RNA sequencing of long-term label-retaining colon cancer stem cells identifies novel regulators of quiescence

Abstract

Summary Recent data suggest that therapy-resistant quiescent cancer stem cells (qCSCs) are the source of relapse in colon cancer. Here, using colon cancer patient-derived organoids and xenografts, we identify rare long-term label-retaining qCSCs that can re-enter the cell cycle to generate new tumors. RNA sequencing analyses demonstrated that these cells display the molecular hallmarks of quiescent tissue stem cells, including expression of p53 signaling genes, and are enriched for transcripts common to damage-induced quiescent revival stem cells of the regenerating intestine. In addition, we identify negative regulators of cell cycle, downstream of p53, that we show are indicators of poor prognosis and may be targeted for qCSC abolition in both p53 wild-type and mutant tumors. These data support the temporal inhibition of downstream targets of p53 signaling, in combination with standard-of-care treatments, for the elimination of qCSCs and prevention of relapse in colon cancer.

Protocol for isolation and functional validation of label-retaining quiescent colorectal cancer stem cells from patient-derived organoids for RNA-seq

“…Cells are stained for F-ACTIN (green), and nuclei are counterstained with DAPI (blue) (scale bars, 20 μm; scale bar for zoomed image, 5 μm). Images used with permission under a CC-BY 4.0 license from ( Regan et al., 2021 ). Culture plates at 37°C for 12 days, changing media every 2 days.…”

“…We have successfully used this protocol to isolate PKH26 LRCs and reported the first whole-transcriptome analyses of functionally validated qCSCs in a panel of colon cancer PDOs ( Regan et al., 2021 ). These cells maintain a large proliferative capacity, persist long term in vivo, and display the molecular hallmarks of quiescent tissue stem cells ( Cheung and Rando, 2013 ).…”

“…Tumor material used for this protocol was obtained with informed consent from CRC patients under approval from the local Institutional Review Board of Charité University Medicine (Charité Ethics Committee, Berlin, Germany) (EA 1/069/11), the ethics committee of the Medical University of Graz (Graz, Austria) and by the ethics committee of the St John of God Hospital Graz (23-015 ex 10/11). For additional information see ( Regan et al., 2021 ).…”

“…This protocol can also be applied to normal tissue-derived organoids. For complete details on the use and execution of this protocol, please refer to Regan et al. (2021) .…”

“…For complete details on the use and execution of this protocol, please refer to Regan et al. (2021) .…”