Macrophages mediate a switch between canonical and non-canonical Wnt pathways in canine mammary tumors

Abstract

Objective: According to the current hypothesis, tumor-associated macrophages (TAMs) are "corrupted" by cancer cells and subsequently facilitate, rather than inhibit, tumor metastasis. Because the molecular mechanisms of cancer cell-TAM interactions are complicated and controversial we aimed to better define this phenomenon.

Methods and results: Using microRNA microarrays, Real-time qPCR and Western blot we showed that co-culture of canine mammary tumor cells with TAMs or treatment with macrophage-conditioned medium inhibited the canonical Wnt pathway and activated the non-canonical Wnt pathway in tumor cells. We also showed that co-culture of TAMs with tumor cells increased expression of canonical Wnt inhibitors in TAMs. Subsequently, we demonstrated macrophage-induced invasive growth patterns and epithelial-mesenchymal transition of tumor cells. Validation of these results in canine mammary carcinoma tissues (n = 50) and xenograft tumors indicated the activation of non-canonical and canonical Wnt pathways in metastatic tumors and non-metastatic malignancies, respectively. Activation of non-canonical Wnt pathway correlated with number of TAMs.

Conclusions: We demonstrated that TAMs mediate a "switch" between canonical and non-canonical Wnt signaling pathways in canine mammary tumors, leading to increased tumor invasion and metastasis. Interestingly, similar changes in neoplastic cells were observed in the presence of macrophage-conditioned medium or live macrophages. These observations indicate that rather than being "corrupted" by cancer cells, TAMs constitutively secrete canonical Wnt inhibitors that decrease tumor proliferation and development, but as a side effect, they induce the non-canonical Wnt pathway, which leads to tumor metastasis. These data challenge the conventional understanding of TAM-cancer cell interactions.

Figure 1. Expression of selected Wnt genes in co-cultured canine mammary tumor cells and macrophages.

Real-time RT-PCR analysis of Wnt genes in co-cultured and monocultured canine mammary neoplastic cells and macrophages. (A) Representative agarose gel electrophoresis of Cd163 and Mmp9 PCR products following real-time SYBR Green amplification in macrophages cultured alone and in co-culture with neoplastic cells. (B) Representative agarose gel electrophoresis of PCR products following real-time SYBR Green amplification (CA, cancer cells grown as monoculture; CA+MQ, co-cultured cancer cells; MQ, mono-cultured macrophages; and MQ+CA, macrophages co-cultured with cancer cells). (C) Fold changes in examined genes in co-cultured macrophages compared with control macrophages. (D) Western blots of cytoplasmic and membrane Wnt proteins from Macrophages grown as monocultures or sorted from co-culture with canine mammary neoplastic cells. The level of examined proteins (E, F, G) was expressed as IOD (Integrated Optical Density) in arbitrary units with the value obtained using the Odyssey Infrared Imaging System (LI-COR Inc., USA). The results are expressed as the mean ±SD. The ANOVA + Tukey post-hoc test were applied (Graph Pad v. 5.0), the values differed significantly (p<0.05) were marked as *, whereas values differed highly significant (p<0.01 or p<0.001) were marked as ** or ***, respectively.

Figure 2. Expression of Wnt proteins in co-cultured canine mammary tumor cells and macrophages.

Fold changes of Wnt genes in CMT-U27, CMT-U309, and P114 canine mammary neoplastic cells cultured in macrophage-conditioned medium or co-cultured with macrophages compared with control cells (A). Analysis of variance and Tukey's test were applied (GraphPad Prism 5.0, USA); the Differences were considered significant when *p<0.05 and highly significant when **p<0.001. Western blots of cytoplasmic and membrane or nuclear (B) Wnt proteins from CMT-U27, CMT-U309, and P114 canine mammary neoplastic cells grown in control medium, macrophage-conditioned medium, or in co-culture with macrophages. (C) Neoplastic cell survival (MTT assays) in control media, macrophage-conditioned medium (CM) and in co-culture with macrophages (MQ).

Figure 3. Growth characteristic on the Matrigel matrix.

