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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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ZA decreases microglia (MG)-induced invasiveness of MCF-7 cells(A) ZA decreases MG-induced invasiveness of MCF-7 cells shown by microinvasion assay of MCF-7 cells alone (white bar) and in coculture with MG 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 < 0.05). (B) MTT assay of organotypic brain slices treated with ZA at the concentrations indicated or methanol (MeOH) reveal no toxic effects of ZA on the tissue. MTT reduction is given as percentage of untreated slices (means ± SD, n = 6, *P < 0.001). (C) ZA decreases the expression of F4/80 and Csf-1R but not Gfap in organotypic brain slices measured by qRT-PCR. Tissue was treated with 1 μM (gray bars), 2 μM (black bars) or 5 μM (black doted bars) ZA for 72 h. Relative expression levels are indicated as fold changes to the untreated control (means ± SD, n = 3, *P < 0.001). (D) ZA decreases MCF-7 cell invasion in organotypic whole-brain slice cocultures. Tumor cell invasion was quantified in the absence (left bar) and presence (right bar) of 2 μM ZA (n = number of slices; +++ = highliy invasive; ++ = moderately invasive; + = low invasive; 0 = non-invasive) (*P = 0.01). (E) Picture of organotypic brain slices cocultured with GFP-labeled MCF-7 cells (green) and stained with isolectin B4-674 (blue) and αGFAP-TRITC (red) in the absence (left) and presence (right) of 2 μM ZA. Scale bars indicate 50 μm.
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Figure 4: ZA decreases microglia (MG)-induced invasiveness of MCF-7 cells(A) ZA decreases MG-induced invasiveness of MCF-7 cells shown by microinvasion assay of MCF-7 cells alone (white bar) and in coculture with MG 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 < 0.05). (B) MTT assay of organotypic brain slices treated with ZA at the concentrations indicated or methanol (MeOH) reveal no toxic effects of ZA on the tissue. MTT reduction is given as percentage of untreated slices (means ± SD, n = 6, *P < 0.001). (C) ZA decreases the expression of F4/80 and Csf-1R but not Gfap in organotypic brain slices measured by qRT-PCR. Tissue was treated with 1 μM (gray bars), 2 μM (black bars) or 5 μM (black doted bars) ZA for 72 h. Relative expression levels are indicated as fold changes to the untreated control (means ± SD, n = 3, *P < 0.001). (D) ZA decreases MCF-7 cell invasion in organotypic whole-brain slice cocultures. Tumor cell invasion was quantified in the absence (left bar) and presence (right bar) of 2 μM ZA (n = number of slices; +++ = highliy invasive; ++ = moderately invasive; + = low invasive; 0 = non-invasive) (*P = 0.01). (E) Picture of organotypic brain slices cocultured with GFP-labeled MCF-7 cells (green) and stained with isolectin B4-674 (blue) and αGFAP-TRITC (red) in the absence (left) and presence (right) of 2 μM ZA. Scale bars indicate 50 μm.

Mentions: To demonstrate that ZA could also affect the resident macrophage-like populations in target organs of metastasis other than the bone, we investigated the effects of ZA on different aspects during cerebral metastasis. In modified Boyden chamber assays, conducting indirect coculture of breast cancer cells and microglia, ZA reduced the microglia-induced invasion of MCF-7 cells nearly to the basal level (Fig. 4 A). Most importantly, these effects were detected at concentrations (1 μM) which showed no influence on the carcinoma cells.


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)

ZA decreases microglia (MG)-induced invasiveness of MCF-7 cells(A) ZA decreases MG-induced invasiveness of MCF-7 cells shown by microinvasion assay of MCF-7 cells alone (white bar) and in coculture with MG 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 < 0.05). (B) MTT assay of organotypic brain slices treated with ZA at the concentrations indicated or methanol (MeOH) reveal no toxic effects of ZA on the tissue. MTT reduction is given as percentage of untreated slices (means ± SD, n = 6, *P < 0.001). (C) ZA decreases the expression of F4/80 and Csf-1R but not Gfap in organotypic brain slices measured by qRT-PCR. Tissue was treated with 1 μM (gray bars), 2 μM (black bars) or 5 μM (black doted bars) ZA for 72 h. Relative expression levels are indicated as fold changes to the untreated control (means ± SD, n = 3, *P < 0.001). (D) ZA decreases MCF-7 cell invasion in organotypic whole-brain slice cocultures. Tumor cell invasion was quantified in the absence (left bar) and presence (right bar) of 2 μM ZA (n = number of slices; +++ = highliy invasive; ++ = moderately invasive; + = low invasive; 0 = non-invasive) (*P = 0.01). (E) Picture of organotypic brain slices cocultured with GFP-labeled MCF-7 cells (green) and stained with isolectin B4-674 (blue) and αGFAP-TRITC (red) in the absence (left) and presence (right) of 2 μM ZA. Scale bars indicate 50 μm.
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Related In: Results  -  Collection

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Figure 4: ZA decreases microglia (MG)-induced invasiveness of MCF-7 cells(A) ZA decreases MG-induced invasiveness of MCF-7 cells shown by microinvasion assay of MCF-7 cells alone (white bar) and in coculture with MG 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 < 0.05). (B) MTT assay of organotypic brain slices treated with ZA at the concentrations indicated or methanol (MeOH) reveal no toxic effects of ZA on the tissue. MTT reduction is given as percentage of untreated slices (means ± SD, n = 6, *P < 0.001). (C) ZA decreases the expression of F4/80 and Csf-1R but not Gfap in organotypic brain slices measured by qRT-PCR. Tissue was treated with 1 μM (gray bars), 2 μM (black bars) or 5 μM (black doted bars) ZA for 72 h. Relative expression levels are indicated as fold changes to the untreated control (means ± SD, n = 3, *P < 0.001). (D) ZA decreases MCF-7 cell invasion in organotypic whole-brain slice cocultures. Tumor cell invasion was quantified in the absence (left bar) and presence (right bar) of 2 μM ZA (n = number of slices; +++ = highliy invasive; ++ = moderately invasive; + = low invasive; 0 = non-invasive) (*P = 0.01). (E) Picture of organotypic brain slices cocultured with GFP-labeled MCF-7 cells (green) and stained with isolectin B4-674 (blue) and αGFAP-TRITC (red) in the absence (left) and presence (right) of 2 μM ZA. Scale bars indicate 50 μm.
Mentions: To demonstrate that ZA could also affect the resident macrophage-like populations in target organs of metastasis other than the bone, we investigated the effects of ZA on different aspects during cerebral metastasis. In modified Boyden chamber assays, conducting indirect coculture of breast cancer cells and microglia, ZA reduced the microglia-induced invasion of MCF-7 cells nearly to the basal level (Fig. 4 A). Most importantly, these effects were detected at concentrations (1 μM) which showed no influence on the carcinoma cells.

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