Lineage tracing reveals the cellular basis of early metastasis in oesophageal squamous carcinoma


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Julia Frede1, Philip Greulich2, Benjamin Simons2, Maria Alcolea1, Philip Jones1
1MRC Cancer Cell Unit, Cambridge, UK, 2University of Cambridge, Cambridge, UK

Background

Oesophageal squamous cell carcinoma has a poor prognosis and is characterised by early invasion and metastasis. Here we apply large scale genetic lineage tracing and statistical physics to resolve cell behaviour in a new model of oesophageal cancer development in transgenic mice.

Method

To generate tumours, we used Sorafenib, a multikinase inhibitor that causes cutaneous squamous cell carcinoma (SCC) as a side effectin cancer patients. Mice carrying a conditional fluorescent protein reporter allele and an inducible form of cre recombinase were treated with a low dose of the cigarette derived carcinogen, diethlynitrosamine (DEN), followed by Sorafenib or vehicle control. Cells were then genetically labelled1. We applied statistical physics to resolve cell behaviour from the clone size distributions.

Results

Treatment with Sorafenib alone produces dramatic increases in normal oesophageal progenitor cell division and stratification rates, but did not alter the normal balanced production of proliferating and differentiating cells by oesophageal progenitors. In combination with DEN, however, Sorafenib produces multiple tumours, whereas none are seen in DEN + vehicle controls. The lesions appear intraepithelial by conventional histology, but confocal imaging reveals stromal inflammation, associated with Stat3 phosphorylation, angiogenesis and lymphangiogenesis, all features of invasive oesophageal SCC. Lineage tracing within tumours reveals a dramatic change in cell dynamics. The lesions grow through a combination of increased proliferation, generating cells at double the rate of uninvolved epithelium, and a block in cell shedding leading to the accumulation of differentiating cells. Remarkably, genetically labelled keratinocytes are seen undergoing epithelial-mesenchymal transition, invading the stroma and entering blood and lymph vessels.

Conclusion

Lymphovascular intravasation occurs fromsquamous tumourswhich areintraepithelial by conventional histology and have marked effects on the underlying stroma usually associated with invasive lesions. These findings explain the early metastasis of oesophageal SCC and validate a new model to test therapeutic interventions.