Abstract
Current preclinical models in tumor biology are limited in their ability to recapitulate relevant (patho-) physiological processes, including autophagy. Three-dimensional (3D) growth cultures have frequently been proposed to overcome the lack of correlation between two-dimensional (2D) monolayer cell cultures and human tumors in preclinical drug testing. Besides 3D growth, it is also advantageous to simulate shear stress, compound flux and removal of metabolites, e.g., via bioreactor systems, through which culture medium is constantly pumped at a flow rate reflecting physiological conditions. Here we show that both static 3D growth and 3D growth within a bioreactor system modulate key hallmarks of cancer cells, including proliferation and cell death as well as macroautophagy, a recycling pathway often activated by highly proliferative tumors to cope with metabolic stress. The autophagy-related gene expression profiles of 2D-grown cells are substantially different from those of 3D-grown cells and tumor tissue. Autophagy-controlling transcription factors, such as TFEB and FOXO3, are upregulated in tumors, and 3D-grown cells have increased expression compared with cells grown in 2D conditions. Three-dimensional cultures depleted of the autophagy mediators BECN1, ATG5 or ATG7 or the transcription factor FOXO3, are more sensitive to cytotoxic treatment. Accordingly, combining cytotoxic treatment with compounds affecting late autophagic flux, such as chloroquine, renders the 3D-grown cells more susceptible to therapy. Altogether, 3D cultures are a valuable tool to study drug response of tumor cells, as these models more closely mimic tumor (patho-)physiology, including the upregulation of tumor relevant pathways, such as autophagy.
Highlights
The success rates for investigational cancer drugs in clinical development are poor
We explored differences between cultured tumor cells and primary tumor tissues by comparing the gene expression profiles of MYCN amplified neuroblastoma cell lines with tissue samples from a publically available data set (Mixed Neuroblastoma – Versteeg; R2 database)
We hypothesized that threedimensional (3D) growth would better recapitulate neuroblastoma physiology
Summary
The success rates for investigational cancer drugs in clinical development are poor. The clinical approval rate of compounds for the treatment of solid tumors is 10% or less.[1,2] Improving basic research models is critical for achieving clinical success. Histone deacetylase (HDAC) inhibitors interfere with key tumor-relevant pathways, including proliferation, apoptosis, differentiation and autophagy in several cancer entities in vitro and in vivo.[17,18,19] The human HDAC family comprises four classes: class I, II, subdivided into classes IIa and IIb, III and IV.[20,21,22] Class IIb family members (HDACs 6 and 10) are linked to cellular stress, protein degradation and autophagy.[12,23,24,25,26] We have previously identified HDAC10 as a mediator of autophagic flux in neuroblastoma and inhibition as well as depletion of HDAC10 sensitized monolayer neuroblastoma cells to cytotoxic drugs.[12] It remains unclear how different cell culture settings influence autophagy, in the context of drug development and therapy resistance. Our data suggest that 3D tumor models are advantageous when studying autophagytargeting treatment and resistance mechanisms
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