USING CLONAL SAMPLING TO ANALYSE OESOPHAGEAL EPITHELIAL HOMEOSTASIS

Maria P. Alcolea & Philip H. JonesMRC Cancer Cell Unit, Hutchison-MRC Research Centre, Cambridge, CB2 0XZ, UK

Oesophageal cancer represents one of the most common and aggressive cancers worldwide; the eighth most frequently diagnosed and the 6 th most common cause of death from cancer. Despite these dramatic figures, the oesophagus still remains a little studied tissue. Understanding the mechanisms by which the maintenance of this tissue is achieved constitutes an important step towards the advance on therapeutic treatments for this disease.

Previous studies on adult skin have allowed the development of a quantitative model that exquisitely explains how the maintenance of this tissue is achieved by balanced cell fate decisions (Clayton E, Nature, 2007; Doupe DP, Dev Cell, 2010). A single progenitor cell population divides generating equal amounts of cells able to proliferate again and cells committed to maturation, which do not divide but acquire more specialized tasks. The current study was designed to investigate the mechanisms of homeostasis in the oesophagus, an epithelium with an embryonic origin different from the skin. This was achieved by counting and analyzing the evolution of thousands of clones that originated from single labelled cells in vivo. Interestingly, the results obtained corroborate the observations from the skin; the average size of the clones increases in direct proportion to the time (Fig. 1). This is in accordance with a single cell population maintaining the tissue under normal conditions.

The conclusions drawn from this in vivo cell fate tracking, diverge from the traditional theory of tissue maintenance by stem cells (SC). Until very recently two different populations of progenitor cells (Stem and Transit Amplifying cells) were thought to have distinct properties and were both required for the homeostasis of an adult epithelium. However, the new model of tissue maintenance by a single cell population is becoming more widely accepted given that recent studies have observed a similar behaviour in other experimental set ups, such as the intestine and spermatogenesis, among others (Lopez-Garcia C, Science, 2010; Klein A, Cell Stem Cell, 2010).

We conclude that the oesophageal epithelium is maintained by a single

progenitor population that divides generating equal numbers of progenitor and differentiated cells. This ensures tissue preservation from cellular turnover without the need to recruit any other cell type, e.g. SC, under normal physiological conditions.

It has been widely assumed that a discrete, slow cycling or quiescent SC population is responsible for tissue regeneration and repair after injury or under stress conditions. We

Fig 1. . Clonal analysis of murine oesophagealepithelium. Clonal fate analysis showing probability offinding clones containing n basal cells at different timepoints (from 3 days to one year). Points representdata; error bars, SEM; curves model the behaviour ofprogenitor cells.

Time (days)

Clon

e Fr

eque

ncy

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1 10 100 1000

Clonal distribution

set out to identify the SC population in the oesophagus and to study its contribution to tissue formation under non-homeostatic conditions. We have used transgenic cell labelling to detect slow cycling cells but find no evidence of slow cycling SC in the oesophagus. Neither can we find any evidence of cells expressing known epithelial stem cell markers.

The question remains- how does oesophagus responds to challenges to homeostasis? To this end we studied a genetic mutation inhibiting the Notch pathway in oesophagus (DNMAML1-EGFP). Notch regulates cell behaviour and the mutation we studied is known to increase the likelihood of developing skin cancer in mouse models. Analysis of clones deriving from mutated cells showed a clear alteration in the progenitor cell maturation program. Mutant cells remain in the proliferative compartment, acquiring a clear growth advantage which explains the role of the mutation in preneoplasia (Fig. 2).

In conclusion, the current project has significantly contributed to the understanding of oesophageal epithelial maintenance and plasticity upon tissue disturbance. The quantitative model developed by Jones’ laboratory constitutes therefore an excellent tool in order to define how specific genetic mutations or pharmacological agents can lead to homeostatic imbalance and epithelial disorders. More importantly, this model also represents a powerful approach to study drugs with the potential to restore normal cell behaviour and hence to develop more effective therapies with minimal off target effects.

Contact information:

Researcher: Dr. Maria P. Alcolea (mpa28@cam.ac.uk)Scientific in charge: Dr. Philip H. Jones (phj20@cam.ac.uk)Affiliation: MRC Cancer Cell Unit, Hutchison-MRC Research Centre, Cambridge, CB2 0XZ, UK

EYFP-6mth DNMAML1-6mth DNMAML1-1yr

100µmDAPIFP

Fig 2. Clonal advantage. Long term induction reveals the clear advantage of cells expressing DNMAML1 (FPpositive). Projections of oesophageal wholemounts at 6 months and 1 year post-induction show the ability ofDNMAML1 cells to take over the whole extension of the tissue at the expense of unrecombined cells. Staining forDAPI (blue), fluorescent protein (FP, yellow). Scale bar, 10µm.