Different Lipid Regulation in Ovarian Cancer: Inhibition of the Immune System

Abstract

Lipid metabolism is altered in several cancer settings leading to different ratios of intermediates. Ovarian cancer is the most lethal gynecological malignancy. Cancer cells disperse in the abdominal space and ascites occurs. T cells obtained from ascites are unable to proliferate after an antigenic stimulus. The proliferation of ascites-derived T cells can be restored after culturing the cells for ten days in normal culture medium. No pathway aberrancies were detected. The acellular fraction of ascites can inhibit the proliferation of autologous as well as allogeneic peripheral blood lymphocytes, indicating the presence of soluble factors that interfere with T cell functionality. Therefore, we analyzed 109 lipid mediators and found differentially regulated lipids in suppressive ascitic fluid compared to normal abdominal fluid. Our study indicates the presence of lipid intermediates in ascites of ovarian cancer patients, which coincidences with T cell dysfunctionality. Since the immune system in the abdominal cavity is compromised, this may explain the high seeding efficiency of disseminated tumor cells. Further research is needed to fully understand the correlation between the various lipids and T cell proliferation, which could lead to new treatment options.

Keywords: lipid intermediates; modified lipid metabolism; paralyzed T cells; tumor microenvironment.

Figure 1

Decreased proliferation, but normal cytokine secretion of ascites-derived T cells. Proliferation and cytokine secretion was measured after stimulating 5 × 10⁴ cells with increasing concentrations of αCD3/28 beads for three days. (A) Proliferation of control PBLs (mean of n = 8), sorted lymphocytes (n = 7) and sorted CD4⁺CD25⁻ T cells (n = 5) from ascites-derived MNCs. (B) Proliferation of patient-derived PBMCs. Data are given as mean of cpm ³H-thymidine incorporation of triplicate cultures ± SEM. (C) IFN-γ; and (D) IL-2 production of ascites-derived lymphocytes (n = 4) and control PBLs (n = 3). Supernatants of triplicate cultures were pooled and tested by an 11-plex for cytokine production. Statistical analysis using one-way ANOVA with Bonferroni post-test (* p < 0.05, ** p < 0.01).

Figure 2

Hypoproliferation of ascites-derived T cells cannot be overcome by IL-2 or IL-12. (A) Proliferation; and (B) IFN-γ secretion of control (n = 3) and ascites-derived CD4⁺CD25⁻ T cells (P61, P68, P73) after three days of stimulation. Cells were cultured with αCD3/28 (2500 beads/well), IL-2 (25 or 125 U/mL), and IL-12 (1000 pg/mL), or a combination of the different stimuli. Values are means ± SD.

Figure 3

Normal expression and phosphorylation of the IL-2 receptor and its downstream signaling components. PBLs of healthy individuals or sorted ascites-derived CD4⁺CD25⁻ T cells (5 × 10⁵ cells/well) were stimulated with 20,000 αCD3/28 beads/well for three days. Subsequently, samples were stained for flow cytometry analysis. (A) Cells were stained for isotype control, CD25, CD122, and CD132. Plots are representative for eight controls and three patients. (B) Control (n = 3) and ascites-derived T cells (n = 3) were stained for JAK3, STAT5, phospho-STAT5, phosphor-AKT and phosphor-ERK. One representative example is shown.

Figure 4

Ascites inhibits proliferation of PBLs from healthy donors: (A) 5 × 10⁴ control PBLs were stimulated with 2500 beads/well in the presence of 20% ascites fluid from ovarian cancer patients. Data are given as mean of cpm ³H-thymidine incorporation of triplicate cultures ± SEM. (B) Summary of PBLs stimulated with αCD3/28 (n = 4) and PBLs stimulated in the presence of ascites (n = 14). Values are given as mean ± SD. Statistical analysis using unpaired student’s t-test with Welch’s correction (*** p < 0.001).

Figure 5

Cyclin D and E expression in control and ascites-derived lymphocytes. Control PBLs or sorted ascites-derived lymphocytes (5 × 10⁵ cells) were stimulated with 20,000 αCD3/28 beads/well. After 48 h, cells were stained for CD4, cyclin D and cyclin E. Plots are representative for three individual controls and four patients.

Figure 6

IL-2 and IL-12 responsiveness of ascites-derived CD4⁺CD25^- can be restored. In total, 5 × 10⁵ sorted CD4⁺CD25^- ascites-derived T cells of P71 were stimulated directly with 5000 αCD3/28 beads/well for three days in the presence or absence of 25 or 125 U/mL IL-2, 1000 pg/mL IL-12 or a combination of both and subsequently cultured for 10 days in normal medium before being re-stimulated. (A) Proliferation was measured before (Day 3) and after culture (Day 10). Data are given as mean of cpm ³HTdR incorporation of triplicate cultures ± SEM. (B) Triplicate cultures were pooled and tested in three different dilutions in an IFN-γ ELISA. The data are representative for three tested patients. (C) Ascites-derived MNCs were sorted using a lymphocyte gate. Subsequently, ascites-derived lymphocytes and PBLs from healthy donors were labeled with CFSE, plated at a concentration of 5 × 10⁵ cells and stimulated with 20,000 αCD3/28 beads. After three days, the cells were stained for CD4 and CD25 and measured with FACS (upper panel). A part of the stimulated cells was kept in culture for ten days and restimulated with αCD3/28 beads, stained for CD4 and CD25 and subsequently measured with FACS (lower panel).

Figure 7

Synthetic Lipid-metabolites partially inhibit T cell proliferation. In total, 50,000 control PBLs from healthy donors were stimulated with αCD3/28 for three days in the presence of 9(R)HODE, 9(S)HODE or 13-OxODE. Only 9(R)HODE decreased the proliferation of control PBLs. Data given as mean of cpm ³HTdR incorporation of triplicate cultures ± SEM. Statistical analysis was performed using one-way ANOVA with Bonferroni post-test (*** p < 0.001).