Fig. 3.

Relaxins enhance brain colonization in whole brain organotypic slice cocultures. a Quantification of cancer cell invasion in murine brain slices. The histogram of the different degrees of invasion (for details see “Materials and methods” section) shows enhanced colonization by human and murine breast cancer cells upon incubation with porcine relaxin. *p = 0.012, **p = 0.042, ***p = 0.026, each versus the respective controls. b RXFP expression in MCF-7 cells (RT-PCR): lane 1 RXFP1 (297 bp), lane 2 RXFP2 (302 bp), lane 3 RXFP3 (189 bp), lane 4 loading control 26S (326 bp), lane 5 neg. control. c RXFP1 protein in MCF-7 cells (Western blot). d RXFPs in SK-BR3 cells (RT-PCR): lane 1 RXFP1, lane 2 RXFP2, lane RXFP3. e–g Confocal microscopy of whole brain organotypic slice cocultures: green GFP-transfected MCF-7 cells; red microglia stained with isolectin IB4. Tumour cells on the left side, embedded in ECM (showing some unspecific diffuse red staining), invade into the brain slice (right side, slice border dashed line) and colocalize with red microglial cells. e Control, showing only few invading cells and no microglial reaction after 72 h. f The RXFP3 agonist R3/I5 (2 μg/ml) does not significantly enhance invasion. g Pronounced invasion and microglia accumulation upon addition of relaxin-3 (2 μg/ml). h Quantification of MCF-7 invasion in the whole brain organotypic slice cocultures presented in e–g. *p = 0.011 versus control, **p = 0.013 versus control. (Color figure online)