Metabolomic characterization of experimental ovarian cancer ascitic fluid

Abstract

Introduction: Malignant ascites (MA) is a major cause of morbidity that occurs in 37% of ovarian cancer patients. The accumulation of MA in the peritoneal cavity due to cancer results in debilitating symptoms and extremely poor quality of life. There is an urgent unmet need to expand the understanding of MA to design effective treatment strategies, and to improve MA diagnosis.

Objective: Our purpose here is to contribute to a better characterization of MA metabolic composition in ovarian cancer.

Method: We determined the metabolic composition of ascitic fluids resulting from orthotopic growth of two ovarian cancer cell lines, the mouse ID8-vascular endothelial growth factor (VEGF)-Defb29 cell line and the human OVCAR3 cell line using high-resolution ¹H MRS. ID8-VEGF-Defb29 tumors induce large volumes of ascites, while OVCAR3 tumors induce ascites less frequently and at smaller volumes. To better understand the factors driving the metabolic composition of the fluid, we characterized the metabolism of these ovarian cancer cells in culture by analyzing cell lysates and conditioned culture media with ¹H NMR.

Results: Distinct metabolite patterns were detected in ascitic fluid collected from OVCAR3 and ID8-VEGF-Defb29 tumor bearing mice that were not reflected in the corresponding cell culture or conditioned medium.

Conclusion: High-resolution ¹H NMR metabolic markers of MA can be used to improve characterization and diagnosis of MA. Metabolic characterization of MA can provide new insights into how MA fluid supports cancer cell growth and resistance to treatment, and has the potential to identify metabolic targeting strategies to reduce or eliminate the formation of MA.

Keywords: Ovarian cancer; ascitic fluid; cancer cells and conditioned culture media; high-resolution proton NMR; metabolites; orthotopic tumor implantation.

Figure 1

(A) Representative CPMG ¹H MR spectra obtained from ascitic fluid of an ID8-VEGF-Defb29 (Red), and an OVCAR3 (Green) tumor bearing mice. Expansions of the spectra from 6.5–8.0 ppm are 4X vertically zoomed. (BHB; betahydroxybutyrate, BCA; branch chain amino acid). (B) Metabolic heat maps representing the significant changes in metabolites between ID8-VEGF-Defb29 and OVCAR3 ascitc fluids. Individual samples are separated by vertical column. Heat maps show a clear separation of metabolic pattern between these two groups with respect amino acid, choline, glutamine-glutamate and glucose metabolism. Mean value of all samples from VEGF-Defb29 and OVCAR3 groups were subjected to unpaired student t-test to measure statistical significance. (* P<0.05, ** P <0.01 and *** P <0.001)

Figure 2

Representative CPMG ¹H MR spectra of ascitic fluids obtained from ID8-VEGF-Def29 and OVCAR3 tumor bearing mice showing presence of choline and phosphocholine in the 3.2 ppm region.

Figure 3

(A) Representative CPMG ¹H MR spectra obtained from conditioned culture media from ID8-VEGF-Defb29 cells (red), OVCAR3 cells (green) and control RPMI media (black). Expansion of the spectra from 1.30–1.35 ppm, 5.20–5.27 ppm and 6.5–8.5 ppm are vertically zoomed for −14X, −4X and 4X respectively. BHB; betahydroxybutyrate, BCA; branch chain amino acids. (B) Metabolic heat maps representing the significant differences in the metabolites present in the conditioned media obtained from ID8-VEGF-Defb29 and OVCAR3 groups. Mean value of all samples from ID8-VEGF-Defb29 and OVCAR3 groups were subjected to unpaired student t-test to measure statistical significance. (* P<0.05, ** P <0.01 and *** P <0.001)

Figure 4

(A) Representative CPMG ¹H MR spectra obtained from ID8-VEGF-Defb29 (red), and OVCAR3 cell lysates (green). Expansion of the spectra from 3.2–3.3 and 6.5–8.0 ppm are vertically zoomed for −4X and 4X respectively. BHB; beta-hydroxybutyrate, BCA; branch chain amino acid. (B) Metabolic heat maps representing significant differences in ID8-VEGF-Defb29 and OVCAR3 cell lysates metabolites. Mean value of all samples from ID8-VEGF-Defb29 and OVCAR3 groups were subjected to unpaired student t-test to measure statistical significance. (* P<0.05, ** P <0.01 and *** P <0.001)

Figure 5

Venn diagrams representing all the metabolites detected in the different compartments in OVCAR3 and ID8-VEGF-Defb29 ascitic fluids.