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Carcinoma cells misuse the host tissue damage response to invade the brain.

Chuang HN, van Rossum D, Sieger D, Siam L, Klemm F, Bleckmann A, Bayerlová M, Farhat K, Scheffel J, Schulz M, Dehghani F, Stadelmann C, Hanisch UK, Binder C, Pukrop T - Glia (2013)

Bottom Line: Here we report that this is a fatal side effect of a physiological damage response of the brain tissue.While the glial damage response intended to protect the brain from intrusion of benign epithelial cells by inducing apoptosis, it proved ineffective against various malignant cell types.They did not undergo apoptosis and actually exploited the local tissue reaction to invade instead.

View Article: PubMed Central - PubMed

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

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CXCR4 and WNT signaling are involved in microglia- and astrocyte-induced invasion. (A) The degree of MCF-7 invasion and microglia (MG) accumulation at the contact area/invasion front was determined in brain slice cocultures (n = 203). The analysis revealed a significant correlation between the degree of tumor invasion and the degree of microglial accumulation (Spearman’s test; P <  0.001). (B and C) qRT-PCR results of cxcr4 and cxcl12 illustrated by the ΔCt method. Coculture with MDCK or MCF-7 cells for 96 h upregulates cxcr4 but not its ligand, cxcl12 in the organotypic brain slices. Line represents the mean value of all experiments and each dot represents the mean value of triplicate analyses with n ≥ 15 from three individual experiments. (D) MDCK, MCF-7, microglia, and astrocytes (AS) express CXCR4 protein detected by immunoblot. (E1 and E2) The glial cells astrocytes and microglia reveal a heterogeneous expression pattern of CXCR4 (red) on immunofluorescence staining counterstained with DAPI (scale bars represent 20 µm). (F) The invasion of the MCF-7-GFP cells into the whole brain slice was significantly reduced after DKK2 as well as AMD3100 treatment. (G) The influence of indirect cocultures with microglia or astrocytes on carcinoma cell invasion measured by modified Boyden chamber assays. Treatment with WNT inhibitor DKK2 as well as CXCR4 inhibitor AMD3100 significantly reduced both the astrocyte- or microglia-induced carcinoma cell invasion. Data represent mean ± SEM with n ≥ 9 of three individual experiments. (H) The CXCR4 inhibitor also partially rescued the glial-induced apoptosis of MDCK at Position 1 (scheme see Fig. 4A) similar to clodronate or DKK2 treatment shown in Fig. 5C. Data represent mean ± SEM with n ≥ 9 from more than three individual experiments. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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fig06: CXCR4 and WNT signaling are involved in microglia- and astrocyte-induced invasion. (A) The degree of MCF-7 invasion and microglia (MG) accumulation at the contact area/invasion front was determined in brain slice cocultures (n = 203). The analysis revealed a significant correlation between the degree of tumor invasion and the degree of microglial accumulation (Spearman’s test; P <  0.001). (B and C) qRT-PCR results of cxcr4 and cxcl12 illustrated by the ΔCt method. Coculture with MDCK or MCF-7 cells for 96 h upregulates cxcr4 but not its ligand, cxcl12 in the organotypic brain slices. Line represents the mean value of all experiments and each dot represents the mean value of triplicate analyses with n ≥ 15 from three individual experiments. (D) MDCK, MCF-7, microglia, and astrocytes (AS) express CXCR4 protein detected by immunoblot. (E1 and E2) The glial cells astrocytes and microglia reveal a heterogeneous expression pattern of CXCR4 (red) on immunofluorescence staining counterstained with DAPI (scale bars represent 20 µm). (F) The invasion of the MCF-7-GFP cells into the whole brain slice was significantly reduced after DKK2 as well as AMD3100 treatment. (G) The influence of indirect cocultures with microglia or astrocytes on carcinoma cell invasion measured by modified Boyden chamber assays. Treatment with WNT inhibitor DKK2 as well as CXCR4 inhibitor AMD3100 significantly reduced both the astrocyte- or microglia-induced carcinoma cell invasion. Data represent mean ± SEM with n ≥ 9 of three individual experiments. (H) The CXCR4 inhibitor also partially rescued the glial-induced apoptosis of MDCK at Position 1 (scheme see Fig. 4A) similar to clodronate or DKK2 treatment shown in Fig. 5C. Data represent mean ± SEM with n ≥ 9 from more than three individual experiments. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Mentions: Considering the severe effects of microglia on malignant invasion as well as the link of microglia activation and infiltrating carcinoma cells (as demonstrated above Fig. 2A2–3), we investigated the correlation between astrocytes/microglia accumulation in the contact area and the degree of MCF-7 invasion in our coculture system. While the degree of astrocyte accumulation did not have any major influence, microglia accumulation significantly correlated with MCF-7 invasion based on a series of 203 coculture experiments (P < 0.001) (Fig. 6A). We further evaluate the candidate genes which are possibly involved in microglia-induced by qRT-PCR. Consistent with our previous microarray results on microglia cells (Pukrop et al., 37), cxcr4 but not its ligand cxcl12 (also known as stromal cell-derived factor-1, SDF1) was upregulated in brain slices upon coculture (Fig. 6B,C). We then scrutinized the protein expression of CXCR4 in MDCK, MCF-7, microglia, and astrocyte. All cell types expressed CXCR4 (Fig. 6D). Interestingly, both epithelial cell types depicted rather homogeneous staining (Supp. Info. Fig. 3), whereas microglia and astrocyte revealed a heterogeneous expression pattern (Fig. 6E). We then analyzed the influence of murine astrocytes on carcinoma cell invasion in comparison with microglia-induced invasion. Astrocyte coculture also induced carcinoma cell invasion in modified Boyden chamber assays, however, to a much lower extent than microglia (Fig. 6G). Additionally, both kinds of glia-induced cancer cell invasion were significantly inhibited by the WNT-inhibitor DKK2 as well as by AMD3100, a CXCR4 inhibitor (Fig. 6G) without cytotoxicity either to the epithelial cells, MCF-7 and MDCK, or the glia cells, microglia and astrocytes (Supp. Info. Fig. 2B). Both inhibitors also reduced invasion of MCF-7 into the whole brain slice to almost the same extent (Fig. 6F). In contrast, the viability of MDCK at Position 1 was only slightly increased by the CXCR4 inhibitor, again comparable to DKK2 treatment and microglia depletion (Fig. 6H). In conclusion, microglia have less influence on MDCK apoptosis than on invasion of the carcinoma cells, for which the effects of WNT or CXCR4 inhibition as well as microglia depletion are more pronounced.


