Changes in the transcriptome profile of breast cancer cells grown as spheroids

https://doi.org/10.1016/j.bbrc.2019.06.155Get rights and content

Highlights

  • Multicellular spheroids presents changes in the transcriptome (mRNA and ncRNAs).

  • Multicellular spheroids active pathways associate to cholesterol metabolism.

  • In multicellular spheroids there is an increase in MIF.

Abstract

Background

Multicellular tumor spheroids mimic the functional organization of tumors in vivo, providing biological readouts that predict the behavior of cancer cells more accurately. The current study aimed to evaluate the transcriptome (mRNAs and long non-coding RNAs) of multicellular tumor spheroids from breast cancer cells.

Methods

MCF-7 cell spheroids were used; the transcriptome was analyzed using RNAseq and RNA microarrays; the secretion of macrophage migration inhibitor (MIF), a cytokine exported by the cholesterol efflux regulatory protein, was measured by ELISA. Linc00052 was inhibited using short-hairpin RNAs (shRNAs).

Results

We found several differentially regulated mRNAs and lncRNAs in MCF-7 cell spheroids. We also found significant enrichment of the Wnt/B-catenin death receptor and the cholesterol metabolic processes. Interestingly, we also found an increased concentration of MIF. Further, at 12 and 20 days of 3D culture we found 221 and 1146 dysregulated lncRNAs, respectively; including linc00052 (long intergenic non-protein coding RNA 52), which has been involved in breast cancer. Linc00052 knock-down experiments suggest that it could be a key regulator of cholesterol pathways in breast cancer.

Conclusions

Our data shows that tumor spheroids can induce changes in the transcriptome of the cultured cells, including both mRNAs and ncRNA. One of the major changes included the deregulation of cholesterol pathways, of which linc00052 is apparently a key regulator.

Introduction

The architecture of the tumor microenvironment contributes significantly to its neoplastic phenotype [1]. The behavior of tumor cells in vivo can be mimicked in vitro through multi-cellular tumor spheroids (MCTS) [2], this is due to the appearance of different cell layers and cell-cell/ECM-cell connections [3] through which distinct microenvironments are formed, most of them hostile due to hypoxia and the lack of nutrients. MCTS display an early phase of exponential cell proliferation followed by decreased cell growth and an increased number of quiescent cells. Thus, three distinct areas appear when tumoral spheroids reach 250 μm in diameter, i.e. an outer zone of proliferating cells, an intermediate zone of quiescent cells, and a necrotic/apoptotic center [4]. Further, various biological processes are modified when cells are grown in 3D cultures, e.g. cells gain greater ability to migrate, proliferate, and have important metabolic changes [5,6].

Long non-coding RNAs (lncRNA) have been recently described as a mechanism for gene regulation [7] despite lacking the capacity to encode proteins [8]. The importance of lncRNAs lies mainly in their ability to form DNA, RNA or protein complexes and regulate gene expression at the transcriptional and posttranscriptional level. Moreover, lncRNA deregulation has been associated with several human diseases, including cancer [8].

It has been shown that breast cancer is linked with alterations in lipid and cholesterol metabolism [9]. Further, cholesterol exerts a functional control of the estrogen receptor ERRα [10] and modulates the expression of chemokines such as CXCL9, CXCL10 [11] and IL-8, which have been associated with proliferation, angiogenesis, migration, and chemosensitivity in cancer cells [12]. Moreover, at least 7 lncRNAs: Malat, H19, LncHR1, Lexis, Mexis, ANRIL, and Chrome, have been linked with cholesterol metabolism [13].

In the present study we analyzed some of the transcriptional changes induced by a 3D microenvironment in breast cancer MCTS through genome-wide RNAseq.

Section snippets

Monolayer and spheroid culture

Breast cancer MCF-7 cells (ATCC, HTB-22, USA) were cultured in RPMI 1640 (10-040-CMR CORNING NY) with 5% FBS (CORNING, USA), in a humid atmosphere at 37 °C in a 5% CO2. MCTS were generated using the liquid overlay technique as reported previously [6]. The cell cultures were maintained with constant orbital motion (60 rpm) at 37 °C. The size of MCTS were determinate collecting a sample at specific time, were fixed used 4% formalin and two orthogonal diameters of 30 MCTS were measured using an

MCF-7 3D cultures

MCTS reached a diameter of ∼250 μm at 12 days of culture, with a maximum growth plateau after 20 days (Fig. 1A and Fig. 1B). A hypoxic gradient formed between 12 and 20 days of culture, leading to an apoptotic/necrotic center [19]. After 20 days, the typical three zone structure had been established within the MCTS (proliferative, quiescent, and apoptotic/necrotic cell layers) (Fig. 1C).

Transcriptome changes in MCF-7 MCTS at 12 and 20 days of growth

The MCTS presented 2942 and 2483 differentially-expressed (DE) mRNAs at 12 and 20 days, respectively,

Discussion

MCTS grow as a homogeneous cellular population which later evolves into a complex structure with a necrotic center induced by hostile conditions, e.g. hypoxia and lack of nutrients. It has been well documented that the microenvironment elicited by 3D culture induces greater capacity for migration and invasion, chemoresistance, and an epithelial/mesenchymal phenotype.

In this study, we used massive RNA sequencing to analyze the transcriptome of MCTS at two time points, finding that 2942 and 2483

Funding

Laura Muñoz Galindo is a doctoral student from: Programa de Doctorado en Ciencias Biológicas, Facultad de Medicina, Universidad Nacional Autónoma de México (UNAM), and this study is part of her doctoral thesis. Laura Muñoz received a fellowship from the National Council of Science and Technology (CONACYT) (CVU 215926). We thank Dr. Daniel Díaz, Ph.D. for his assistance in proofreading this manuscript.

Conflicts of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.

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