**7. Cell culture models**

Traditionally animal models were commonly employed for carrying out study of different types of cancer for the past three decades [74]. These animal studies have many drawbacks including lack of high-throughput drug screening, longer time consumption to conduct tests and ethical controversies concerning animal testing. Cell culture is the most widely used alternative to animal studies and cell culture techniques can broadly be classified into 2D and 3D methods [75]. The potentialities of primary cancer cell models' cultures in preclinical studies for cancer research and drug discovery has amplified over the past few years. Primary cell cultures provide a good model system to understand normal and malignant biological activities. Carcinogenesis-related behavior such as apoptosis, proliferation, adhesion, differentiation, migration, senescence, invasion, angiogenesis, and other metabolic pathways have been studied in recent years. One of the major advantages is that the heterogeneity of cell populations composing a primary culture mimics the tumor microenvironment, crosstalk, and interactions between malignant and healthy cells, neither of which is possible with cell lines [76].

Most studies have shown that the cellular responses to drug treatments in 3D cell culture are significant and more similar to that of *in vivo* architecture when compared to 2Dcell culture. One of the most improved successful assays using 3D culture for cell-based screening in the early phase of drug discovery is cancer cell viability assessment. This assay is particularly useful to test the cytotoxic effects of compounds that may lead to cell death. It plays an essential role in checking how many cells are viable at the end of each experiment. Cell viability assay is closely followed by cell proliferation, cell migration and then cell signaling assays [77–79].

Currently a number of anticancer drugs belonging to different classes chemically are available. To be used as a potential anticancer agent, the testing compounds need to inhibit the growth and proliferation of cancer cell lines. This will further inhibit the signaling pathways by knocking in or knocking out a candidate gene thereby stopping the progression of tumor to fatal stages. For instance, antiproliferative investigations were performed on prostate cancer cell line DU-145 *in vitro* and *in vivo* using salvia miltiorrhizabunge [80]. Another example to justify this concept will be a study performed on PC-3 cell line. Generally, cancer cells express higher amount of Transferrin Receptors (TfR) for an increased uptake of iron in relative to normal cells. This higher amount of intracellular free iron is required for the growth and proliferation of cancer cells. Anhydrodihydroartemisinin (ADHA) was used to inhibit PC-3 cell lines through caspase-dependent pathway [81].
