Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Peripheral nerve injury-induced remodeling of the tumor-associated macrophages promotes immune evasion in breast cancer

doi: 10.1186/s13046-025-03545-x

Figure Lengend Snippet: Peripheral nerve destruction restrained antitumor immunity. ( A ) Area under the curve of tumor size in NOD-SCID mice upon treatment with 6-OHDA or normal saline. ( n = 8 per group). ( B ) The image of tumors from the control and 6-OHDA treatment groups.in NOD-SCID mice. ( C ) Representative immunofluorescent images of Granzyme B (GZMB), CD8, and panCK staining in tumor tissues from mice treated with 6-OHDA or normal saline (left). Right panel shows the proportion of GZMB + cells among total CD8 + T cells. ( n = 3 each group). ( D ) Percentage of GZMB + cells among CD45 + CD8 + T cells, as measured by flow cytometry. ( n Control = 3, n 6 − OHDA = 6. ( E ) t-SNE map of immune cell populations following treatment with nerve lysate or normal saline (left). Right panel shows t-SNE map of immune cell population distributions, grouped by treatment. ( F ) Proportional distribution of immune cells in tumor tissues from mice treated with LYSATE or NS. ( G ) Percentage of CD8 + T cells, as measured by mIHC, statistic by HALO software. ( H ) Proportion of GZMB + cells within total CD8 + T cells in the LYSATE and NS treatment groups, as measured by mIHC, statistic by HALO software. ( I ) Representative immunofluorescent images of ( G ) and ( H ). Data in ( A ) are presented as mean ± SEM, while other data are presented as mean ± SD. Data in ( G ) and ( H ) are statistic by HALO software. Statistical significance was determined using a t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: CD11b+ cells and CD8+ T cells were then separated using the EasySepTM Mouse CD11b Positive Selection Kit II (stemcell, 18970) and REAlease ® CD8a MicroBead Kit, mouse (Miltenyi, 130-126-707), respectively, following the manufacturer’s instructions, and immediately used for downstream functional assays and experimental analyses.

Techniques: Saline, Control, Staining, Flow Cytometry, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Peripheral nerve injury-induced remodeling of the tumor-associated macrophages promotes immune evasion in breast cancer

doi: 10.1186/s13046-025-03545-x

Figure Lengend Snippet: Nerve Injury Promoted Breast Cancer Immune Suppression via NFL. ( A ) The Venn diagram analysis of the top 20 high-scoring proteins (> 10 kDa) from 2 neural lysate samples and 3 tumor supernatant samples identified neurofilament light chain (NFL) as the unique intersection protein present in nerve injury conditions but absent in tumor microenvironments. ( B ) Experimental design of NFL treatment in a PY8119 tumor mouse model on C57BL/6 background. ( C ) Tumor growth curve(up) in C57BL/6 mice treated with NFL or PBS (up), and the image(down) of tumors from the PBS and NFL treatment group in C57BL/6 mice. ( n = 6 per group). ( D ) Tumor growth curve(left) in NOD-SCID mice treated with NFL or PBS (left), and the image(right) of tumors from the PBS and NFL treatment groups in NOD-SCID mice. ( n = 6 per group). ( E ) t-SNE map showing the distribution of immune cell populations, grouped by treatment. ( F ) t-SNE map of different T cell subtypes. ( G ) t-SNE map showing T cell subtype distributions, grouped by treatment. ( H ) Proportional distribution of T cells in tumor tissues from mice treated with NFL or PBS. ( I ) Representative flow cytometry plot showing percentage of CD8 + or CD4 + cells among CD45 + CD3 + live cells from tumor tissues of mice treated with NFL or PBS. ( J ) A scatter plot represents the percentage of CD8 + T cells in ( I ), ( n = 3 pergroup). ( K ) Violin plot showing the expression level of Mki67 in the NFL and PBS treatment groups. ( L ) Representative immunofluorescent images of Granzyme B and CD8 staining in tumor tissues from mice treated with NFL or PBS (left). Right panel shows the proportion of GZMB + cells among total CD8 + T cells, as measured by mIHC, statistic by HALO software, ( n NFL = 4, n PBS = 3). All data are presented as mean ± SD. Statistical significance was determined using a t-test. * p < 0.05

Article Snippet: CD11b+ cells and CD8+ T cells were then separated using the EasySepTM Mouse CD11b Positive Selection Kit II (stemcell, 18970) and REAlease ® CD8a MicroBead Kit, mouse (Miltenyi, 130-126-707), respectively, following the manufacturer’s instructions, and immediately used for downstream functional assays and experimental analyses.

