Journal: bioRxiv

Article Title: Spatial epitope barcoding reveals subclonal tumor patch behaviors

doi: 10.1101/2021.06.29.449991

Figure Lengend Snippet: Validation of barcode expression in NCI-H82 SCLC cells, related to . ( A ) Schematic of the expected epitopes (rows) for each combination (columns) in a 6-choose-3 strategy. A filled square indicates presence of the epitope in the combination. An empty square indicates absence of the epitope in the combination. ( B ) GFP and iridium ( 191 Ir)-based DNA intercalator were measured by mass cytometry in a pooled population of 20 barcoded NCI-H82 cell lines. The dashed red box indicates live cells expressing high levels of GFP that were selected for further analysis. ( C ) Biaxial plots showing positive and negative populations for each of the epitopes, validating epitope expression and antibody staining. GFP and epitopes were measured by mass cytometry in the pooled population of 20 barcoded NCI-H82 cell lines (n = 45,700). ( D ) UMAP of cells (n = 45,700) grouped by epitope expression (AU1, FLAG, HA, StrepII, Prot C, and VSVg). Each point represents a cell. Each number indicates a barcode combination (see epitope column in “F”). ( E ) Schematic of the palladium staining. Each of the 20 epitope combinations is labeled by one unique palladium combination. ( F ) UMAP of cells (n = 45,700) grouped by epitope expression and colored by epitope (AU1, FLAG, HA, StrepII, Prot C, and VSVg) or palladium isotope intensity. Pairs of palladium isotope and epitope in each row show similar patterns indicating a unique palladium and epitope combination for each of the 20 cell lines. ( G ) Representative overlay of MIBI images (AU1: red; FLAG: green; HA: blue; Strep II: cyan; Prot C: magenta; VSVg: yellow) of a cell pellet consisting of 50% wild-type NCI-H82 cells and 50% of a pooled population of 20 barcoded NCI-H82 cell lines. The white squares are enlarged in and their associated numbers indicate the barcode combination. The multicolor image in is an enlarged version of the bottom right of this image. Scale bars: 100 μm.

Article Snippet: After staining, cells were washed twice with CSM, once with 1X PBS, and incubated with 1X PBS containing 1.6% PFA and 1 M iridium-based DNA intercalator (Fluidigm, #201192B) for 16 h at 4 °C.

Techniques: Expressing, Mass Cytometry, Staining, Labeling

Journal: bioRxiv

Article Title: Spatial epitope barcoding reveals subclonal tumor patch behaviors

doi: 10.1101/2021.06.29.449991

Figure Lengend Snippet: Validation of barcode expression in NCI-H82 SCLC xenografts, related to . ( A ) Workflow for mass cytometry analysis of NCI-H82 xenografts. (1) Cell lines expressing unique combinations of epitopes are generated based on an 6-choose-3 strategy and grown in vitro in an independent flask. (2) Barcoded cells are mixed and injected subcutaneously into the flanks of a mouse. (3) Tumors are processed to generate a single-cell suspension, stained with an isotope-conjugated antibody cocktail, and analyzed by mass cytometry. ( B ) GFP and iridium ( 191 Ir)-based DNA intercalator measured by mass cytometry in a single-cell suspension from barcoded NCI-H82 tumors. The dashed red ellipse indicates live cells that express high levels of GFP that were selected for further analysis ( and ; n = 39,630). ( C ) Biaxial plots showing positive and negative populations for each of the epitopes, validating epitope expression and antibody staining in cells from barcoded NCI-H82 tumors. GFP and epitopes were measured by mass cytometry in the single-cell suspensions from tumors. UMAP of all cells from the dashed red ellipse in panel B (n = 39,630) colored by epitope expression (AU1, FLAG, HA, StrepII, Prot C, and VSVg). Each point represents a cell. Each epitope barcode is a unique group. ( E ) UMAP of cells from a pooled population of 40 barcoded NCI-H82 cell lines analyzed by mass cytometry grouped by protein and epitope (GFP, mCherry, AU1, FLAG, HA, StrepII, Prot C, and VSVg) and colored by their intensity. Each point represents a cell. Each number indicates a barcode combination. The dashed lines separate GFP-positive from mCherry-positive cells. ( F ) Overlay of 40 barcoded NCI-H82 cell lines (red) and a pooled population of 38 barcoded NCI-H82 cell lines (blue). The latter includes the same cell lines as were mixed in the 40-line pool except for the line expressing GFP with a tag consisting of Prot C, StrepII, and VSVg (combination 20) and the line expressing mCherry with a tag consisting of AU1, FLAG, and HA (combination 21). Each number indicates a barcode combination. The dashed lines separate GFP-positive from mCherry-positive cells.

Article Snippet: After staining, cells were washed twice with CSM, once with 1X PBS, and incubated with 1X PBS containing 1.6% PFA and 1 M iridium-based DNA intercalator (Fluidigm, #201192B) for 16 h at 4 °C.

Techniques: Expressing, Mass Cytometry, Generated, In Vitro, Injection, Staining