Spontaneous Cell Detachment and Reattachment in Cancer Cell Lines: An In Vitro Model of Metastasis and Malignancy.

Elena Vargas-Accarino,Trond Aasen,Carlos Herrera-Montávez,Santiago Ramón Y Cajal

doi:10.3390/ijms22094929

Abstract

There is an unmet need for simplified in vitro models of malignancy and metastasis that facilitate fast, affordable and scalable gene and compound analysis. “Adherent” cancer cell lines frequently release “free-floating” cells into suspension that are viable and can reattach. This, in a simplistic way, mimics the metastatic process. We compared the gene expression profiles of naturally co-existing populations of floating and adherent cells in SW620 (colon), C33a (cervix) and HeLa (cervix) cancer cells. We found that 1227, 1367 and 1333 genes were at least 2-fold differentially expressed in the respective cell lines, of which 122 were shared among the three cell lines. As proof of principle, we focused on the anti-metastatic gene NM23-H1, which was downregulated both at the RNA and protein level in the floating cell populations of all three cell lines. Knockdown of NM23-H1 significantly increased the number of floating (and viable) cells, whereas overexpression of NM23-H1 significantly reduced the proportion of floating cells. Other potential regulators of these cellular states were identified through pathway analysis, including hypoxia, mTOR (mechanistic target of rapamycin), cell adhesion and cell polarity signal transduction pathways. Hypoxia, a condition linked to malignancy and metastasis, reduced NM23-H1 expression and significantly increased the number of free-floating cells. Inhibition of mTOR or Rho-associated protein kinase (ROCK) significantly increased cell death specifically in the floating and not the adherent cell population. In conclusion, our study suggests that dynamic subpopulations of free-floating and adherent cells is a useful model to screen and identify genes, drugs and pathways that regulate the process of cancer metastasis, such as cell detachment and anoikis.

Highlights

IntroductionMetastasis is a process by which cancer cells leave their primary site and spread to other tissues and organs to form new tumors [1]
We observed that the colon cancer cell line SW620 and the cervical cell line C33a produce many floating cells (Figure 1a,b) even at low density
As a proof of principle of our model, we focused on NM23-H1, as this anti-metastatic gene has been robustly linked to metastasis, and was downregulated in the floating cell population of all three cell lines

Summary

Introduction

Metastasis is a process by which cancer cells leave their primary site and spread to other tissues and organs to form new tumors [1]. Ninety percent of deaths from solid tumors are due to metastasis. Key features during epithelial cancer cell metastasis are detachment, adaptation and prevention of detachment-induced cell death (anoikis) in the absence of extracellular matrix (ECM), and subsequent reattachment [1]. This is a highly complex process regulated by numerous genes including adhesion molecules, receptor tyrosine kinases, and pathways such as the mechanistic target of rapamycin (mTOR) and RhoA signalling [1]

Methods

Results

Discussion

Conclusion