Research data supporting “Metabolic imaging distinguishes ovarian cancer subtypes and detects their early and variable responses to treatment”

Overview The dataset includes files containing raw values that correspond to the figures in the manuscript. Specifically, files are organized by figure number, including Main Figures 1-8 and additional Supplemental Information. Each file includes a description detailing the dataset's contents, format, and alignment with specific figure panels. Data files Fig. 1a: Contains values for the time taken for each PDO model tumor to reach 300 mm3. Fig. 2: Contains values used to generate each sub figure 2a-h. a)Tumor lactate labelling following injection of hyperpolarized [1-13C]pyruvate. Ratios of the areas under the lactate and pyruvate labelling curves (AUC). b) Lactate dehydrogenase (LDH) activity measured in tumor extracts. c) Expression of lactate dehydrogenase A (LDHA), relative to -actin, determined by western blot of tumor extracts. d) Quantitative analysis of MCT 4 membrane expression in tumor sections. e) Quantitative analysis of GLUT 1 membrane expression in tumor sections. f) Mean fluorescence intensity of organoids 1 hour after incubation with 40 M 2-NBDG in glucose-free media. Organoids were plated at 150,000/well the day before incubation with 2-NBDG. g) PET measurements of [18F]FDG uptake. Tumor SUVmax at 90 min following i.v. injection of [18F]FDG. h) Expression of hexokinase II (HKll) relative to -actin in tumor extracts. Fig. 3: Contains values used to generate each sub figure 3b,d,e. b) Densitometric determination of the concentrations of c-Myc relative to -actin on western blots of tumor extracts. d) Quantitative analysis of EGFR staining in tumor sections. Percentage of positive stain. e) Quantitative analysis of phosphorylated ERK (pERK) staining on tumor sections. Percentage of positive stain. Fig. 4: Contains values used to generate each sub figure 4a-f. a) Effect on cell viability. Organoids were plated at 10,000/well and treated for 120 hours with 2 nM- 200 nM Myci975 and cell viability assessed using the AlarmarBlue assay. Viabilities relative to controls in paired wells are shown. Each biological replicate included 2-4 technical replicates. b) LDH activity following 3 days of treatment with 200 nM Myci975 or matched DMSO control. Each biological replicate included 2-3 technical replicates. Treatment of organoids with an EGFR inhibitor (Erlotinib). c) Effect of Erlotinib on cell viability. Organoids were plated at 10,000/well and treated for 120 hours with 2 - 200 nM Erlotinib and cell viability assessed using AlarmarBlue. Viability ratios relative to controls in paired wells of the same experiment. Each biological replicate included 2-4 technical replicates. For measurements of phosphorylated EGFR (pEGFR), HKII expression and LDH activity organoids were plated at 150,000/well the day before the experiment. Posttreatment measurements were made 3 days after treatment with 200 nM Erlotinib. Each biological replicate included 2-3 technical replicates. d) P-EGFR expression relative to -actin. e) HKll expression relative to -actin. f) LDH activity. Fig. 5: Contains values used to generate each sub figure 5a-h. Effect of Erlotinib treatment on tumor lactate labelling from hyperpolarized [1-13C]pyruvate in a) PDO2 and b) PDO5 tumors. Mice were treated by oral gavage daily for 7 days after baseline imaging on day 0. Control group: 6% Captisol in water, 10 ml/kg/day. Treatment group: Erlotinib 50 mg/kg/day in 6% Captisol. The ratio of the areas under the lactate and pyruvate labelling curves (AUC) were recorded in the same mouse at baseline and after 7 days of treatment and the difference between the 7 day time point and baseline were calculated for Erlotinib-treated and control mice. Change in tumor volumes (mm3) between baseline and after 7 days of treatment in individual treated and control mice with c) PDO2 and d) PDO5 tumors, corresponding to the mice shown in (a) and (b) respectively. Effect of Erlotinib treatment on [18F]FDG uptake in e) PDO 2 and f) PDO 5 tumors. The change in tumor SUVmax between baseline and after 7 days of treatment are shown in individual treated and control mice. Change in tumor volumes (mm3) between baseline and after 7 days of treatment in individual treated and control mice with g) PDO 2 and h) PDO 5 tumors, corresponding to the mice shown in (e) and (f) respectively. Fig. 6: Contains values used to generate each sub figure 6a-f. Mice were treated with 50 mg CBT/kg/week dissolved in mannitol/water for injection (10 mg/ml). Controls were treated with mannitol alone. Times shown are the number of weeks after the start of treatment. a) H2AX staining in PDO 2 tumor sections. b) H2AX staining in PDO 5 tumor sections. c) CC3 staining in PDO 2 tumor sections. d) CC3 staining in PDO 5 tumor sections. e) TUNEL staining in PDO 2 tumor sections. f) TUNEL staining in PDO 5 tumor sections. Fig. 7: Contains values used to generate each sub figure 7a-h. Imaging response to CBT treatment using hyperpolarized [1-13C]pyruvate in a) CBT-sensitive PDO 2 tumors and b) CBT-resistant PDO 5 tumors. The ratio of the areas under the lactate and pyruvate labelling curves (AUC) were recorded in the same mouse at baseline and at the indicated times after the start of treatment and the difference calculated for CBT-treated and control mice. c) Change in PDO 2 tumor volumes (mm3) from baseline in individual treated and control mice, corresponding