Journal: Angiogenesis

Article Title: A novel lymphatic pattern promotes metastasis of cervical cancer in a hypoxic tumour-associated macrophage-dependent manner

doi: 10.1007/s10456-020-09766-2

Figure Lengend Snippet: Hypoxic TAMs promote lymphangiogenesis and LVEM formation. a The function of CM from different macrophage-treated HDLECs on M2-polarized THP-1 macrophages and tumour cells (SiHa) was detected by transwell array in vitro. b Statistical analysis showing the length of tube formation in vitro. Average length of tubes per field were calculated. c Statistical analysis showing the expression of LV and LVEM in mouse footpad tumour. d Representative micrographs showing tube formation assay in vitro of HDLECs pretreated with different macrophage CM for 48 h. Scale bar, 50 μm. e – i Popliteal lymphatic metastasis model was established in female C57BL/6 mice by inoculating the footpad with TC-1 cells (5 × 10 6 ). When footpad tumour size reached 50 mm 3 , macrophage supernatants of different treatment conditions (10 μl) were 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 mm 3 , and then footpad tumours and popliteal LNs were collected for study. e Representative images of LYVE-1 + lymphatic vessel (red), CD206 + TAMs (green) and DAPI (blue) fluorescence staining in footpad tumour. Images are shown at × 400 magnification (Scale bar, 50 μm). f IHC Staining of CK7 in popliteal LNs. Representative micrographs are shown (Scale bar, 100 μm). Metastasis-positive LNs were identified by staining for epithelial marker CK7. g Photos of mouse popliteal LNs in different macrophage CM-primed tumour ( n = 5/group). h Statistical analysis showing the volume (mm 3 ) of the LNs. i The ratio of metastasis-positive to total dissected popliteal LNs from mice treated with different macrophage supernatants. Error bars represent the mean ± SD of three independent experiments. * P < 0.05. N: Normoxia; H: Hypoxia

Article Snippet: Human dermal LECs (HDLECs) were purchased from ScienCell and cultured in endothelial cell medium (ScienCell, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum and endothelial growth medium supplements (ScienCell).

Techniques: In Vitro, Expressing, Tube Formation Assay, Injection, Fluorescence, Staining, Immunohistochemistry, Marker

Journal: Angiogenesis

Article Title: A novel lymphatic pattern promotes metastasis of cervical cancer in a hypoxic tumour-associated macrophage-dependent manner

doi: 10.1007/s10456-020-09766-2

Figure Lengend Snippet: 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 × 10 6 ). When footpad tumour size reached 50 mm 3 , 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 mm 3 , 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

Article Snippet: Human dermal LECs (HDLECs) were purchased from ScienCell and cultured in endothelial cell medium (ScienCell, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum and endothelial growth medium supplements (ScienCell).

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay, In Vitro, In Vivo, Injection, Fluorescence, Staining, Immunohistochemistry, Marker

Journal: Angiogenesis

Article Title: A novel lymphatic pattern promotes metastasis of cervical cancer in a hypoxic tumour-associated macrophage-dependent manner

doi: 10.1007/s10456-020-09766-2

Figure Lengend Snippet: CCL1 mediates the migration of TAMs towards LECs in the formation of LVEM. a Multiple related chemokines were screened in IL-10-activated LECs by qRT-PCR. b Levels of CCL1 secreted by IL-10-activated LECs were measured by ELISA. c The migration effects of CCL1 on tumour cells (SiHa) and M2-polarized THP-1 macrophages were significantly decreased by blockade of CCR8 on receptor cells (siRNA or CCR8 antagonist MC148) or adding anti-CCL1 neutralization antibody in IL-10-activated LEC CM. d – f Popliteal lymphatic metastasis model was established in female C57BL/6 mice by inoculating the footpad with TC-1 cells (5 × 10 6 ). When footpad tumour size reached 50 mm 3 , CCL1 (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 mm 3 , and then popliteal LNs were collected for study. d Representative images of LYVE-1 + lymphatic vessel (red), CD206 + TAMs (green) and DAPI (blue) fluorescence staining in footpad tumour. e IHC Staining of CK7 in popliteal LNs from mice treated with CCL1 (Scale bar, 100 μm). f Statistical analysis showing the ratio of LNM. Error bars represent the mean ± SD of three independent experiments. ** P < 0.01. * P < 0.05

Article Snippet: Human dermal LECs (HDLECs) were purchased from ScienCell and cultured in endothelial cell medium (ScienCell, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum and endothelial growth medium supplements (ScienCell).

Techniques: Migration, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Neutralization, Injection, Fluorescence, Staining, Immunohistochemistry

Journal: Angiogenesis

Article Title: A novel lymphatic pattern promotes metastasis of cervical cancer in a hypoxic tumour-associated macrophage-dependent manner

doi: 10.1007/s10456-020-09766-2

Figure Lengend Snippet: Sp1 high LECs are fundamental to LVEM formation and lymphatic metastasis. a Bioinformatic prediction and screening of potential transcription factors of CCL1. b RNA sequence of the predicted Sp1-binding site to CCL1 was TCCCCTCCCCC. A dual-luciferase reporter assay system was performed to demonstrate the direct binding of Sp1 to the CCL1 promoter region. Transient co-transfection of pcDNA3.1-Sp1 construct with CCL1 promoter into both 293T cells and HDLECs resulted in a significant increase in firefly luciferase activity relative to co-transfection of pcDNA3.1-NC construct with CCL1 promoter or the negative control. c EILSA analysis showed that CCL1 upregulation was detected in Sp1-transduced HDLEC CM, and CCL1 was decreased in Sp1-silenced HDLEC CM. d Representative micrographs of the tube formation assay in vitro (upper panel) and in vivo (lower panel) of HDLECs with stable overexpression or silencing of Sp1 (Scale bar, 100 μm). e Statistical analysis showing the length of tube formation. Average length of tubes per field were calculated. f The migration effects of M2-polarized THP-1 macrophages and tumour cells (SiHa) towards CM of HDLECs with stable overexpression or silencing of Sp1 were detected by transwell array in vitro. g Immunofluorescence staining was applied to analyse Sp1 (purple), LYVE-1 (red), CD163 (green) and DAPI (blue) expression in CSCC tissues (Scale bar, 100 μm). h The correlation between Sp1 expression and LVEM density was statistically analysed by Pearson’s coefficient test (red line, r = 0.5846, P = 0.0009; blue line, r = 0.1018, P = 0.2465). i Immunofluorescence staining was applied to analyse Sp1 (purple), CCL1 (red), CD163 (green) and DAPI (blue) expression in CSCC tissues (Scale bar, 50 μm). j The correlation between Sp1 and CCL1 expression was statistically analysed by Pearson’s coefficient test ( r = 0.4126, P < 0.001). Error bars represent the mean ± SD of three independent experiments. *, P < 0.05

Article Snippet: Human dermal LECs (HDLECs) were purchased from ScienCell and cultured in endothelial cell medium (ScienCell, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum and endothelial growth medium supplements (ScienCell).

Techniques: Sequencing, Binding Assay, Luciferase, Reporter Assay, Cotransfection, Construct, Activity Assay, Negative Control, Tube Formation Assay, In Vitro, In Vivo, Over Expression, Migration, Immunofluorescence, Staining, Expressing