A Experimental procedure stem cell clones were marked with an individual combination of fluorochromes, mixed and injected into mice for multiplex competitive in vivo experiments. Depicted are major chromosomal changes and AML related mutations at each intersection (black), numbers of individual clones (colored boxes), and name of clusters (colored letters)Ī transcriptome based score from cytarabine resistant PDX clones predicts clinical outcome in AML patients. 50 SNVs of the trunk refer to the complete remission control. H 424 single nucleotide variants (SNVs) were identified from exome sequencing and used to calculate a phylogenetic tree the length of each branch correlates to number of SNV changes (grey boxes). G Gene expression profile was analysed via prime-seq from 3–4 biological replicates per clone and a t-distributed stochastic neighbor embedding (t-SNE) plot built by unsupervised clustering. Mean (solid line) ± 95% CI (dashed line) is depicted. Frequency of LIC and statistical significance was calculated using the ELDA software. F Leukemia initiating cell (LIC) frequency of clone 4 ( NRAS Q61K) and clone 8 ( NRAS wt) cells were injected into mice in limiting dilutions and positive engraftment analyzed. E NRAS Q61K was determined in PDX clones and compared to proportion of NRAS Q61K cells within bulk REL1 and REL2 PDX cells (mean ± SD, see B). PDX populations consisting of a single barcode were defined as single stem cell clones (red box). D Numbers of barcodes within REL1 or REL2 populations one dot represents one mouse. At advanced leukemia, PDX cells were re-isolated and barcodes quantified. C Experimental procedure passage-1 bulk REL1 or REL2 PDX cells were transduced with a genetic barcode and marker + cells injected into mice in limiting dilutions (REL1: 1100–33,000 cells, n = 18 REL2: 100–10,000 cells, n = 11).
C–H Generation and characterization of single PDX AML stem cell clones. Variant allele frequency (VAF) is depicted. B Primary tumor ( n = 1), REL1 PDX ( n = 9) and REL2 PDX ( n = 3) cells were analyzed by targeted sequencing. REL1 and REL2, but not ID, allowed engraftment. A Primary AML cells from a 52-year-old female patient at time of initial diagnosis (ID), first (REL1) and second relapse (REL2) were transplanted into NSG mice. Sequencing divided 12 PDX AML single stem cell clones according to first and second relapse.
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