Although we have shown that matrix stiffness and cell density both affect uptake, we have yet to determine the reason for this observation. We hypothesized that the actin cytoskeleton might be responsible for the differential uptake between tissue culture plastic and polyacrylamide gels. Actin stress fibers are BIBW2992 customer reviews affected by substrate stiffness. Actin stress fibers are also regulated by cell-cell contact. Cells on soft substrates do not typically exhibit stress fibers, however, when cells on soft substrates are in cell-cell contact, stress fibers reappear. The actin cytoskeleton is critical in caveolae-mediated endocytosis, and is necessary for the closure and initial uptake of caveolar vesicles. Other investigators have shown that increased density of organized stress fibers impedes clathrin-mediated endocytosis. Furthermore, stress fibers are not prominent in cells in vivo. However, our data suggests that YARA uptake is independent of the state of actin polymerization since neither LPA nor cytochalasin D affected YARA uptake. Microtubules are also important in endosome trafficking. In this study microtubules were shown to be important in YARA uptake or trafficking since nocodazole treatment significantly enhanced YARA uptake. Microtubules are confirmed to affect endosome trafficking including recycling to the plasma membrane; thus, it is likely that disruption of microtubules does not increase the rate of endocytosis of YARA, but delays recycling of YARA to the membrane. The end result of microtubule disruption and delayed recycling is accumulation of YARA within the cells. Uptake appears to be independent of actin polymerization, while accumulation of YARA within the cell is dependent upon microtubule polymerization. Understanding how substrate stiffness affects intracellular uptake has broad implications in the design of drug screening platforms, both in screening potential drugs for evidence of efficacy and for understanding how uptake might differ in cells within a 
diseased state. Several different disease states are characterized by changes in tissue rigidity due to inflammation, fibrosis, calcification, or other biochemical changes within the tissue. Understanding OTX015 whether a drug is influenced by tissue rigidity can help physicians choose therapies that may be more effective for the patient, depending on the stage of the disease. Pancreatic cancer is the fourth leading cause of cancer death in the Western world. Pancreatic cancer is a disease of insidious progression and high lethality, with a 5-year survival rate of just 6%. In the United States alone, an estimated 43,920 patients are expected to be diagnosed with the disease in 2012, and 37,390 patients are expected to die from it. The vast majority of these cases are pancreatic ductal adenocarcinomas, which develop in the ducts of the pancreas. These highly invasive tumors consist of an abundant desmoplastic stroma, in which are embedded malignant cancer cells expressing markers of pancreatic ductal cells. For patients with pancreatic ductal adenocarcinoma, the only curative option is surgery. The standard procedure is a pancreaticoduodenectomy, a surgical operation that removes the head of the pancreas but spares the remaining tissue. Unfortunately, most pancreatic cancer patients present with unresectable metastatic or locally advanced disease. In fact, only 20% of patients have resectable tumors at the time of diagnosis. But even for those patients who undergo surgery, the overall 5year survival rate is of just 20%, as most of these patients will relapse within a year of their surgery. Hence, there is a critical need for novel drugs that can more efficaciously target these tumor cells and/or reduce the incidence of recurrence.