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Melanoma cells homing to the brain: an in vitro model.

Rizzo A, Vasco C, Girgenti V, Fugnanesi V, Calatozzolo C, Canazza A, Salmaggi A, Rivoltini L, Morbin M, Ciusani E - Biomed Res Int (2015)

Bottom Line: M2 cells showed a statistically significant higher capability to pass across the in vitro BBB model, compared to M1.PCR array data showed that M2 had a higher expression of several matrix metalloproteinase proteins (MMPs) compared to M1.Future studies will be necessary to deepen the mechanisms of central nervous system invasion.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Clinical Pathology and Medical Genetics, Foundation IRCCS Neurological Institute Carlo Besta, Via Celoria 11, 20133 Milano, Italy.

ABSTRACT
We developed an in vitro contact through-feet blood brain barrier (BBB) model built using type IV collagen, rat astrocytes, and human umbilical vein endothelial cells (HUVECs) cocultured through Transwell porous polycarbonate membrane. The contact between astrocytes and HUVECs was demonstrated by electron microscopy: astrocytes endfeet pass through the 8.0 μm pores inducing HUVECs to assume a cerebral phenotype. Using this model we evaluated transmigration of melanoma cells from two different patients (M1 and M2) selected among seven melanoma primary cultures. M2 cells showed a statistically significant higher capability to pass across the in vitro BBB model, compared to M1. Expression of adhesion molecules was evaluated by flow cytometry: a statistically significant increased expression of MCAM, αvβ3, and CD49b was detected in M1. PCR array data showed that M2 had a higher expression of several matrix metalloproteinase proteins (MMPs) compared to M1. Specifically, data suggest that MMP2 and MMP9 could be directly involved in BBB permeability and that brain invasion by melanoma cells could be related to the overexpression of many MMPs. Future studies will be necessary to deepen the mechanisms of central nervous system invasion.

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Related in: MedlinePlus

(a) The endothelial cells (E) were plated on the upper side of polycarbonate membrane at a density of 2 · 105/well and the astrocytes (A) were cultured on the bottom side of the membrane at a density of 2 · 105/well. The arrowhead shows the contact between astrocyte endfeet and endothelial cells monolayer across a pore (P) of the membrane. (b) Contact (arrowhead) between astrocyte endfeet (A) and endothelial cells (E) across a pore (P) at higher magnification. (c) Transversal section of endothelial cells monolayer (E) after four days of cocultures with astrocytes (see Material and Methods). Arrows indicate the tight junction (TJ) between two adjacent endothelial cells.
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fig1: (a) The endothelial cells (E) were plated on the upper side of polycarbonate membrane at a density of 2 · 105/well and the astrocytes (A) were cultured on the bottom side of the membrane at a density of 2 · 105/well. The arrowhead shows the contact between astrocyte endfeet and endothelial cells monolayer across a pore (P) of the membrane. (b) Contact (arrowhead) between astrocyte endfeet (A) and endothelial cells (E) across a pore (P) at higher magnification. (c) Transversal section of endothelial cells monolayer (E) after four days of cocultures with astrocytes (see Material and Methods). Arrows indicate the tight junction (TJ) between two adjacent endothelial cells.

Mentions: We developed an in vitro BBB model which is a “contact through-feet” model. It is built with human endothelial cells deriving from HUVECs and rat astrocytes cocultured in Transwell 8.0 μm porous polycarbonate membrane (see Materials and Methods for further details). We obtained a model that satisfied some of the in vivo BBB characteristics; by electron microscopy we demonstrated that our model allowed astrocytes endfeet to physically contact HUVECs through membrane pores (Figure 1(a)) inducing the formation of tight junctions (Figure 1(b)) which is a morphological feature indicating the acquisition of functional properties of the cerebral endothelium.


Melanoma cells homing to the brain: an in vitro model.

Rizzo A, Vasco C, Girgenti V, Fugnanesi V, Calatozzolo C, Canazza A, Salmaggi A, Rivoltini L, Morbin M, Ciusani E - Biomed Res Int (2015)

(a) The endothelial cells (E) were plated on the upper side of polycarbonate membrane at a density of 2 · 105/well and the astrocytes (A) were cultured on the bottom side of the membrane at a density of 2 · 105/well. The arrowhead shows the contact between astrocyte endfeet and endothelial cells monolayer across a pore (P) of the membrane. (b) Contact (arrowhead) between astrocyte endfeet (A) and endothelial cells (E) across a pore (P) at higher magnification. (c) Transversal section of endothelial cells monolayer (E) after four days of cocultures with astrocytes (see Material and Methods). Arrows indicate the tight junction (TJ) between two adjacent endothelial cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321090&req=5

fig1: (a) The endothelial cells (E) were plated on the upper side of polycarbonate membrane at a density of 2 · 105/well and the astrocytes (A) were cultured on the bottom side of the membrane at a density of 2 · 105/well. The arrowhead shows the contact between astrocyte endfeet and endothelial cells monolayer across a pore (P) of the membrane. (b) Contact (arrowhead) between astrocyte endfeet (A) and endothelial cells (E) across a pore (P) at higher magnification. (c) Transversal section of endothelial cells monolayer (E) after four days of cocultures with astrocytes (see Material and Methods). Arrows indicate the tight junction (TJ) between two adjacent endothelial cells.
Mentions: We developed an in vitro BBB model which is a “contact through-feet” model. It is built with human endothelial cells deriving from HUVECs and rat astrocytes cocultured in Transwell 8.0 μm porous polycarbonate membrane (see Materials and Methods for further details). We obtained a model that satisfied some of the in vivo BBB characteristics; by electron microscopy we demonstrated that our model allowed astrocytes endfeet to physically contact HUVECs through membrane pores (Figure 1(a)) inducing the formation of tight junctions (Figure 1(b)) which is a morphological feature indicating the acquisition of functional properties of the cerebral endothelium.

Bottom Line: M2 cells showed a statistically significant higher capability to pass across the in vitro BBB model, compared to M1.PCR array data showed that M2 had a higher expression of several matrix metalloproteinase proteins (MMPs) compared to M1.Future studies will be necessary to deepen the mechanisms of central nervous system invasion.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Clinical Pathology and Medical Genetics, Foundation IRCCS Neurological Institute Carlo Besta, Via Celoria 11, 20133 Milano, Italy.

ABSTRACT
We developed an in vitro contact through-feet blood brain barrier (BBB) model built using type IV collagen, rat astrocytes, and human umbilical vein endothelial cells (HUVECs) cocultured through Transwell porous polycarbonate membrane. The contact between astrocytes and HUVECs was demonstrated by electron microscopy: astrocytes endfeet pass through the 8.0 μm pores inducing HUVECs to assume a cerebral phenotype. Using this model we evaluated transmigration of melanoma cells from two different patients (M1 and M2) selected among seven melanoma primary cultures. M2 cells showed a statistically significant higher capability to pass across the in vitro BBB model, compared to M1. Expression of adhesion molecules was evaluated by flow cytometry: a statistically significant increased expression of MCAM, αvβ3, and CD49b was detected in M1. PCR array data showed that M2 had a higher expression of several matrix metalloproteinase proteins (MMPs) compared to M1. Specifically, data suggest that MMP2 and MMP9 could be directly involved in BBB permeability and that brain invasion by melanoma cells could be related to the overexpression of many MMPs. Future studies will be necessary to deepen the mechanisms of central nervous system invasion.

Show MeSH
Related in: MedlinePlus