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Mesenchymal stromal cells loaded with paclitaxel induce cytotoxic damage in glioblastoma brain xenografts.

Pacioni S, D'Alessandris QG, Giannetti S, Morgante L, De Pascalis I, Coccè V, Bonomi A, Pascucci L, Alessandri G, Pessina A, Falchetti ML, Pallini R - Stem Cell Res Ther (2015)

Bottom Line: In adjacent brain regions, we injected green fluorescent protein-expressing murine MSCs, either loaded with PTX or unloaded.In rats grafted with PTX-MSCs, the nuclei of U87MG cells showed changes that are typically induced by PTX, including multi-spindle mitoses, centrosome number alterations, and nuclear fragmentation.MSCs appear particularly suited for anti-neoplastic drug delivery in the brain since PTX-specific damage of GBM cells can be achieved avoiding side effects to the normal tissue.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurosurgery, Università Cattolica del Sacro Cuore, Largo Agostino Gemelli 8, 00168, Rome, Italy. simone.pacioni@ibcn.cnr.it.

ABSTRACT

Introduction: The goal of cancer chemotherapy is targeting tumor cells and/or tumor-associated microvessels with the lowest systemic toxicity. Mesenchymal stromal cells (MSCs) are promising vehicles for selective drug delivery due to their peculiar ability to home to pathological tissues. We previously showed that MSCs are able to uptake and subsequently to release the chemotherapeutic compound Paclitaxel (PTX) and to impair the growth of subcutaneous glioblastoma multiforme (GBM) xenografts. Here we used an orthotopic GBM model 1) to assess whether PTX-loaded MSCs (PTX-MSCs) retain a tropism towards the tumor cells in the brain context, and 2) to characterize the cytotoxic damage induced by MSCs-driven PTX release in the tumor microenvironment.

Methods: U87MG GBM cells were fluorescently labeled with the mCherry protein and grafted onto the brain of immunosuppressed rats. In adjacent brain regions, we injected green fluorescent protein-expressing murine MSCs, either loaded with PTX or unloaded. After 1 week survival, the xenografted brain was assessed by confocal microscopy for PTX-induced cell damage.

Results: Overall, MSCs showed remarkable tropism towards the tumor. In rats grafted with PTX-MSCs, the nuclei of U87MG cells showed changes that are typically induced by PTX, including multi-spindle mitoses, centrosome number alterations, and nuclear fragmentation. Multi-spindle mitoses resulted in multinucleated cells that were significantly higher in tumors co-grafted with PTX-MSCs than in controls. Nuclear changes did not occur in astrocytes and neurons surrounding the tumor.

Conclusions: MSCs appear particularly suited for anti-neoplastic drug delivery in the brain since PTX-specific damage of GBM cells can be achieved avoiding side effects to the normal tissue.

No MeSH data available.


Related in: MedlinePlus

Cytotoxic effect of PTX on cultured U87MG cells. U87MG cells treated with PTX undergo aberrant mitoses with monopolar or multipolar spindles, as assessed by immunostaining of mitotic spindles and centrosomes by anti-α-tubulin and anti-γ-tubulin antibodies, respectively (a). The percentage of abnormal spindles is dose dependent. Changes in the percentage of monopolar and multipolar spindle mitoses are significant at 50 and 100 nM PTX (*p <0.05 and **p <0.0005) (b) (left panel). n >1000 cells were counted from each of three independent experiments. Exposure of U87MG cells to the CM of PTX-loaded MSCs induces a strong cytotoxic effect, quantitatively similar to 100 nM PTX, with significant increase of multipolar spindle mitoses (***p <0.0001) (b) (right panel). As a consequence of multispindle mitoses, the percentage of multinucleated U87MG cells (c) (lower panel) versus mononucleated U87MG cells (c) (upper panel) significantly increases (***p <0.0001) both after direct PTX treatment (d) (left panel) and after exposure to CM from PTX-loaded MSCs (d) (right panel). n >2000 cells were counted from each of three independent experiments. Scale bars = 10 μm. CM conditioned medium, CTRL control, DAPI 4′,6-diamidino-2-phenylindole, MSC mesenchymal stem/stromal cell, PTX paclitaxel
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Fig1: Cytotoxic effect of PTX on cultured U87MG cells. U87MG cells treated with PTX undergo aberrant mitoses with monopolar or multipolar spindles, as assessed by immunostaining of mitotic spindles and centrosomes by anti-α-tubulin and anti-γ-tubulin antibodies, respectively (a). The percentage of abnormal spindles is dose dependent. Changes in the percentage of monopolar and multipolar spindle mitoses are significant at 50 and 100 nM PTX (*p <0.05 and **p <0.0005) (b) (left panel). n >1000 cells were counted from each of three independent experiments. Exposure of U87MG cells to the CM of PTX-loaded MSCs induces a strong cytotoxic effect, quantitatively similar to 100 nM PTX, with significant increase of multipolar spindle mitoses (***p <0.0001) (b) (right panel). As a consequence of multispindle mitoses, the percentage of multinucleated U87MG cells (c) (lower panel) versus mononucleated U87MG cells (c) (upper panel) significantly increases (***p <0.0001) both after direct PTX treatment (d) (left panel) and after exposure to CM from PTX-loaded MSCs (d) (right panel). n >2000 cells were counted from each of three independent experiments. Scale bars = 10 μm. CM conditioned medium, CTRL control, DAPI 4′,6-diamidino-2-phenylindole, MSC mesenchymal stem/stromal cell, PTX paclitaxel

