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

Maetzig, Tobias; Rüschmann, Jens; Lai, Courteney K.; Ngom, Mor; Imren, Suzan; Rosten, Patty; Norddahl, Gudmundur L.; Krosigk, Niklas von; Milde, Lea Sanchez; May, Christopher G.; Selich, Anton; Rothe, Michael; Dhillon, Ishpreet; Schambach, Axel; Humphries, R. Keith

doi:10.1016/j.ymthe.2016.12.005

Cited by 17 publications

(39 citation statements)

References 45 publications

(68 reference statements)

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“…That approach nicely demonstrated that hepatocytic and T cell lines can be fluorescently labeled with three colors and can subsequently be single-cell sorted to generate individual clones barcoded by different fluorescence intensities. 14 Notably, Maetzig et al 26 also recently introduced a further advancement and extension of the FGB approach based on lentiviral vectors.…”

Section: Discussionmentioning

confidence: 99%

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

et al. 2017

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Intratumoral heterogeneity has been identified as one of the strongest drivers of treatment resistance and tumor recurrence. Therefore, investigating the complex clonal architecture of tumors over time has become a major challenge in cancer research. We developed a new fluorescent "optical barcoding" technique that allows fast tracking, identification, and quantification of live cell clones in vitro and in vivo using flow cytometry (FC). We optically barcoded two cell lines derived from malignant glioma, an exemplary heterogeneous brain tumor. In agreement with mathematical combinatorics, we demonstrate that up to 41 clones can unambiguously be marked using six fluorescent proteins and a maximum of three colors per clone. We show that optical barcoding facilitates sensitive, precise, rapid, and inexpensive analysis of clonal composition kinetics of heterogeneous cell populations by FC. We further assessed the quantitative contribution of multiple clones to glioblastoma growth in vivo and we highlight the potential to recover individual viable cell clones by fluorescence-activated cell sorting. In summary, we demonstrate that optical barcoding is a powerful technique for clonal cell tracking in vitro and in vivo, rendering this approach a potent tool for studying the heterogeneity of complex tissues, in particular, cancer.

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Section: Discussionmentioning

confidence: 99%

Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

et al. 2017

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“…Seminal studies utilized stochastic combinatorial expression methods of various fluorescent proteins in transgenic animals or in retrovirally transduced cells for creating defined hues compatible with fluorescent microscopy-assisted clonal cell tracking 26, 27, 28. Refinement of these techniques enabled the flow cytometric characterization of multiple uniquely labeled cell populations in multiplex assays by employing pre-defined fluorescent color codes as recently demonstrated by the in vivo tracking of transgenic murine HSC and lentivirally marked glioblastoma clones 29, 30, 31, 32. In our work, we further expanded the scope of flow cytometric tracking applications by developing three lentiviral fluorescent genetic barcoding (FGB) vector systems 32 .…”

Section: Introductionmentioning

confidence: 99%

Lentiviral Fluorescent Genetic Barcoding for Multiplex Fate Tracking of Leukemic Cells

Maetzig

Rüschmann

Milde

et al. 2017

Molecular Therapy - Methods & Clinical Development

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Tracking the behavior of leukemic samples both in vitro and in vivo plays an increasingly large role in efforts to better understand the leukemogenic processes and the effects of potential new therapies. Such work can be accelerated and made more efficient by methodologies enabling the characterization of leukemia samples in multiplex assays. We recently developed three sets of lentiviral fluorescent genetic barcoding (FGB) vectors that create 26, 14, and 6 unique immunophenotyping-compatible color codes from GFP-, yellow fluorescent protein (YFP)-, and monomeric kusabira orange 2 (mKO2)-derived fluorescent proteins. These vectors allow for labeling and tracking of individual color-coded cell populations in mixed samples by real-time flow cytometry. Using the prototypical Hoxa9/Meis1 murine model of acute myeloid leukemia, we describe the application of the 6xFGB vector system for assessing leukemic cell characteristics in multiplex assays. By transplanting color-coded cell mixes, we investigated the competitive growth behavior of individual color-coded populations, determined leukemia-initiating cell frequencies, and assessed the dose-dependent potential of cells exposed to the histone deacetylase inhibitor Entinostat for bone marrow homing. Thus, FGB provides a useful tool for the multiplex characterization of leukemia samples in a wide variety of applications with a concomitant reduction in workload, processing times, and mouse utilization.

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“…Technologies for real-time in vivo monitoring of multiple cell populations, such non-invasive three-dimensional visualization of single cells, are still at their infancy. However, in the March issue of Molecular Therapy , two independent studies present substantially improved multiplex fluorescent barcoding strategies to mark and visualize clonal cell subpopulations 3, 4…”

Section: Main Textmentioning

confidence: 99%

“…Future scaling of the multiplicity of individual traceable clones can then only be achieved by increasing the color space or, more likely, by the use of random color-specific genetic barcodes introduced into the vector as in Cornils et al 7 Figure 1Schematic Comparison of the In Vivo Clonal Tracking Strategies Presented by Maetzig et al 3 . and Mohme et al 4 …”

Section: Main Textmentioning

confidence: 99%