In vitro characterization of tumor cell biology or of potential anticancer drugs is usually performed using tumor cell lines cultured as a monolayer. However, it has been previously shown that three-dimensional (3D) organization of the tumor cells is important to provide insights on tumor biology and transport of therapeutics. Several methods to create 3D tumors in vitro have been proposed, with hanging drop technique being the most simple and, thus, most frequently used. However, in many cell lines this method has failed to form the desired 3D tumor structures. The aim of this study was to design and test an easy-to-use and highly reproducible modification of the hanging drop method for tumor sphere formation by adding methylcellulose polymer. Most pancreatic cancer cells do not form cohesive and manageable spheres when the original hanging drop method is used, thus we investigated these cell lines for our modified hanging drop method. The spheroids produced by this improved technique were analyzed by histology, light microscopy, immunohistochemistry, and scanning electron microscopy. Results show that using the proposed simple method; we were able to produce uniform spheroids for all five of the tested human pancreatic cancer cell lines; Panc-1, BxPC-3, Capan-1, MiaPaCa-2, and AsPC-1. We believe that this method can be used as a reliable and reproducible technique to make 3D cancer spheroids for use in tumor biology research and evaluation of therapeutic responses, and for the development of bio-artificial tissues.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic/stromal reaction, which contributes to the poor clinical outcome of this disease. Therefore, greater understanding of the stroma development and tumor-stroma interactions is highly required. Pancreatic stellate cells (PSC) are myofibroblast-like cells that located in exocrine areas of the pancreas, which as a result of inflammation produced by PDAC migrate and accumulate in the tumor mass, secreting extracellular matrix components and producing the dense PDAC stroma. Currently, only a few orthotopic or ectopic animal tumor models, where PDAC cells are injected into the pancreas or subcutaneous tissue layer, or genetically engineered animals offer tumors that encompass some stromal component. Herein, we report generation of a simple 3D PDAC in vitro micro-tumor model without an addition of external extracellular matrix, which encompasses a rich, dense and active stromal compartment. We have achieved this in vitro model by incorporating PSCs into 3D PDAC cell culture using a modified hanging drop method. It is now known that PSCs are the principal source of fibrosis in the stroma and interact closely with cancer cells to create a tumor facilitatory environment that stimulates local and distant tumor growth. The 3D micro-stroma models are highly reproducible with excellent uniformity, which can be used for PDAC-stroma interaction analysis and high throughput automated drug-screening assays. Additionally, the increased expression of collagenous regions means that molecular based perfusion and cytostaticity of gemcitabine is decreased in our Pancreatic adenocarcinoma stroma spheroids (PDAC-SS) model when compared to spheroids grown without PSCs. We believe this model will allow an improved knowledge of PDAC biology and has the potential to provide an insight into pathways that may be therapeutically targeted to inhibit PSC activation, thereby inhibiting the development of fibrosis in PDAC and interrupting PSCPDAC cell interactions so as to inhibit cancer progression.

The evaluation of heat production from gold nanoparticles (AuNPs) irradiated with radiofrequency (RF) energy has been problematic due to Joule heating of their background ionic buffer suspensions. Insights into the physical heating mechanism of nanomaterials under RF excitations must be obtained if they are to have applications in fields such as nanoparticle-targeted hyperthermia for cancer therapy. By developing a purification protocol which allows for highly-stable and concentrated solutions of citrate-capped AuNPs to be suspended in high-resistivity water, we show herein, for the first time, that heat production is only evident for AuNPs of diameters ≤ 10 nm, indicating a unique size-dependent heating behavior not previously observed. Heat production has also shown to be linearly dependent on both AuNP concentration and total surface area, and severely attenuated upon AuNP aggregation. These relationships have been further validated using permittivity analysis across a frequency range of 10 MHz to 3 GHz, as well as static conductivity measurements. Theoretical evaluations suggest that the heating mechanism can be modeled by the electrophoretic oscillation of charged AuNPs across finite length scales in response to a time-varying electric field. It is anticipated these results will assist future development of nanoparticle-assisted heat production by RF fields for applications such as targeted cancer hyperthermia.