Mentions: To assess the cytotoxic effect of PTX, we exposed the U87MG cells for 24 hours to increasing doses of PTX, ranging from 20 to 100 nM. Exposure of U87MG cells to PTX results in a dramatic increase of multispindle mitoses, as assessed by immunohistochemical staining of spindles and centrosomes with anti-α-tubulin and anti-γ-tubulin antibodies respectively (Fig. 1a). The percentage of monopolar and multipolar spindle mitoses increases in a dose-dependent manner (Fig. 1b). While untreated U87MG cells show 0.13 ± 0.09 and 0.06 ± 0.06 % (mean ± SEM) monopolar and multipolar spindle mitoses, respectively, following treatment with 100 nM PTX the percentage of abnormal spindle mitoses in the U87MG cells increases to 1.41 ± 0.34 and 4.63 ± 0.94 % (mean ± SEM) of monopolar and multipolar spindle mitoses, respectively. Of note, the tumor cells exposed to PTX showed a higher number of spindles with respect to control cells (Fig. 1b). For example, the percentage of mitotic figures was significantly higher in U87MG cells treated with 100 nM PTX than in untreated control cells (6.25 ± 1.14 and 0.95 ± 0.27 %; p <0.0005, Wilcoxon test). This is probably because PTX stabilizes microtubules preventing their depolymerization, therefore increasing the number of spindled cells, owing to the active mitotic checkpoint which freezes cells in metaphase. As a consequence of multispindled mitoses, the percentage of multinucleated U87MG cells derived from mitotic slippage increased, in a dose-dependent manner, from 8.87 ± 1.35 % (mean ± SEM) of control cells to 62.49 ± 2.06 % (mean ± SEM) of U87MG treated with 100 nM PTX (Fig. 1d; p <0.0001, ANOVA test).Fig. 1


Mesenchymal stromal cells loaded with paclitaxel induce cytotoxic damage in glioblastoma brain xenografts.

Pacioni S, D'Alessandris QG, Giannetti S, Morgante L, De Pascalis I, Coccè V, Bonomi A, Pascucci L, Alessandri G, Pessina A, Falchetti ML, Pallini R - Stem Cell Res Ther (2015)

