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: For NFL treatment, NFL (CUSABIO, CSB-YP015688MO, 200 μg/200 g body weight) or PBS (control) was mixed with the tumor cell suspension prior to implantation.

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 treatment increased the proportion of tumor-infiltrating macrophages. ( A ) Proportional distribution of myeloid cells in tumor tissues from mice treated with NFL or PBS. ( B ) Feature plot showing the expression of Cd14 in immune cells from tumor tissues. ( C ) Representative flow cytometry plot showing immune cells from tumor tissues of mice treated with PBS or NFL. ( D ) The proportion of TAM (CD11b + F4/80 + ) cells within CD45 + live cells in ( C ), ( n PBS = 4, n NFL = 7). ( E ) Representative immunofluorescent images of F4/80 and panCK staining in tumor tissues from mice treated with NFL or PBS. ( F ) shows the proportion of F4/80 + cells among total cells in ( E ), statistic by HALO software, ( n PBS = 3, n NFL = 4). ( G ) Representative immunofluorescent images of F4/80 and panCK staining in tumor tissues from mice treated with NS (control) or 6-OHDA. ( H ) The proportion of TAM (CD45 + /F4/80 + ) cells within cells in ( G ), ( n Control = 3, n 6 − OHDA = 3). ( I ) Representative flow cytometry plot showing TAM (F4/80 + ) cells from tumor tissues of mice treated with NS (control) or 6-OHDA. ( J ) The proportion of TAM (CD11b + F4/80 + ) cells within live cells in ( I ), ( n Control = 3, n 6 − OHDA = 3). All data are presented as mean ± SD. Data in ( F ) and ( H ) were statistically analyzed using HALO software. Statistical significance was determined using a t-test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For NFL treatment, NFL (CUSABIO, CSB-YP015688MO, 200 μg/200 g body weight) or PBS (control) was mixed with the tumor cell suspension prior to implantation.

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

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: For NFL treatment, NFL (CUSABIO, CSB-YP015688MO, 200 μg/200 g body weight) or PBS (control) was mixed with the tumor cell suspension prior to implantation.

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: For NFL treatment, NFL (CUSABIO, CSB-YP015688MO, 200 μg/200 g body weight) or PBS (control) was mixed with the tumor cell suspension prior to implantation.

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