Kl200

OPM-2^eGFP multiple myeloma cells (2.5 × 10⁵) were mixed with rat-tail collagen and human mesenchymal stromal cells (0.5 × 10⁵) and the 1 nmol of the respective compounds. Collagen drops (30 μl) were placed on parafilm for 30 min to allow polymerization of the extracellular matrix at 37 °C. Then onplants were transferred to the CAM of 7-day-old chick embryos. After 5 days of in vivo growth, onplants were documented by the Olympus SZX10 stereomicroscope (Olympus) equipped with an Olympus DFPL 2-4x objective lens connected with a digital camera (Olympus E410) and flexible cold light (KL200; Olympus). Excised xenografts were transferred into 0.5 ml RIPA Buffer (Sigma Aldrich, Linz, Austria) and homogenized with an Ultra Turrax homogenizer three times for 5 s on ice. Thereafter, homogenate underwent three freezing/thawing-cycles in liquid nitrogen and 37 °C water bath. After centrifugation, supernatants were diluted in assay buffer. GFP levels were measured by Cell Biolabs’ GFP ELISA Kit (San Diego, CA, USA), using biotinylated anti-GFP antibodies, according to the manufacturer’s protocol.

Transgenic MM cell lines (OPM-2^eGFP and RPMI-8226^eGFP; 250,000 per spheroid) were mixed into the collagen together with human mesenchymal stromal cells (50,000 cells/spheroid) or with the respective concentrations of compounds (1-100 nM). Aliquots of the collagen/cell mixture (30 μl) were distributed over a paraffin film in a 12-well plate and allowed to polymerize for 30 min at 37°C, generating so-called collagen spheroids. Thereafter, spheroids were overlaid with culture medium containing the respective concentrations of compounds (1-100 nM) and cultivated for three days. After 72 hours of incubation at 37%, the spheroids were documented with the Olympus SZX10 stereomicroscope (Olympus) equipped with an Olympus DFPL 2X-4 objective lens (numerical aperture 0.2) connected to a digital camera (Olympus E410) and flexible cold light (KL200; Olympus).

The chick chorioallantoic membrane (CAM) assay is an established in vivo model that enables the investigation of tumor angiogenesis, metastasis and invasion without the need to sacrifice mature animals [21 (link), 22 (link)]. In brief, fertilized white Leghorn chicken eggs (SPF eggs; each group n=5) were purchased from Charles River and cultivated in an egg incubator at 37°C and 70% humidity (Compact S84) for three days. Thereafter, embryos were transferred to a plastic dish and incubated “ex ovo” for further four days, so that the CAM was able to develop. OPM-2^eGFP and RPMI-8226^eGFP myeloma cells (2.5 × 10⁵) were mixed with rat-tail collagen and human bone-marrow mesenchymal stromal cells (0.5 × 10⁵) and the 1 nM of the respective compounds. Collagen drops were placed on parafilm for 30 min to allow polymerization of the extracellular matrix at 37°C. Then onplants were transferred to the CAM of 7-day-old chicken embryos. After five days of in vivo growth, onplants were documented with the Olympus SZX10 stereomicroscope (Olympus) equipped with an Olympus DFPL 2X-4 objective lens (numerical aperture 0.2) connected to a digital camera (Olympus E410) and flexible cold light (KL200; Olympus).