Phase contrast micrographs of CMT-U27, CMT-U309, and P114 cells grown on the Matrigel matrix for 72 h under control conditions or in macrophage-conditioned medium. Control neoplastic cells formed colonies, whereas those treated with macrophage-conditioned medium invaded the Matrigel matrix and formed branches.

Figure 4. Confocal and immunohistochemical analysis of canine mammary tumor cell lines grown in co-culture with macrophages.

Confocal images show expression, distribution, and co-localization of actin and p-FSCN1 and formation of stress fibers in control (A), conditioned medium-treated (B), and co-cultured (C) CMT-U27 canine mammary cancer cells. Graph (D) and representative pictures (E) of cytokeratin and vimentin expression in control canine mammary cancer cells (CMT-U27) and after culture in macrophage-conditioned medium (MC-medium) or co-culture with macrophages. Monocultured neoplastic cells showed strong cytokeratin expression and weak vimentin expression. Cytokeratin expression was significantly weaker due to culture in macrophage-conditioned medium or co-culture with macrophages. On the other hand, vimentin expression was found to be significantly stronger due to neoplastic cell culture in macrophage-conditioned medium or co-cultured with macrophages. Cytokeratin (CK) and vimentin (VIM) expression was examined in control macrophages (F; graph and representative pictures). They showed no expression of cytokeratin whereas they showed vimentin expression at the same level as neoplastic cells grown in macrophage-conditioned medium (no significant difference). Images were generated using an Olympus BX60 microscope (200×); Cytokeratin and vimentin are indicated by brown precipitates.

Figure 5. Analysis of β-catenin localization and macrophage numbers in metastatic and non-metastatic canine mammary cancer tissues.

Images of canine mammary metastatic and non-metastatic tumors were obtained using an Olympus BX60 microscope (400×). Tissue sections were treated with specific anti-β-catenin and anti-MAC387 antibodies and stained using an Envision kit (Dako, Denmark). Labeled antigens are indicated by brown precipitates. Cytoplasmic and membrane-bound β-catenin is observed in metastatic tumor cells, whereas nuclear β-catenin is prevalent in non-metastatic tumor cells (indicated as asterix). Macrophage infiltration is much greater in metastatic tumors than in non-metastatic tumors. The colorimetric intensities of 10–20 images of immunohistochemical-stained antigen spots were counted using a computer-assisted image analyzer (Olympus Microimage™ Image Analysis version 4.0 software for Windows, USA) and were expressed as mean pixel integrated optical density (IOD). Statistical analysis was performed using Prism version 5.00 software (GraphPad Software, USA). Analysis of variance and Tukey's post-hoc tests were applied to identify significant differences in optical density between cell lines. Differences were considered significant when *p<0.05 and highly significant when **p≤0.01 or ***p≤0.001. Spearman's coefficient was used to assess correlations.

Figure 6. Analysis of Wnt proteins expression in metastatic and non-metastatic canine mammary cancer tissues.

Images showing Wnt-2, Wnt-5a, Dkk-1, and ROR-2 expression in metastatic and non-metastatic canine mammary tumors were obtained using an Olympus BX60 microscope (400×). Tissue sections were treated with specific antibodies and stained using an EnVision kit (Dako, Denmark). Labeled antigens are indicated by brown precipitates. Metastatic tumors show significantly higher expression of all examined antigens compared with non-metastatic tumors. Colorimetric intensities of immunohistochemical-stained antigen spots on 10–20 images were counted using a computer-assisted image analyzer (Olympus Microimage™ Image Analysis version 4.0 software for Windows, USA) and were expressed as mean pixel integrated optical density (IOD). Statistical analysis was performed using Prism version 5.00 software (GraphPad Software, USA). Analysis of variance and Tukey's post-hoc tests were used to identify differences in optical density. Differences were considered significant when *p<0.05, and highly significant when **p≤0.01 or ***p≤0.001.

Figure 7. Canonical and non-canonical Wnt pathways.

Scheme of the interactions observed between canonical and non-canonical Wnt pathways.