Background

Three-dimensional (3D) tumor spheroids are spherical self-assembled tumor cell aggregates, which resemble micrometastases and replicate many features of solid tumors. As in the non-proliferating regions of avascular solid tumors, tumor cells within the inner regions of spheroids usually demonstrate perturbed gene and protein expression, altered metabolism, cell cycle arrest and necrotic death (Sant and Johnston, 2017). However, most currently available techniques for generating 3D spheroids are time-consuming, difficult and expensive. A simple, fast and easy method for generating 3D spheroids is through the use of the hanging drop assay (Foty, 2011; Berens et al., 2015). Within the hanging drop, cells have no direct contact with substratum and due to gravity accumulate at the free liquid-air interface to form a single spheroid. In light of the increasingly recognized role of interactions between tumor and immune cells, pre-established spheroids can be co-cultured either directly or indirectly with different populations of immune cells, as shown in Figure 1.

Direct co-culture can be performed by culturing pre-established spheroids with immune cells in close proximity in the same culture environment, for example by layering two components one on top of the other. In contrast, indirect co-culture employs cell culture inserts with porous membranes, to keep immune cells separated from spheroids. Co-culturing pre-established spheroids with immune cells directly can provide information on cell-cell interactions based on physical contact, whereas indirect co-culture can be used to study the effects of secreted signaling molecules.

In our recent work, we generated spheroids from B16 melanoma cells and demonstrated their potential to impair proliferation of recently identified innate immune cells, group 2 innate lymphoid cells (ILC2s), through the production of lactic acid (Moro et al., 2010 and 2015; Wagner and Koyasu, 2019; Wagner et al., 2020). Here we provide a detailed workflow necessary for the generation of 3D spheroids from B16 melanoma cells that can be co-cultured with an immune cell population of interest. Using a hanging drop assay, B16 spheroids were generated over a period of 7 days, as shown in Figure 2. The optimal number of uniformly shaped spherical tumor cell aggregates with a smooth surface was obtained using cultures initiated with 10,000 cells per 25 μl. At least 24 spheroids can be produced per dish. Pre-established spheroids can be co-cultured with an immune cell population of interest to assess an impact of tumor cells on immune cell effector functions and vice versa. In order to address specific research questions, alterations to this condition including cell transfection, transduction and cell culture media modification with different therapeutic agents can be easily performed.

The possibility to reproduce the complex interactions between tumor and immune cells represents an important area of unfulfilled need towards the design of efficient therapeutic strategies. This protocol describes a simple, fast, and easy method for generating 3D spheroids that can be used to study the mechanisms of tumor and immune cell interactions.


Figure 1. Scheme displaying the principle of the hanging drop assay. Tumor cells per drop of cell culture medium are placed on the lid of a PBS containing dish and incubated for 7 days. Pre-established spheroids are collected and co-cultured directly or indirectly with an immune cell population of interest.