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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

Surface adhesion molecules expression in M1 (black bars) and M2 cells (grey bars). Cultured cells were stained with the fluorochrome-conjugated specific antibody for the listed molecules and analyzed by flow cytometry (see Material and Method). Data are expressed as means ± SD (error bars) of the ratio between the mean fluorescence intensity (MFI) of specific antibody and the MFI of the relative isotypic control. Values greater than 1 indicate expression of the marker (see Material and Methods). Data refers to at least three independent experiments. Statistics: M1 versus M2 (t-test): □P = 0.019; *P = 0.0003; ▲P = 0.00005.
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fig6: Surface adhesion molecules expression in M1 (black bars) and M2 cells (grey bars). Cultured cells were stained with the fluorochrome-conjugated specific antibody for the listed molecules and analyzed by flow cytometry (see Material and Method). Data are expressed as means ± SD (error bars) of the ratio between the mean fluorescence intensity (MFI) of specific antibody and the MFI of the relative isotypic control. Values greater than 1 indicate expression of the marker (see Material and Methods). Data refers to at least three independent experiments. Statistics: M1 versus M2 (t-test): □P = 0.019; *P = 0.0003; ▲P = 0.00005.

Mentions: To investigate putative molecular mechanisms responsible for the different transmigration behaviour observed in M1 and M2, we analyzed the expression of some proteins potentially involved in adhesion and transmigration process like integrins and adhesion molecules by cytofluorometry: CD49b, ICAM1, MCAM, CD56, αvβ3, VCAM1, ICAM2, and CCR7 were analysed and the data obtained are summarized in Figure 5. A statistically different expression was detected for αvβ3, CD49b, and MCAM, which were more expressed in M1 compared to M2 cells (Figures 6(a) and 6(b); P < 0.05, Student's t-test). On the other hand, a trend to decreased expression of ICAM-1 and CD56 was detected in M1 cells even if no statistically significant differences were found (Figure 6(a)).


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)

Surface adhesion molecules expression in M1 (black bars) and M2 cells (grey bars). Cultured cells were stained with the fluorochrome-conjugated specific antibody for the listed molecules and analyzed by flow cytometry (see Material and Method). Data are expressed as means ± SD (error bars) of the ratio between the mean fluorescence intensity (MFI) of specific antibody and the MFI of the relative isotypic control. Values greater than 1 indicate expression of the marker (see Material and Methods). Data refers to at least three independent experiments. Statistics: M1 versus M2 (t-test): □P = 0.019; *P = 0.0003; ▲P = 0.00005.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Surface adhesion molecules expression in M1 (black bars) and M2 cells (grey bars). Cultured cells were stained with the fluorochrome-conjugated specific antibody for the listed molecules and analyzed by flow cytometry (see Material and Method). Data are expressed as means ± SD (error bars) of the ratio between the mean fluorescence intensity (MFI) of specific antibody and the MFI of the relative isotypic control. Values greater than 1 indicate expression of the marker (see Material and Methods). Data refers to at least three independent experiments. Statistics: M1 versus M2 (t-test): □P = 0.019; *P = 0.0003; ▲P = 0.00005.
Mentions: To investigate putative molecular mechanisms responsible for the different transmigration behaviour observed in M1 and M2, we analyzed the expression of some proteins potentially involved in adhesion and transmigration process like integrins and adhesion molecules by cytofluorometry: CD49b, ICAM1, MCAM, CD56, αvβ3, VCAM1, ICAM2, and CCR7 were analysed and the data obtained are summarized in Figure 5. A statistically different expression was detected for αvβ3, CD49b, and MCAM, which were more expressed in M1 compared to M2 cells (Figures 6(a) and 6(b); P < 0.05, Student's t-test). On the other hand, a trend to decreased expression of ICAM-1 and CD56 was detected in M1 cells even if no statistically significant differences were found (Figure 6(a)).

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