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Zoledronic acid inhibits macrophage/microglia-assisted breast cancer cell invasion.

Rietkötter E, Menck K, Bleckmann A, Farhat K, Schaffrinski M, Schulz M, Hanisch UK, Binder C, Pukrop T - Oncotarget (2013)

Bottom Line: In line with this, manipulation of microglia by ZA in organotypic brain slice cocultures reduced the tissue invasion by carcinoma cells.Thus, anti-metastatic effects of ZA are predominantly caused by modulating the microenvironment.Most importantly, our findings demonstrate that ZA reduced microglia-assisted invasion of cancer cells to the brain tissue, indicating a potential therapeutic role in the prevention of cerebral metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology/Oncology, University Medical Center, 37099 Göttingen, Germany.

ABSTRACT
The bisphosphonate zoledronic acid (ZA) significantly reduces complications of bone metastasis by inhibiting resident macrophages, the osteoclasts. Recent clinical trials indicate additional anti-metastatic effects of ZA outside the bone. However, which step of metastasis is influenced and whether thisis due to directtoxicity on cancer cells or inhibition of the tumor promoting microenvironment, is unknown. In particular, tumor-associated and resident macrophages support each step of organ metastasis and could be a crucial target of ZA. Thus, we comparatively investigate the ZA effects on: i) different types of macrophages, ii) on breast cancer cells but also iii) on macrophage-induced invasion. We demonstrate that ZA concentrations reflecting the plasma level affected viability of human macrophages, murine bone marrow-derived macrophages as well as their resident brain equivalents, the microglia, while it did not influence the tested cancer cells. However, the effects on the macrophages subsequently reduced the macrophage/microglia-induced invasiveness of the cancer cells. In line with this, manipulation of microglia by ZA in organotypic brain slice cocultures reduced the tissue invasion by carcinoma cells. The characterization of human macrophages after ZA treatment revealed a phenotype/response shift, in particular after external stimulation. In conclusion, we show that therapeutic concentrations of ZA affect all types of macrophages but not the cancer cells. Thus, anti-metastatic effects of ZA are predominantly caused by modulating the microenvironment. Most importantly, our findings demonstrate that ZA reduced microglia-assisted invasion of cancer cells to the brain tissue, indicating a potential therapeutic role in the prevention of cerebral metastasis.

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Characterization of ZA-treated human macrophages (MAC)(A) Phalloidin-TRITC staining of untreated MAC (left picture) and MAC treated with 1 μM ZA (right picture) showing the same cell morphology under both conditions. Scale bars indicate 100 μm. (B) Migration assay for MAC in coculture with MCF-7 in the absence (gray bar) and presence of 1 μM ZA (black bar) reveal no difference in the number of migrated cells (means ± SD, n = 8). (C) ZA decreases the MAC-induced invasiveness of MCF-7 cells shown by microinvasion assay of MCF-7 cells alone (white bar) and in coculture with MAC in the absence (gray bar) or presence of 1 μM ZA (black bar). Invasiveness is indicated as percentage of the control MCF-7 cells alone (means ± SD, n = 3, *P < 0.01). (D) Gelatine zymography of cell culture supernatants from MCF-7 cells alone, + ZA, + MAC, + MAC and ZA, MAC alone and MAC +ZA reveal no difference in the secretion of MMP-9 after ZA treatment. (E) ZA decreases T-MV uptake in MAC shown by FACS analysis. Cells were treated with 1 μM ZA (square) or diluent (circle) for 48 h before adding 5 μg PKH26-labeled T-MV for another 24 h. T-MV uptake is indicated as mean fluorescent intensity (MFI) of MAC (means ± SD, n = 7, *P < 0.05). (F) ZA alters LPS-induced expression of TNFα, WNT5A, IL-10 and IL-1β measured by qRT-PCR in MAC treated with 100 μg/ml LPS alone (gray bars) or 100 μg/ml LPS + 1 μM ZA (black bars). Relative expression levels are indicated as percentage of the condition MAC + 100 μg/ml LPS (means ± SD, n ≥ 4, *P ≤ 0.05).
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Figure 3: Characterization of ZA-treated human macrophages (MAC)(A) Phalloidin-TRITC staining of untreated MAC (left picture) and MAC treated with 1 μM ZA (right picture) showing the same cell morphology under both conditions. Scale bars indicate 100 μm. (B) Migration assay for MAC in coculture with MCF-7 in the absence (gray bar) and presence of 1 μM ZA (black bar) reveal no difference in the number of migrated cells (means ± SD, n = 8). (C) ZA decreases the MAC-induced invasiveness of MCF-7 cells shown by microinvasion assay of MCF-7 cells alone (white bar) and in coculture with MAC in the absence (gray bar) or presence of 1 μM ZA (black bar). Invasiveness is indicated as percentage of the control MCF-7 cells alone (means ± SD, n = 3, *P < 0.01). (D) Gelatine zymography of cell culture supernatants from MCF-7 cells alone, + ZA, + MAC, + MAC and ZA, MAC alone and MAC +ZA reveal no difference in the secretion of MMP-9 after ZA treatment. (E) ZA decreases T-MV uptake in MAC shown by FACS analysis. Cells were treated with 1 μM ZA (square) or diluent (circle) for 48 h before adding 5 μg PKH26-labeled T-MV for another 24 h. T-MV uptake is indicated as mean fluorescent intensity (MFI) of MAC (means ± SD, n = 7, *P < 0.05). (F) ZA alters LPS-induced expression of TNFα, WNT5A, IL-10 and IL-1β measured by qRT-PCR in MAC treated with 100 μg/ml LPS alone (gray bars) or 100 μg/ml LPS + 1 μM ZA (black bars). Relative expression levels are indicated as percentage of the condition MAC + 100 μg/ml LPS (means ± SD, n ≥ 4, *P ≤ 0.05).

