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: Establishment of NB60-expressing HEK293T cell line. (A) The vector map of the plasmid construct, pNB60. (B) The specific components of the plasmid construct essential for the expression of Trop2 (NB60) on the surface of the EVs. (C) Expression of NB60 in HEK293T cells after transfection. (D) Expression of NB60 in HEK293T cells after geneticin (200 μg/mL) selection for 30 days. Legend: HEK293t—un-transfected HEK293T cells, NB60-HEK293t—pNB60-transfected HEK293t cells.

Fig 5: In vivo knockdown of Trop2 by NB60 E. BALB/c athymic nude mice were subcutaneously injected with LTLT-Ca cells on their right flank region. When tumor growth becomes visible (550 mm3), NB60 E (1 × 1011 particles/animal) were injected intraperitoneally. After 24 h of EVs treatment, the tumor was excised, and protein lysates were collected for Western blotting. Tumors collected from mice not treated with NB60 E serve as the control (***, p < 0.001 vs. control). Legend: LTLT-Ca (Control)—tumor without EVs treatment, LTLT-Ca (NB60 E)—tumor treated with NB60 E.