High-complexity cellular barcoding identifies clonal substitution as a hallmark of local recurrence in a surgical model of head and neck cancer

The survival prognosis for head and neck squamous cell carcinoma (HNSCC) remains dismal mainly because of insufficient local and loco-regional control resulting in a significant number of treatment failures. Improving this outcome requires a better understanding of the invading cancer cells that re-grow from postsurgical minimal residual disease (MRD). Using cells tagged with multiple fluorescent proteins and a highly complex library of DNA barcodes, we followed clonal dynamics in a surgical mouse model of HNSCC. High-resolution clonal deconvolution of tumors demonstrated a consistent substitution of dominant clones from primary to matched recurrent tumors by initially rare clones tending to be accumulated at the primary tumor periphery. These recurrent tumor-initiating cells (RTICs) were characterized by their ability to disseminate and undergo a progressing epithelial-mesenchymal transition (EMT), thus suggesting that therapies targeting invasion and EMT (e.g., inhibitors of c-MET) could limit the development of recurrences. RTICs pre-existed in a complex population of cancer cells and may share a common origin with tumor-initiating cells (TICs). Overall, our data suggest that clonal mechanisms drive the growth of recurrent HNSCC from MRD, and demonstrate that the reported approach is well suited to explore the biology of RTICs.