Techniques: Flow Cytometry, Expressing, Staining, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Peripheral nerve injury-induced remodeling of the tumor-associated macrophages promotes immune evasion in breast cancer

doi: 10.1186/s13046-025-03545-x

Figure Lengend Snippet: NFL-induced TAM enrichment promoted CD8 + T cell senescence. ( A ) t-SNE map showing macrophage populations following NFL or PBS treatment. ( B ) Proportional distribution of each macrophage cluster after treatment with NFL or PBS. ( C ) Expression of Tnf (left) and Il6 (right) in macrophages following NFL or PBS treatment. ( D ) Relative expression (normalized to GAPDH) of Tnf (left) and Il6 (right) in macrophages following NFL or PBS treatment, detected by RT-qPCR. ( E ) Expression of Gzmk (left) and Cd28 (right) in CD8 + T cells following NFL or PBS treatment. ( F ) Positive proportion of Ki67, CD28 and GZMB in CD8 + T cells following NFL or PBS treatment, detected by flow cytometry. ( G ) Schematic of non-contact co-culture between macrophages and CD8 + T cells, and proportion of GZMB + CD8 + T cells in co-culture with macrophage-conditioned media after NFL or PBS treatment (left). The IFN-γ levels in the supernatant of CD8 + T cells, which were quantified by ELISA (right). ( H )Representative flow cytometry plot (left) showing positive proportion of Ki67, CD28, GZMB in live CD8 + T cells treated with PBS or NFL. The right panel shows the proportion of positive cells within CD8 + live cells. ( I ) Representative flow cytometry plot (left) showing positive proportion of Ki67 in CD8 + T cells treated with macrophages’ supernatant with NFL treatment or additionally added neutralizing antibodies against TNF-α and IL-6. The right panel shows the proportion of Ki67 + cells within CD8 + live cells. ( J ) Flow cytometry histogram illustrating the expression of the senescence marker β-galactosidase (β-gal) in CD8 + T cells (left). Bar graph comparing the mean fluorescence intensity (MFI) of CD8 + T cell senescence marker induced by PBS-and NFL-treated macrophages (right). All data are presented as mean ± SD. Statistical significance was determined using a t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: CD11b+ cells and CD8+ T cells were then separated using the EasySepTM Mouse CD11b Positive Selection Kit II (stemcell, 18970) and REAlease ® CD8a MicroBead Kit, mouse (Miltenyi, 130-126-707), respectively, following the manufacturer’s instructions, and immediately used for downstream functional assays and experimental analyses.

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Marker, Fluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Peripheral nerve injury-induced remodeling of the tumor-associated macrophages promotes immune evasion in breast cancer

doi: 10.1186/s13046-025-03545-x

Figure Lengend Snippet: The supernatant of macrophages treated with NFL activates the NF-κB pathway in T cells, promoting cellular senescence. ( A ) GSEA enrichment analysis reveals that supernatants from macrophages treated with NFL activate the NF-κB pathway in CD8 + T cells. ( B ) Immunofluorescence representative images shows the nuclear translocation of NF-κB in CD8 + T cells treated with supernatants from macrophages treated or untreated with NFL. ( C ) Quantitative analysis of ( B ) showing the percentage of CD8 + T cells with NF-κB nuclear translocation (%) in the NFL-treated macrophage supernatant group versus untreated controls ( n = 3 per group). ( D ) Flow cytometry histogram illustrating the expression of the senescence marker β-galactosidase (β-gal) in CD8 + T cells treated with: supernatants from NFL-treated macrophages, supernatants from untreated macrophages, NF-κB inhibitor alone, or NFL-treated macrophage supernatants combined with NF-κB inhibitor. Right panel compares the mean fluorescence intensity (MFI) of β-gal in CD8 + T cells across these treatment groups ( n = 3 per group). Data are presented as mean ± SD; statistical significance was determined by one-way ANOVA. ( E ) Flow cytometry histogram illustrating the expression of the senescence marker β-galactosidase (β-gal) in CD8 + T cells isolated from tumor tissues of mice treated with PBS or NFL (left), right panel shows the mean fluorescence intensity (MFI) of CD8 + T cell senescence marker induced by PBS-and NFL-treatment. All data are presented as mean ± SD. Statistical significance was determined using Student’s t-test for all panels except ( D ), where one-way ANOVA was applied ( p < 0.05, * p < 0.01, ** p < 0.001, *** p < 0.0001)

Article Snippet: CD11b+ cells and CD8+ T cells were then separated using the EasySepTM Mouse CD11b Positive Selection Kit II (stemcell, 18970) and REAlease ® CD8a MicroBead Kit, mouse (Miltenyi, 130-126-707), respectively, following the manufacturer’s instructions, and immediately used for downstream functional assays and experimental analyses.

Techniques: Immunofluorescence, Translocation Assay, Flow Cytometry, Expressing, Marker, Fluorescence, Isolation