Rapid Clearing for High Resolution 3D Imaging of Ex Vivo Pancreatic Cancer Spheroids.

The currently accepted imaging methods have been a central hurdle to imaging the finer details of tumor behavior in three-dimensional (3D) ex vivo multicellular culture models. In our search for an improved way of imaging tumor behavior in its physiological-like niche, we developed a simple, efficient, and straightforward procedure using standard reagents and imaging equipment that significantly enhanced 3D imaging up to a ~200-micron depth. We tested its efficacy on pancreatic spheroids, prototypes of high-density tissues that are difficult to image. We found we could both save time with this method and extract information about pancreatic tumor spheroids that previously was difficult to obtain. We were able to discern clear differences in the organization of pancreatic tumor spheroids generated from different origins, suggesting cell-specific, inherent, bottom-up organization with a correlation to the level of malignancy. We also examined the dynamic changes in the spheroids at predetermined time points, providing important information related to tissue morphogenesis and its metabolic state. Lastly, this process enabled us to assess a drug vehicle's potential to penetrate dense tumor tissue by improving our view of the inert particles' diffusion in the 3D spheroid. This clearing method, a simple procedure, can open the door to more accurate imaging and reveal more about cancer behavior.

Analyzing force measurements of multi-cellular clusters comprising indeterminate geometries.

Multi-cellular biomimetic models often comprise heterogenic geometries. Therefore, quantification of their mechanical properties-which is crucial for various biomedical applications-is a challenge. Due to its simplicity, linear fitting is traditionally used in analyzing force-displacement data of parallel compression measurements of multi-cellular clusters, such as tumor spheroids. However, the linear assumption would be artificial when the contact geometry is not planar. We propose here the integrated elasticity (IE) regression, which is based on extrapolation of established elastic theories for well-defined geometries, and is free, extremely simple to apply, and optimal for analyzing coarsely concave multi-cellular clusters. We studied here the quality of the data analysis in force measurements of tumor spheroids comprising different types of melanoma cells, using either the IE or the traditional linear regressions. The IE regression maintained excellent precision also when the contact geometry deviated from planarity (as shown by our image analysis). While the quality of the linear fittings was relatively satisfying, these predicted smaller elastic moduli as compared to the IE regression. This was in accordance with previous studies, in which the elastic moduli predicted by linear fits were smaller compared to those obtained by well-established methods. This suggests that linear regressions underestimate the elastic constants of bio-samples even in cases where the fitting precision seems satisfying, and highlights the need in alternative methods as the IE scheme. For comparison between different types of spheroids we further recommend to increase the soundness by regarding relative moduli, using universal reference samples.

Newly synthesized methionine aminopeptidase 2 inhibitor hinders tumor growth.

Methionine aminopeptidase 2 (MetAp2) inhibition has been recognized as a promising approach for suppressing angiogenesis and cancer progression. Small molecule fumagillol derivatives with adamantane side groups were synthesized and evaluated for MetAp2 inhibition activity, and a lead molecule with superior abilities to inhibit the enzymatic activity of MetAp2 was identified. The compound, referred to as AD-3281, effectively suppressed proliferation of cancer and endothelial cells and impaired tube formation of endothelial cells in vitro. When administered systemically, AD-3281 was well tolerated and led to a significant suppression of human melanoma and mammary tumor xenografts grown in mice. The activity in vivo was associated with reduced angiogenesis and tumor proliferation as detected histologically. In order to develop a formulation that can solubilize AD-3281 with a minimal content of organic solvents, biodegradable nanoparticles comprised of poly-lactic-co-glycolic acid (PLGA) were fabricated and characterized. Compared with the free compound, AD-3281-loaded nanoparticles showed an advantageous cellular availability and uptake, leading to higher activity in cells and better transport in three-dimensional (3D) cultures. Taken together, we introduce a novel MetAp2 inhibitor with high anti-cancer activity and a stable nano-formulation with a high potential for future clinical translation.

Surface physical cues mediate the uptake of foreign particles by cancer cells.

Cancer phenotypes are often associated with changes in the mechanical states of cells and their microenvironments. Numerous studies have established correlations between cancer cell malignancy and cell deformability at the single-cell level. The mechanical deformation of cells is required for the internalization of large colloidal particles. Compared to normal epithelial cells, cancer cells show higher capacities to distort their shapes during the engulfment of external particles, thus performing phagocytic-like processes more efficiently. This link between cell deformability and particle uptake suggests that the cell's adherence state may affect this particle uptake, as cells become stiffer when plated on a more rigid substrate and vice versa. Based on this, we hypothesized that cancer cells of the same origin, which are subjected to external mechanical cues through attachment to surfaces with varying rigidities, may express different capacities to uptake foreign particles. The effects of substrate rigidity on cancer cell uptake of inert particles (0.8 and 2.4 µm) were examined using surfaces with physiologically relevant rigidities (from 0.5 to 64 kPa). Our data demonstrate a wave-like ("meandering") dependence of cell uptake on the rigidity of the culture substrate explained by a superposition of opposing physical and biological effects. The uptake patterns were inversely correlated with the expression of phosphorylated paxillin, indicating that the initial passive particle absorbance is the primary limiting step toward complete uptake. Overall, our findings may provide a foundation for mechanical rationalization of particle uptake design.

Enhanced Biomechanically Mediated "Phagocytosis" in Detached Tumor Cells.

Uptake of particles by cells involves various natural mechanisms that are essential for their biological functions. The same mechanisms are used in the engulfment of synthetic colloidal drug carriers, while the extent of the uptake affects the biological performance and selectivity. Thus far, little is known regarding the effect of external biomechanical stimuli on the capacity of the cells to uptake nano and micro carriers. This is relevant for anchorage-dependent cells that have detached from surfaces or for cells that travel in the body such as tumor cells, immune cells and various circulating stem cells. In this study, we hypothesize that cellular deformability is a crucial physical effector for the successful execution of the phagocytosis-like uptake in cancer cells. To test this assumption, we develop a well-controlled tunable method to compare the uptake of inert particles by cancer cells in adherent and non-adherent conditions. We introduce a self-designed 3D-printed apparatus, which enables constant stirring while facilitating a floating environment for cell incubation. We reveal a mechanically mediated phagocytosis-like behavior in various cancer cells, that was dramatically enhance in the detached cell state. Our findings emphasize the importance of including proper biomechanical cues to reliably mimic certain physiological scenarios. Beyond that, we offer a cost-effective accessible research tool to study mixed cultures for both adherent and non-adherent cells.

Low dose amiodarone reduces tumor growth and angiogenesis.

Amiodarone is an anti-arrhythmic drug that was approved by the US Food and Drug Administration (FDA) in 1985. Pre-clinical studies suggest that Amiodarone induces cytotoxicity in several types of cancer cells, thus making it a potential candidate for use as an anti-cancer treatment. However, it is also known to cause a variety of severe side effects. We hypothesized that in addition to the cytotoxic effects observed in cancer cells Amiodarone also has an indirect effect on angiogensis, a key factor in the tumor microenvironment. In this study, we examined Amiodarone's effects on a murine tumor model comprised of U-87 MG glioblastoma multiforme (GBM) cells, known to form highly vascularized tumors. We performed several in vitro assays using tumor and endothelial cells, along with in vivo assays utilizing three murine models. Low dose Amiodarone markedly reduced the size of GBM xenograft tumors and displayed a strong anti-angiogenic effect, suggesting dual cancer fighting properties. Our findings lay the ground for further research of Amiodarone as a possible clinical agent that, used in safe doses, maintains its dual properties while averting the drug's harmful side effects.