Three-dimensional multicellular spheroid-induced 5-fluorouracil drug
resistance provokes global alterations of HeLa carcinoma cells: a
multi-omics analysis
Abstract
Cervical cancer is a serious health problem in women around the globe,
with 600 thousand new cases each year. However, the use of clinical drug
is seriously dampened by the development of drug resistance, which has
been evidenced to be associated with metabolic reprogramming and
heterogeneity in tumor cells. Efficient in vitro tumor model is
essential to improve the efficiency of drug screening and the accuracy
of clinical application. Multicellular tumor spheroids (MTSs) can in a
way recapitulates tumor traits in vivo, thereby representing a powerful
transitional model between 2D monolayer culture and xenograft. In this
study, based on the liquid overlay method, a protocol for rapid
generation of the MTSs with uniform size and high reproducibility in a
high-throughput manner was established. As expected, the cytotoxicity
results showed that there was enhanced 5-FU resistance of HeLa carcinoma
cells in 3D MTSs than 2D monolayer culture with a resistance index of
5.72. In the presence of both glucose and glutamine, HeLa carcinoma
cells preferentially used glutamine as a bioenergetic substrate to
support cell proliferation and maintenance under all conditions, while
the ubiquitous by-product ammonium might be only recycled in the 3D
MTSs. Furthermore, in order to obtain a holistic view of the molecular
mechanisms that drive 5-FU resistance in 3D HeLa carcinoma cells, a
multi-omics study was applied to discover hidden biological
regularities. We found that in the 3D MTSs mitochondrial
function-related proteins and the metabolites of the tricarboxylic acid
cycle (TCA cycle) were significantly decreased, and the cellular
metabolism was shifted towards glycolysis. The differences in the
protein synthesis, processing, and transportation between 2D monolayer
cultures and 3D MTSs was significant, mainly in the heat shock protein
family, with the upregulation of protein folding function in endoplasmic
reticulum (ER) which promoted the maintenance of ER homeostasis in the
3D MTSs. In addition, at the transcript and protein level, the
expression of extracellular matrix (ECM) proteins (e.g., laminin and
collagen) were up-regulated in the 3D MTSs, which enhanced the physical
barrier of drug penetration. Summarizing, this study formulates a rapid,
scalable and reproducible in vitro model of MTS for drug screening
purposes, and the findings establish a critical role of glycolytic
metabolism, ER hemostasis and ECM proteins expression profiling in tumor
chemoresistance of HeLa carcinoma cells towards 5-FU.