A live single-cell reporter system reveals drug-induced plasticity of a cancer stem cell-like population in cholangiocarcinoma

Cancer stem cells (CSC) play an important role in carcinogenesis and are acknowledged to be responsible for chemoresistance in cholangiocarcinoma (CCA). Studying CCA CSC has been challenging, due to lack of consensus CSC markers, and to their plastic nature. Since dual expression of the core pluripotent factors SOX2/OCT4 has been shown to correlate with poor outcome in CCA patients, we selected the SOX2/OCT4 activating short half-life GFP-based live reporter (SORE6-dsCopGFP) to study CSC dynamics at the single-cell level. Transduction of five human CCA cell lines resulted in the expression of 1.8-13.1% GFP-positive (SORE6^POS) cells. By live imaging, we found that SORE6^POS CCA cells possess self-renewal capacity and that they can be induced to differentiate. Significantly, the SORE6^POS cells were highly tumorigenic, both in vitro and in vivo, thus implicating the characteristics of primary CSCs. When we then analyzed for selected CSC-related markers, we found that the majority of both CD133⁺/CD44⁺, and CD133⁺/LGR5⁺ CCA cells were SORE6^POS cells. Exposing transduced cells to standard CCA chemotherapy revealed higher growth rate inhibition at 50% (GR₅₀s) for SORE6^POS cells compared to GFP-negative (SORE6^NEG) ones indicating that these CSC-like cells were more resistant to the treatment. Moreover, the chemotherapy induced SORE6^POS from SORE6^NEG cells, while retaining the existing SORE6^POS population. Finally, treatment of transduced cells with CDK4/6 inhibitors in vitro for 3 days resulted in a lowered CSC number in the culture. Thus, applying a live reporter system allowed us to elucidate the stem cell diversity and drug-induced plasticity of CCA CSCs. These findings have clear implications for future management of such patients.