Background:  Quiescent/slow cycling cells have been identified  in several tumors and correlated with therapy resistance. However, the features of chemoresistant populations and the molecular factors linking quiescence to chemoresistance are largely unknown.

Methods: A population of chemoresistant quiescent/slow cycling cells was isolated through  PKH26 staining  (which allows to separate cells on the basis of their proliferation rate) from colorectal cancer (CRC) xenografts and subjected to global  gene expression and pathway  activation  analyses. Factors expressed by the quiescent/slow

cycling population  were analyzed through  lentiviral overexpression approaches for their ability to induce a dormant chemoresistant state both in vitro and in mouse xenografts. The correlation between  quiescence-associated factors, CRC consensus molecular subtype and cancer prognosis was analyzed in large patient datasets.

Results: Untreated colorectal tumors contain a population of quiescent/slow  cycling cells with stem cell features (quiescent cancer stem cells, QCSCs) characterized by a predetermined  mesenchymal-like chemoresistant phenotype. QCSCs expressed  increased  levels of ZEB2, a transcription factor involved in stem cell plasticity and epithelial-mesenchymal transition (EMT), and  of antiapototic factors pCRAF and pASK1. ZEB2 overexpression upregulated pCRAF/pASK1 levels resulting  in increased chemoresistance, enrichment  of cells with stemness/EMT traits and proliferative slowdown of tumor xenografts. In parallel, chemotherapy  treatment  of tumor xenografts induced the prevalence of QCSCs with a stemness/EMT phenotype  and activation of the ZEB2/pCRAF/pASK1  axis, resulting in a chemotherapy-unresponsive  state. In CRC patients,  increased ZEB2 levels correlated  with worse relapse-free survival and were strongly associated to the consensus molecular subtype 4 (CMS4) characterized  by dismal prognosis, decreased proliferative rates and upregulation  of EMT genes.

Conclusions: These results show that chemotherapy-naive tumors contain a cell population  characterized by a coordinated program of chemoresistance, quiescence, stemness and EMT. Such population becomes prevalent upon drug treatment  and is responsible for chemotherapy resistance, thus representing  a key target for more effective therapeutic approaches.