Fig 2: High-throughput drug combination screens with ATRi and TROP2 expression in patient biopsies and cell lines(A) The heatmap is a ranked average ExcessHSA from a 10 × 10 combination screen of ATRi (ceralasertib) with various drugs selected from a 6 × 6 screen (Figure S1), in PARPi-sensitive PEO1, PARPi-resistant PEO1-olaJR, and PEO1-olaR and de novo PARPi-resistant PEO4. This experiment was performed once.(B) Expression values (Log2 transcripts per million, TPM) for TROP2 mRNA (TACSTD2) from both BRCA-mutant (BRCAmut) cohort (n = 15) and BRCA wild-type (BRCAwt) cohort (n = 20) of patients were compared against TPM values of TROP2 from normal ovarian tissues (normal) obtained from the GTEx database (n = 180). TROP2 values for the no-clinical benefit (<6 months PFS) or clinical benefit (≥6 months PFS) groups for cohorts BRCAwt (n = 9 and n = 11 respectively) and BRCAmut (n = 11 and n = 4 respectively) were compared as well. Graphs show TROP2 expression as median TPM (Log2) ± 95% confidence interval (CI) for each cohort or group. Statistical significance was determined using the unpaired Mann-Whitney U-test and is shown as p-values ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001, ns, not significant.(C) Flow cytometric analysis for TROP2 expression (gray) was performed after incubating cells with recombinant anti-TROP2 hRS7 antibody followed by goat anti-human F(ab)2-AF488 secondary antibody as given in STAR Methods. In control group (white), cells were treated only with secondary antibody. A2780 cell line was used as a TROP2-negative control. Graphs show the value of mean fluorescence intensity (Mean FI) of TROP2 expression peaks divided by that of control peak (n = >8000 events) for each cell line. Figure is representative of 3 biologically independent experiments.(D) Immunoblotting was performed to screen the baseline levels of TROP2 protein in the same cell lines. Blots were overexposed (bottom panel) to highlight low TROP2 expression in PEO1-olaR. Densitometric quantitation of TROP2 relative to Actin are included below. Closest molecular weight marker positions are shown on the right. Figure is representative of 3 biologically independent experiments.(E) Quantitative PCR (qPCR) analysis of total RNA from both TROP2 positive and negative cell lines. Graph is representative of 2 biologically independent experiments and shows TROP2 expression values normalized against that of GAPDH for each cell line. A negative control without RNA template is also included as quality control. Data are represented as mean ± SD.

Fig 3: Effect of antibody drug conjugates (ADCs) on survival and viability in HGSOC cells(A) Cell growth assays (XTT) were performed on TROP2-positive (PEO1 and PEO1-olaJR) HGSOC cells in triplicate wells, both in normal media or after pretreatment with a nontargeting antibody drug conjugate control (cADC) or SG (10 μg/mL, 30 min at 37°C and washed thrice in PBS), prior to adding ATRi berzosertib (0–2 μM) for 48 h. Graphs show mean absorbance values at 490 nm corrected for background ±SD for each treatment (n = 3 biological replicates) against a concentration gradient (Log2) of ATRi. For all treatments we replaced 0 values for ATRi with 0.01 to enable plotting of absorbance values on a logarithmic X axis. Statistical determinations were done with Student’s t test and shown as p-values. The p-values for SG treatment versus untreated are indicated by asterisks (∗) while SG versus cADC are indicated by hashes (#). Slopes for each curve are derived from linear regression formula “y = mx+b” where y = Y-intercept, x = ATRi (μM) and b = correlation coefficient, calculated from the linear part of the respective curves.(B) Graphs show mean cell growth values ±SD of cells with cADC or SG alone relative to untreated, derived from panel A (n = 3 biological replicates). Statistical significance (p-values) was determined with Student’s t test.(C) Clonogenic assays were performed over 7 days with increasing concentrations of berzosertib (ATRi). Graphs show mean ± SD of stained area (n = 3 biological replicates). Statistical significance (p-values) determined using Student’s t test.(D) Representative images from Matrigel plates after crystal-violet staining are shown. Graphs below image panels show total number of crystal-violet-stained invaded cells from imaged panels ±SD (n = 3 biological replicates). Statistical significance (p-values) was determined using Student’s t test. Experiment was performed twice. Scale bar is 100 μm.(E) Flow cytometric analysis was conducted to assess cell viability with AnnexinV-FITC and 7AAD reagents on cells pretreated with SG or ATRi berzosertib (1 μM) alone or SG pretreatment followed by ATRi treatment overnight. Percentage of cells positive for AnnexinV alone (early apoptosis) or both AnnexinV and 7AAD (dead) are shown within respective quadrants. Graphs below show percentage of live (gray) and dead cells (black), while numbers above bars indicate fold increase in dead cells for each treatment relative to untreated. Images are representative of 3 biologically independent experiments.(F) Cells with or without SG pretreatment were treated with ATRi berzosertib (1 μM) for 48 h and then stained with propidium iodide for DNA quantification and cell cycle analysis. Cell populations as fractions of total cells are shown over appropriately marked G1, S, or G2/M phases. Images are representative of 2 experimental replicates. For all images, p-values are ∗, #p < 0.05, ∗∗, ##p < 0.01, and ∗∗∗, ###p < 0.001, ns, not significant.

Fig 4: Working model of TROP2-mediated ferroptosis resistance in NSCLC. In NSCLC, high TROP2 expression promotes tumor progression by enhancing proliferation and survival. Mechanistically, TROP2 inhibits ferroptosis—an iron-dependent form of regulated cell death—by sequestering YAP in the cytoplasm, thereby suppressing its nuclear translocation and subsequent transcriptional activation of the pro-ferroptotic gene HMOX1. Targeted inhibition of TROP2 restores YAP/HMOX1 signaling, sensitizing tumors to ferroptosis inducers (e.g., RSL3), and supporting a potential therapeutic strategy for NSCLC

Fig 5: TROP2 regulates the malignant phenotypes of NSCLC cells. (A, B) Knockdown efficiency of TROP2 in PC9 and H292 cells transfected with TROP2-targeting siRNAs was confirmed at the mRNA level with qRT-PCR (A) and at the protein level by western blot (B). Quantitative analysis (bar graph) of the bands was shown adjacent to the representative blots. (C, D) The cell viability was suppressed in PC9 and H292 cells with TROP2 down-regulation. Cell proliferation was evaluated by CCK8. (E) The colonies were reduced in PC9 and H292 cells with TROP2 down-regulation using colony formation assay. (F) Represent spheres of lung cancer cells with TROP2 down-regulation. (G, H) The cell viability was assayed in PC9 and A549 cells with TROP2 overexpression by CCK8. (I) Colony formation assay was used to evaluate cell viability in PC9 and A549 cells with TROP2 over-expression. (J) Representative images of tumor spheres formed of lung cancer cells with TROP2 over-expression. Data are presented as mean ± SD (n = 3). Statistical significance was determined by two-tailed unpaired Student’s t-test (A, B, E, F, I, J) and two-way ANOVA followed by Sidak’s post hoc test (C, D, G, H). ∗P < 0.05, ∗∗P < 0.01