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

Journal: MedComm

Article Title: Targeting chronic lymphocytic leukemia with B‐cell activating factor receptor CAR T cells

doi: 10.1002/mco2.716

Figure Lengend Snippet: Cytotoxicity of B‐cell activating factor receptor (BAFF‐R) chimeric antigen receptor (CAR) T cells against three chronic lymphocytic leukemia (CLL) cell lines in vitro. BAFF‐R (A) or CD19 (B) expression in MEC‐1 WT, HG‐3, or CII cell lines were characterized by BAFF‐R‐AF647 or CD19 APC antibodies staining and assessed using flow cytometry. (C and D) BAFF‐R CAR T cells were incubated with MEC‐1 WT, HG‐3, or CII cells at an E:T ratio of 2:1. CD107a‐positive cell analysis, which was utilized to indicate antigen‐specific cytotoxicity, was focused on CD8 + (C) or CD4 + CAR T cells (D); non‐CAR T cells were used as negative controls. (E) Additionally, cytotoxicity of BAFF‐R CAR T cells was observed with the co‐incubated with either MEC‐1 WT, HG‐3, or CII cell lines at an E:T ratio of 4:1. Interferon‐gamma (IFN‐γ) was measured from the harvested supernatant after 72 h. Non‐CAR T cells were used as controls. The results were showed as mean ± SEM from four replicates and representative from three independent experiments ( ∗∗∗ p < 0.001).

Article Snippet: Nalm‐6, MEC‐1, HG‐3, and CII cell lines were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig).

Techniques: In Vitro, Expressing, Staining, Flow Cytometry, Incubation, Cell Analysis

Journal: Bioinformatics

Article Title: SurfaceGenie: a web-based application for prioritizing cell-type-specific marker candidates

doi: 10.1093/bioinformatics/btaa092

Figure Lengend Snippet: GenieScore components and application to two proteomic analyses of four lymphocyte lines. (A) The features of a protein that were hypothesized to predominate the capacity of a protein to serve as a cell surface marker are shown with the names of the mathematical terms derived to represent them. The marker potential features are annotated by the applied approach (i.e. predictive or experimental) to answer the relevant questions. The remaining panels depict the distribution of the individual components and GenieScores calculated from the data acquired from application of whole-cell lysate (WCL) or Cell Surface Capture (CSC) to four lymphocyte cell lines (n = 3 per cell line, N = 485 data points for WCL, N = 325 data points for CSC). (B) A histogram depicting the distribution of SPC scores within predicted surface proteins (SPC score >0) identified by application of WCL and CSC. (C) A violin plot depicting the distribution of signal dispersion for the predicted surface proteins identified by WCL and CSC. (D) A violin plot depicting the distribution of signal strength for the predicted surface proteins identified by WCL and CSC. (E) Plot of GenieScore against rank-order of candidate cell surface markers for predicted surface proteins identified by WCL. (F) Plot of GenieScore against rank-order of candidate cell surface markers for predicted surface proteins identified by CSC. (G) GenieScores calculated using either WCL or CSC data are plotted against each other the 91 proteins identified by both approaches along with the Spearman’s Correlation for those scores

Article Snippet: 2.1 Cell culture Human lymphocyte cell lines (Ramos, HG-3, RCH-ACV, Jurkat) were cultured and passaged as described previously ( Haverland et al. , 2017 ). α-TC1 clone 6 (CRL-2934; ATCC, Manassas, VA) and β-TC-6 (CRL-11506; ATCC) cells were maintained at 37 ̊C and 5% CO 2 , cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum containing 16.6 or 5.5 mM glucose, respectively.

Techniques: Marker, Derivative Assay, Dispersion

Journal: Bioinformatics

Article Title: SurfaceGenie: a web-based application for prioritizing cell-type-specific marker candidates

doi: 10.1093/bioinformatics/btaa092

Figure Lengend Snippet: Distributions of observed abundance for selected proteins in the lymphocyte data with a range of GenieScores. The number of peptide-spectrum matches (PSMs) assigned to selected proteins for both Cell Surface Capture (CSC) and whole-cell lysate (WCL) experiments. Biological replicates (n = 3) are shown as data points and averages are shown as columns. The ranks assigned to each protein, according to the set of calculated GenieScores, are shown for both CSC and WCL datasets

Article Snippet: 2.1 Cell culture Human lymphocyte cell lines (Ramos, HG-3, RCH-ACV, Jurkat) were cultured and passaged as described previously ( Haverland et al. , 2017 ). α-TC1 clone 6 (CRL-2934; ATCC, Manassas, VA) and β-TC-6 (CRL-11506; ATCC) cells were maintained at 37 ̊C and 5% CO 2 , cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum containing 16.6 or 5.5 mM glucose, respectively.

Techniques: