Multi-color lineage tracing reveals clonal dynamics of squamous carcinoma evolution from initiation to metastasis

Tumor cells are subjected to evolutionary selection pressures during progression from initiation to metastasis. We analyzed the clonal evolution of squamous skin carcinomas induced by DMBA/TPA treatment using the K5CreER-Confetti mouse and stage-specific lineage tracing. We show that benign tumors are polyclonal, but only one population contains the Hras driver mutation. Benign papillomas are therefore monoclonal in origin, but recruit neighboring epithelial cells during growth. Papillomas that never progress to malignancy retain several distinct clones, whereas progression to carcinoma is associated with a clonal sweep. Newly generated clones within carcinomas demonstrate intratumoral invasion and clonal intermixing, often giving rise to metastases containing two or more distinct clones derived from the matched primary tumor. These data demonstrate that late stage tumor progression and dissemination are governed by evolutionary selection pressures that operate at a multicellular level, and thus differ from the clonal events that drive initiation and the benign-malignant transition.