Migration of MSC to growing tumour and normal cells, VEGF, PDGF, and EGF. (A) Established cell lines from pancreatic cancer (Capan-1, Colo357, BxPc-3, and MIA-PaCa-2), kidney (T293), and primary cell lines from fibroblasts and endothelial cells were cultured in medium containing 2% FCS for 48 h. Supernatant was transferred to the lower well and migration of MSC placed to the upper well was measured in a ChemoTx system as described in Materials and methods. Pos Co, cell-free medium with 20% FCS; Neg Co, cell-free medium with 2% FCS. (B) Dose-dependent migration of MSC towards medium containing 2% FCS alone (CO) or to VEGF, PDGF, and EGF in 2% FCS and in concentrations indicated. (C) Migration of MSC to growth factors alone (GF alone) or to growth factors in the presence of the inhibitor of PDGF receptor (Glivec, 3 μM), or blocking antibodies to EGF receptor (Erbitux, 3 μM), or VEGF (Avastin, 25 μg/ml). (D) Induction of HIF-1α and secretion of VEGF by pancreatic cancer cells following hypoxia. For the induction of hypoxia, the pancreatic cancer cell line BxPc-3 was treated with CoCl₂ (100 μM). Two to 16 h later, protein expression of HIF-1α was examined by western blot analysis. β-Actin served as a control for equal conditions. For the evaluation of VEGF secretion, a six-well plate with growing BxPc-3 cells was placed in a modular incubator chamber and hypoxia was induced by floating with a preanalysed air mixture (89.25% N₂, 10% CO₂, 0.75% O₂) at 37°C for 16 h. Immediately thereafter, VEGF secretion into the supernatant was analysed by the ELISA assay, as described in Materials and methods. Results presented are from three independent experiments and s.d. are shown.

Mesenchymal stem cells induce sprouting of endothelial cells by VEGF expression but do not differentiate into endothelial cells. (A) Vascular endothelial growth factor protein in supernatant of MSC cultured under hypoxic (black bars) or normoxic (white bars) conditions in the presence or absence of Avastin (25 μg ml⁻¹) was analysed by the ELISA assay. (B) RNA expression of VEGF was analysed by RT-PCR in MSC. The expression of α-SMA served as control for equal conditions. (C) A volume of 100 or 200 μl supernatant from MSC (as indicated), which have been cultured in medium containing 2% FCS for 48 h or recombinant VEGF (50 ng ml⁻¹), was transferred to spheroids consisting of 1 × 10³ HUVEC growing in wells of a 24-well plate 48 spheroids per well. The length of sprouts was analysed by cell^B 2.3 software and median lengths are given. Representative spheroids have been visualised by phase-contrast microscopy using an Olympus CKX41 microscope, × 10 magnification, and a colorview camera soft imaging system. (D) Mesenchymal stem cells were seeded on chamber slights covered with fibronectin and cultured in endothelial cell medium containing VEGF (50 ng ml⁻¹) for 2 weeks. Immunohistochemistry was performed using specific antibodies for the detection of the specific endothelial cell markers von-Willebrand factor (α-vWF) and CD31 (α-CD31). Human umbilical vein endothelial cells were used as positive control. A Leica DMRB microscope and × 1000 magnification were used.

Recruitment of MSC in mice by intravital microscopy reveals attachment to normal and tumour blood vessels. Mice with MIA-PaCa-2 orthotopic pancreatic cancer xenografts were injected with lentiviral GFP-transduced MSC (four consecutive injections of 10⁵ MSC per 0.2 ml normal saline in time intervals of about 15 min) into the carotid artery. Photographs of circulating MSC in (A) cremaster muscle and (B) tumour vessels were taken and analysed as described in Materials and methods. Circulating (white arrow) and attached (white asterisk) green fluorescent MSC appeared during the first minute after injection. Red arrows indicate the direction of flow. a, artery; v, venule. Pictures are freezed images from video sequences provided in the supplement. (C) Immunofluorescence staining of a GFP-expressing MSC incorporated in vWF-positive endothelial cells of a vessels was detected in an orthotopic pancreatic xenograft of nude mice, which have been injected with MSC as described in Figure 4.

Migration of MSC to spheroids of pancreatic tumour cells. (A) Green-labeled spheroids (composed of 5 × 10² MIA-PaCa-2, 2.5 × 10² human primary fibroblasts, and 2.5 × 10² HUVEC) and red-labeled MSC were seeded in opposite edges of a well of a 24-well plate and covered with methocel/collagen solution as described in Materials and methods. (B) Cells were analysed using a fluorescence microscope (Olympus IX 70) with a red and green filter, 20-fold magnification and two-fold binning immediately after seeding. The focus was put to spheroids. Migration of red fluorescent MSC to green fluorescent spheroids was monitored by time-lapse photography during a period of 12 h and a picture was taken every 5 min. Representative data of one experiment out of three similar is shown.

Migration of MSC in orthotopic MIA-PaCa-2 pancreatic xenografts in nude mice and the incorporation in tumour blood vessels. (A) Four mice with MIA-PaCa-2 orthotopic pancreatic cancer xenografts were injected with lentiviral eGFP-labeled MSC (4 × 10⁵ in tail vein). Four xenografted mice received PBS injection and served as controls. Three days after injection, mice were killed followed by the resection of organs and xenografts. Cells were isolated from tissue pieces and examined by flow cytometry for expression of green fluorescence of eGFP-expressing MSC. Mean bars±s.e. are shown. (B) Four xenografted mice per group were injected with PBS only (no MSC) or with MSC transduced with lentiviral control vector (VEC-CO), or with MSC transduced with lentiviral siRNA towards VEGF (siVEGF). Three days later, microvessel density in cryosections of xenografts was analysed by immunohistochemistry for CD31 in a Leica DMRB microscope with 250-fold magnification. Microvessel density was quantified using eight images from each of four different tumours per group. Microvessels per field of 1 mm² were counted. (C) Data from one representative staining per group are shown. (D) Vascular endothelial growth factor protein in supernatant of MSC transduced with lentiviral control vector (VEC-CO) or with siRNA towards VEGF (siVEGF) cultured under normoxic (white bars) or hypoxic (black bars) conditions was analysed by the ELISA assay. Statistical significance was determined by t-test (P<0.05) and is indicated by an asterisk.