to the mice shown in (a). d) Change in PDO 5 tumor volumes (mm3) from baseline in individual treated and control mice, corresponding to the mice shown in (b). Imaging response to CBT treatment using [18F]FDG-PET in e) CBT-sensitive PDO2 tumors and f) CBT-resistant PDO 5 tumors. Change in SUVmax from baseline in individual treated and control mice and at the indicated times after the start of treatment. g) Change in PDO 2 tumour volumes (mm3) from baseline in individual treated and control mice, corresponding to the mice shown in (e). h) Change in PDO 5 tumour volumes (mm3) from baseline in individual treated and control mice, corresponding to the mice shown in (f). Fig. 8: Contains values used to generate each sub figure 8a-h. Liquid Chromatography - Mass Spectrometry measurements of NAD+ and NADH in tumor extracts before and at the indicated times after the start of treatment. Changes in peak areas show the relative changes in NAD+ and NADH concentrations. Peak areas in extracts of PDO 2 tumors, a) NAD+ and b) NADH and in PDO 5 tumors, c) NAD+ and d) NADH. LDH activity (units/mg tumor protein) in e) PDO 2 and f) PDO 5 tumor extracts before and at the indicated times after the start of treatment. g) RT-qPCR measurements of GLUT 1 expression in fine needle aspirates from PDO 2 tumors in treated and control mice. Paired values were recorded in the same mouse at baseline and at the indicated times following treatment. The fold change from the baseline value was calculated using the 2(-Delta Delta C(T)) method. h) Fluorescence measurements of NBDG uptake in organoids at 3 days post treatment with 50 M Carboplatin. Mean NBDG fluorescence from paired control and treatment organoids were recorded and the data expressed relative to the paired control. Final Statistics Report: Contains full statistical analysis of every dataset published in the manuscript. GSVA Results for Fig. 1b, S3bi and S3bii: Contains gene set variation analysis (GSVA) values used to generate figure 1b, S3bi and S3bii based on the following genesets: ETCsig REACTOME_EGFR_DOWNREGULATION REACTOME_SIGNALING_BY_EGFR_IN_CANCER These genesets were sourced from the Gene Set Enrichment Analysis (GSEA) database, which provides open-access resources for gene set collections. Main RNA seq dataset for analysis incl Fig. 3a and 3c: The RNA sequencing data used in this analysis is available at the Gene Expression Omnibus (GEO) under accession number GSE208216 and has been described previously in the following reference: Vias M, Morrill Gavarró L, Sauer CM, Sanders DA, Piskorz AM, Couturier DL et al. High-grade serous ovarian carcinoma organoids as models of chromosomal instability. Elife 2023; 12. Specifically PDO 1,2 5 and 11’s data was extracted to this folder for use in the RNA sequencing analysis shown in the current manuscript. Index of mice used for imaging: This Excel sheet contains categorized data on mice used in the imaging experiments. Each entry represents an individual mouse tracked across multiple imaging sessions, with data segmented by imaging modality, time points of imaging and drug treatment (if any). Imaging techniques used included localized hyperpolarized [1-13C]pyruvate spectroscopy (13C), [18F]FDG PET (PET) and Dynamic Contrast Enhanced (DCE) MRI. Supplementary information figures: Contains values used to generate the supplementary figures S1- S7. Figure S1. c) Average areas under the tumor contrast agent uptake curves (AUC) during the first 5 minutes after contrast agent injection. d) Tumor volumes (mm3) at the time the hyperpolarized [1-13C]pyruvate experiments were performed. e) Tumor volumes (mm3) at the time the PET measurements were performed. f) Tumor volumes (mm3) at the time the perfusion measurements were made. Figure S2. e) Quantitative analysis of the percentage of human and mouse cells in tumor sections. Percentage composition of mouse cells (%). f) Tumor cell density in the tumour sections. Figure S3 c) GSEA of EGFR target gene expression. Figure S4. Treatment of organoid models with an EGFR inhibitor (Erlotinib). Western blot analysis of P-EGFR, HKII and LDHA expression in organoids that had been plated at 150,000 cells/well the day before the experiment and then treated for 3 days with 200 nM Erlotinib or a volume-matched DMSO solvent control. b) LDHA expression relative to -actin. Figure S5. Response of drug-sensitive and drug-resistant organoids to standard-of-care treatment. Organoids were plated at 10,000 cells/well and treated for 120 hours with 10 nM Paclitaxel (PTX) or 50 M Carboplatin (CBT) or a combination of the two. Cell viability was assessed using an AlarmarBlue cell viability assay. Cell viabilities relative to control in paired wells are shown for a) PDO 1, b) PDO 2, c) PDO 5 and d) PDO 11. Figure S6. Metabolic changes in PDO 2 and PDO 5 tumors following Carboplatin treatment. Hexokinase activity (mU/mg tumor protein) in extracts of a) PDO 2 and b) PDO 5 tumors at the indicated times after the start of treatment. Figure S7. Vascularity in PDO 2 tumors following treatment with Carboplatin. Two serial 3 m sections, one stained for the endothelial cell marker CD31 and one for  smooth muscle actin (ASMA), a marker of mature blood vessels, were co-registered (representative tumor sections are shown). Areas with positive CD31 staining (e) were identified and the selected areas classified as CD31 positive. From the selected red areas, the percentage of cells staining for ASMA (f) were determined.