Journal: bioRxiv

Article Title: Actionable biological programs to enhance EGFR-targeted therapy response unveiled by single-cell lineage tracing in clinically relevant lung cancer models

doi: 10.1101/2025.05.19.654081

Figure Lengend Snippet: (a) Time points of single-cell suspension collection from barcoded TH107 PDOs upon EGFR targeted therapy using osimertinib. (b, c) Assessment of osimertinib sensitivity by 3D-CTG (b) and high-magnification images (c) of TH107 PDOs at D0-D30 upon osimertinib or DMSO treatments (Parental: treatment-naive TH107 PDOs; Persisters: osimertinib persisters TH107 PDOs; p-value calculated using Two-way ANOVA with Sidak’s multiple comparison test). (d) Identification of four distinct lineage populations (L.A, L.B, L.C, L.D) differentially enriched at D7 and D14 in osimertinib-treated TH107 PDOs normalized to DMSO control. (e) Genomic barcode frequency scores for L.A-L.D populations highlight a gradual enrichment at D14 of the L.D population upon osimertinib treatment (p-values calculated using the Mann-Whitney-Wilcoxon test). (f) Lineage population abundances over the entire time course of the experiment show the distinctiveness behavior of the four lineage populations among osimertinb and DMSO-treated TH107 PDOs. (g) Pathway analysis of gene expression (iPAGE) comparing L.D population versus rest (L.A-L.C) at D0. All individual sequenced genes are sorted by log2FC and divided into 9 equally populated bins from lowest (negative) log2FC (left) to greatest log2FC (right). The red bar within the black header indicates the range of log2FC values in each bin. The heatmap is colored by the enrichment or depletion of the gene in each bin. The top/right-most iPAGE bin, containing genes with the greatest log2FC values, is significantly enriched for genes in the Hallmark Hedgehog Signaling gene set. The significance of the mutual information value (bits) is calculated using a non-parametric randomization test (see Methods). (h) Dotplot of Hallmark Hedgehog Signaling Gene Set genes present in the top iPAGE bin. See also Supplementary Figures 1, and 2.

Article Snippet: Statistical significance for the functional tests was calculated using Two-way ANOVA with Sidak’s multiple comparison test, one-way ANOVA with Tukey’s multiple comparison test, or Student T-test (GraphPad Prism vs 10.3.0).

Techniques: Suspension, Comparison, Control, MANN-WHITNEY, Gene Expression

Journal: bioRxiv

Article Title: Actionable biological programs to enhance EGFR-targeted therapy response unveiled by single-cell lineage tracing in clinically relevant lung cancer models

doi: 10.1101/2025.05.19.654081

Figure Lengend Snippet: (a, b) Functional crystal violet assay quantification in 2D- (a) and 3D soft agar colony formation (b) cultures with lineage A enriched (L.A) or unsorted control (U.C.) H1975 cells; quantification of the crystal violet OD (a) or the number of 3D colonies (b) is provided (n = 2-3 replicates per experimental group; p-value calculated using Student’s t-test). (c) Functional in vivo experiments with lineage A enriched (L.A) or unsorted control (U.C.) H1975 CDXs treated with osimertinib or vehicle control (p-value calculated using One-way ANOVA with Tukey’s multiple comparison test). (d, e) The tumor volume fold change was calculated from each xenograft (d) and response to osimertinib classified with RECIST criteria (e) (p-value calculated using Two-way ANOVA with Sidak’s multiple comparison test). (f, g) Functional tests using CRISPR-Cas9 lentiviral infection with sgRNA targeting FOXD1 protein and 3D-soft agar colony formation assay using unsorted control (U.C.) (f) or lineage A enriched (L.A) (g) H1975 cells treated with a dose escalation of osimertinib (Osi., 2.5-10 nM). Colonies were stained with crystal violet at day 21 and quantified (f, g) (n = 3 replicates per experimental group; p-value calculated using Two-way ANOVA with Sidak’s multiple comparison test). (Osi.: osimertinib; CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease). See also Supplementary Figures 6 and 7.

Article Snippet: Statistical significance for the functional tests was calculated using Two-way ANOVA with Sidak’s multiple comparison test, one-way ANOVA with Tukey’s multiple comparison test, or Student T-test (GraphPad Prism vs 10.3.0).

Techniques: Functional Assay, Crystal Violet Assay, Control, In Vivo, Comparison, CRISPR, Infection, Soft Agar Assay, Staining