Assay and Drug Development Technologies - 28

EGLEN AND RANDLE
As several authors8-10 have argued, drug discovery using 3D
cell culture better reflects compound interaction with cells and
tissues in vivo. There are several published reports comparing the
pharmacology of compounds screened at cells grown under 2D
versus 3D conditions, and a summary of these can be found in
Edmondson et al.5 and citations therein. A particularly relevant
example has been reported12 in studies with cancer cells overexpressing
human epidermal growth factor receptor (HER2). In
this study, cells grown in spheroids were compared to 2D culture.
In the latter, it was seen that HER2 and HER3 formed heterodimers,
whereas in 3D spheroids, homodimerization of HER2
occurred. This latter association resulted in an enhanced antiproliferative
potency of trastuzumab (Herceptin)-a monoclonal
antibody specifically targeting HER2. 3D culture, therefore, facilitated
HER2 homodimerization, leading to enhanced activation
of HER2 and induced a signaling pathway switch from
phosphoinositide 3-kinase (PI3K) to mitogen-activated protein
kinase (MAPK). The authors of this study concluded that human
multicellular tumor spheroids allow identification of novel targets
for treatment of HER2-positive breast cancer patients.12
Advanced 3D culture technologies, coupled with improved
detection technologies (notably confocal optical imaging), allow
drug screening in protocols that are not feasible with 2D
cultures.6-8 SLAS/SBS standard microtiter plates (96 or 384
well) are widely used in conjunction with 3D culture models.
This is most relevant when screening for novel antitumor
agents using cell spheroids in multiwell plate-based assays in
HTS/HCS protocols.5 Such cell assemblies are formed using
hangingdropmethods,
rotatingwall vessels, or
surfaces
modified to reduce cell attachment (Fig. 1).13 Ofthese, hanging
drop and low-attachment microtiter plates have found broad
use due to their high compatibility with automated screening
instrumentation and detection systems. 3D Spheroids are also
widely used for functional assays that exploit key features of
tumor physiology. These include coculture assays using stromal
and immune cells tomodulate tumor growth, migration, or
invasion. Spheroids can also be cocultured with embryoid
bodies containing differentiated endothelial cells, providing a
means to screen for tumor angiogenesis inhibitors.13 It is
probable that compound screening ofpatient-derived cells will
be amajor application of3D culture. As an example, circulating
tumor cells derived from patients with breast cancer proliferate
in 3D culture as tumor spheres, providing an approach to screen
compounds on cells from individual patients.14
3D CULTURE, SURFACES, SCAFFOLDS,
AND STEM CELLS
Cells used in 3D culture are acquired from many sources,
including autologous or allogeneic cells, human- or animalderived
primary cells, genetically engineered cells and stem
cells. Pluripotent stemcells (PSCs), in particular, can be directed
to differentiate into specific somatic cell lineages by inductive
signals that mimic changes occurring during embryogenesis.
Stem cells self-organize, self-pattern and undergo selfmorphogenesis,
all of which depend on the spatial location of
the cell. Therefore, it is not surprising that many current applications
of 3D cell culture specifically involve stem cells.15
The majority of3D cell culture involves the use ofhydrogelbased
matrices or solid scaffolds.16 Emerging techniques
include bioprinting, which allows for precision stem cell
Fig. 1. AR splice variant screening assay. (A) Schematic representation of the AR-FL and splice variants AR-v7 and AR-v12 lacking the LBD.
The figure illustrates binding of testosterone, and the antiandrogen enzalutamide to the AR-FL, but not AR-vs. LBD, DBD, and NTD. (B) Twostep
design for the generation of double-stable cell lines expressing a receptor-dependent reporter gene and an inducible AR-v polypeptide.
AR-FL, full-length androgen receptor; AR-v, androgen receptor splice variant; DBD, DNA binding domain; LBD, ligand binding
domain; NTD, amino terminal domain.
28 ASSAY and Drug Development Technologies
ª 2022 MARY ANN LIEBERT, INC.

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