Differentiation and Malignant Transformation of Epithelial Cells: 3D Cell Culture Models

Project Leader
Docent Sinikka Eskelinen, Ph.D.

Biocenter Oulu and Unit of Cancer Research and Translational Medicine, Faculty of Medicine, University of Oulu

Background and Significance

Secretory as well as absorptive organs, such as lung, salivary gland, kidney and mammary gland have a specialized luminal space within an epithelial tube where fluid is transported to its destination. The luminal space is formed during embryogenesis and begins by segregation of transmembrane proteins such as ion channels to distinct membrane domains. Cell junctions are responsible for maintenance of these specialised domains and differentiated phenotype of the epithelium. Tools to control cell adhesion in tissues range from gene expression to cell proliferation, migration and apoptosis. In addition to cell–cell contacts, integrin-mediated cell–ECM contacts are crucial for the differentiation of epithelial cells.

In the kidney, water and electrolytes are secreted and reabsorbed in different segments of nephrons in a highly regulated manner. In renal failure, glomerular filtration may be reduced, leaving only tubular secretory mechanisms in nephrons as a means to form urine. Hence, efficient secretory and absorptive mechanisms are especially needed when the organism is facing drastic changes in the environment and kidney epithelium must have the capacity to regulate transcellular transport as well as lumen size in order to keep the epithelium functional. 

The differentiated architecture of an epithelial layer is disrupted in carcinogenesis and filling of the luminal spaces of epithelial glands is a hallmark of early epithelial tumours. Transformed tumour cells populate the lumen and this process requires both enhanced proliferation induced by oncogenes and inhibition of apoptosis by antiapoptotic signals. Three-dimensional cell culture systems can be of utmost importance in testing the therapeutic responses of tumour cells to various compounds or in evaluating the invasive capacity of transformed cells. We have developed methods to monitor epithelial–mesenchymal and mesenchymal–epithelial transformation in a highly regulated manner and to analyse the sequence of events with state-of-the-art spinning disc confocal and light sheet microscopes.

Recent Progress

Lumen formation by MDCK cells in a 3D environment

Canine kidney epithelial MDCK cells can differentiate in a 3D environment into spherical cysts with a lumen inside and a basal membrane facing the extracellular matrix. We have monitored cyst formation of MDCK cells stably transfected with Venus-tagged CD59 protein (kind gift from Reika Watanabe and Guillaume Castillon, Dept. Biochem., Univ. Geneva) as a function of time using state-of-the-art spinning disc microscopy techniques. Since CD59 is a lipid-anchored, secreted protein, it served as a marker for the apical membranes and lumina. MDCK cells grown in matrigel for 2–3 days form preapical patches (PAPs) between adjacent cells which develop into a large lumen 2–3 days later. Time lapse imaging of Venus-CD59 MDCK cells grown within matrigel under the microscope resulted in the conclusion that there is a third mechanism of lumen formation besides hollowing and cavitation. We have called it “coalescence”. In this process, the cells initially divide irregularly, but rapidly reorganize themselves along the cyst periphery in a dynamic way. Hence, at least in vitro, the differentiation process is flexible and the cells have the capacity to correct erroneous cell divisions and restructure the multilumina into a well-organized epithelial structure with one lumen surrounded by a single layer of cells having uniform apico-basal orientation.

Factors regulating transepithelial water transport and generation of hydrostatic pressure within the lumen

We have built up an MDCK cell culture model where we can analyse the mechanics of lumen formation and enlargement within short periods of time, without interference by alterations in cell number through proliferation or apoptosis, by using high-resolution confocal microscopy and imaging software. We have used the method to elucidate the capacity of cells to respond to changes in the composition of the extracellular milieu or changes in membrane potential within short time intervals. This gives valuable information on the behaviour of kidney cells under extreme situations or under the influence of drugs affecting ion pumps or channels. 

