A persister phenotype in afatinib-treated tumor xenografts is driven by YAP activation.

(A) Mice harboring HCC827 tumor xenografts were treated with 20mg/kg afatinib for 5d (n = 4) or vehicle (n = 4). (B) Immunofluorescence images of YAP, pan-cytokeratin, and DAPI in afatinib-treated remnant and vehicle tumors (magnification, 20x). (C) Quantification of nuclear YAP in vehicle- or afatinib-treated HCC827 xenografts. (D) Gene-set enrichment (GSEA) of YAP signatures from differential expression of 83 matched sets of patient adenocarcinoma or non-malignant tissue using YAP_conserved_signature [Normalized Enrichment Score = −1.76, p = 0.0039] (left) or YAP_up [Normalized Enrichment Score = −1.69, p = 0.0068] (right) gene signature in the ranked genes. Average Z-scores for each gene set were compared between conditions using a t-test [P = 8.5e-16 and P < 2.2e-16, respectively]. (E) Representative images of Masson’s trichrome and quantification of percentage area coverage of collagen for vehicle-treated tumors and afatinib-treated remnant tumors are shown (magnification, 10x). (F) Representative images of Picrosirius red staining and quantification of percentage area coverage per field of view (magnification, 10x). All scale bars, 50 μm. Data are shown as means ± s.e.m. (n = 3 biological replicates). * P < 0.05, *** P < 0.001, **** P < 0.0001 as determined by a two-tailed t-test.

Cell line EC50 and % Max Inh for targeted therapies

EGFR-mutated NSCLC spheroids, but not monolayers, acutely undergo massive apoptosis in response to EGFRi.

(A) HCC827 spheroids (96 hr) and monolayers (48 hr) were harvested for western blot analysis against indicated proteins. Ponceau S was used as a total protein loading control. (B) Dose response curves for HCC827 cells cultured as spheroids or monolayers and treated with afatinib. Curves normalized to DMSO. LogEC50 (ns) P value = 0.4 between monolayer (EC50 = 2.36 nM) and spheroid (EC50 = 1.52 nM). * P value = 0.0199 between monolayer (Bottom value = 44.16 relative ATP) and spheroid (Bottom value = 10.06 relative ATP). (C) Dose-response curves for HCC827 cells cultured as spheroids or monolayers and treated with cisplatin. Curves normalized to 0.9% NaCl vehicle. (D) Representative Hoechst (monolayer) or brightfield (spheres) and corresponding Propidium Iodide fluorescence of DMSO- or afatinib-treated HCC827 monolayers and spheroids at indicated doses (magnification, 4x). All scale bars, 50 μm. Total, normalized RFP+ fluorescence pixels across each representative image shown. (E) Dose-response curves of % Propidium Iodide pixels / total Hoechst (monolayer) or (F) brightfield (spheroid) pixel area in DMSO- or afatinib-treated HCC827 monolayers and spheroids. (n = 3 biological replicates). (G) Quantification of total cell numbers across indicated DMSO- or afatinib-treated HCC827 monolayer cultures. Relative ATP was measured by a Cell Titer Glo assay. Data are shown as means ± s.e.m. (n = 3 biological replicates). * P < 0.05, ** P < 0.01, **** P < 0.0001 as determined by Extra sum-of-squares F Test; n.s., not significant.

Distinct KRASG12C-mutant cell lines exhibit varying EC50 values in response to KRASG12C inhibition.

(A) Dose response curves of HCC60 spheroids or monolayers treated with ARS-1620 for 72h. Relative ATP was measured by a Cell Titer Glo assay. Curves normalized to DMSO. LogEC50 **** P value = < 0.0001 between monolayer (EC50 = 7.70 µM) and spheroid (EC50 = 1.62 µM). (B) Dose response curves of H358 spheroids or monolayers treated with ARS-1620 for 72h. Relative ATP was measured by a Cell Titer Glo assay. Curves normalized to DMSO. LogEC50 (ns) P value = 0.415 between monolayer (EC50 = 0.32 µM) and spheroid (EC50 = 0.23 µM). (C-D) Dose-response curves of % Propidium Iodide pixels / total Hoechst (monolayer) or brightfield (spheroid) pixel area in DMSO- or ARS-1620-treated HCC60 monolayers and spheroids. (E-F) Dose-response curves of % Propidium Iodide pixels / total Hoechst (monolayer) or brightfield (spheroid) pixel area in DMSO- or ARS-1620-treated H358 monolayers and spheroids. Data are shown as means ± s.e.m. (n = 3 biological replicates). **** P < 0.0001 as determined by Extra sum-of-squares F Test; n.s., not significant.

Vemurafenib resistant BRAFV600E mutant line, RPMI-7951, is sensitive to vemurafenib treatment in spheroid culture.

