Journal: Nature cancer

Article Title: Multifunctional barcoding with ClonMapper enables high-resolution study of clonal dynamics during tumor evolution and treatment

doi: 10.1038/s43018-021-00222-8

Figure Lengend Snippet: a–c, Experimental workflow for the multifunctional ClonMapper system. Barcoding and selection of tagged cells (a). Characterization of clonal dynamics through targeted barcode sequencing and clone-resolved scRNA-seq (b). Live clone retrieval and functional analysis of clones of interest as determined in b (c). d, Parallel drug treatment replicates of eight ClonMapper-tagged HG3 and REC1 cell lines. TP, time point. Red indicates treatment window (top). Unique barcode counts at TP0 (n = 1 barcoded parental cell culture population) and in TP1 (n = 8 parallel barcoded cell culture replicates) replicates for the HG3 and REC1 cell lines (error bars denote mean ± s.d.) (bottom). e, Percentage of observed unique barcodes present in each TP1 replicate and shared across replicates (n = 8 parallel barcoded cell culture replicates) (left). Shared barcodes weighted by population abundance (right). f–i, Uniform manifold approximation and projection (UMAP) of 8,975 cells from scRNA-seq of the HG3 barcoded population at TP0 and TP1. Sample annotations are green, TP0; blue, TP1 replicate 1; and purple, TP1 replicate 7 (f). Leiden cluster annotations reveal substantial pre- and post-treatment heterogeneity (g). Annotation of the ten most abundant clones reveals their exclusive presence in TP1 clusters 0, 1, 3, 4, 7 and in TP0 cluster 5 (each color represents a unique clone) (h). Clonal survivorship classification based on clonal residence in either TP1 clusters 0, 1, 3, 4 and 7 (red, HS) or cluster 6 (blue, LS) (i). Clones not detected in TP1 are labeled in gray (unassigned).

Article Snippet: Cell lines utilized in this study were cultured as follows: HG3 (DSMZ, ACC 765): RPMI 1640 (Gibco, 11875–093), 15% fetal bovine serum (FBS) (Sigma-Aldrich), 1% GlutaMAX (Gibco, 35050–061), 1% penicillin–streptomycin (PS) (Life Technologies).

Techniques: Selection, Sequencing, Functional Assay, Clone Assay, Cell Culture, Labeling

Journal: Nature cancer

Article Title: Multifunctional barcoding with ClonMapper enables high-resolution study of clonal dynamics during tumor evolution and treatment

doi: 10.1038/s43018-021-00222-8

Figure Lengend Snippet: a, UMAP of expression profiles derived from bulk RNA-seq of 6 untreated HG3 samples (TP0) and 610 treatment-naïve primary CLLs annotated by expression cluster (left) and IGHV mutation status (right)39. All 616 samples were pre-processed together by using PEER factors to remove technical effects69. b, Hierarchical clustering of the expression profiles used in (a) further supports the similarity between HG3 and IGHV-unmutated primary CLLs as noted by the dendrogram sub-tree highlights (red, HG3; yellow, IGHV-unmutated). c, Diversity of the ClonMapper library as estimated by deep sequencing. Green dots correspond to total unique read counts at corresponding read depths. Approximately 68 million unique barcodes were identified at a read depth of 138 million reads (red line), and fitting of the sampled data (blue line) yields unique barcode estimates of ~7.6 × 107. d, Barcode frequency distribution of the ClonMapper library as quantified by deep sequencing (depth of 138 million reads with no base below Q30). e, CellTiter-Glo analysis of 72-hour dose curves using each chemotherapeutic alone (n = 2 cell culture replicates from one independent experiment, respectively) or (f) in combination at molar ratios equivalent to each drug’s respective LD50 concentrations (HG3 n = 2, REC1 n = 3 cell culture replicates as in d above). g, Ridgeline plots representing the log2 fold change of barcode proportions from TP0 to TP1, where barcodes are grouped by their presence across 8 barcoded cell culture replicates. Only barcodes at or above 0.005% (HG3) and 0.0005% (REC1) abundance at TP0 are shown. h, Unique barcode read counts of TP0 and TP1 barcoded cell culture replicates 1–8 normalized to log10 counts per million. Barcodes are sorted in descending order by the sum of their counts across TP1 columns. i, Venn diagrams of clones occupying HS versus LS clusters at TP0 and TP1.

Article Snippet: Cell lines utilized in this study were cultured as follows: HG3 (DSMZ, ACC 765): RPMI 1640 (Gibco, 11875–093), 15% fetal bovine serum (FBS) (Sigma-Aldrich), 1% GlutaMAX (Gibco, 35050–061), 1% penicillin–streptomycin (PS) (Life Technologies).

Techniques: Expressing, Derivative Assay, RNA Sequencing Assay, Mutagenesis, Sequencing, Cell Culture, Clone Assay