Fig. 1. Establishing an inducible knockdown system in PDX acute leukemia cells in vivo.

a Overview of the experimental setup: Primary acute leukemia (AL) cells were amplified in NSG mice and serially passaged PDX cells lentivirally transduced twice in a row; first to constitutively express Cre-ER^T2 together with mCherry and a luciferase (Luc); second to express inducible knockdown vectors containing (i) a constitutively expressed fluorochrome marker (either iRFP or mTagBFP) and (ii), placed in antisense orientation, a miR30-based knockdown cassette coupled to a second inducible fluorochrome (either T-Sapphire or eGFP). After amplification in mice, purified transgenic PDX cells were mixed 1:1 and transplanted into next recipient mice for competitive in vivo experiments. In mice with established leukemias, TAM was administered to induce Cre-ER^T2-mediated recombination. Recombination inverted the knockdown cassette and induced (i) expression of the shRNA; (ii) deletion of the constitutive fluorochrome (either iRFP or mTagBFP) and (iii) expression of the inducible fluorochrome (either T-Sapphire or eGFP; see Fig. S1b for detailed description). As result, T-Sapphire positivity indicated cells expressing the shRNA targeting a control (shCTRL), while eGFP positivity indicated cells expressing the shRNA targeting the gene-of-interest (shGOI). If the GOI harbors an essential function, the eGFP-positive population gets lost over time in vivo, indicating that the patient might profit from drugs targeting the GOI. b Switch in fluorochrome expression upon Cre-ER^T2-recombination: Double transgenic PDX AML-388 cells expressing Cre-ER^T2 together with either iRFP/shCTRL or mTagBFP/shGOI (shMCL1) were mixed 1:1 and injected into the tail vein of NSG mice (3×10⁵ cells/mouse; n = 14). 7 days after injection, 2 mice were sacrificed and PDX cells analyzed by flow cytometry for all 4 fluorochromes. In the remaining mice, 50 mg/kg TAM was administered by oral gavage to induce Cre-ER^T2-mediated recombination. Resulting increase in T-Sapphire or eGFP expression, indicating expression of shCTRL and shGOI, respectively, was measured in PDX cells isolated from mice at the indicated time points (24, 36, 52 and 72 h after TAM; n = 3 per time point). Representative histograms and plots are shown. c Typical results for a GOI with essential function: Upper scheme depicts the experimental procedure: For pairwise competitive assays, mice were injected with either of two mixtures: a control mixture of iRFP/shCTRL and mTagBFP/shCTRL (short shCTRL/shCTRL) or the experimental mixture iRFP/shCTRL and mTagBFP/shGOI (short shCTRL/shGOI); as GOI, the apoptosis regulator MCL1 was chosen (shGOI = shMCL1) (3×10⁵ cells/mouse, data from 4 exemplary mice are shown). TAM was administered 7 days after injection (day 0). Mice were sacrificed 3 and 26 days after TAM and PDX cells analyzed for expression of the inducible fluorochromes T-Sapphire and eGFP. Density plots show representative results for both mixtures on day 3 (left) and day 26 (right). Right panels show quantification as percentage of [eGFP/shGOI positive cells divided by (the sum of T-Sapphire/shCTRL and eGFP/shGOI positive cells)]; the shCTRL/shCTRL mixture is analyzed and depicted, respectively.