Fig 2: Uptake of SIINFEKL loaded CNPs by DCs efficiently stimulates CD8+ OT-1 T cell activation and expansion.A) DC2.4 cells were either left untreated or treated with 100 µg/ml SIINFEKL-loaded 90/10-CNPs for 1, 5, 9 and 24 hours or B) DC2.4 cells were treated with 100 µg/ml empty (90/10) or SIINFEKL-loaded 90/10-CNPs or 1 µg/ml SIINFEKL for 5 hours. SIINFEKL bound to H-2Kb molecules was determined by flow cytometric analysis. Median fluorescence intensity (MFI) ratio was calculated by normalizing MFI of staining with anti-SIINFEKL-H-2Kb antibody to MFI detected in staining with its respective isotype control (n-fold MFI). In A) MFI ratio of treated cells was also normalized to MFI of 1h treated cells and expressed as n-fold MFI of untreated cells. C) CD8+ OT-1 T lymphocytes were isolated from spleens of OT-1 mice. Purity of CD8+ OT-1 T cells was determined after negative MACS selection by CD8 staining and flow cytometric analysis. One representative dot blot is shown. D-F) DC2.4 cells were either left untreated or were treated with 100 µg/ml empty (90/10) or 100 µg/ml SIINFEKL-loaded 90/10-CNPs or 1 µg/ml SIINFEKL for 5 hours. Afterwards, differentially treated DC2.4 cells were cocultured with CD8+ OT-1 T lymphocytes for 72 hours. D) Representative phase contrast images of CD8+ OT-1 T cells cocultured with DC2.4 cells that had been either left untreated or subjected to the indicated treatment. Scale bar = 1000 µm. E) Numbers of vital CD8+ OT-1 T lymphocytes were determined after the indicated coculture setting. Data are presented as mean + SEM of three independent experiments. F) Flow cytometric analysis of T cell activation by staining of cell surface CD25, CD44 and CD69 of CD8+ OT-1 T cells directly after isolation and after coculture with DC.24 cells that had been pretreated with SIINFEKL or 90/10-SIINFEKL CNPs. Representative histograms from one out of 3 independent experiments are shown and bar charts present the % of CD8+ OT-1 T cells positive for cell surface expression of CD25, CD44 and CD69, respectively. Data are presented as mean +SEM of three independent experiments. *p < 0.05.

Fig 3: Mesenchymal and cancer stem-cell like features in TU-BcX-2 K1 cells. a Panel of epithelial (CD24, CDH1) and mesenchymal (CDH2, VIM, FRA1, SNAI1, TWIST, cFOS, cMYC and SLUG) genes analyzed by qRT-PCR in TU-BcX-2 K1 tumors passaged in mice (T2, T3, T4, T6). There were no endogenous levels of CD44 in any TU-BcX-2 K1 tumors passaged in mice and very low levels of CDH2 in T3 and T6 and CD24 in T3. Due to limited tissue availability, one sample was obtained per passage and analyzed. Data was normalized to ß-actin. b Immunohistochemistry staining for CDH1 protein expression in the TU-BcX-2 K1 T3 tumor. Representative images in inserts are shown at 200X magnification. c Flow cytometry of circulating tumor cells and matched TU-BCx-2 K1 tumor explants. Mouse cells are HLA- and human cells are HLA+; N = 2

Fig 4: Flow cytometry results of umbilical cord blood mesenchymal stem cells (MSCs). (A) Blank control; (B) CD29 single labeling; (C) CD44 single labeling; (D) CD29+CD44 double labeling. Among all the samples of umbilical cord blood MSCs, CD44+CD29+ double-labeled cells accounted for 50.8%.

Fig 5: CD31int/CD45dim/CD44+ Cells Enriched in Lung Metastases Are CD41+/Hoechst-/Pf4+ Platelets(A–D) Lung metastases induced by E0771-LG i.v. injection were analyzed after 11 days for CD41 expression (A and B) or Hoechst (Hst) staining (C and D).(E) Lung metastases were dissected from 14.5-week-old PyMT mice for similar analyses.(F) The indicated cell populations from the experimental metastasis model were sorted and fixed on a glass slide for imaging. Arrows indicate CD31+/Hst+ ECs; arrowheads indicate CD31+/Hst- platelets. Scale bars, 25 µm.(G) WT mice that received Pf4-tdT BMT were either untreated or i.v. injected with E0771-LG. After 11 days, the lungs and lung metastases were analyzed.(H) Quantification of (G). All error bars represent mean ± SEM.See also Figure S4.