Lentiviral Fluorescent Genetic Barcoding for Multiplex Fate Tracking of Leukemic Cells

Abstract

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.

A Multiplex CRISPR-Screen Identifies PLA2G4A as Prognostic Marker and Druggable Target for HOXA9 and MEIS1 Dependent AML

“…Conceivably, this approach does not allow for a direct comparison of multiple targets, and, thus, consumes cell culture reagents and time, limiting the number of candidate genes that can be characterized in parallel. To alleviate this obstacle, we and others developed the fluorescent genetic barcoding (FGB) technique [21][22][23][24]. FGB utilizes lentiviral vectors to introduce genetically encoded color codes into dedicated cell samples.…”

Longitudinal Monitoring of Intra-Tumoural Heterogeneity Using Optical Barcoding of Patient-Derived Colorectal Tumour Models

“…Although this, in theory, greatly extends the colour repertoire reported in previous techniques such as the Brainbow [18] and Confetti [46] mice, due to technical limitations associated with FC analysis, the quantification was restricted to a maximum of 15 colours per animal. Similarly, Maetzig and colleagues [47] recently described a method of multiplexed fluorescent barcoding that can generate up to 26 different colours; however, only 12 of these were traced in vivo. Although the SILU-based analyzes may need to be revised for ex vivo or intravital colour deconvolution, the FC pipeline presented herein is suitable for the analysis of optically barcoded cells cultured in vitro as well as dissociated primary tumour material recovered from animal models.…”

Fluorescent genetic barcoding for cellular multiplex analyses

“…Therefore, six cell dilutions could be assayed in sixfold repetitions in just six mice compared with 36 mice required for conventional LDA. Likewise, multiplexing reduced the number of recipient mice of in vitro D 1 2 X Xdrugtreated cells for the assessment of anti-leukemic properties in short-term in vivo D 1 3 X Xassays by 12-fold [25]. However, it should be clear that MLDA and short-term drug assays both have limitations in that MLDA will preferentially identify populations with the highest LSC frequency that outcompete smaller input populations and that the potential of multiplex drug assays to quantify long-term in vivo D 1 4 X Xeffects of in vitroD 1 5 X X drugtreated cells must still be demonstrated.…”

“…In contrast to cell lines, genetic modification of HSCs ideally requires vector libraries consisting of single vectors for each color code, such as the 14xFGB and 6xFGB systems, which circumvents the need for additional sorting steps. Additionally, these vectors allow for the direct coupling of color codes to factors that influence cell fate decisions (e.g., single-guide RNA, microRNA, or short-hairpin RNA) [21], and DNA barcode elements as an alternative approach for population tracing and quality control by polymerase chain reaction or sequencing [21,25].…”

“…As a first step toward closing the gap between flow cytometric and sequencing-based multiplexing, Cornils et al developed a lentiviral RGB vector platform in which each of the three fluorescent markers was combined with a unique DNA barcode library [44]. Therefore, advanced FGB vector designs in combination with complex DNA libraries may guide the way toward the development of next-generation cell-tracking tools that should be accompanied by the implementation of algorithm-assisted automated gating routines to fully unleash the potential of high-definition FGB in hematopoietic research [19,25,45,46].…”