Optical Barcoding for Single-Clone Tracking to Study Tumor Heterogeneity

Abstract

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.

Lentiviral Fluorescent Genetic Barcoding for Multiplex Fate Tracking of Leukemic Cells

“…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 .…”

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

“…37,38 To further extend the power of FGB approaches, multiplexing of up to 12 populations can readily be achieved with the FGB-BC vector platform by mixing of FGB-BC color-coded cells derived from CD45.1 and CD45.2 murine cell lines for competitive in vitro tracking or transplantation into CD45.1xCD45.2 F1 mice. Importantly, in an independent study published in this issue of Molecular Therapy, Mohme et al 39 present an extended color-coding strategy based on a set of six lentiviral vectors expressing unique fluorescent proteins for the combinatorial transduction of cells with up to three fluorescent markers. Although this approach resulted in 41 traceable clones, complex immunophenotyping potential might be limited due to the occupation of at least 6 channels in flow cytometers by genetically encoded fluorescent markers, and clonal identities may change due to vector silencing in co-transduced cells.…”

The Clonal Fate of Live Cells

“…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…”

“…Both fluorescent barcoding strategies rely on a lentiviral vector system expressing the distinct fluorophores. Mohme et al 4. used a binary codification of fluorescent signal patterns reaching 41 color combinations to track tumorgenic cells and its progeny.…”

“…In contrast, Mohme et al 4 . employed optical barcoding in a strategy that made use of up to six different fluorophores—EBPF2, eGFP, mOrange2, T-Sapphire, Venus, dKatushka—to monitor the fate of clonal subpopulations in vivo (Figure 1).…”