The rationale for 3D cell culture models is quite simple; biology does not occur in two dimensions and thus traditional adherent cell culture is limited in its ability to replicate the complex characteristics of the in vivo microenvironment, such as diffusion gradients, receptor expression, etc. Because of these differences, the technique in which cells are cultured (2D vs. 3D) can substantially alter the drug’s effect on the cells. There are many examples in the literature regarding the substantial differences between cells cultured in 2D vs 3D format, a few of which are highlighted below.
Differences in receptor expression
“Cells grown as 3D spheroids using p-HEMA-coated plates had HER2 homodimers form; while in 2D cultures, HER2 formed heterodimers with HER3. Furthermore, in 3D versus 2D culture, epidermal growth factor receptor (EGFR) levels were slightly reduced; phosphorylation of HER2, HER3 and EGFR was enhanced; and activated Akt was downregulated” [1, 2]
We recently demonstrated that when the primary cell-derived Wood breast cancer cell line (Cellaria) was grown in 2D it did not express estrogen receptor (ER) as expected from immunohistochemical analysis of patient biopsy, whereas when grown in 3D, ER expression was recapitulated.
Differences in response to drug treatment
“When a fixed concentration of trastuzumab (a monoclonal antibody that targets HER2) was added to the cells, 2D cultures underwent approximately 16% reduction in proliferation, whereas proliferation in 3D spheroids was reduced by a considerable 48%. This indicates that the architecture of 3D spheroids differs from that in 2D and that the targeted anti-cancer drug, trastuzumab, has a dramatically different effect on cells grown in monolayer as compared to those in 3D, due to differences in the cell surface molecules and thus intracellular signalling events.” [1, 2]
“Cell viability at 10 mM 5-FU 96 h after drug treatment for 7500-cell A431.H9 spheroids and 2D culture condition. At the same 5-FU concentration, there is only 5% viability relative to untreated control for 2D cultures, but still 75% viability relative to control for 3D spheroids. This clearly shows that A431.H9 cells are more resistant to 5-FU in 3D than 2D cultures. In contrast, [treatment with] TPZ, a hypoxia-activated cytotoxin, shows that at 10 mM TPZ 96 h after drug treatment, there is still 75% viability relative to control for 2D cultures, but only 40% viability for 7500-cell A431.H9 spheroids. The IC50 of A431.H9 cells cultured in 2D is about 50 mM, while the IC50 of the A431.H9 3D spheroids […] is about 8 mM” 
Further study on the primary cell-derived Wood breast cancer cell line (Cellaria) indicated specific differences in the sensitivity of Wood cells to common antineoplastic agents indicated for breast cancer depending on whether they were cultured in 2D or 3D. Specifically, the Wood line showed little sensitivity to fulvestrant, an ER-targeting drug, in 2D format, but was exquisitely sensitive to the drug in 3D.
Thus, there is ample evidence to suggest that 3D cell culture models may be preferable for use in drug screening applications, when cost and throughput requirements permit. For many research questions, 3D cell culture can improve the translational gap between in vitro results and what is expected in vivo.