Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Single-cell transcriptome analysis reveals the malignant characteristics of tumour cells and the immunosuppressive landscape in HER2-positive inflammatory breast cancer

doi: 10.1186/s13046-025-03454-z

Figure Lengend Snippet: B cells in the IBC tumour microenvironment promote inflammatory responses through high expression of tumour necrosis factor. a Comparison of 6 TNF-related pathway signals in B cells and endothelial cells. b Comparison of 15 in death signalling scores in endothelial cells. c After co-culturing IBC-educated B cell supernatant with endothelial cells, the viability of endothelial cells was assessed by Calcein-AM/EthD-1 staining. The experimental schematic is shown. d, f Nec-1 or PTN blocking peptide was used to inhibit endothelial cell death induced by IBC-educated B cell supernatant. The bar graph ( d ) shows the percentage of endothelial cell death, and immunofluorescence imaging ( f ) shows the Calcein-AM + and EthD-1 + endothelial cells. e, g sIn endothelial tube formation assays, Nec-1 or PTN blocking peptide was used to block endothelial cell death induced by IBC-educated B cell supernatant. Quantification of tube formation is presented as bar graphs ( e ), and representative tube structures are shown by phase-contrast microscopy ( g ). i TNF + B cells were found around the necrotic endothelial cells based on multiplex immunofluorescence. j Under normal culture, B cell supernatant co-culture, or IBC tumor cell-treated B cell supernatant conditions, Nec-1 or PTN blocking peptide was used to block endothelial cell death induced by IBC-educated B cell supernatant. Western blotting was used to detect phosphorylated MLKL. k Under normal culture, B cell supernatant co-culture, or PTN-treated B cell supernatant co-culture conditions, RIPK3 expression in endothelial cells was knocked down or TNF neutralizing antibody was used to block endothelial cell death induced by IBC-educated B cell supernatant. q-PCR was used to measure the expression levels of IL-6 and IL-8 in endothelial cells. l M0 macrophages were cultured under normal conditions, endothelial cell supernatant co-culture, or supernatant of endothelial cells educated with IBC-treated B cells. IgG or IL-6 neutralizing antibody was added, and q-PCR was used to measure the expression of M2 macrophage markers (IL-10, TGF-β, and CD206) in M0 macrophages. m Diagram of the mechanism of interactions between tumour cells, B cells and endothelial cells in the IBC tumour microenvironment

Article Snippet: HUVECs from control and experimental groups were incubated with 2 μM Calcein-AM and 4 μM Ethidium Homodimer-1 (EthD-1) (MCE, HY-D0093) for 10 min in the dark.

Techniques: Expressing, Comparison, Staining, Blocking Assay, Immunofluorescence, Imaging, Microscopy, Multiplex Assay, Co-Culture Assay, Western Blot, Cell Culture