A marker-independent lineage-tracing system to quantify clonal dynamics and stem cell functionality in cancer tissue

Marker-free lineage tracing reveals an environment-instructed clonogenic hierarchy in pancreatic cancer

“…All lines were transduced with the LVind-LS2 vector to allow marker-independent and permanent labeling of cells and their offspring with mStrawberry following induction with tamoxifen (Figure 1A). Cultures were single-cell cloned as previously described to exclude clonal variation and to ensure homogeneous labeling (Lenos et al, 2018(Lenos et al, , 2019. We confirmed a dose-dependent induction of mStrawberry by tamoxifen in these cultures and ensured that label-positive cells were stable over time, excluding a specific cell population to be preferentially labeled or that mStrawberry expression impacts on expansion rates (Figures S1A and S1B).…”

“…Importantly, we did not observe any induction of apoptosis following the administration of the dose of tamoxifen appropriate for clone induction in vivo (Figure S1E). For PANC-1, we selected 0.025 mg per injection, as this dose resulted in very low clone induction (<1%), to avoid clone collision that would hamper clone size quantification as we previously described (Lenos et al, 2019). For the other four cell lines, we selected tamoxifen concentrations based on the comparison of the in vitro tamoxifen concentration of PANC-1 and the respective line to obtain similar degrees of clonal labeling in vivo (Figure S1F).…”

“…To define the mode of growth of the PDAC xenografts and investigate the contribution of CSCs to this process, we employed the analytical tumor growth framework we previously used for colon cancer (Lenos et al, 2018(Lenos et al, , 2019. Briefly, this quantitative model assumes the existence of both clonogenic and non-clonogenic cells and encompasses three variables that together define the mode of growth of solid cancers (Figure 2A).…”

“…The daughter cells are either stem cells (probability a) or differentiated cells that lost clonogenic capacity (probability 1 À a)). Further, parameter a also defines the mode of growth of (A) Graphical representation of lentiviral vector LV-indLS2 (Rodriguez et al, 2014) and experimental design for marker-free labeling in established pancreatic cancer tumors (Lenos et al, 2018(Lenos et al, , 2019. After subcutaneous tumor formation (T0), a low dose of 4-OH-tamoxifen is administrated and permanent sporadic labeling is followed by tumor isolation at five different time points (T1-T5 the xenograft, which is either exponential growth (a R 0.6) or expansion at the surface (a $0.5).…”

“…We previously demonstrated the utility of a marker-free lineage tracing system to elucidate the clonal dynamics in colon cancer (Lenos et al, 2018(Lenos et al, , 2019. Here we employ a similar unbiased tracing strategy in combination with a quantitative framework to uncover the clonal dynamics that drive tumor growth of human PDAC xenografts.…”