Defining the mode of tumour growth by clonal analysis

Abstract

Recent studies using the isolation of a subpopulation of tumour cells followed by their transplantation into immunodeficient mice provide evidence that certain tumours1,2, including squamous skin tumours3-5, contain cells with high clonogenic potential that have been referred to as cancer stem cells (CSCs). Until now, CSC properties have only been investigated by transplantation assays, and their existence in unperturbed tumour growth is unproven. Here we make use of clonal analysis of squamous skin tumours using genetic lineage tracing to unravel the mode of tumour growth in vivo in its native environment. To this end, we used a genetic labelling strategy that allows individual tumour cells to be marked and traced over time at different stages of tumour progression. Surprisingly, we found that the majority of labelled tumour cells in benign papilloma have only limited proliferative potential, whereas a fraction has the capacity to persist long term, giving rise to progeny that occupy a significant part of the tumour. As well as confirming the presence of two distinct proliferative cell compartments within the papilloma, mirroring the composition, hierarchy and fate behaviour of normal tissue, quantitative analysis of clonal fate data indicates that the more persistent population has stemcell-like characteristics and cycles twice per day, whereas the second represents a slower cycling transient population that gives rise to terminally differentiated tumour cells. Such behaviour is shown to be consistent with double-labelling experiments and detailed clonal fate characteristics. By contrast, measurements of clone size and proliferative potential in invasive squamous cell carcinoma show a different pattern of behaviour, consistent with geometric expansion of a single CSC population with limited potential for terminal differentiation. This study presents the first experimental evidence for the existence of CSCs during unperturbed solid tumour growth.Different models have been proposed to explain tumour heterogeneity and growth1,6,7. In the stochastic model, all tumour cells are considered equipotent and only a fraction (due to Correspondence Here we sought to investigate the fate of individual tumour cells within their natural microenvironment in the early stage of squamous skin tumours. Chemical-induced carcinogenesis is the most commonly used tumour model in mice9,10. Although this model may only imperfectly mimic the occurrence of human skin cancers, it has proved to be extremely useful in dissecting the different steps of tumour initiation, promotion and malignant progression, and has been instrumental in defining many paradigms in cancer biology relevant to human pathology9,11,12. In this protocol, mice are first treated with the carcinogen DMBA (7,12-dimethylbenz[α]anthracene) followed by continuous administration of the mitogen TPA (12-O-tetradecanoylphorbol-13-acetate). During the course of TPA administration, benign tumours, known as papilloma, appear (initiation) and grow steadily (promotion), w...

Cancer stem cell definitions and terminology: the devil is in the details

“…In this case, the role of specific genes in the spontaneous generation of tumours can be examined using transgenic technology, as well as transplantation assays that can use syngeneic recipients of any age. More recently, lineage tracing has been exploited to add further strong evidence of CSCs in glioblastomas, intestinal adenomas and epidermal tumours developing de novo in mice and, in the case of the glioblastoma, their anticipated drug resistance was also demonstrated [61][62][63] . These findings highlight how mouse models of tumorigenesis may facilitate the future development of methods for interrogating CSCs in patient samples for clinical purposes.…”

Cancer Stem Cells: Formidable Allies of Cancer

“…This suggested that the former subset included the differentiated/progenitor cells, while the later might be stem cells which maintain the tumour growth. Additionally, the growth characteristic of the tumour was reminiscent of the normal skin stratified architecture [57]. Taken together, these three mouse models, employing skillful imaging and lineage tracking studies, proved beyond doubt the existence of CSC and their role in tumour initiation and relapse.…”

“…Schepers et al used the intestinal adenoma (a premalignant precursor lesion; driver mutation is APC null mutation)-prone mouse model and Driessens et al used a benign papilloma mouse model to drive home an important point: tumours are maintained by dedicated cancer stem cells, and tumour growth is analogous to normal tissue renewal and architecture [56,57]. Schepers et al brought a loxP cassette containing four fluorescent labels in the background of adenoma-prone mouse which conditionally express Cre recombinase and in which intestinal stem cells are labelled with GFP.…”

Emerging Roles of Krüppel-Like Factor 4 in Cancer and Cancer Stem Cells

“…In most cases, they are referred to as tumor-initiating cells (TICs). The existence of CSC population remains a controversial topic due to questions about the robustness of CSC markers (Magee et al, 2012), however, three independent studies successfully identified and tracked CSCs in brain, skin and intestinal tumors of mice Driessens et al, 2012;Schepers et al, 2012). These findings might provide further evidence to support that CSCs are the driving force of tumor development (Gilbertson and Graham, 2012).…”