This study investigated tumor-associated macrophages activity in the microenvironment of renal cell carcinoma. Via a co-culture with macrophage-like cells differentiated from human monocyte cell line THP-1 and U937 cells, the migration ability of ACHN and Caki-1 cells, which are human renal cell carcinoma cell line cells, was significantly increased, as was the epithelial–mesenchymal transition change. A chemokine array identified the CCL20-CCR6 axis as a concentration-dependent signal in ACHN and Caki-1 cell migration. Akt in the ACHN and Caki-1 cells was activated by macrophage-like cells, and the CCL20 neutralizing antibody suppressed migration ability, epithelial–mesenchymal transition, and Akt phosphorylation in the ACHN and Caki-1 cells. Akt inhibitor AZD5363 also decreased the epithelial–mesenchymal transition change and migration ability in the ACHN and Caki-1 cells. In 42 renal cell carcinoma tissues, patients with CCR6 and macrophage infiltration indicated poor prognoses. In the tumor microenvironment of renal cell carcinoma, cancer cells are activated by CCL20 secreted by tumor-associated macrophages through Akt activation, followed by epithelial–mesenchymal transition and an acquired migration ability. Thus, inhibition of the CCL20-CCR6 axis may be a potential therapeutic strategy for renal cell carcinoma.

We investigated the mechanisms of interaction between bladder cancer (BC) cells and tumor-associated macrophages (TAMs). Coculturing BC cell lines (UMUC3 and T24) with macrophage-like cells differentiated from THP-1 into M2-like TAMs revealed a decrease in Cluster of Differentiation (CD) 68 expression and an increase in CD206 expression. This differentiation enhanced BC cell migration and invasion. Additionally, M2-like TAMs significantly increased the secretion of C-C motif chemokine ligand (CCL) 20, which promotes BC cell migration and invasion via the MEK/ERK signaling pathway through its paracrine effects. Coculturing with TAMs also elevated the expression of CC chemokine receptor (CCR) 6 in BC cells, indicating increased sensitivity to CCL20. Immunohistochemistry analysis of human BC tissues showed a significant correlation between CCR6 expression levels and BC prognosis. Inhibition of CCR6 reduced BC cell metastasis both in vitro and in vivo. Additionally, CXCL1 secretion from BC cells was found to contribute to the M2-like polarization of macrophages and to enhance BC cell migration and invasion through autocrine and indirect effects. In summary, CCL20 and CXCL1 play crucial roles in the interaction between BC cells and TAMs.

We appreciate Zins and Abraham [1] commenting on our paper studying the role of the CCL20-CCR6 axis on renal cell carcinoma (RCC) cells [2]. As they pointed out, our study has certain limitations. Although M1- and M2-types cannot be separated clearly and a consecutive change of character might exist between them, it has been reported that plural specific markers express on M1- and M2-types. Unfortunately, a definite difference between M1 and M2 macrophages was not confirmed in our study. For more differentiation, multiple stimulations, such as suggested in the comments of Zins and Abraham, might be needed. Hence, we needed to expediently use “M1-like” and “M2-like” to mention specific status of these macrophage-like cells. Meanwhile, CCL20 expression levels of M2-like-THP-1 cells co-cultured with RCC cells were dramatically increased compared with parental THP-1 cells, indicating that certain stimulations within the tumor microenvironment rather than theoretical stimulations make macrophages differentiated; however, further studies are needed to clarify this mechanism using a more appropriate co-culture system mimicking the tumor microenvironment. Immunohistochemistry of CCL20 and M2 markers will help to better understand the role of tissue infiltrating macrophages, even tissue CD68 staining intensity itself was reported to correlate with prognosis of RCC patients [3].

Renal cancer remains a major global health burden, and current targeted and immunotherapeutic strategies are frequently limited by toxicity, therapeutic resistance, and suboptimal response rates. Natural bioactive compounds such as epigallocatechin gallate (EGCG) and cafestol exhibit anticancer activity; however, their therapeutic utility is constrained by limited potency and dose-dependent adverse effects. In this study, the antiproliferative and synergistic effects of cafestol and a hyaluronic acid (HA)–EGCG conjugate were investigated in renal cancer cells. HA conjugation significantly enhanced the antiproliferative efficacy of EGCG in both ACHN and A498 human renal cancer cells, whereas unmodified HA exhibited no intrinsic anticancer activity. Importantly, the HA–EGCG conjugate enabled a pronounced synergistic interaction with cafestol, particularly in ACHN cells, as confirmed by combination index analysis, while free EGCG and cafestol failed to achieve synergistic inhibition. In addition, the HA–EGCG conjugate and its combination with cafestol exhibited favorable selectivity toward renal cancer cells compared with normal renal proximal tubule epithelial cells (RPTECs). This combination robustly enhanced apoptosis and was associated with significant downregulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL at the transcriptional level, together with suppression of the epithelial–mesenchymal transition-associated transcription factor SNAIL at both transcriptional and protein levels. Notably, the enhanced antiproliferative effects were achieved at reduced concentrations, highlighting the potential to mitigate dose-related toxicity. Collectively, these findings support HA-based conjugation as an effective strategy to potentiate the anticancer activity of natural bioactive compounds and to enable synergistic, multi-targeted therapeutic effects against renal cancer.