Fig. 5: Characterizing tumor spheroids in lung adenocarcinoma.

a Morphological composition of tumor cells in LUAD samples, sorted by spheroid fraction. b Example images (gray: brightfield, pink: DAPI/nuclei) of MSE samples with low and high fractions of tumor cell spheroids. Images are representative of 59 biological replicates and 400 images each. c Fraction of spheroids stratified by the response to the treatment following the biopsy, for n = 24 samples obtained prior to therapy start. d Fraction of spheroids stratified by actionable genomic alterations (BRAF V600E: 11 samples, 3 patients; EGFR: 18 samples, 9 patients; other: 34 samples, 24 patients). P values from Tukey’s honestly significant difference (HSD) test. e Example images of drug-induced spheroid dissociation (gray: brightfield, pink: DAPI/nuclei). Images are representative of 59 biological replicates with 100 images per drug treatment. f Integration of spheroid abundances from PCY and transcriptional responses from DRUG-seq (n = 5 LUAD samples and 48 drugs). TIMP1 (left) represents a gene whose expression was associated with spheroid abundance across ex vivo treatments. g Volcano plot for DRUG-seq analysis of spheroid fractions (see Methods). h Top 10 pathways (GSEA) for genes associated with spheroid fraction. Density plots and P values as in Fig. 4b. NMD nonsense-mediated decay, n-t nuclear-transcribed, PTTM protein targeting to membrane. i Pathway scores (singscore⁹¹, see Methods) for mitochondrial translation (GO:0032543) and cell adhesion (GO:0007155) associated with spheroid fraction. P value from linear regression (two-sided t test). j Spheroid dissociation in response to EGFR inhibition in 43 spheroid-containing LUAD MSE samples, stratified by the presence of any mutation in EGFR (EGFR mutant: n = 12 samples from 8 patients, EGFR wild type: n = 31 samples from 20 patients). The values correspond to the mean spheroid dissociation response across four tested EGFR inhibitors (see Supplementary Fig. 14). All box-plots as in Fig. 3e; P values in (c) and (j) from two-sided Wilcoxon rank-sum test; P values in (f) and (g) from edgeR quasi-likelihood F-test with BH correction. Lines and shaded areas in (f) and (i) correspond to a linear regression fit with 95% confidence bands.