Novel biomarker discovery requires high quality biospecimens and proper maintenance of cell phenotype. However, patient samples have reduced availability rendering not practical for large-scale drug screening and biomarker discovery. This impracticality has stimulated the use of cell lines in many "-omics" research studies to discover biomarkers. In addition, cells in these in vitro models are typically grown on 2-dimensional (2D) culture dishes, which is obviously different from native in vivo microenvironments. Data extraction from cells grown in 2D has less relevance, and thus, the biomarkers derived from studies using 2D platforms will likely have less clinical value. Thus, implementation of in vitro models that take into account primary patient samples and in vivo-like factor represent a paradigm shift in cancer biomarker discovery. This review emphasizes on current 3D cell culture platforms used to recreate in vivo conditions and their ability to adapt towards demanding conditions of biological relevance. OverviewSince the discovery of the cell as the basic unit of tissues, cell culture has been traditionally defined by simple approximations. The most common are two-dimensional (2D) cell culture and separating disease cells from their native microenvironment. Because these limitations result in challenges for the discovery of clinically relevant biomarkers, many 3-dimensional (3D) cell culture methods have been introduced over the past decades. Models that have recently been used in tissue engineering include 3D geometry and cell co-culture to better represent tissue homeostasis in in vitro settings [1]. Adopting the success of Tissue Engineering models, the same principles can also be used for biomarker discovery projects for cancer. As mounting evidence indicates that gene expression and signaling pathways of tumor tissues depend on context or their native microenvironment, these 3D co-culture models more closely resemble cancers [2]. In the tumor microenvironment, normal and cancer cells interact in a 3D setting and influence each other to positively enhance oncogenic potential [3]. For example, during cancer progression and metastasis, tumor cells up regulate oncogenes to increase proliferation while actively modify their cellular microenvironment to control angiogenesis, extracellular matrix (ECM) stiffness, proliferation, and oxygen levels [4][5][6]. This highly orchestrated sequence of events defines the innate plasticity of cancer cells that allows them to exert control at molecular and tissue levels to maintain a malignant phenotype [7][8][9]. Clearly, the conditions that surround tumor cells must be replicated in an in vitro setting to resemble the tumor microenvironment, and the traditional 2D tumor cell culture method is an oversimplified in vitro cell-based model that cannot recreate the environment of cancer homeostasis [10]. As a consequence, biomarkers identified in 2D culture often lack critical in vivo signatures, whereas biomarkers identified in 3D culture are more likely to ult...