Characterization of autoimmune inflammation induced prostate stem cell expansion.

BACKGROUND: The presence of inflammation in prostate cancer (PCa) and benign prostate hyperplasia (BPH) has been well described but the cellular mechanisms by which inflammation modulates the prostate are currently unclear. Prostate stem cells (PSC) not only maintain prostate homeostasis but also are considered to be the cell of origin of PCa and an important contributor to BPH. However, the impact of inflammation on PSC is not well understood. Therefore, we initiated studies to evaluate the effect of inflammation on PSC. METHOD: Ovalbumin specific CD8(+) T cells were intravenously delivered to intact and castrated prostate ovalbumin expressing transgenic-3 (POET-3) mice to induce inflammation. Lin (CD45/CD31)(-) Sca1(+) CD49f(+) cells (LSC) and progenitor cells within LSC were determined by flow cytometry. Sorted LSC were subjected to a prostate sphere forming assay to evaluate PSC clonal propagation, proliferation, immediate differentiation, and self-renewal ability. Density of individual spheres was measured by a cantilever-based resonator weighing system. Morphology and characterization of prostate spheres was determined by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). Finally, immediate PSC differentiation in sphere formation was determined by immunofluorescence for epithelial cytokeratin markers cytokeratin (CK) 5 and CK8. RESULT: Data presented here demonstrate a significant expansion of the proliferative (BrdU(+) ) LSC population, including CK5(+) , p63(+) , CK18(+) cells, as well as intermediate cells (CK5(+) /CK8(+) ) in inflamed prostates. Histological images reveal that PSC from inflamed prostates produce significantly larger spheres, indicating that the enhanced proliferation observed in LSC is sustained in vitro in the absence of inflammatory mediators. In addition, cultures from inflamed PSC yielded increased number of tubule-like spheres. These tube-like spheres grown from PSCs isolated from inflamed mice exhibited stratification of a CK8(+) luminal-like layer and a CK5(+) basal-like layer. Notably, the numbers of spheres formed by inflamed and non-inflamed PSC were equal, suggesting that even though proliferation is enhanced by inflammation, the homeostatic level of PSC is maintained. CONCLUSION: Induction of inflammation promotes PSC expansion and immediate differentiation through highly proliferative progenitor cells while the homeostasis of PSC is maintained.

Concurrent Detection of Cellular and Molecular Cancer Markers Using an Immunomagnetic Flow System.

We report a detection system for simultaneous measurement of cellular and molecular markers of cancer. Magnetic beads conjugated with antibodies against a specific antigen are used to capture both free molecules and whole cells overexpressing the antigen. The target-bound beads then flow through a microfluidic chamber where they are drawn to a glass surface by an external magnetic field. The cells and molecules captured on the surface are quantitatively analyzed using fluorescent microscopy. The system was characterized by detecting free folate receptor (FR) and an FR+ cancer cell line (KB) in culture media. The system detected as low as 10 pM of FR and captured 87% of the spiked KB cells at a volumetric throughput of 3 mL/min. We further demonstrated the detection of 100 KB cells and 200 pM FR spiked into healthy human blood to simulate detection of rare cells and protein biomarkers present in a cancer patient's blood sample. The FR concentration was measured to be 244 pM (including the intrinsic FR present in the blood), and the total number of KB cells in the sample was estimated to be 98. The potential of this approach in clinical diagnostics was also demonstrated by detecting both FR+ cells and free FR in an ascites sample obtained from an ovarian cancer patient. Because of the system's capability to detect multiple targets at the same time, its high throughput, and its overall simplicity, we expect it to be highly useful in a wide range of research settings.

Circulating tumor cell detection using a parallel flow micro-aperture chip system.

We report on-chip isolation and detection of circulating tumor cells (CTCs) from blood samples using a system that integrates a microchip with immunomagnetics, high-throughput fluidics and size-based filtration. CTCs in a sample are targeted via their surface antigens using magnetic beads functionalized with antibodies. The mixture is then run through a fluidic chamber that contains a micro-fabricated chip with arrays of 8 µm diameter apertures. The fluid runs parallel to the microchip while a magnetic field is generated underneath to draw the beads and cells bound to them toward the chip surface for detection of CTCs that are larger than the apertures and clear out free beads and other smaller particles bound to them. The parallel flow configuration allows high volumetric flow rates, which reduces nonspecific binding to the chip surface and enables multiple circulations of the sample fluid through the system in a short period of time. In this study we first present models of the magnetic and fluidic forces in the system using a finite element method. We then verify the simulation results experimentally to determine an optimal flow rate. Next, we characterize the system by detecting cancer cell lines spiked into healthy human blood and show that on average 89% of the spiked MCF-7 breast cancer cells were detected. We finally demonstrate detection of CTCs in 49 out of 50 blood samples obtained from non-small cell lung cancer (NSCLC) patients and pancreatic cancer (PANC) patients. The number of CTCs detected ranges from 2 to 122 per 8 mL s of blood. We also demonstrate a statistically significant difference between the CTC counts of NSCLC patients who have received therapy and those who have not.

On-demand weighing of single dry biological particles over a 5-order-of-magnitude dynamic range.

We report a simple and highly versatile system to select and weigh individual dry biological particles. The system is composed of a microtweezer to pick and place individual particles and a cantilever-based resonator to weigh them. The system can weigh entities that vary from a red blood cell (~10(-11) g) to the eye-brain complex of an insect (~10(-6) g), covering a 5-order-of-magnitude mass range. Due to its versatility and ease of use, this weighing method is highly compatible with established laboratory practices. The system can provide complementary mass information for a wide variety of individual particles imaged using scanning electron microscopy and determine comparative weights of individual biological entities that are attached to microparticles as well as weigh fractions of individual biological entities that have been subjected to focused ion beam milling.

A humidity-sensitive hydrogel-Bacillus spore composite for micropatterning of biomolecular gradients.

A composite material consisting of Bacillus subtilis spores suspended in a humidity sensitive hydrogel can be used to pattern biomolecules in different concentrations directly onto glass surfaces using a mechanical micromanipulator. By altering the relative humidity surrounding the composite gel during deposition, surface concentration of patterned biomolecules can be controlled and varied to create user-defined, biomolecular surface concentrations.