cell aggregates derived from the tumor fluid (ascites) of individuals with late-stage stage disease range in quantity (from two to more than 20) and size (from 30C200 m, even up to 750 m in diameter) and contain up to 100 cells, suggesting a high patient to patient variability [35,55,56,57]. upon modulation of protease manifestation, integrin engagement and anti-cancer treatment providing a micro-scaled yet high-throughput technique to assess malignant progression and drug-resistance. culture methods mimic more closely the physiological cell-cell and cell-extracellular matrix (ECM) relationships seen [1,2,3,4,5,6,7]. We have shown that biomimetic hydrogels can be used as 3D cell tradition platform to investigate the interplay of ovarian malignancy cells with the ECM [8]. Within these synthetic microenvironments ovarian malignancy cells form multi-cellular spheroids, an integral step leading to metastatic outgrowth and ultimately malignant progression The fabrication of hydrogel microwell arrays was a multistep smooth lithography process as reported previously [36]. Briefly, a topographically organized silicon wafer was fabricated, and then polydimethylsiloxane (PDMS; Dow Corning Corporation, Midland, MI, USA) was solid onto this structure, and finally, hydrogel films were patterned inside a stamping step using the PDMS template. A 4-in . silicon wafer was designed using the layout editor of CleWin (PhoeniX, Enschede, The Netherlands). A pattern was selected consisting of eight squares; each square matched the sizes of a standard 96-well plate, comprising 33 33 = 1,000 microwells, having a diameter of 100 m and a depth of 50 m per microwell. Additionally, fresh silicon wavers were designed to create microwells of varying sizes of 50 50, 100 100, 150 150, 200 200 m. Microwell arrays were created from polyethylene glycol (PEG) hydrogel precursors by cross-linking PF-04457845 two multi-arm PEG macromers (NOF Corporation, Tokyo, POLDS Japan), end-functionalized with either thiol (SH) or vinylsulfone (VS) organizations [36]. The 8arm-PEG-VS was dissolved in 0.3 M triethanolamine (Sigma-Aldrich, Buchs, Switzerland), and the 4arm-PEG-SH was dissolved in bi-distilled water to obtain 100 m thin hydrogel films (5% (w/v)) coated onto 8-well chamber -slides (ibidi GmbH, Munich, Germany) for any microwell size of 50 100 m or onto 48-well cells culture plates (Thermo Fisher Scientific Inc., Lausanne, Switzerland) for any microwell size of 50C200 50C200 m. Optional, hydrogel microwell arrays were coated with laminin (0.1 mg/mL; BD Biosciences, Allschwil, Switzerland) or type I collagen (0.1 mg/mL; Sigma-Aldrich), both revised with an N-hydroxylsuccinimide (NHS)-PEG-maleimide linker (JenKem Technology, Allen, TX, USA) as explained previously [38]. The human being epithelial ovarian carcinoma cell collection OV-MZ-6 was founded from malignant tumor fluid (ascites) [39], and stable transfectants, PF-04457845 with human being KLK4, KLK5, KLK6, and KLK7 full-length cDNA (OV-KLK) derived from ovarian malignancy tissue and an empty vector plasmid (OV-Vector), provided by Viktor Magdolen (Complex University or college of Munich, Munich, Germany), were cultured as reported previously [29]. At a confluency PF-04457845 of 60C80%, cells were harvested with EDTA (0.48 mmol/L; Invitrogen, Lucerne, PF-04457845 Switzerland). For cell aggregate cultures, cells (5 104 cells/mL) were seeded on top PF-04457845 of each square, centrifuged at 800 rpm for 5 min and cultivated over 120 h in 0.25 mL media (Number 1(A)). Cell denseness was adapted accordingly to microwells of varying sizes (100 50 m: 5 104 cells/mL, 50 50 m: 5 104 cells/mL, 100 100 m: 10 104 cells/mL, 150 150 m: 15 104 cells/mL, 200 200 m: 20 104 cells/mL). For exposure to paclitaxel, a microtubule-stabilizing agent that mediates cell cycle arrest and apoptosis [40], cell aggregates were treated with press comprising paclitaxel (0, 1, 10, 100 nM; Invitrogen). Integrin inhibition was accomplished.