Figure 1.
High-grade serous ovarian tumors express the vasculature marker CD31.
(A) An image of high (left panel) or low (right panel) CD31 immunohistochemistry staining scanned at 10× magnification. Scale bar = 50 µm. Inset images show high (left panel) or low (right panel) CD31 staining when scanned at 40× magnification. Arrowheads indicate brown regions of CD31 staining (B) Kaplan-Meier analysis of disease-specific survival in high-grade serous ovarian cancer patients. Statistical significance was assessed using a log-rank test. (C) Mean CD31 vascular density comparisons with patient age (split on the median) and disease stage (p = not significant). Statistical significance was assessed using a Mann Whitney rank-sum test. (D) Contingency analysis of CD31 and VEGF. Statistical significance was assessed by Fisher's exact test.
Table 1.
Patient characteristics, follow-up time and survival characteristics for high-grade serous ovarian carcinoma cases.
Figure 2.
High-grade serous tumors containing TIL have higher vascular density scores than those without TIL.
Mean CD31 vascular density scores were compared in relation to expression of (A) CD8, (B) CD4 and (C) FoxP3. Error bars indicate standard error of the mean. Statistical significance was assessed by Mann Whitney test. *p<0.05, ** p<0.01.
Figure 3.
High-grade serous tumors containing markers of immune function have higher vascular density scores.
Mean CD31 vascular density scores were compared in relation to the expression of (A) TIA-1 and (B) granzyme B (GzmB) tumor infiltrates in high-grade serous tumors. Error bars indicate standard error of the mean. Statistical significance was assessed by Mann Whitney test. ** p<0.01, ***p<0.001.
Figure 4.
Hypoxia suppresses CD8 T cell effector function.
(A) Representative histogram plots for the mean fluorescence intensity of IFNγ (top panel) and TNFα (bottom panel) produced by CD8 T cells cultured under normoxic conditions or 1.5% oxygen. (B) The average geometric mean fluorescence and standard error of the mean for 3 independent experiments is shown. Statistical significance was determined by paired t-tests. (C) OT-I T cells were co-cultured with E.G7 (top panel) or EL4 (bottom panel) target cells at various effector∶target ratios for 4 hours under normoxia (norm) or 1.5% oxygen (hyp). The mean and standard error of the mean of 4 or 5 independent cytotoxicity experiments is shown. Statistical significance was determined by paired t-tests on log transformed values. *p<0.05, ** p<0.01, ***p<0.001.
Figure 5.
Hypoxia activates autophagy in CD8 T cells.
(A) A representative Western blot indicating protein expression of HIF-1α, p62, LC3-II and β-actin is shown for OT-I CD8 T cells cultured under 1.5% oxygen. Cobalt chloride (CoCl2) treatment was used as a positive control for HIF-1α protein expression. (B) Autophagy induction is shown by quantification of decreasing p62 levels without chloroquine (CQ) treatment and (C) LC3-II accumulation with CQ treatment under normoxia (norm) or 1.5% oxygen (hyp). The mean and standard error of the mean for the fold change of 3 independent experiments is reported. Statistical significance was determined using a one-sample t-test on log transformed values compared to a hypothetical mean of zero. *p<0.05.
Figure 6.
TIL and markers of immune function are associated with improved patient outcome.
Kaplan-Meier analysis of disease-specific survival of high-grade serous ovarian carcinoma patients categorized by CD31, VEGF or the indicated immune markers: (A) CD31 and FoxP3, (B) CD31 and TIA-1, (C) CD31 and CD8, (D) VEGF and Granzyme B (GzmB), (E) VEGF and CD8. The statistical significance was determined using a log-rank test.