OBJECTIVE: We recently demonstrated that the formation of CLSs in vitro, which are thought to be a reconstitution of VM, is controlled by VEGFA. CLS formation also requires the extracellular matrix signals, presumably transduced by integrins. Both pathways are affected by Ca(2+). Therefore, we directly tested the roles of Ca(2+) and integrin in melanoma VM. METHODS: The investigation was performed by immunocytochemical, histochemical, and 3D co-culture assays. We have also used an in vivo animal model. RESULTS: The extracellular and intracellular Ca(2+) chelators, EGTA and BAPTA-AM, prevented CLS formation on Matrigel, caused actin rearrangement, and completely destroyed the preformed CLS. Addition of colcemid or cytochalasin D prevented the CLS formation and destroyed the preformed CLS network. Herein, we also show that blocking antibodies to ανβ3 and ανβ5 integrins disrupted the CLS network. Control blocking antibody to β1 integrin had no effect. In vivo experiments indicated that Ca(2+) chelation dramatically reduced the signs of VM in melanoma tumors grafted in mice. CONCLUSIONS: Our results indicate that the formation of CLS is tightly regulated by extracellular and intracellular Ca(2+) levels; ανβ3 and ανβ5 integrins are primarily responsible for CLS formation, whereas β1 integrin does not participate in CLS formation.
OBJECTIVE: We recently demonstrated that the formation of CLSs in vitro, which are thought to be a reconstitution of VM, is controlled by VEGFA. CLS formation also requires the extracellular matrix signals, presumably transduced by integrins. Both pathways are affected by Ca(2+). Therefore, we directly tested the roles of Ca(2+) and integrin in melanoma VM. METHODS: The investigation was performed by immunocytochemical, histochemical, and 3D co-culture assays. We have also used an in vivo animal model. RESULTS: The extracellular and intracellular Ca(2+) chelators, EGTA and BAPTA-AM, prevented CLS formation on Matrigel, caused actin rearrangement, and completely destroyed the preformed CLS. Addition of colcemid or cytochalasin D prevented the CLS formation and destroyed the preformed CLS network. Herein, we also show that blocking antibodies to ανβ3 and ανβ5 integrins disrupted the CLS network. Control blocking antibody to β1 integrin had no effect. In vivo experiments indicated that Ca(2+) chelation dramatically reduced the signs of VM in melanoma tumors grafted in mice. CONCLUSIONS: Our results indicate that the formation of CLS is tightly regulated by extracellular and intracellular Ca(2+) levels; ανβ3 and ανβ5 integrins are primarily responsible for CLS formation, whereas β1 integrin does not participate in CLS formation.
Authors: Francesca Spinella; Valentina Caprara; Valeriana Di Castro; Laura Rosanò; Roberta Cianfrocca; Pier Giorgio Natali; Anna Bagnato Journal: J Mol Med (Berl) Date: 2012-09-11 Impact factor: 4.599