Cytotoxic effect of PTX on cultured U87MG cells. U87MG cells treated with PTX undergo aberrant mitoses with monopolar or multipolar spindles, as assessed by immunostaining of mitotic spindles and centrosomes by anti-α-tubulin and anti-γ-tubulin antibodies, respectively (a). The percentage of abnormal spindles is dose dependent. Changes in the percentage of monopolar and multipolar spindle mitoses are significant at 50 and 100 nM PTX (*p <0.05 and **p <0.0005) (b) (left panel). n >1000 cells were counted from each of three independent experiments. Exposure of U87MG cells to the CM of PTX-loaded MSCs induces a strong cytotoxic effect, quantitatively similar to 100 nM PTX, with significant increase of multipolar spindle mitoses (***p <0.0001) (b) (right panel). As a consequence of multispindle mitoses, the percentage of multinucleated U87MG cells (c) (lower panel) versus mononucleated U87MG cells (c) (upper panel) significantly increases (***p <0.0001) both after direct PTX treatment (d) (left panel) and after exposure to CM from PTX-loaded MSCs (d) (right panel). n >2000 cells were counted from each of three independent experiments. Scale bars = 10 μm. CM conditioned medium, CTRL control, DAPI 4′,6-diamidino-2-phenylindole, MSC mesenchymal stem/stromal cell, PTX paclitaxel
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig1: Cytotoxic effect of PTX on cultured U87MG cells. U87MG cells treated with PTX undergo aberrant mitoses with monopolar or multipolar spindles, as assessed by immunostaining of mitotic spindles and centrosomes by anti-α-tubulin and anti-γ-tubulin antibodies, respectively (a). The percentage of abnormal spindles is dose dependent. Changes in the percentage of monopolar and multipolar spindle mitoses are significant at 50 and 100 nM PTX (*p <0.05 and **p <0.0005) (b) (left panel). n >1000 cells were counted from each of three independent experiments. Exposure of U87MG cells to the CM of PTX-loaded MSCs induces a strong cytotoxic effect, quantitatively similar to 100 nM PTX, with significant increase of multipolar spindle mitoses (***p <0.0001) (b) (right panel). As a consequence of multispindle mitoses, the percentage of multinucleated U87MG cells (c) (lower panel) versus mononucleated U87MG cells (c) (upper panel) significantly increases (***p <0.0001) both after direct PTX treatment (d) (left panel) and after exposure to CM from PTX-loaded MSCs (d) (right panel). n >2000 cells were counted from each of three independent experiments. Scale bars = 10 μm. CM conditioned medium, CTRL control, DAPI 4′,6-diamidino-2-phenylindole, MSC mesenchymal stem/stromal cell, PTX paclitaxel
Mentions: To assess the cytotoxic effect of PTX, we exposed the U87MG cells for 24 hours to increasing doses of PTX, ranging from 20 to 100 nM. Exposure of U87MG cells to PTX results in a dramatic increase of multispindle mitoses, as assessed by immunohistochemical staining of spindles and centrosomes with anti-α-tubulin and anti-γ-tubulin antibodies respectively (Fig. 1a). The percentage of monopolar and multipolar spindle mitoses increases in a dose-dependent manner (Fig. 1b). While untreated U87MG cells show 0.13 ± 0.09 and 0.06 ± 0.06 % (mean ± SEM) monopolar and multipolar spindle mitoses, respectively, following treatment with 100 nM PTX the percentage of abnormal spindle mitoses in the U87MG cells increases to 1.41 ± 0.34 and 4.63 ± 0.94 % (mean ± SEM) of monopolar and multipolar spindle mitoses, respectively. Of note, the tumor cells exposed to PTX showed a higher number of spindles with respect to control cells (Fig. 1b). For example, the percentage of mitotic figures was significantly higher in U87MG cells treated with 100 nM PTX than in untreated control cells (6.25 ± 1.14 and 0.95 ± 0.27 %; p <0.0005, Wilcoxon test). This is probably because PTX stabilizes microtubules preventing their depolymerization, therefore increasing the number of spindled cells, owing to the active mitotic checkpoint which freezes cells in metaphase. As a consequence of multispindled mitoses, the percentage of multinucleated U87MG cells derived from mitotic slippage increased, in a dose-dependent manner, from 8.87 ± 1.35 % (mean ± SEM) of control cells to 62.49 ± 2.06 % (mean ± SEM) of U87MG treated with 100 nM PTX (Fig. 1d; p <0.0001, ANOVA test).Fig. 1

Bottom Line: In adjacent brain regions, we injected green fluorescent protein-expressing murine MSCs, either loaded with PTX or unloaded.In rats grafted with PTX-MSCs, the nuclei of U87MG cells showed changes that are typically induced by PTX, including multi-spindle mitoses, centrosome number alterations, and nuclear fragmentation.MSCs appear particularly suited for anti-neoplastic drug delivery in the brain since PTX-specific damage of GBM cells can be achieved avoiding side effects to the normal tissue.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurosurgery, Università Cattolica del Sacro Cuore, Largo Agostino Gemelli 8, 00168, Rome, Italy. simone.pacioni@ibcn.cnr.it.

ABSTRACT

Introduction: The goal of cancer chemotherapy is targeting tumor cells and/or tumor-associated microvessels with the lowest systemic toxicity. Mesenchymal stromal cells (MSCs) are promising vehicles for selective drug delivery due to their peculiar ability to home to pathological tissues. We previously showed that MSCs are able to uptake and subsequently to release the chemotherapeutic compound Paclitaxel (PTX) and to impair the growth of subcutaneous glioblastoma multiforme (GBM) xenografts. Here we used an orthotopic GBM model 1) to assess whether PTX-loaded MSCs (PTX-MSCs) retain a tropism towards the tumor cells in the brain context, and 2) to characterize the cytotoxic damage induced by MSCs-driven PTX release in the tumor microenvironment.

Methods: U87MG GBM cells were fluorescently labeled with the mCherry protein and grafted onto the brain of immunosuppressed rats. In adjacent brain regions, we injected green fluorescent protein-expressing murine MSCs, either loaded with PTX or unloaded. After 1 week survival, the xenografted brain was assessed by confocal microscopy for PTX-induced cell damage.

Results: Overall, MSCs showed remarkable tropism towards the tumor. In rats grafted with PTX-MSCs, the nuclei of U87MG cells showed changes that are typically induced by PTX, including multi-spindle mitoses, centrosome number alterations, and nuclear fragmentation. Multi-spindle mitoses resulted in multinucleated cells that were significantly higher in tumors co-grafted with PTX-MSCs than in controls. Nuclear changes did not occur in astrocytes and neurons surrounding the tumor.

Conclusions: MSCs appear particularly suited for anti-neoplastic drug delivery in the brain since PTX-specific damage of GBM cells can be achieved avoiding side effects to the normal tissue.

No MeSH data available.


Related in: MedlinePlus