3D Spheroids to Study the Impact of Immune Checkpoint Proteins in Solid Tumors.

In vitro 3D cultures are commonly used in cancer research, as they mimic the in vivo tumor growth better than conventional 2D monolayer cultures. Among the various 3D culture models, spheroids are suitable for experimental cancer research due to their simplicity and capacity to reproduce the organization and microenvironment of the tumor. The liquid overlay technique (LOT), based on the use of culture plates with non-adherent surfaces, is a low-cost and easy-handling method, commonly used to generate spheroids in the laboratory. LOT is a reliable technique in terms of reproducibility and high yield, representing a relevant 3D model for drug response screening studies in cancer research. B7-family proteins are immune checkpoint molecules involved in the regulation of antitumor immune responses. Some B7 proteins are highly expressed in different types of tumors, which often correlates with cancer progression and poor prognosis. Thus, B7 proteins are relevant immunotherapeutic targets for cancer treatment. In this chapter, we show the methodology to generate spheroids from different cancer cell lines by LOT. We illustrate the application of this technique to study the functional properties of B7 proteins.