(A) Dose response curves of RPMI-7951 spheroids or monolayers treated with vemurafenib for 72h. Relative ATP was measured by a Cell Titer Glo assay. Curves normalized to DMSO. LogEC50 **** P value = < 0.0001 between monolayer (EC50 = 9.46 µM) and spheroid (EC50 = 0.08 µM). (B-C) Representative Hoechst (monolayer) or brightfield (spheres) and corresponding Propidium Iodide fluorescence of DMSO- or vemurafenib treated RPMI-7651 monolayers and spheroids at indicated doses (magnification, 4x). Total, normalized RFP+ fluorescence pixels across each image shown. (D) Dose-response curves of % Propidium Iodide pixels / total Hoechst (monolayer) or brightfield (spheroid) pixel area in DMSO- or vemurafenib-treated RPMI-7951 monolayers and spheroids. (E) Quantification of total cell numbers across indicated DMSO- or vemurafenib-treated RPMI-7951 monolayer cultures. Data are shown as means ± s.e.m. (n = 3 biological replicates). **** P < 0.0001 as determined by Extra sum-of-squares F Test.

YAP nuclear translocation mediates resistance to EGFR/RAS/RAF pathway inhibitors.

(A) Immunofluorescence images of YAP, β-catenin, and DAPI in EGFR mutant sphere (magnification, 20x) and monolayer (magnification, 10x) cultures. All scale bars 50μm. (B) Quantification of nuclear YAP to DAPI ratios in HCC827 and H1975 adherent monolayers and spheroids. Data are shown as means ± s.d. (C-D) qPCR analysis (CTGF, TRAIL,CYR61) in EGFR mutant spheroids normalized to monolayers. (E) Immunofluorescence images of YAP, pan-Cytokeratin (HCC60) or β-catenin (H358), and DAPI in KRASG12C mutant sphere and monolayer cultures (magnification, 20x). All scale bars 50μm. (F) Quantification of nuclear YAP to DAPI ratios in HCC60 and H358 adherent monolayers and spheroids. Data are shown as means ± s.d. (G-H) qPCR analysis (CTGF, TRAIL, CYR61) in KRAS mutant spheroids normalized to monolayers. (I) Immunofluorescence images of YAP, pan-cytokeratin, and DAPI in RPMI-7951 (BRAFV600E mutant) sphere and monolayer cultures (magnification, 20x). All scale bars 50μm. (J) Quantification of nuclear YAP to DAPI ratios in RPMI-7951 adherent monolayers and spheroids. Data are shown as means ± s.d. (K) qPCR analysis (CTGF, TRAIL, CYR61) in BRAF mutant spheroids normalized to monolayers. Data are shown as means ± s.d. (n = 3 biological replicates). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 as determined by a two-tailed t-test; n.s., not significant.

Increased YAP activity is sufficient to protect HCC827 spheroids from afatinib-mediated cell death.

(A) HCC827-control and HCC827-YAPS127A monolayers (48 hr) were harvested for western blot analysis against indicated proteins. (B) qPCR analysis (CTGF, TRAIL, CYR61) in HCC827-YAPS127A spheroids normalized to HCC827-control. Data are shown as means ± s.e.m. (n = 3 biological replicates). (C) Dose response curves for HCC827-control compared to HCC827-YAPS127A cells cultured as spheroids and treated with afatinib for 72 hr. Relative ATP was measured by a Cell Titer Glo assay. Curves normalized to DMSO. LogEC50 (ns) P value = 0.91 between control (EC50 = 1.21 nM) and YAPS127A (EC50 = 1.27 nM). * P value = 0.021 between HCC827-control (Bottom value = 25.98 relative ATP) and HCC827-YAPS127A (Bottom value = 38.21 relative ATP). (D) Dose-response curves of % GFP pixels / total brightfield pixel area in HCC827-control and HCC827-YAPS127A normalized to DMSO % GFP pixels / total brightfield pixel area. Data are shown as means ± s.e.m. (n = 3 biological replicates). (E) H358-parental, H358-control, and H358-YAPS127A monolayers (48 hr) were harvested for western blot analysis against indicated proteins. (F) qPCR analysis (CTGF, TRAIL, CYR61) in H358-YAPS127A monolayers normalized to H358-control. Data are shown as means ± s.e.m. (n = 3 biological replicates). (G) Dose response curves for H358-control compared to H358-YAPS127A cells cultured as spheroids and treated with ARS-1620 for 72 hr. Relative ATP was measured by a Cell Titer Glo assay. Curves normalized to DMSO. LogEC50 ** P value = 0.005 between H358-control (EC50 = 0.47 µM) and YAPS127A (EC50 = 1.18 µM). * P < 0.05, ** P < 0.01 as determined by Extra sum-of-squares F Test; n.s., not significant.

