Introduction The detection of circulating tumor cells (CTC) is prognostic in several cancer types. This trial examines the incidence and prognostic value of CTCs in urothelial cancer (UC). Materials and Methods 44 subjects with UC were assessed for CTCs using CellSearch® Technology (Veridex, LLC, Raritan, NJ), using 7.5 ml of peripheral blood, sorted by magnetic separation (EpCaM positive, and immunofluorescent staining, (Cytokeratin 8, 18, or 19 positive, CD 45 negative, DAPI positive cells) to identify CTCs. Results Five of 30 (17%) subjects with clinically localized and 7 of 14 (50%) patients with metastatic UC had at least 1 detectable CTC, with a range of 1–177 CTCs. Six subjects had 5 or more CTCs. FISH analysis was performed in 20 samples from 18 unique subjects, using the UroVysion probe set. Copy number gains consistent with neoplasm were observed in cases with measurable CTCs, but not in any of the CTC-negative samples tested. With a median follow-up of 337 days, all 7 metastatic subjects with detectable CTCs had died, versus 3 of 7 (43%) metastatic subjects without detectable CTCs. Conclusions CTCs were observed in 50% of the metastatic UC patients tested. FISH analysis confirmed the aneusomic chromosomal content in the CTCs. The findings suggest measurable CTCs may be prognostic for shortened survival in metastatic UC patients, although the optimal threshold for a “positive” finding is unknown. CTCs were also detected in a subset of patients with clinically-localized disease, identifying a potential high-risk, pre-operative group for future study.

The high water content of hydrogels allows these materials to closely mimic the native biological extracellular conditions, but it also makes difficult the histological preparation of hydrogel-based bioengineered tissue. Paraffin-embedding techniques require dehydration of hydrogels, resulting in substantial collapse and deformation, whereas cryosectioning is hampered by the formation of ice crystals within the hydrogel material. Here, we sought to develop a method to obtain good-quality cryosections for the microscopic evaluation of hydrogelbased tissue-engineered constructs, using polyethylene glycol (PEG) as a test hydrogel. Conventional sucrose solutions, which dehydrate cells while leaving extracellular water in place, produce a hydrogel block that is brittle and difficult to section. We therefore replaced sucrose with multiple protein-based and nonprotein-based solutions as cryoprotectants. Our analysis demonstrated that overnight incubation in bovine serum albumin (BSA), fetal bovine serum (FBS), polyvinyl alcohol (PVA), optimum cutting temperature (OCT Ò ) compound, and Fisher HistoPrep frozen tissue-embedding media work well to improve the cryosectioning of hydrogels. The protein-based solutions give background staining with routine hematoxylin and eosin, but the use of nonprotein-based solutions PVA and OCT reduces this background by 50%. These methods preserve the tissue architecture and cellular details with both in vitro PEG constructs and in constructs that have been implanted in vivo. This simple hydrogel cryosectioning technique improves the methodology for creation of good-quality histological sections from hydrogels in multiple applications.

Medtronic’s INFUSE Bone Graft provides surgeons with a potent tool for stimulating bone formation. Current delivery vehicles that rely on Absorbable Collagen Sponges (ACS) require excessive quantities of the active ingredient in INFUSE, recombinant human Bone Morphogenic Protein-2 (rhBMP2), to achieve physiologically relevant concentrations of the growth factor, driving up the cost of the product and increasing the likelihood of undesirable side effects in neighboring tissues. We demonstrate that a simple light-mediated, thiol-ene chemistry can be used to create an effective polymer delivery vehicle for rhBMP2, eliminating the use of xenografic materials and reducing the dose of rhBMP2 required to achieve therapeutic effects. Comprised entirely of synthetic components, this system entraps rhBMP2 within a biocompatible hydrogel scaffold that is degraded by naturally occurring remodeling enzymes, clearing the way for new tissue formation. When tested side-by-side with ACS in a critical-sized bone defect model in rats, this polymeric delivery system significantly increased bone formation over ACS controls.

1α, (1,25(OH) 2 D 3 ) has shown strong promise as an anti-proliferative agent in several malignancies, yet its therapeutic use has been limited by its toxicity leading to search for analogs with anti-tumor property and low toxicity. In this study we evaluated the in vitro and in vivo properties of 1,25-dihydroxyvitamin D 3 -3-bromoacetate (1,25(OH) 2 D 3 -3-BE), an alkylating derivative of 1,25(OH) 2 D 3 as a potential therapeutic agent for renal cancer. Doseresponse of 1,25(OH) 2 D 3 -3-BE in two kidney cancer cell-lines was evaluated for its antiproliferative and apoptotic properties, and mechanisms were evaluated by Western Blot and FACS analyses. Therapeutic potential of 1,25(OH) 2 D 3 -3-BE was assessed by determining its stability in human serum, and evaluating its efficacy in a mouse xenograft model of human renal tumor. We observed that 1,25(OH) 2 D 3 -3-BE is significantly more potent than an equivalent concentration of 1,25(OH) 2 D 3 in inhibiting growth of A498 and Caki 1 human kidney cancer cells. 1,25(OH) 2 D 3 -3-BE-mediated growth inhibition was promoted through inhibition of cell cycle progression by down-regulating cyclin A and induction of apoptosis by stimulating caspase activity. Moreover, 1,25(OH) 2 D 3 -3-BE strongly inhibited Akt phosphorylation and phosphorylation of its downstream target, caspase 9. 1,25(OH) 2 D 3 -3-BE appeared to be stable in human serum. In xenograft mouse model of human renal tumor, 1,25(OH) 2 D 3 -3-BE was more potent at reducing tumor size compared to 1,25(OH) 2 D 3 which was accompanied by an increase in apopotosis and reduction of cyclin A staining in the tumors. These results suggest a translational potential of this compound as a therapeutic agent in renal cell carcinoma. Data from this study and extensive studies of vitamin D for the prevention of many malignancies support the potential of 1,25(OH) 2 D 3 -3-BE for preventing renal cancer and the development of relevant in-vivo prevention models for assessing this potential, which do not exist at present.