Carcinoma cells misuse the host tissue damage response to invade the brain.

Chuang HN, van Rossum D, Sieger D, Siam L, Klemm F, Bleckmann A, Bayerlová M, Farhat K, Scheffel J, Schulz M, Dehghani F, Stadelmann C, Hanisch UK, Binder C, Pukrop T - Glia (2013)

CXCR4 and WNT signaling are involved in microglia- and astrocyte-induced invasion. (A) The degree of MCF-7 invasion and microglia (MG) accumulation at the contact area/invasion front was determined in brain slice cocultures (n = 203). The analysis revealed a significant correlation between the degree of tumor invasion and the degree of microglial accumulation (Spearman’s test; P <  0.001). (B and C) qRT-PCR results of cxcr4 and cxcl12 illustrated by the ΔCt method. Coculture with MDCK or MCF-7 cells for 96 h upregulates cxcr4 but not its ligand, cxcl12 in the organotypic brain slices. Line represents the mean value of all experiments and each dot represents the mean value of triplicate analyses with n ≥ 15 from three individual experiments. (D) MDCK, MCF-7, microglia, and astrocytes (AS) express CXCR4 protein detected by immunoblot. (E1 and E2) The glial cells astrocytes and microglia reveal a heterogeneous expression pattern of CXCR4 (red) on immunofluorescence staining counterstained with DAPI (scale bars represent 20 µm). (F) The invasion of the MCF-7-GFP cells into the whole brain slice was significantly reduced after DKK2 as well as AMD3100 treatment. (G) The influence of indirect cocultures with microglia or astrocytes on carcinoma cell invasion measured by modified Boyden chamber assays. Treatment with WNT inhibitor DKK2 as well as CXCR4 inhibitor AMD3100 significantly reduced both the astrocyte- or microglia-induced carcinoma cell invasion. Data represent mean ± SEM with n ≥ 9 of three individual experiments. (H) The CXCR4 inhibitor also partially rescued the glial-induced apoptosis of MDCK at Position 1 (scheme see Fig. 4A) similar to clodronate or DKK2 treatment shown in Fig. 5C. Data represent mean ± SEM with n ≥ 9 from more than three individual experiments. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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fig06: CXCR4 and WNT signaling are involved in microglia- and astrocyte-induced invasion. (A) The degree of MCF-7 invasion and microglia (MG) accumulation at the contact area/invasion front was determined in brain slice cocultures (n = 203). The analysis revealed a significant correlation between the degree of tumor invasion and the degree of microglial accumulation (Spearman’s test; P <  0.001). (B and C) qRT-PCR results of cxcr4 and cxcl12 illustrated by the ΔCt method. Coculture with MDCK or MCF-7 cells for 96 h upregulates cxcr4 but not its ligand, cxcl12 in the organotypic brain slices. Line represents the mean value of all experiments and each dot represents the mean value of triplicate analyses with n ≥ 15 from three individual experiments. (D) MDCK, MCF-7, microglia, and astrocytes (AS) express CXCR4 protein detected by immunoblot. (E1 and E2) The glial cells astrocytes and microglia reveal a heterogeneous expression pattern of CXCR4 (red) on immunofluorescence staining counterstained with DAPI (scale bars represent 20 µm). (F) The invasion of the MCF-7-GFP cells into the whole brain slice was significantly reduced after DKK2 as well as AMD3100 treatment. (G) The influence of indirect cocultures with microglia or astrocytes