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

Abstract

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.

Keywords: NK cells; flow cytometry; live cell imaging; microscopy; tissue sectioning; tumor microenvironment; tumor organoids; tumor spheroids.

Figure 1

Overview of 3D cultures. (A) Illustration of scaffold-based 3D cell culture. Scaffolds resemble the ECM composition and 3D architecture of human tissues, providing support for cells to grow and differentiate in vitro. (B) Illustration of scaffold-free 3D cell culture. Often the substrate (gray) is coated by a non-adhesive, inert chemical (beige), which promotes cell-to-cell interaction, ECM and growth factor release, leading to the formation of self-sustained 3D cell cultures in vitro. (C) The illustration depicts the general features of organoids, such as multicellular composition, defined cellular distribution, cell differentiation and basolateral specialization of functions, such as EMC production in the basal area and fluid release in the apical area of cells.

Figure 2

Overview of NK cell functions.

Figure 3

Methods of spheroid formation applied to NK cell research. (A) Spinner cultures. Tumor cell suspensions are transferred into spinner flasks that provide constant agitation and promote cell aggregation. Spheroid formation is reached within days or a few weeks, depending on the adhesive properties of each cell type. Spheroids generated by spinner cultures have been exposed to allogenic lymphocyte cultures (upper panel to the right) and implanted in allogenic mice (lower panel to the right) to study NK cell infiltration and clearance of tumors (–91). (B) Liquid-overlay method. Cell suspensions are seeded into standard culture plates pre-treated with non-adhesive coatings, such as agarose or poly-HEMA, that indirectly promote spheroid formation by preventing cell adhesion to the plate. Gravity and cell confinement in wells facilitate cell aggregation (spheroids are obtained within a few days). NK cells can be co-cultured with tumor spheroids to study their infiltration and cytotoxic capacity in vitro. (C) Hanging-drop cultures. Drops of cell suspension is dispensed onto a standard culture plate, that is turned upside-down. Surface tension and gravity enforce the formation of a single spheroid per drop within a few days. (D) Illustrations of hanging-drop platforms compatible with long-term and high-throughput spheroid cultures, based on the general features of 3D Biomatrix and InSphero plates. Multiple drops are formed by transferring cell suspension into the inlets of an array plate. Liquid reservoirs prevent drop evaporation. After spheroid formation, each drop is displaced into a single well of an ULA plate by pipetting additional medium on top of the inlets or by centrifugation. From this point, the hanging drop-derived spheroids can be used as described in the liquid-overlay section. (E) Schematic representation of the microfluidic platform developed by Ayuso et al. (92). The microfluidic device consists of a central chamber filled with collagen type I hydrogel, hanging drop-derived spheroids and NK cells. Antibody solutions are perfused through two lateral channels covered with endothelial cells. (F) Schematic representation of the MIVO device developed by Marrella et al. (93, 94). Alginate-derived spheroids are transferred into the top chamber of the MIVO device, which resembles a trans-well insert. NK cell suspensions are perfused through capillaries running under the chambers containing spheroids, allowing NK cells to migrate through the trans-well membrane and infiltrate the tumor spheroid (95). (G) USW-induced spheroid formation in microwell chip. Left panel: cells are seeded into a glass-bottom microwell array chip coated with non-adhesive coating. USW exposure induce the formation of single spheroids in the center of each well. This step is followed by a period of spheroid stabilization in the absence of USW. Once spheroids are formed, NK cells are seeded into the wells under USW exposure to promote NK cell-spheroid interaction. The characterization of NK cell infiltration and killing in tumor spheroids can be performed directly in the chip by imaging, or off-chip by retrieving the samples from the chambers. Right panel: illustration of the multichambered microwell chip with 16 chambers, each containing 36 microwells, giving a total of 576 microwells per chip.

Figure 4

Dynamics of NK cell cytotoxicity against tumor spheroids. IL-15 activated NK cells were incubated with pre-formed renal carcinoma spheroids and imaged for 72 hours. (A) Time-lapse sequence of NK cell killing of renal carcinoma spheroids analyzed by live imaging. TMRM (in magenta) and a caspase-3/7 activity reporter (in green) were used to detect spheroid viability and apoptosis, respectively. NK cells can be seen in the brightfield channel. Scale bar: 100 μm. (B, C) Time-course of spheroid viability (B) and apoptotic index (C) from a single microwell chip chamber (n=36). The data were presented in Carannante et al. (186).

Figure 5

Visualization of spheroid-infiltrating NK cells by light sheet microscopy and confocal microscopy. (A) Light sheet microscopy image showing NK92 cells (yellow) infiltrating a renal carcinoma spheroid (magenta). RFP⁺ A498 renal carcinoma spheroids were incubated with GFP⁺ NK92 cells for 2 hours before undergoing tissue expansion in deionized water and imaged by light sheet microscopy. (B) YFP⁺ A498 renal carcinoma spheroids were incubated with resting NK for 48 hours before being imaged by confocal microscopy. NK cells were detected in both extra-tumoral and intra-tumoral areas. However, the signal was progressively lost at increased depth. Left panel: 3D rendered confocal stack of an A498 renal carcinoma spheroid (magenta) co-cultured with NK cells (yellow). Central panel: optical section of the spheroid showed in the left panel (z = 30 μm). Central panel: optical section of the spheroid showed in the left panel (z = 60 μm).