Mentions: One μM ZA already decreased the cell index of human macrophages. This led to the question whether the morphology of human macrophages is influenced by ZA. Therefore, we stained human macrophages with PKH26 and analyzed their cell shape by fluorescence microscopy. Obviously, the morphology of human macorphages was not affected by ZA (Fig. 3 A). Next, we wanted to know if the migratory capacity of human macrophages is altered by ZA and performed a coculture-based migration assay. Human macrophages were cocultivated with MCF-7 cells alone or in the presence of 1 μM ZA. The number of migrated cells was determined after 11 h of coculture. As shown in Fig. 3 B, ZA did not affect the migratory capacity of the macrophages.


Zoledronic acid inhibits macrophage/microglia-assisted breast cancer cell invasion.

Rietkötter E, Menck K, Bleckmann A, Farhat K, Schaffrinski M, Schulz M, Hanisch UK, Binder C, Pukrop T - Oncotarget (2013)

Characterization of ZA-treated human macrophages (MAC)(A) Phalloidin-TRITC staining of untreated MAC (left picture) and MAC treated with 1 μM ZA (right picture) showing the same cell morphology under both conditions. Scale bars indicate 100 μm. (B) Migration assay for MAC in coculture with MCF-7 in the absence (gray bar) and presence of 1 μM ZA (black bar) reveal no difference in the number of migrated cells (means ± SD, n = 8). (C) ZA decreases the MAC-induced invasiveness of MCF-7 cells shown by microinvasion assay of MCF-7 cells alone (white bar) and in coculture with MAC in the absence (gray bar) or presence of 1 μM ZA (black bar). Invasiveness is indicated as percentage of the control MCF-7 cells alone (means ± SD, n = 3, *P < 0.01). (D) Gelatine zymography of cell culture supernatants from MCF-7 cells alone, + ZA, + MAC, + MAC and ZA, MAC alone and MAC +ZA reveal no difference in the secretion of MMP-9 after ZA treatment. (E) ZA decreases T-MV uptake in MAC shown by FACS analysis. Cells were treated with 1 μM ZA (square) or diluent (circle) for 48 h before adding 5 μg PKH26-labeled T-MV for another 24 h. T-MV uptake is indicated as mean fluorescent intensity (MFI) of MAC (means ± SD, n = 7, *P < 0.05). (F) ZA alters LPS-induced expression of TNFα, WNT5A, IL-10 and IL-1β measured by qRT-PCR in MAC treated with 100 μg/ml LPS alone (gray bars) or 100 μg/ml LPS + 1 μM ZA (black bars). Relative expression levels are indicated as percentage of the condition MAC + 100 μg/ml LPS (means ± SD, n ≥ 4, *P ≤ 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824537&req=5

