New frontiers in anti-cancer drug testing: The need for a relevant In vitro testing model

• Reviews methods for generating 3D structured spheroids for anticancer drug testing. • Explores role of spheroids in understanding cancer cell to cell and cell to matrix interaction. • Highlights advances in spheroid models for mimicking tumor microenvironment. • Discusses cytotoxicity assays and apoptosis analysis using flow cytometry technique for characterizing spheroids. • Explores advancements in bioink formulations for 3D bioprinting of spheroids. Drug development is a complex process involving multiple stages, including discovery, preclinical testing and clinical trials to ensure safety and efficacy. In vitro testing is crucial for early-stage anti-cancer drug toxicity and efficacy evaluation, often utilizing traditional monolayer cell cultures. Traditional cell cultures involve growing cells in a two-dimensional monolayer, providing a controlled environment for initial drug testing. It does not entirely replicate or mimic human in vivo microenvironment conditions, where the cell-to-cell interaction and cell-to-matrix (Extra Cellular Matrix) interaction which are vital in cellular function are limited in the traditional cell culture method and the preclinical animal testing models. Also, there are ethical considerations to be faced while using animal models, and the spheroid model can be a potential in vitro model that can reduce the use of animal testing. Hence, the spheroid models, which offer a three-dimensional structure more accurately mimic the natural tumor environment enhancing the study of drug interactions and cellular responses. This review focuses on the various anti-cancer drugs such as 5-Fluorouracil (5-FU), Tirapazamine (TPZ), Cisplatin, Resveratrol, Irinotecan and Doxorubicin that are tested in traditional monolayer cell culture and Spheroid models for comparing the efficacy and toxicity results in both using various analytical methods which helps us understand each method's advantages and limitations in drug development.