Sulfobetaine polymers for effective permeability into multicellular tumor spheroids (MCTSs)

Multicellular tumor spheroids (MCTSs) are attractive for drug screening before animal tests because they emulate an in vivo microenvironment. The permeability of the MCTSs and tumor tissues by the candidate...

Confocal Microscopy in Conjunction With Haemocytometry to Evaluate the Imperative Physical Characteristics of Multicellular Tumor Spheroids

Background: Tumor tissues resist penetration of therapeutic molecules. Multicellular tumor spheroids (MCTSs) were used as an in vitro tumor model. The aim of this study was to determine the growth of MCTSs with the age of spheroids, which could be applied and compared with in vivo drug uptake and penetration. Method: Spheroids were generated by liquid overlay techniques, and their diameter was measured by confocal microscopy for up to two weeks. The trypan blue exclusion method was used to count dead and live cells separately via a hemocytometer. Results: The pentaphysical characteristics of spheroids, including diameter, cell number, volume per cell, viability status, and estimated shell of viable and core of dead cells, were determined. The growth of spheroids was linear over the first week but declined in the 2nd week, which may be due to an overconcentration of dead cells and degraded products inside the spheroids, hence lowering the ratio of live cells in spheroids. Compaction of spheroids occurs from day 3 to day 7, with the mature spheroids having a low amount of extracellular space compared to intracellular volume. Conclusion: Age-oriented growth of MCTSs provides a rationale to predict less rapid penetration as spheroids get older and could be correlated with in vivo tumors to predict pharmaceutical and therapeutic intervention.

Paclitaxel - Combined Curcumin - Loaded PLA-TPGS - Based Nanoparticles Induce Apoptosis and Inhibit the Growth of Breast Cancer Cells and Multicellular Tumor Spheroids

Paclitaxel and curcumin have been reported as anti-cancer drugs. Here we presented a novel combination of paclitaxel and curcumin-loaded PLA-TPGS (PTX-Cur/PLA-TPGS) nanoparticles prepared by a modified solvent extraction/evaporation technique. These nanoparticles were well distributed and stable in water. This combination of paclitaxel and curcumin gave a higher efficiency of both drugs in cytotoxicity; induced apoptosis; and effect on cell cycles of KPL4 cell line in compare with the use of paclitaxel or curcumin alone or even a normal mixture of these two drugs. Furthermore, PTX-Cur/PLA-TPGS nanoparticles exhibited a powerful ability in preventing MCF7 spheroids growth. Interestingly, curcumin also functioned as both a drug and a label. Base on the autofluorescence of curcumin, the absorption of PTX-Cur/PLA-TPGS nanoparticles into MCF7 spheroids could be followed and calculated. These results suggested that the nanoparticle-drug combination may provide a promising multifunctional delivery system for anti-cancer drugs.

Single cell organization and cell cycle characterization of DNA stained multicellular tumor spheroids

Multicellular tumor spheroids (MCTSs) can serve as in vitro models for solid tumors and have become widely used in basic cancer research and drug screening applications. The major challenges when studying MCTSs by optical microscopy are imaging and analysis due to light scattering within the 3-dimensional structure. Herein, we used an ultrasound-based MCTS culture platform, where A498 renal carcinoma MCTSs were cultured, DAPI stained, optically cleared and imaged, to connect nuclear segmentation to biological information at the single cell level. We show that DNA-content analysis can be used to classify the cell cycle state as a function of position within the MCTSs. We also used nuclear volumetric characterization to show that cells were more densely organized and perpendicularly aligned to the MCTS radius in MCTSs cultured for 96 h compared to 24 h. The method presented herein can in principle be used with any stochiometric DNA staining protocol and nuclear segmentation strategy. Since it is based on a single counter stain a large part of the fluorescence spectrum is free for other probes, allowing measurements that correlate cell cycle state and nuclear organization with e.g., protein expression or drug distribution within MCTSs.