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M. Chapellier et al.
labeling of leukemia cell populations with distinct molec- ular barcodes followed by exposure to separate experi- mental conditions. Each labeled cell population was stim- ulated with one cytokine ex vivo and after culture, leukemia cells from multiple cytokine conditions were pooled prior to in vivo competition. To trace the effects of the cytokines to the leukemia-initiating capacity of bar- coded cells, the representation of individual barcodes in vivo was assessed using NGS (Figure 1A).
To allow for in vivo competition of up to 11 barcoded cell populations, we generated 11 lentiviral vectors with unique molecular barcodes and GFP as a marker gene (Online Supplementary Table S1). The barcodes were used to mark c-Kit+ murine MLL-AF9 AML cells generated on a dsRed transgenic background (Figure 1A and Online Supplementary Figure S1A). We have previously used these cells in screens because they have a well-defined leukemia-initiating cell population and initiate AML with a short latency, enabling rapid follow-up experiments in syngeneic hosts.4,9,10,14,15
To validate the robustness of the new methodology, 11 barcoded c-Kit+ leukemia cell populations were exposed separately for three days to stem cell factor (SCF), which binds c-Kit and activates signaling that promotes leukemia-initiating cells.16 Leukemia cells from the 11 bar- coded cell populations were then pooled and injected into sublethally irradiated mice for in vivo competition of
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leukemia-initiating activity. Seven days post transplanta- tion, mice were sacrificed, their BM was harvested, and DNA was extracted. We found that the 11 barcoded vari- ants showed a similar distribution with less than 2-fold variability in the input pool relative to the in vivo pool (range: 0.51- to 1.65-fold), demonstrating that the arrayed barcoding methodology was robust in assessing the leukemia-initiating activity of the AML cells following ex vivo culture (Figure 1B).
Identification of TNFSF13 as a positive regulator of acute myeloid leukemia-initiating cells
To assess the impact of a library of 114 murine cytokines on c-Kit+ MLL-AF9 leukemia cells, we per- formed two arrayed ex vivo cytokine screens, in which each cytokine was assessed in triplicate wells (Online Supplementary Table S2). After three days of ex vivo cytokine stimulation, up to 11 cytokine conditions of bar- coded leukemia cells were pooled and injected into recip- ient mice (Figure 1A). As an internal positive control, for each pool, we stimulated one of the barcoded cell popula- tions with SCF. The mice were sacrificed on day 7 (screen I) or day 12 (screen II) post transplantation, and engraft- ment of transduced leukemia cells was confirmed by assessing the frequency of GFP positive cells within dsRed+ cells (Online Supplementary Figure S1B and C). As predicted, barcoded leukemia cells stimulated with SCF
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Figure 1. A barcoded cytokine screen identifies TNFSF13 as a positive regulator of acute myeloid leukemia (AML)-initiating cells. (A) Schematic flowchart depicting the arrayed barcoded ex vivo cytokine screen with AML cells. In total, 12 pools in triplicate were used to screen the entire cytokine library. After seven (screen I) or 12 (screen II) days, mice were sacrificed. Data were normalized to the input representation for each barcode. (B) Pie chart displaying the contribution of each bar- coded cell population in vivo following ex vivo cultures with stem cell factor (SCF) only for all barcoded cell populations. (C) Scatter plot showing fold-change in vivo versus input of barcoded cell populations for the two screens. Dotted lines represent a fold-change threshold of 2 to identify cytokines that promote leukemia-initi- ating cells. Red dots: barcoded cell populations stimulated ex vivo with SCF as the positive control within each pool; blue dots: cells stimulated with TNFSF13. NGS: next-generation sequencing.
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haematologica | 2019; 104(10)