Afatinib-treated remnant tumors demonstrate reduced mitotic activity compared to vehicle-treated tumors.

(A) Representative immunofluorescence images of vehicle or afatinib-treated remnant tumors stained with phospho-HistoneH3 (pHH3), EGFRdel19, or DAPI (magnification, 20x). (B) Quantification of pHH3 staining in the vehicle compared to afatinib-treated remnant tumors. All scale bars, 50 μm. Data are shown as means ± s.e.m. (n = 3 biological replicates). * P < 0.05 as determined by a two-tailed t-test.

Standard loading controls for western blot analysis cannot be used for comparative analysis of monolayers and spheroids.

(A) Ponceau staining of n=3 replicates of paired monolayer and spheroid protein lysate. (B) Quantification of ponceau band intensity. (C) Representative images of Vinculin and Histone H3 western blot bands in monolayer and sphere lysate. EGFR and pEGFR blots in Figure 2 were stripped and re-probed with vinculin. (D) Quantification of Vinculin and Histone H3 bands relative to Ponceau band intensity.

Cells do not demonstrate compromised overall fitness resulting from spheroid culture.

(A) Dose-response curves for A549 cells cultured as spheroids or monolayers and treated with afatinib. Curves normalized to DMSO. LogEC50 **P value = 0.0023 between monolayer (EC50 = 4.55 μM) and spheroid (EC50 = 75.33 μM). (B) Dose-response curves for A549 cells cultured as spheroids or monolayers and treated with cisplatin. Curves normalized to 0.9% NaCl vehicle. Relative ATP was measured by a Cell Titer Glo assay. Data are shown as means ± s.e.m. (n = 3 biological replicates). * P < 0.05, ** P < 0.01, **** P < 0.0001 as determined by Extra sum-of-squares F Test; n.s., not significant.

EGFR-mutated NSCLC monolayers undergo G1 arrest as an acute response to EGFR inhibition.

(A) FUCCI (mKO2-hCdt1 and mAG-hGeminin) expressing HCC827 monolayers were treated with DMSO or afatinib at indicated doses for 72h and analyzed by immunofluorescence. Representative images shown (magnification, 4x). Scale bar 50μm. (B) Cell counts of mKO2-hCdt1 and mAG-hGeminin HCC827 expressing to quantify dose-dependent cell-cycle distribution from FUCCI expressing HCC827 monolayers following 72h of DMSO or afatinib treatment. (C) Percentage of mKO2-hCdt61 expressing cells (G1 cell cycle marker) over total cells. Data are shown as means ± s.e.m. (n = 4 biological replicates). * P < 0.05, *** P < 0.001, **** P < 0.0001 as determined by a two-tailed t-test.

Differential sensitivity between EGFR-mutated spheroids and monolayer to EGFRi is not limited to a single agent or cell line.

(A) Dose response curves of H-1975 spheroids or monolayers treated with osimertinib for 72h. Curves normalized to DMSO. Relative ATP was measured by a Cell Titer Glo assay. LogEC50 (ns) P value = 0.50 between monolayer (EC50 = 3.17 nM) and spheroid (EC50 = 4.00 nM). * P value = 0.023 between monolayer (Bottom value = 39.29 relative ATP) and spheroid (Bottom value = 7.01 relative ATP). (B) Representative Hoechst (monolayer) or brightfield (spheres) and corresponding Propidium Iodide fluorescence of DMSO- or 100 nM osimertinib-treated H-1975 monolayers and spheroids at indicated dosages (magnification, 4x). Total, normalized RFP+ fluorescence pixels across each image shown. (C) Dose-response curves of % Propidium Iodide pixels / total Hoechst (monolayer) or brightfield (spheroid) pixel area in DMSO- or osimertinib-treated H-1975 monolayers and spheroids. (n = 3 biological replicates). (D) Quantification of total cell numbers across indicated DMSO- or afatinib-treated HCC827 monolayer cultures. Relative ATP was measured by a Cell Titer Glo assay. Data are shown as means ± s.e.m. (n = 3 biological replicates). ns = not significant, * P < 0.05 as determined by Extra sum-of-squares F Test.

YAP exhibits cytoplasmic localization in H-1975 spheroids.

(A) Immunofluorescence images of YAP, β-catenin, and DAPI in EGFR mutant sphere (magnification, 20x) and monolayer (magnification, 10x) cultures. All scale bars 50μm.

Dose-response assays using CytoToxic Green show HCC827-YAPS127A cells are partially protected from EGFRi-mediated cell death.

(A) Representative brightfield images and corresponding CytoToxic Green fluorescence of DMSO- or afatinib-treated HCC827-control and HCC827-YAPS127A spheroids at indicated doses (magnification, 4x). Total, normalized green fluorescence pixels across each image shown.