Fig 2: Loss of BGN in BCSCs leads to reduced CSC populations and tumorsphere forming ability. (A) Immunoblots showing BGN expression in MMTV-PyMT tumor cells (FF99) were stably expressing mouse BGN sgRNA and Control sgRNA (Ctrl sgRNA). Immunoblots and immunofluorescence depicting BGN expression in (B,C) ALDH+ BCSCs and (D,E) CD29hi CD61+ BCSCs. (F) Dot plots of ALDH assay of ALDH+ cells in BGN sgRNA cells compared to Ctrl sgRNA. (G) Quantification of ALDH assay of ALDH+ BCSCs with BGN depletion. (H) FACS analysis of CD29 and CD61 marker expression in FF99 cells transduced with control and BGN sgRNA. (I) Quantification of H depicting the percentage of CD29hi CD61+ BCSCs of BGN depleted cells. Representative images showing tumorsphere formation of (J) CD29hi CD61+ and (L) ALDH+ BCSCs and their (K,M) quantification. Scale bar = 50 µm. Data shown as mean ± SEM of three or four independent experiments. Statistical significance was determined using two-tailed t test; * p = 0.05 as compared to control sgRNA. Representative images of number of spheres were quantified using ImageJ.

Fig 3: VCAM-1 blockade reduces lymphatic permeability. (a, b) Representative FACS histograms (left panels) and staining quantification (right panels) of the classic VCAM-1 receptors integrin a4 (Itga4), integrin a9 (Itga9) and integrin ß1 (Itgb1) in 4T1-1F8 cells (a) and cultured mouse LECs with or without TNF-a treatment for 24 hr (b; n = 3). (c) Adhesion of fluorescently labeled 4T1-1F8 cells to monolayers of cultured mouse LECs pretreated with TNF-a or not and in the presence of control IgG or VCAM-1 blocking antibodies (n = 3 wells/condition; 1 representative of 6 individual experiments is shown). (d) In vitro permeability of mouse LEC monolayers grown on transwell inserts (0.4 µm pore size) toward 70 kD FITC-dextran. LEC monolayers were pretreated with TNF-a overnight and subsequently incubated with control or 4T1 CM in presence of control IgG or anti-VCAM-1 antibodies (n = 3 wells/condition; one representative of three individual experiments is shown). Statistical test refers to 4T1-1F8 CM—rat IgG1 compared to 4T1-1F8 CM—anti-VCAM-1. (e) Representative images (left panels) and quantification (right panel) of VE-cadherin staining in mouse LEC monolayers that were pretreated with TNF-a overnight and subsequently incubated with control or 4T1 CM in presence of control IgG or anti-VCAM-1 antibodies (n = 10 random images/condition; *p < 0.05). [Color figure can be viewed at wileyonlinelibrary.com]

Fig 4: Uptake of B16F10-derived EVs by LN LECs is partly dependent on VCAM-1. (A, B) Representative histogram (left panels) and quantification (right panels) of a4 integrin (ITGA4, A, N = 5) and ß1 integrin (ITGB1, B, N = 3)) on the surface of B16F10-derived EVs detected by small particle flow cytometry. * p < 0.05, ** p < 0.01, Student's t-test. (C) Representative immunofluorescence images of subcapsular (left) and medullary (right) regions of naïve and B16F10 tumour-draining inguinal LNs stained for Lyve-1 (red) and VCAM-1 (green). (D) Example histograms and quantification of VCAM-1 expression in LN LEC subsets by flow cytometry (N = 3). *** p < 0.001, **** p < 0.0001, one-way ANOVA with Sidak's post-test. (E) Re-analysed single-cell RNA sequencing data of human LN LECs (Takeda et al., 2019) demonstrating enrichment of VCAM-1 transcripts in fLECs (LEC II) and mLECs (LEC IV). (F, G) B16F10-derived EVs labelled with DiD were injected into tamoxifen-treated Prox1-CreERT2 x Vcam1fl/fl mice. Example histograms (left panels) and quantification (right panels) of DiD uptake in popliteal LN LECs (F) and MSMs (G) (Control = wildtype mice injected with control liposomes; N = 3–4 pools of 2–3 mice each / condition). * p < 0.05, unpaired Student's t-test

Fig 5: Wound healing accelerated by ASCs seeding into ABCcolla® Collagen Matrix in the first week. (A) Surface marker analysis of ASCs. Flow cytometry results of rat ASCs. CD29+/CD31-/ CD90+/ CD45- expression indicated the presence of ASCs. (B) Macroscopic wound healing photographs. (C) The ASC-ABCcolla® Collagen Matrix (DM+A/A) group showed significantly decreased unhealed wound percentage compared with other groups in the first week (* p < 0.05, ** p < 0.01). (D) In the DM wound without treatment (DM-), the wound healing rate was significantly lower than that in the other groups in the first week (* p < 0.05, ** p < 0.01).