Fig 1.
Bcl-2 positive circulating endothelial cell (CEC) levels are higher in cancer patients.
CECs were isolated from the blood samples of head and neck cancer patients (n = 15) and healthy volunteers (n = 10) using anti-CD146 antibody and Easy Step magnetic nanoparticles (Stem Cell Technology). A; The purified CECs were counted and statistical significance was analyzed by Mann-Whitney test (p value <0.0001). CECs were then cytospun on glass slides and stained for factor VIII (vWf, B) or Bcl-2 (C). *, represent a significant difference (p<0.01) in Bcl-2 positive CECs in head and neck cancer patients as compared to healthy volunteers.
Fig 2.
Bcl-2 positive tumor-associated endothelial cells co-migrate to lungs.
Green fluorescent protein (GFP)-labeled EC-Bcl-2 or EC-VC were co-implanted with tumor cells at the floor of the mouth. After 14 days, blood samples and lungs were collected. A-B; Circulating endothelial cells (CECs, GFP-labeled) were purified by negative selection method. A; GFP-labeled CECs were counted and *, represent a significant difference (p<0.01) in CEC numbers in animals bearing tumor cells and EC-Bcl-2 as compared to animals bearing tumor cells and EC-VC. B; A representative picture showing CTC (red) bound to CEC (green). Separate pictures for GFP-EC-Bcl-2 (green) and CTC (cytokeratin staining, red) were taken by Nikon Eclipse 80i microscope with DS-Ril camera and overlaid using NIS-Elements-Basic Research software (Nikon). The scale bar represents 10 μm. C; Circulating tumor cells (CTCs) bound to EC-VC or EC-Bcl-2 were counted and expressed as %CTC bound. D-E; Lung sections were analyzed by IHC for co-migration of EC-GFP and tumor cells. D; Number of GFP-CECs present in lung sections were counted. E; A representative picture showing GFP-labeled ECs (black arrow) along with tumor nodules in lung. The scale bar represents 50 μm.
Fig 3.
Bcl-2 upregulates adhesion molecules expression.
A; Adhesion molecule expression on endothelial cells expressing Bcl-2 (EC-Bcl-2) or vector control (EC-VC) was examined by flow cytometry. B; siRNA was used to knocked down Bcl-2 expression in EC-Bcl-2 cells. Equal protein loading was verified by stripping the blots and reprobing with tubulin antibody C; Adhesion molecule expression on EC-Bcl-2 cells or Bcl-2 knock-down (KD) cells was examined by flow cytometry. The results are expressed as % positive cells ± S.E. *p<0.05.
Fig 4.
Endothelial cells expressing Bcl-2 (EC-Bcl-2) show enhanced tumor cell binding.
A-B; EC-Bcl-2 or EC-VC cells were cultured in 8-well chamber slides to form a uniform mono-layer. Tumor cells (CAL27 and UM-SCC-74B) were then pre-labeled with fluorescent dye (calcein-acetoxymethyl ester) and then added to endothelial cells and incubated for an additional 3 hours. At the end of incubation, cells were gently washed and photographed using Nikon Eclipse 80i microscope with DS-Ril camera. The scale bar represents 100 μm. C-D; tumor cell binding assays were performed in the presence or absence of neutralizing E-selectin, ICAM-1 or VCAM-1 antibodies. *, represent a significant increase and **, represent a significant decrease in adhesion molecule expression (p<0.05).
Fig 5.
Endothelial cells expressing (EC-Bcl-2) protect tumor cells from anoikis via the Src-FAK signaling pathway.
A-C; CAL27 cells were co-cultured along with EC-VC or EC-Bcl-2 cells in a non-adherent 6 well plates for 8 hours. At the end of incubation, cells were carefully retrieved and analyzed for anoikis by TUNEL staining (A) or cell lysate was prepared and Western blotted for pSrc, pFAK, Src and FAK (B). C; tumor cell anoikis assay was performed by either adding neutralizing anti-E-selectin antibody or knocking down FAK in tumor cells.
Fig 6.
Endothelial cells expressing Bcl-2 (EC-Bcl-2) enhances tumor metastasis by chaperoning tumor cells to lungs.
Tumor cells (UM-SCC-74B or CAL27) along with GFP-labeled EC-VC or EC-Bcl-2 were injected into the tail veins of SCID mice. After 3 weeks, tumor metastasis to lungs was analyzed. A; Representative images of lungs at day 21. B; Histological evaluation of lungs in the same animals at day 21. The scale bar represents 100 μm. C; A representative picture showing GFP-labeled ECs (black arrows) along with tumor nodules in lung. The scale bar represents 50 μm. D-E; the number of metastatic nodules in the lungs were counted under microscope. *, represents a significant difference (p<0.05).
Fig 7.
E-selectin knockdown in EC-Bcl-2 or FAK/FUT3 knockdown in tumor cells reverses EC-Bcl-2-mediated tumor metastasis.
A; E-selectin expression was knocked down in EC-Bcl-2 cells by siRNA. B; FUT3 and FAK expression was knocked down in tumor cells (UM-SCC-74B or CAL27) by siRNA. EC-Bcl-2 transduced with scrambled control siRNA (EC-Bcl-2-SC) or tumor cells transduced with scrambled control siRNA (CAL27-SC) were used as control. C-F; Tumor cells along with EC-Bcl-2 cells were co-injected into the tail veins of SCID mice. C; Representative images of lungs from animals injected with (a) CAL27 and EC-Bcl-2 cells, (b) CAL27-FUT3 knockdown and EC-Bcl-2- E-selectin knockdown cells and (c) CAL27- FAK knockdown and EC-Bcl-2 cells. D; Histological evaluation of lungs from animals injected with (a) CAL27 and EC-Bcl-2 cells, (b) CAL27-FUT3 knockdown and EC-Bcl-2- E-selectin knockdown cells and (c) CAL27- FAK knockdown and EC-Bcl-2 cells. Metastatic nodules in lungs are highlighted by black arrows. The scale bar represents 100 μm. E-F; the number of metastatic nodules in the lungs were counted under microscope. *, represents a significant difference (p<0.05).