Multicellular tumor spheroid models to explore cell cycle checkpoints in 3D

Abstract

BackgroundMultiCellular Tumor Spheroid (MCTS) mimics the organization of a tumor and is considered as an invaluable model to study cancer cell biology and to evaluate new antiproliferative drugs. Here we report how the characteristics of MCTS in association with new technological developments can be used to explore the regionalization and the activation of cell cycle checkpoints in 3D.MethodsCell cycle and proliferation parameters were investigated in Capan-2 spheroids by immunofluorescence staining, EdU incorporation and using cells engineered to express Fucci-red and -green reporters.ResultsWe describe in details the changes in proliferation and cell cycle parameters during spheroid growth and regionalization. We report the kinetics and regionalized aspects of cell cycle arrest in response to checkpoint activation induced by EGF starvation, lovastatin treatment and etoposide-induced DNA damage.ConclusionOur data present the power and the limitation of spheroids made of genetically modified cells to explore cell cycle checkpoints. This study paves the way for the investigation of molecular aspects and dynamic studies of the response to novel antiproliferative agents in 3D models.

Comparison of cancer cells cultured in 2D vs 3D reveals differences in AKT/mTOR/S6-kinase signaling and drug response

“…This might explain the drastic decrease of RPS6 phosphorylation -from high levels in the outer two-cell-layer-thick zone to low levels in the deeper areas. Of note, cell proliferation also decreased gradually in the inner zones of the spheroids, as demonstrated previously (Grimes et al, 2014;Laurent et al, 2013). However, when mTOR activity was inhibited, RPS6 phosphorylation was lost but Ki67 remained, indicating that there is no direct correlation of RPS6 phosphorylation with cell proliferation.…”

“…6E). The proliferation marker Ki67 exhibited a well-described decrease of Ki67 + cells from the outside to the core of the spheroid (Grimes et al, 2014;Laurent et al, 2013). Ki67 + cells were significantly reduced in the treated samples as compared to the control ( Fig.…”

Spheroid-based 3D Cell Cultures Enable Personalized Therapy Testing and Drug Discovery in Head and Neck Cancer

“…Furthermore, in 3D models, cells seem to express distinct genes usually associated with drug resistance that were also found in in vivo animal studies (58). Factors, such as alteration of chromatin structure, apoptosis inhibition, cell cycle and permeability, make the MCTS a more reliable and predictable drug testing in vitro model compared to 2D monolayer models (29,37,(62)(63)(64). Radiation resistance has also been linked to spheroid models in the past.…”

Reversible growth arrest of 3D tumor spheroids stored in oxygen absorber-induced anoxia

“…HCT116 colon adenocarcinoma cells (ATCC) were cultured in DMEM (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% foetal calf serum (FCS), 2 mM/l glutamine and penicillin/streptomycin in a tissue culture incubator (humidified atmosphere of 5% CO 2 at 37˚C). Spheroids were prepared as previously described (9). Briefly, 500 cells/well were distributed in ultra-low attachment 96-round bottom well plates.…”

“…Several reports have shown that spheroid growth results in the generation of a hypoxia and nutrient gradients (7)(8)(9) where cells localized in the inner region exit the cell cycle and enter a G0 quiescent state. The induced cell quiescence in the spheroid central region mimics the situation observed in vivo in microtumor domains and the subsequent resistance to classical chemotherapeutic agents (10,11).…”