Macrophages Mediate a Switch between Canonical and Non-Canonical Wnt Pathways in Canine Mammary Tumors

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.

研究目标
当前主流假说认为，肿瘤相关巨噬细胞（tumor-associated macrophages, TAMs）会被癌细胞"策反"，进而促进而非抑制肿瘤转移。鉴于癌细胞与TAMs互作的分子机制复杂且存在争议，本研究旨在更清晰地阐明这一现象。

研究方法与结果
我们通过微RNA微阵列（microRNA microarrays）、实时定量聚合酶链反应（real-time quantitative PCR, qPCR）以及蛋白质印迹（Western blot）实验发现，将犬乳腺肿瘤细胞与TAMs共培养，或用巨噬细胞条件培养基（macrophage-conditioned medium）处理肿瘤细胞，均可抑制肿瘤细胞内的经典Wnt信号通路（canonical Wnt signaling pathway），并激活非经典Wnt信号通路（non-canonical Wnt signaling pathway）。
我们还证实，TAMs与肿瘤细胞共培养可上调TAMs内经典Wnt信号通路抑制剂的表达水平。后续实验进一步证明，巨噬细胞可诱导肿瘤细胞形成侵袭性生长表型并发生上皮间质转化（epithelial–mesenchymal transition, EMT）。
在50例犬乳腺腺癌组织（n=50）及异种移植瘤（xenograft tumors）中验证上述结果后发现，转移性肿瘤与非转移性恶性肿瘤分别激活了非经典Wnt信号通路与经典Wnt信号通路，且非经典Wnt信号通路的激活程度与TAMs的浸润数量呈正相关。

研究结论
我们证实，在犬乳腺肿瘤中，TAMs可介导经典与非经典Wnt信号通路之间的"转换"，进而促进肿瘤侵袭与转移。
值得注意的是，在添加巨噬细胞条件培养基或共培养活巨噬细胞的环境中，肿瘤细胞均出现了相似的表型变化。上述结果表明，TAMs并非被癌细胞"策反"，而是持续分泌经典Wnt信号通路抑制剂以抑制肿瘤增殖与发生，但这一过程会附带诱导非经典Wnt信号通路激活，最终促进肿瘤转移。
本研究数据挑战了学界对TAMs与癌细胞互作的传统认知。