Figure 3: TNF-α in human tumour lymphangiogenesis and metastasis.

(a) ELISA assay of TNF-α protein levels in the conditioned medium of human OVCAR-8 and IGROV-1 ovarian cancer cells. (b) Confocal image of LYVE-1⁺ tumour lymphatic vessels (red) in OVCAR-8 and IGROV-1 tumours. Dashed line marks the borders between tumour tissues and neighbouring healthy tissues. Arrowheads point to intratumoral lymphatics. Intratumoral lymphatic vessels were quantified (eight randomized fields per group). PTL, peritumoral lymphatics; ITL, intratumoral lymphatics. Scale bar, 100 μm. (c) TNFα shRNA inhibited TNF-α mRNA and protein expression in stably transfected IGROV-1 tumour cells (n=3 samples per group). Real-time PCR and ELISA were used for detection. Growth rates of TNFα shRNA- and scrambled shRNA-transfected IGROV-1 cells. (d) Lymphatic vessel (red) localization and quantification in TNFα shRNA-transfected and control IGROV-1 tumours. Dashed line marks the borders between tumour tissues and neighbouring healthy tissues. Arrowheads point to intratumoral lymphatics. Lymphatic vessels were quantified from eight randomized fields per group. Localization and quantification of TAMs (green) in TNFα shRNA-transfected and control IGROV-1 tumours. Macrophages were quantified from eight randomized fields of six samples per group. Scale bar, 100 μm (upper panels); 50 μm (lower panels). (e) Left: representative sentinel lymph nodes (LNs) of TNFα shRNA-transfected and control IGROV-1 tumour-bearing mice. Scale bar, 1 cm. Middle: hematoxylin and eosin stain (H&E) and fluorescence images of LNs. TNFα shRNA-transfected IGROV-1 metastases were detected in LNs. Dashed line marks the rim of a metastatic tumour in LN. T, tumour. Scale bar, 50 μm (upper panels); 100 μm (lower panels). Right: quantification of LN volume and weight in TNFα shRNA-transfected and control IGROV-1 tumour-bearing mice (n=10 mice per group). *P<0.05; **P<0.01. All error bars represent s.e.m. All P values were analysed according to Student’s t-test.