Fig 1: IL-10 derived from hypoxic TAMs is required to maintain LVEM. a The different cytokines expression profiles among M0-N, M0-H, TAM-N and TAM-H were analysed by cytokine array (RayBio GSM-CAA-4000). b Screening and analysis of the differentially expressed cytokines. c The expressions of the five significant cytokines were analysed by qRT-PCR. d The secretions of the five significant cytokines were analysed by ELISA. e The migration effects of hypoxic TAMs-treated HDLECs on tumour cells (SiHa) and M2-polarized THP-1 macrophages were analysed by transwell assay in vitro. “Blank” represents the medium group. f Representative micrographs showing the tube formation in vitro (Scale bar, 50 μm). g Representative images showing the tube formation in vivo (Scale bar, 100 μm). h Statistical analysis showing the length of tube formation. Average length of tubes per field were calculated. i–l Popliteal lymphatic metastasis model was established in female C57BL/6 mice by inoculating the footpad with TC-1 cells (5 × 106). When footpad tumour size reached 50 mm3, IL-10 (50 ng/ml) or PBS was then injected into the centre of the tumours (n = 5/group, repeated twice) for 2 weeks daily. After 2 weeks of induction, primary tumours reached a comparable size of ~ 150 mm3, and then footpad tumours and popliteal LNs were collected for study. i Representative images of LYVE-1+ lymphatic vessel (red), CD206+ TAMs (green) and DAPI (blue) fluorescence staining in footpad tumour. j Metastasis-positive LNs were identified by IHC staining for epithelial marker CK7. k Statistical analysis showing the expression of peritumoural LV and LVEM in footpad tumour. l Statistical analysis showing the ratio of LNM. Error bars represent the mean ± SD of three independent experiments. **P < 0.01
Fig 2: IL-10 derived from hypoxic TAMs is required to maintain LVEM. a The different cytokines expression profiles among M0-N, M0-H, TAM-N and TAM-H were analysed by cytokine array (RayBio GSM-CAA-4000). b Screening and analysis of the differentially expressed cytokines. c The expressions of the five significant cytokines were analysed by qRT-PCR. d The secretions of the five significant cytokines were analysed by ELISA. e The migration effects of hypoxic TAMs-treated HDLECs on tumour cells (SiHa) and M2-polarized THP-1 macrophages were analysed by transwell assay in vitro. “Blank” represents the medium group. f Representative micrographs showing the tube formation in vitro (Scale bar, 50 µm). g Representative images showing the tube formation in vivo (Scale bar, 100 µm). h Statistical analysis showing the length of tube formation. Average length of tubes per field were calculated. i–l Popliteal lymphatic metastasis model was established in female C57BL/6 mice by inoculating the footpad with TC-1 cells (5 × 106). When footpad tumour size reached 50 mm3, IL-10 (50 ng/mL) or PBS was then injected into the centre of the tumours (n = 5/group, repeated twice) for 2 weeks daily. After 2 weeks of induction, primary tumours reached a comparable size of ~ 150 mm3, and then footpad tumours and popliteal LNs were collected for study. i Representative images of LYVE-1+ lymphatic vessel (red), CD206+ TAMs (green) and DAPI (blue) fluorescence staining in footpad tumour. j Metastasis-positive LNs were identified by IHC staining for epithelial marker CK7. k Statistical analysis showing the expression of peritumoural LV and LVEM in footpad tumour. l Statistical analysis showing the ratio of LNM. Error bars represent the mean ± SD of three independent experiments. **P < 0.01
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