In vitro models to study natural killer cell dynamics in the tumor microenvironment

Immunotherapy is revolutionizing cancer therapy. The rapid development of new immunotherapeutic strategies to treat solid tumors is posing new challenges for preclinical research, demanding novel in vitro methods to test treatments. Such methods should meet specific requirements, such as enabling the evaluation of immune cell responses like cytotoxicity or cytokine release, and infiltration into the tumor microenvironment using cancer models representative of the original disease. They should allow high-throughput and high-content analysis, to evaluate the efficacy of treatments and understand immune-evasion processes to facilitate development of new therapeutic targets. Ideally, they should be suitable for personalized immunotherapy testing, providing information for patient stratification. Consequently, the application of in vitro 3-dimensional (3D) cell culture models, such as tumor spheroids and organoids, is rapidly expanding in the immunotherapeutic field, coupled with the development of novel imaging-based techniques and -omic analysis. In this paper, we review the recent advances in the development of in vitro 3D platforms applied to natural killer (NK) cell-based cancer immunotherapy studies, highlighting the benefits and limitations of the current methods, and discuss new concepts and future directions of the field

Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level

Ultrasound-Based Scaffold-Free Core-Shell Multicellular Tumor Spheroid Formation

In cancer research and drug screening, multicellular tumor spheroids (MCTSs) are a popular model to bridge the gap between in vitro and in vivo. However, the current techniques to culture mixed co-culture MCTSs do not mimic the structural architecture and cellular spatial distribution in solid tumors. In this study we present an acoustic trapping-based core-shell MCTSs culture method using sequential seeding of the core and shell cells into microwells coated with a protein repellent coating. Scaffold-free core-shell ovarian cancer OVCAR-8 cell line MCTSs were cultured, stained, cleared and confocally imaged on-chip. Image analysis techniques were used to quantify the shell thickness (23.2 ± 1.8 µm) and shell coverage percentage (91.2 ± 2.8%). We also show that the shell thickness was evenly distributed over the MCTS cores with the exception of being slightly thinner close to the microwell bottom. This scaffold-free core-shell MCTSs formation technique and the analysis tools presented herein could be used as an internal migration assay within the MCTS or to form core-shell MCTS co-cultures to study therapy response or the interaction between tumor and stromal cells

Femtosecond linear dichroism of DNA-intercalating chromophores: Solvation and charge separation dynamics of [Ru(phen)(2)dppz](2+) systems

The DNA-intercalating chromophore [Ru(phen)(2)dppz](2+) has unique photophysical properties, the most striking of which is the “light-switch” characteristic when binding to DNA. As a dimer, it acts as a molecular staple for DNA, exhibiting a remarkable double-intercalating topology. Herein, we report femtosecond dynamics of the monomeric and the covalently linked dimeric chromophores, both free in aqueous solution and complexed with DNA. Transient absorption and linear dichroism show the electronic relaxation to the lowest metal-to-ligand charge-transfer (CT) state, and subpicosecond kinetics have been observed for this chromophore for what is, to our knowledge, the first time. We observe two distinct relaxation processes in aqueous solution with time constants of 700 fs and 4 ps. Interestingly, these two time constants are very similar to those observed for the reorientational modes of bulk water. The 700-fs process involves a major dichroism change. We relate these observations to the change in charge distribution and to the time scales involved in solvation of the CT state. Slower processes, with lifetimes of ≈7 and 37 ps, were observed for both monomer and dimer when bound to DNA. Such a difference can be ascribed to the change of the structural and electronic relaxation experienced in the DNA intercalation pocket. Finally, the recombination lifetime of the final metal-to-ligand CT state to the ground state, which is a key in the light-switch process, is found in aqueous solution to be sensitive to structural modification, ranging from 260 ps for [Ru(phen)(2)dppz](2+) and 360 ps for the monomer chromophore derivative to 2.0 ns for the dimer. This large change reflects the direct role of solvation in the light-switch process