We observed that when the cells were subjected to a hyperpolarizing environment, i.e. external medium with no sodium or potassium, apical secretion of chloride ions and transepithelial water transport is initiated. As a consequence the lumen is rapidly expanded. In contrast, a depolarizing environment, i.e. equalizing internal and external potassium levels, leads to cell swelling and ultimately death. Hence, water influx to the lumen could be induced solely by hyperpolarization, which activates the cellular machinery, cystic fibrosis transmembrane receptors (CFTRs) and aquaporins. An antioxidant molecule, N-acetylcysteine (NAC) has been widely used in the treatment of cystic fibrosis patients and in contrast-induced nephropathy. We wished to elucidate the mechanism of NAC action in our experimental set-up. It completely prevented water flow and luminal expansion in hyperpolarizing conditions and depolarized the cells analogously to ionophores to monovalent cations. These findings open new perspectives for pharmacological treatment of increased or decreased water secretion.

Malignant transformation of epithelial cells by the Src oncogene

In order to study malignant transformation in cultured cells, an inducible culture system is required. A good cell culture model consists of ts-Src-transformed canine kidney MDCK cells which, when cultivated at 40.5 oC, behave as normal epithelial cells, whereas after a shift to 35 oC, Src tyrosine kinase is activated and the transformation process begins. In a 2D environment, the cells become mesenchymal with poor cell–cell contacts and a flattened shape. In a 3D environment they form irregular clusters without lumina. This feature gives us a tool to analyse the regulatory factors of the transformation process induced by the v-Src oncogene just by changing the culture temperature. With this model we have monitored changes in gene expression, protein phosphorylation, cadherin internalization, mitochondrial activity and apoptosis. Recently we have focused on the physical mechanisms behind lumen filling and epithelial–mesenchymal transition. Preliminary results show that Src activation leads to rapid luminal collapse as a result of opening of tight junctions and release of intraluminal hydrostatic pressure, and subsequent lumen filling by cell migration and proliferation. The spinning disc confocal microscope enables visualization of the time course and mechanism of lumen filling once Src is activated.

Role of survivin in malignant transformation

So that cancer cells can grow freely and metastasize, they need to overcome their own proliferation regulation and inhibit apoptosis. A member of the inhibitor of apoptosis (IAP) protein family, survivin, is expressed in most tumour cells and is considered to be a promising therapeutic target. We have observed downregulation of survivin in untransformed MDCK cells and elevated expression in Src-transformed MDCK cells grown in a 3D environment (Töyli et al. 2010). There are controversial views on the mechanism of how survivin promotes the transformation process. In addition to inhibition of apoptosis, it regulates proliferation and promotes migration. We aim to discover whether there is a correlation and causal relationship between expression of survivin and cell migration or proliferation.

Future Goals

We aim to understand the mechanisms behind the morphogenesis, functionality and malignant transformation of epithelial MDCK cysts in 3D model systems. To achieve these goals we are trying to find answers to the following questions:

  1. What determines the ultimate size of the lumen and cyst in 3D culture of MDCK cells?
  2. What is the source of the lumen negative potential in kidney cells and how can it be regulated in vitro and in vivo?
  3. What are the roles of survivin and matrix composition in 3D differentiation?
  4. What are the targets of Src tyrosine kinase in two-dimensional and three-dimensional cell cultures?  5. What are the mutual roles of reactive oxygen species (ROS) and antioxidants in malignant transformation and its inhibition in Src-transformed MDCK cells in a 3D environment?

The main methods are visualization of cells grown in extracellular matrix (matrigel), using spinning disc confocal microscopy and light sheet confocal microscopy for living cells and point scanning confocal microscopy for fixed cells.

Research Group Members

Project Leader:
Sinikka Eskelinen, Ph.D. (Research Services and Biocenter Oulu)

Ph.D. Students:
Janne Capra, M.Sc. (Biocenter Oulu)

Main source of salary in brackets.

Last updated: 13.10.2016