Slide angiogenesis chamber

The interplay of CAFs and AsPC-I during infiltration of the surrounding matrix was studied with heterospheroids embedded in 3D gels made of ECM components. The iCAF and α3KO-iCAFs were transduced with lentivirus containing a mCherry vector, and AsPC-I and PANC-I cells with a lentivirus containing a GFP vector. Both vectors were kindly provided by Dr. Stephan Huveneers. The transduction was performed as detailed in Section 4.2, and the virus was produced as described in Section 4.9 and concentrated using the kit Lenti-X™ Concentrator (Takara Bio Inc., Shiga, Japan). The transfected iCAFs, AsPC-I and PANC-I cells were selected with puromycin (see Section 4.9). Being already resistant to puromycin, α3KO-iCAFs were sorted by fluorescence-assisted cell sorting. The heterospheroids comprised 400 mCherry-iCAFs or mCherry-α3KO-iCAFs as well as 400 GFP-AsPC-I or GFP-PANC-I cells. The homospheroids also comprised 400 of each cell type. The spheroids were embedded into a gel composed of 2 mg/mL of rat tail collagen I, 30% Matrigel (Thermo Fisher Scientific) and 2.5 µg/mL of laminin-332, on a µ-Slide angiogenesis chamber (Ibidi, Martinsried, Germany). The gel was overlaid with cell culture medium and images were acquired using the confocal microscope (LSM 800, Zeiss). The experiment was analysed for the number of invading AsPC-I cancer cells (Figure 6D).

Human umbilical vein endothelial cells (HUVEC) were cultured in the Endothelial Growth Medium-2 (EGM-2, Lonza), and cells in the passage 2–5 were used in this study. Some of the experiments were performed with HUVECs stably transduced with LifeAct-Green Fluorescent Protein (GFP). Spheroids (500 cells per spheroid) were formed with the use of an AggreWell 400 device (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer’s instructions. After a 24 h incubation, the spheroids were harvested and embedded in the growth factor-reduced Matrigel (Corning, New York, NY, USA) within a µ-Slide Angiogenesis chamber (Ibidi, Gräfelfing, Germany). EGM-2 medium containing microparticles was added to the samples after Matrigel gelation. Endothelial cell sprouting was monitored for up to 3 days. Alternatively, single cell HUVECs were seeded on top of the Matrigel and the effect of microparticles on capillary network formation in 2D was evaluated after 24 h. Image analysis was performed using ImageJ and the Sprout Morphology plugin [24 (link)]. We estimated the sprouting features including sprout number, length, and density of endothelial cells grown in a bead sprouting assay. Formation of cellular capillary-like networks was evaluated with the Angiogenesis Analyzer ImageJ plugin [25 ].