Figure 3: Characterization of ZA-treated human macrophages (MAC)(A) Phalloidin-TRITC staining of untreated MAC (left picture) and MAC treated with 1 μM ZA (right picture) showing the same cell morphology under both conditions. Scale bars indicate 100 μm. (B) Migration assay for MAC in coculture with MCF-7 in the absence (gray bar) and presence of 1 μM ZA (black bar) reveal no difference in the number of migrated cells (means ± SD, n = 8). (C) ZA decreases the MAC-induced invasiveness of MCF-7 cells shown by microinvasion assay of MCF-7 cells alone (white bar) and in coculture with MAC in the absence (gray bar) or presence of 1 μM ZA (black bar). Invasiveness is indicated as percentage of the control MCF-7 cells alone (means ± SD, n = 3, *P < 0.01). (D) Gelatine zymography of cell culture supernatants from MCF-7 cells alone, + ZA, + MAC, + MAC and ZA, MAC alone and MAC +ZA reveal no difference in the secretion of MMP-9 after ZA treatment. (E) ZA decreases T-MV uptake in MAC shown by FACS analysis. Cells were treated with 1 μM ZA (square) or diluent (circle) for 48 h before adding 5 μg PKH26-labeled T-MV for another 24 h. T-MV uptake is indicated as mean fluorescent intensity (MFI) of MAC (means ± SD, n = 7, *P < 0.05). (F) ZA alters LPS-induced expression of TNFα, WNT5A, IL-10 and IL-1β measured by qRT-PCR in MAC treated with 100 μg/ml LPS alone (gray bars) or 100 μg/ml LPS + 1 μM ZA (black bars). Relative expression levels are indicated as percentage of the condition MAC + 100 μg/ml LPS (means ± SD, n ≥ 4, *P ≤ 0.05).
Mentions: One μM ZA already decreased the cell index of human macrophages. This led to the question whether the morphology of human macrophages is influenced by ZA. Therefore, we stained human macrophages with PKH26 and analyzed their cell shape by fluorescence microscopy. Obviously, the morphology of human macorphages was not affected by ZA (Fig. 3 A). Next, we wanted to know if the migratory capacity of human macrophages is altered by ZA and performed a coculture-based migration assay. Human macrophages were cocultivated with MCF-7 cells alone or in the presence of 1 μM ZA. The number of migrated cells was determined after 11 h of coculture. As shown in Fig. 3 B, ZA did not affect the migratory capacity of the macrophages.

Bottom Line: In line with this, manipulation of microglia by ZA in organotypic brain slice cocultures reduced the tissue invasion by carcinoma cells.Thus, anti-metastatic effects of ZA are predominantly caused by modulating the microenvironment.Most importantly, our findings demonstrate that ZA reduced microglia-assisted invasion of cancer cells to the brain tissue, indicating a potential therapeutic role in the prevention of cerebral metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology/Oncology, University Medical Center, 37099 Göttingen, Germany.

ABSTRACT
The bisphosphonate zoledronic acid (ZA) significantly reduces complications of bone metastasis by inhibiting resident macrophages, the osteoclasts. Recent clinical trials indicate additional anti-metastatic effects of ZA outside the bone. However, which step of metastasis is influenced and whether thisis due to directtoxicity on cancer cells or inhibition of the tumor promoting microenvironment, is unknown. In particular, tumor-associated and resident macrophages support each step of organ metastasis and could be a crucial target of ZA. Thus, we comparatively investigate the ZA effects on: i) different types of macrophages, ii) on breast cancer cells but also iii) on macrophage-induced invasion. We demonstrate that ZA concentrations reflecting the plasma level affected viability of human macrophages, murine bone marrow-derived macrophages as well as their resident brain equivalents, the microglia, while it did not influence the tested cancer cells. However, the effects on the macrophages subsequently reduced the macrophage/microglia-induced invasiveness of the cancer cells. In line with this, manipulation of microglia by ZA in organotypic brain slice cocultures reduced the tissue invasion by carcinoma cells. The characterization of human macrophages after ZA treatment revealed a phenotype/response shift, in particular after external stimulation. In conclusion, we show that therapeutic concentrations of ZA affect all types of macrophages but not the cancer cells. Thus, anti-metastatic effects of ZA are predominantly caused by modulating the microenvironment. Most importantly, our findings demonstrate that ZA reduced microglia-assisted invasion of cancer cells to the brain tissue, indicating a potential therapeutic role in the prevention of cerebral metastasis.

Show MeSH
Related in: MedlinePlus