Tracing Dynamics and Clonal Heterogeneity of Cbx7-Induced Leukemic Stem Cells by Cellular Barcoding

Abstract

SummaryAccurate monitoring of tumor dynamics and leukemic stem cell (LSC) heterogeneity is important for the development of personalized cancer therapies. In this study, we experimentally induced distinct types of leukemia in mice by enforced expression of Cbx7. Simultaneous cellular barcoding allowed for thorough analysis of leukemias at the clonal level and revealed high and unpredictable tumor complexity. Multiple LSC clones with distinct leukemic properties coexisted. Some of these clones remained dormant but bore leukemic potential, as they progressed to full-blown leukemia after challenge. LSC clones could retain multilineage differentiation capacities, where one clone induced phenotypically distinct leukemias. Beyond a detailed insight into CBX7-driven leukemic biology, our model is of general relevance for the understanding of tumor dynamics and clonal evolution.

Clonal selection and asymmetric distribution of human leukemia in murine xenografts revealed by cellular barcoding

“…Single cell suspensions were prepared by crushing in lysis solution (NH 4 Cl) and/or filtering through a 100-M filter, as previously described. 29,32 Cells were analyzed by flow cytometry ( Figure 3B; supplemental File 5) and stored in pellets of 1 to 5 3 10 6 for barcode analysis.…”

“…27 In contrast, in vivo, leukemia induced by artificial oncogenic overexpression rapidly evolved toward mono-or oligoclonality. 28,29 It is unclear to what extent the murine environment contributes to these differences 30 and whether these models reflect the competitive biological behavior of patient-derived leukemia clones.…”

A Lentiviral Fluorescent Genetic Barcoding System for Flow Cytometry-Based Multiplex Tracking

“…This design would allow for flow cytometric tracking of color-coded populations as well as next generation sequencing-based assessment of clonality in multiplexed samples, which may be especially powerful when working with HSCs and leukemic stem cells. [2][3][4]33 To this end, we established color coding conditions for murine HSCs and human CB derived CD34 + HSPC and evaluated the in vivo color code distribution in different BM subpopulations at the end of the observation period (Figures 5 and 6). To neglect cell type-specific differences in CSF promoter activity as well as potential variations in expression intensity due to the influence of the activation status of the (e.g., T) cells, gates were first set based on surface marker expression before analyzing color-coded populations therein.…”

“…While gammaretroviral or lentiviral delivery of readily detectable reporters has initially proven valuable for enrichment and tracking of transduced populations, viral integration site analysis and retroviral barcoding have further improved clonal resolutions. [1][2][3][4][5][6][7] The power of integration site analysis and DNA barcoding lies in parallel cell fate tracking, which opens up numerous experimental opportunities including in vivo applications in a reduced number of mice enabling decreased variability and improved efficiencies. 8 Application of such tracking and fate mapping approaches have for example been extensively and elegantly exploited in the context of studies in the hematopoietic system both in vivo and in vitro.…”

“…8 Application of such tracking and fate mapping approaches have for example been extensively and elegantly exploited in the context of studies in the hematopoietic system both in vivo and in vitro. [2][3][4]9,10 Despite these opportunities, molecular multiplexing generally imposes a substantial delay between sample acquisition and data mining due to the requirement for PCR amplification steps, next generation sequencing, and bioinformatics analyses. 8 For accelerated multiplex characterization of a smaller number of cell populations as required for the comparison of culture conditions, drug treatment responses, or replicate experiments in the same mouse or culture well, a real-time readout system would be desirable.…”