Journal: Nature Communications

Article Title: Breast cancer remodels lymphatics in sentinel lymph nodes

doi: 10.1038/s41467-025-64981-z

Figure Lengend Snippet: a Experimental setup for the in vitro experiments. Cells were seeded at Day 0 (D0), CM was generated on D1 to D2 (24 hours) and receiving cells were exposed on D2 to D4 (48 h). b Venn diagrams showing the number of upregulated and downregulated genes obtained by RNA-seq of CM-exposed LECs. c Heatmap showing the top DEGs in the CM-exposed LECs. Gray triangles indicate the result of unsupervised clustering for genes and cell types behaving in a similar way. d Volcano plots depicting genes upregulated and downregulated in LECs after CM exposure (two-sided Wald test). Genes indicated in red are considered mostly pro-inflammatory, whereas genes in blue are anti-inflammatory. e Pathway analysis showing the most significantly downregulated and upregulated pathways (the integrated two-sided Fisher’s exact test). LECs are from 4 different sources ( n = 4) ( a – e ). f Gene expression changes in CM-exposed LECs ( n = 3–9 HLECs). Data were analyzed with a one-way ANOVA. g Protein expression levels of MGP in LECs following CM exposure ( n = 6 HLECs). Data were analyzed with a one-way ANOVA linear mixed model and Dunnett’s correction. h Protein expression of MGP and podoplanin on LECs after coculture of T47D cells with LECs compared to culture with CM. Data were analyzed with two-way ANOVA and Sidak’s multiple comparison test ( n = 4 HLECs). i mRNA Expression of CDH2 , CDH5 , and SNAI1 in CM-exposed LECs. Data were shown as box plots ( n = 5–7 HLECs). Data were analyzed using a two-way ANOVA linear mixed model and statistical significance was adjusted with Dunnett multiplicity correction. Source data, non-significant p values and detailed experiment and n -numbers (biologically independent samples of cultured cells) are provided in the Source Data file.

Article Snippet: Cells were blocked for 20 min with human Ig on ice, permeabilized with the BD Cytofix/Cytoperm Kit (554714), and sequentially stained for 30 min with the following antibodies: MGP (NBP2-45844) from Novus (1:150); negative control, mouse IgG2a (553454) from BD and anti-mouse IgG2a AF546 (A21133) from Invitrogen.

Techniques: In Vitro, Generated, RNA Sequencing, Gene Expression, Expressing, Comparison, Cell Culture

Journal: Nature Communications

Article Title: Breast cancer remodels lymphatics in sentinel lymph nodes

doi: 10.1038/s41467-025-64981-z

Figure Lengend Snippet: a Expression of MGP in LECs after direct exposure to recombinant VEGF165 (5, 25, or 100 ng/mL), VEGF-C (5, 50, or 100 ng/mL), TGF-β (5, 25, or 100 ng/mL), or EGF (5, 25, or 100 ng/mL) are shown ( n = 4 HLECs). Data were depicted as Tukey box plots and analyzed using one-way ANOVA linear mixed models (Sidak correction) fitted separately for each parameter with group (recombinant vs control), dose and their interaction as fixed effects. b Gene expression changes in modified CM-exposed LECs are shown as determined by qPCR. CM was generated in the presence of antibodies against VEGFR3 (1 or 10 μg/mL, n = 4 HLECs), TGF-β (2 or 20 μg/mL, n = 8–10 HLECs), and EGF (1 or 10 μg/mL, n = 3–5 HLECs), compared to isotype control exposed samples and data were depicted as Tukey box plots showing relative gene changes and analyzed using a one-way ANOVA linear mixed models (Sidak) fitted separately for each parameter with group (antibody vs control) and dose and their interaction as fixed effects. c MGP expression determined by qPCR. CM generated with culture media devoid of VEGF supplement was used. Data were shown as Tukey box plots ( n = 9 HLECs). Data were analyzed using a one-way ANOVA linear mixed model (Sidak). The center line of the box plots represents the median, the box the 25th to 75th percentiles and the whiskers the inner fences. Statistics of group and dose effects are presented within the boxes; significant differences in comparison to the controls (defined as 1) are indicated by p values. Source data, non-significant p values and detailed experiment and n -numbers (biologically independent samples of cultured cells) are provided in the Source Data file.

Article Snippet: Cells were blocked for 20 min with human Ig on ice, permeabilized with the BD Cytofix/Cytoperm Kit (554714), and sequentially stained for 30 min with the following antibodies: MGP (NBP2-45844) from Novus (1:150); negative control, mouse IgG2a (553454) from BD and anti-mouse IgG2a AF546 (A21133) from Invitrogen.

Techniques: Expressing, Recombinant, Control, Gene Expression, Modification, Generated, Comparison, Cell Culture

Journal: Nature Communications

Article Title: Breast cancer remodels lymphatics in sentinel lymph nodes

doi: 10.1038/s41467-025-64981-z

Figure Lengend Snippet: a MGP expression in LECs after CM treatment with anti-MGP antibody ( n = 5–6 HLECs), analyzed using one-way ANOVA linear mixed models (Sidak correction). b MGP expression in LECs exposed to anti-MGP antibody or recombinant MGP ( n = 6–10 HLECs), analyzed using two-way ANOVA linear mixed models (Sidak correction). c MGP expression in siRNA-silenced LECs ( n = 5 HLECs), analyzed by two-sided Mann–Whitney U -test. d Tube formation quantified by number of nodes, junctions and branches in MGP-silenced vs. control LECs, shown as geometric mean with 95% CI ( n = 17 HLECs), analyzed with a two-sided paired t -test. e Scratch assay of MGP-silenced and control LECs over 2 days (mean ± SEM; n = 13 HLECs), analyzed by repeated measures two-way ANOVA (matched full mixed model) with Sidak’s multiple comparisons. f Soluble MGP binding to cancer cells: MGP (His) was added with anti-His antibody (red), while the control (blue) contained only cancer cells with antibody. Histograms represent two independent experiments. g Ex vivo adhesion assay of T47D cells binding to lymphatic sinuses of six metastatic and five non-metastatic LNs from four and five patients, respectively (two-sided ratio paired t -test). LN sections were treated with anti-MGP or control antibody. The binding after control antibody was defined as 100% due to day-to-day variation. Example images show T47D cells binding after control vs. anti-MGP treatment to the same metastatic LN area. Adherent cells (some marked by yellow arrowheads) lie on top of tissue sections, focus adjusted to highlight cell adhesion. Scale bar: 50 μm. For box plots: center line = median; box = 25th–75th percentiles; whiskers = inner fences. Source data, non-significant p values and detailed experiment and n -numbers (biologically independent samples of cultured cells) are provided in the Source Data file.

Article Snippet: Cells were blocked for 20 min with human Ig on ice, permeabilized with the BD Cytofix/Cytoperm Kit (554714), and sequentially stained for 30 min with the following antibodies: MGP (NBP2-45844) from Novus (1:150); negative control, mouse IgG2a (553454) from BD and anti-mouse IgG2a AF546 (A21133) from Invitrogen.

Techniques: Expressing, Recombinant, MANN-WHITNEY, Control, Wound Healing Assay, Binding Assay, Ex Vivo, Cell Adhesion Assay, Cell Culture