Visualizing Extracellular Vesicles and Their Function in 3D Tumor Microenvironment Models.

Extracellular vesicles (EVs) are cell-derived nanostructures that mediate intercellular communication by delivering complex signals in normal tissues and cancer. The cellular coordination required for tumor development and maintenance is mediated, in part, through EV transport of molecular cargo to resident and distant cells. Most studies on EV-mediated signaling have been performed in two-dimensional (2D) monolayer cell cultures, largely because of their simplicity and high-throughput screening capacity. Three-dimensional (3D) cell cultures can be used to study cell-to-cell and cell-to-matrix interactions, enabling the study of EV-mediated cellular communication. 3D cultures may best model the role of EVs in formation of the tumor microenvironment (TME) and cancer cell-stromal interactions that sustain tumor growth. In this review, we discuss EV biology in 3D culture correlates of the TME. This includes EV communication between cell types of the TME, differences in EV biogenesis and signaling associated with differing scaffold choices and in scaffold-free 3D cultures and cultivation of the premetastatic niche. An understanding of EV biogenesis and signaling within a 3D TME will improve culture correlates of oncogenesis, enable molecular control of the TME and aid development of drug delivery tools based on EV-mediated signaling.

Phosphate regulates chondrogenesis in a biphasic and maturation-dependent manner.

Inorganic phosphate (Pi) has been recognized as an important signaling molecule that modulates chondrocyte maturation and cartilage mineralization. However, conclusive experimental evidence for its involvement in early chondrogenesis is still lacking. Here, using high-density monolayer (2D) and pellet (3D) culture models of chondrogenic ATDC5 cells, we demonstrate that the cell response to Pi does not correlate with the Pi concentration in the culture medium but is better predicted by the availability of Pi on a per cell basis (Pi abundance). Both culture models were treated with ITS+, 10mM ß-glycerophosphate (ßGP), or ITS+/10mM ßGP, which resulted in three levels of Pi abundance in cultures: basal (Pi/DNA <10ng/µg), moderate (Pi/DNA=25.3 - 32.3ng/µg), and high abundance (Pi/DNA >60ng/µg). In chondrogenic medium alone, the abundance levels were at the basal level in 2D culture and moderate in 3D cultures. The addition of 10mM ßGP resulted in moderate abundance in 2D and high abundance in 3D cultures. Moderate Pi abundance enhanced early chondrogenesis and production of aggrecan and type II collagen whereas high Pi abundance inhibited chondrogenic differentiation and induced rapid mineralization. Inhibition of sodium phosphate transporters reduced phosphate-induced expression of chondrogenic markers. When 3D ITS+/ßGP cultures were treated with levamisole to reduce ALP activity, Pi abundance was decreased to moderate levels, which resulted in significant upregulation of chondrogenic markers, similar to the response in 2D cultures. Delay of phosphate delivery until after early chondrogenesis occurs (7 days) no longer enhanced chondrogenesis, but instead accelerated hypertrophy and mineralization. Together, our data highlights the dependence of chondroprogenitor cell response to Pi on its availability to individual cells and the chondrogenic maturation stage of these cells and suggest that appropriate temporal delivery of phosphate to ATDC5 cells in 3D cultures represents a rapid model for mechanistic studies into the effects of exogenous cues on chondrogenic differentiation, chondrocyte maturation, and matrix mineralization.

Magnetic Particle Imaging Reveals that Iron-Labeled Extracellular Vesicles Accumulate in Brains of Mice with Metastases.

The incidence of breast cancer remains high worldwide and is associated with a significant risk of metastasis to the brain that can be fatal; this is due, in part, to the inability of therapeutics to cross the blood-brain barrier (BBB). Extracellular vesicles (EVs) have been found to cross the BBB and further have been used to deliver drugs to tumors. EVs from different cell types appear to have different patterns of accumulation and retention as well as the efficiency of bioactive cargo delivery to recipient cells in the body. Engineering EVs as delivery tools to treat brain metastases, therefore, will require an understanding of the timing of EV accumulation and their localization relative to metastatic sites. Magnetic particle imaging (MPI) is a sensitive and quantitative imaging method that directly detects superparamagnetic iron. Here, we demonstrate MPI as a novel tool to characterize EV biodistribution in metastatic disease after labeling EVs with superparamagnetic iron oxide (SPIO) nanoparticles. Iron-labeled EVs (FeEVs) were collected from iron-labeled parental primary 4T1 tumor cells and brain-seeking 4T1BR5 cells, followed by injection into the mice with orthotopic tumors or brain metastases. MPI quantification revealed that FeEVs were retained for longer in orthotopic mammary carcinomas compared to SPIOs. MPI signal due to iron could only be detected in brains of mice bearing brain metastases after injection of FeEVs, but not SPIOs, or FeEVs when mice did not have brain metastases. These findings indicate the potential use of EVs as a therapeutic delivery tool in primary and metastatic tumors.