on carcinoma cell invasion measured by modified Boyden chamber assays. Treatment with WNT inhibitor DKK2 as well as CXCR4 inhibitor AMD3100 significantly reduced both the astrocyte- or microglia-induced carcinoma cell invasion. Data represent mean ± SEM with n ≥ 9 of three individual experiments. (H) The CXCR4 inhibitor also partially rescued the glial-induced apoptosis of MDCK at Position 1 (scheme see Fig. 4A) similar to clodronate or DKK2 treatment shown in Fig. 5C. Data represent mean ± SEM with n ≥ 9 from more than three individual experiments. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Mentions: Considering the severe effects of microglia on malignant invasion as well as the link of microglia activation and infiltrating carcinoma cells (as demonstrated above Fig. 2A2–3), we investigated the correlation between astrocytes/microglia accumulation in the contact area and the degree of MCF-7 invasion in our coculture system. While the degree of astrocyte accumulation did not have any major influence, microglia accumulation significantly correlated with MCF-7 invasion based on a series of 203 coculture experiments (P < 0.001) (Fig. 6A). We further evaluate the candidate genes which are possibly involved in microglia-induced by qRT-PCR. Consistent with our previous microarray results on microglia cells (Pukrop et al., 37), cxcr4 but not its ligand cxcl12 (also known as stromal cell-derived factor-1, SDF1) was upregulated in brain slices upon coculture (Fig. 6B,C). We then scrutinized the protein expression of CXCR4 in MDCK, MCF-7, microglia, and astrocyte. All cell types expressed CXCR4 (Fig. 6D). Interestingly, both epithelial cell types depicted rather homogeneous staining (Supp. Info. Fig. 3), whereas microglia and astrocyte revealed a heterogeneous expression pattern (Fig. 6E). We then analyzed the influence of murine astrocytes on carcinoma cell invasion in comparison with microglia-induced invasion. Astrocyte coculture also induced carcinoma cell invasion in modified Boyden chamber assays, however, to a much lower extent than microglia (Fig. 6G). Additionally, both kinds of glia-induced cancer cell invasion were significantly inhibited by the WNT-inhibitor DKK2 as well as by AMD3100, a CXCR4 inhibitor (Fig. 6G) without cytotoxicity either to the epithelial cells, MCF-7 and MDCK, or the glia cells, microglia and astrocytes (Supp. Info. Fig. 2B). Both inhibitors also reduced invasion of MCF-7 into the whole brain slice to almost the same extent (Fig. 6F). In contrast, the viability of MDCK at Position 1 was only slightly increased by the CXCR4 inhibitor, again comparable to DKK2 treatment and microglia depletion (Fig. 6H). In conclusion, microglia have less influence on MDCK apoptosis than on invasion of the carcinoma cells, for which the effects of WNT or CXCR4 inhibition as well as microglia depletion are more pronounced.

Bottom Line: Here we report that this is a fatal side effect of a physiological damage response of the brain tissue.While the glial damage response intended to protect the brain from intrusion of benign epithelial cells by inducing apoptosis, it proved ineffective against various malignant cell types.They did not undergo apoptosis and actually exploited the local tissue reaction to invade instead.

View Article: PubMed Central - PubMed

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

Show MeSH
Related in: MedlinePlus