Cancer Spheroids and Organoids as Novel Tools for Research and Therapy: State of the Art and Challenges to Guide Precision Medicine

Abstract

Spheroids and organoids are important novel players in medical and life science research. They are gradually replacing two-dimensional (2D) cell cultures. Indeed, three-dimensional (3D) cultures are closer to the in vivo reality and open promising perspectives for academic research, drug screening, and personalized medicine. A large variety of cells and tissues, including tumor cells, can be the starting material for the generation of 3D cultures, including primary tissues, stem cells, or cell lines. A panoply of methods has been developed to generate 3D structures, including spontaneous or forced cell aggregation, air-liquid interface conditions, low cell attachment supports, magnetic levitation, and scaffold-based technologies. The choice of the most appropriate method depends on (i) the origin of the tissue, (ii) the presence or absence of a disease, and (iii) the intended application. This review summarizes methods and approaches for the generation of cancer spheroids and organoids, including their advantages and limitations. We also highlight some of the challenges and unresolved issues in the field of cancer spheroids and organoids, and discuss possible therapeutic applications.

Keywords: 3D cell culture; cell and gene therapy; drug screening; immunotherapy; personalized medicine; tissue engineering.

Figure 1

Summary of the procedures used to establish cancer spheroids and organoids. Patients’ primary cells, CRISPR/Cas9 engineered cells, pluripotent stem cells, and a mixture of different cell types and co-culture with immune cells can be used to establish organoids. This figure was created with BioRender.com (accessed on 14 March 2023).

Figure 2

Schematic representation of the different 3D cell culture techniques: (A) In the hanging method, the cells form a single spheroid by accumulating at the free liquid interface formed by their suspension due to the inversion of the dish. (B) The forced floating method can be carried out using uncoated polystyrene plates or plates coated with a hydrophilic polymer that suppresses cell-substrate interactions, e.g., ultra-low attachment (ULA) plates. (C) In the modeling culture system, the cells are seeded and allowed to self-aggregate into non-adhesive micro-molds. (D) The magnetic levitation employed magnetic beads assembly technique. Cells treated with magnetic beads aggregate into spheroids or organoids under magnetic forces within a few hours after a magnet is placed on top of the lid. (E) The Rotary Cell Culture System is another agitation-based technique used to obtain a higher number of large spheroids with a small number of starting cells. (F) Organoids can be cultured in the submerged method by disrupting tissue mechanically and enzymatically into single-cell suspensions, followed by embedding them in basement membrane extract (BME) and submerging them in the culture media. This figure was created with BioRender.com (accessed on 14 March 2023).

Figure 3

Application of organoids in Cancer Immunotherapy. The TME can be generated in PDOs by two types of approaches: the reconstituted TME (Upper panel, pink color) and the native TME (Below panel, blue color). In the reconstituted system, PDOs were exclusively generated from tumor cells after physical or enzymatical digestion. Organoids are cultured in extracellular matrix dome (Matrigel or BME (Basement Membrane Extract)). Organoids were co-cultured with autologous PBMC isolated from peripheral blood, lymph node, or from tumor. In native TME model, tumors were minced or enzymatically digested, then filtered to obtain appropriately sized fragments, embedded into collagen-based extracellular matrix with medium in the top. Alternatively, in air–liquid interface (ALI) culture, native tumor fragments are embedded in collagen matrix on the top of a transwell insert exposed to air with culture medium below it. Numerous downstream applications of organoids in immunotherapy followed by systemic functional and quantitative analysis are summarized in the right panel. It allows the interaction between immune cells and tumor cells to be defined, in order to identify and predict the clinically relevant immunotherapy strategy for patients. Abbreviations: CAR, chimeric antigen receptor; NK, Natural killer; ICI, immune checkpoint inhibitors, CTLA4, cytotoxic T-lymphocyte associated protein4; PD-1, programmed cell death-1; LAG-3, Lymphocyte-activation gene 3; TIM-3, T-cell immunoglobulin and mucin containing protein-3. This figure was created with BioRender.com (accessed on 14 March 2023).