Three-dimensional tumor cell growth stimulates autophagic flux and recapitulates chemotherapy resistance

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.

Dissecting pharmacological effects of chloroquine in cancer treatment: interference with inflammatory signaling pathways

“…In addition to blocking entry of free CQ into cells, the acidic extracellular microenvironment triggers activation of the transcription factor TFEB that induces lysosomal biogenesis. 264 In many cell types, though, especially in normal macrophage cells, TFEB is mutually antagonistic to STAT3 activity, and would thereby limit the protection of a tumor by macrophages. Deregulated TFEB leads to metabolic reprogramming of the cancer microenvironment.…”

“…263 In vitro, cancer cell autophagy and TFEB activity can be modeled in three-dimensional growth conditions. 264 In many cell types, though, especially in normal macrophage cells, TFEB is mutually antagonistic to STAT3 activity, and would thereby limit the protection of a tumor by macrophages. 265 It was shown that TFEB is activated and translocated to the nucleus via inhibition of mammalian target of rapamycin complex by CQ.…”

Mass spectrometry imaging of endogenous metabolites in response to doxorubicin in a novel 3D osteosarcoma cell culture model

“…The study of drug distributions and effects on downstream pathways via metabolomics approaches has successfully been used to predict antitumour cytotoxic responses by the analysis of extracellular metabolites in cancer patients . These metabolomic studies have been extended to three‐dimensional (3D) cell culture studies, whereby the environment recapitulates the drug resistance seen in vivo , and have further shown that metabolomic profiles may predict the malignant potential of cells grown in 3D cell culture …”

Implementation of a dynamic culture condition to the heterotypic 3D breast cancer model

“…In contrast, to flow within the blood vessels, the interstitial flow moves in all directions. The studies on flow‐induced shear stress showed its influence on the cell molecular signaling, gene expression, cell cycle, autophagy, and chemotherapy resistance (Bingel et al, ; Chang et al, ; Hakkinen, Harunaga, Doyle, & Yamada, ; Ham et al, ; Massai et al, ; Santoro, Lamhamedi‐Cherradi, Menegaz, Ludwig, & Mikos, ). Accordingly, to develop an appropriate in vitro model of cancer that would more closely imitate the pathophysiological conditions, at least three requirements should be implemented such as the cells heterogeneity, three‐dimensionality, and the stress forces.…”