Cancer is one of the top five main causes of death worldwide, creating the need for new diagnostic and therapeutic tools to ensure early diagnosis and effective treatment. Drugs that enter human clinical trials often prove ineffective due to person-to-person heterogeneity, the absence of proper preclinical models, ineffective therapeutic strategies, and limited knowledge about the tumor microenvironment. The tumor microenvironment comprises microglia, astrocytes, macrophages, and extracellular matrix (ECM), playing a major role in the disease's progression, invasion, and resistance.

Researchers worldwide are exploring various cancer models, among which the 3D culture model is particularly popular and widely used. With a focus on the 3Rs (Replacement, Reduction, and Refinement) principle of animal use in preclinical trials, the 3D spheroid model emerges as a promising alternative testing platform for in vivo cancer models and for understanding physiology at the tissue level. The 3Rs guide the use of animals in scientific research by advocating for the replacement of animal testing with more humane methods, reduction in the number of animals used, and refinement of methods to minimize animal suffering. When animal use is unavoidable, these principles aim to enhance both scientific quality and animal welfare.

Three-dimensional (3D) cell cultures offer numerous advantages over 2D cell cultures. 3D tumor models more accurately mimic the complexity of tumor tissue architecture and biology, bridging the gap between in vitro and in vivo assessments of anticancer drugs. Multicellular tumor spheroids (MTS) within 3D culture systems contain heterogeneous cell populations that exhibit varying biological characteristics and therapeutic responses. Therefore, 3D tumor models play a crucial role in elucidating the microenvironment of solid tumors.

The primary goal of this project was to establish and characterize three-dimensional heterocellular spheroids for glioblastoma that closely resemble tumors. We characterized the spheroids based on their growth, viability, compactness, and circularity.