Background

Esophageal cancer ranked the sixth among malignant tumors worldwide. Over 90% of the tumor cases are esophageal squamous cell carcinoma (ESCC) [1, 2]. Despite recent advances in the personalized therapies, molecular subty** and development of targeted drugs such as epithermal growth factor receptor (EGFR) inhibitor Gefitinib and anti-programed cell death protein-1 (anti-PD-1) pembrolizumab [47] which may be overlooked by previous studies focusing on only one or several types of TME cells [48, 49]. In ESCC tumors, the increased cell interactions occurred mostly within and between the epithelial, fibroblasts and immune cells. Moreover, differential cell interaction analysis identified signaling from macrophages, DCs, and monocytes to epithelial/tumor and T cells was strengthened in ESCC tumors (Fig. 3D).

Combing clinical information in the ESCC microarray cohort, we were able to uncover prognostically relevant cell subtypes and cell communication ligands by dissecting the ESCC TME. In multiple solid tumors, TAMs are actively recruited to the TME through paracrine communication and chemotaxis with the tumor cells [16, 19, 36]. CCL18 is mainly secreted by antigen-presenting cells of the innate immune system, such as DCs, monocytes, and macrophages. It can act on the adaptive immune cells, and recruit naïve T cells, Tregs, or even B cells [50, 51]. TAMs were thought to closely resemble the M2 phenotype; however, recent findings suggested that this binary polarization model was oversimplified and a spectrum model of TAM phenotypes has been proposed instead [52]. TAM is highly plastic within TME, where they display different characteristics and functions and has mixed expression profiles ranging from M1 to M2 [53].

Comparative analysis of cell communication further screened prognostic ligand receptor pairs regulating ESCC TME including CCL3/CCR1, CCL3/CCR5 and CCL18/PITPNM3 (Fig. S1). We finally confirmed CCL18-PITPNM3 signaling network showed strongest interaction occurred between epithelial/tumor cells and macrophages based on cell interaction network inferred from ESCC scRNA-seq data (Fig. 4E). Chemokines play important roles during the cell migration between different organs and tissues. Therefore, it would be helpful to develop potential cancer treatments involving the immunostimulatory action of cytokines in order to address cytokine imbalance. They can regulate host response to cancer by directing leukocytes or other cells into the TME to elicit anti-tumor or tumor-promoting effects [33, 34]. The role of CCL18 in cancer progression is controversial, it was reported that CCL18 could directly promote invasion, metastasis and angiogenesis in breast cancer, pancreatic cancer and ovarian cancer [16, 17], but CCL18 was associated with prolonged survival in patients with gastric cancer [54]. Here we found that CCL18 was elevated in tumor tissues of ESCC, negatively correlated with the patient survival, and positively correlated with tumor develo** stage (Table 1), which corresponded with its high expression level on TAMs infiltration (Fig. 2B). As a receptor of CCL18, PITPNM3 mainly expressed in human retina, brain, spleen, and breast cancer cells [55]. It was reported that CCL18/PITPNM3 interaction could also promote liver cancer migration, invasion, EMT, and the progression of pancreatic ductal carcinoma [56]. Similarly, we discovered that CCL18 could promote proliferation of esophageal cancer cells via its interaction with PITPNM3. Although we discovered the CCL18 was mainly expressed by M1 macrophages, the role of M2 macrophages shall not be fully ruled out. It was uncovered that CCL18 caused the maturation of cultured monocytes to macrophages in the M2 spectrum [57]. Therefore, CCL18 secreted by macrophages in the tumor tissues of ESCC could promote the proliferation of esophageal cancer cells and led the polarization of macrophages toward M2 phenotype.

Considering the high similarities of structures and sequences of CCL3 and CCL18, we designed and evaluated blocking peptides based on the functional fragments of both chemokines. The Pep3 constructed from the N terminus of hCCL18 was selected due to its greatest inhibition effect on EC-109 cell proliferation (Fig. 6). The antitumor efficacy of CCL18 blocking peptide Pep3 was further validated in vitro and in vivo, using a spontaneous ESCC mice model induced by 4-NQO. Pep3 is, to our knowledge, the first peptide to inhibit CCL18 and greatly reduce the ESCC cancer progression. This peptide may reduce esophageal carcinogenesis by inhibiting TAM infiltration and recruitment via CCL18/PITPNM3 or CCL3/CCR1/CCR5 signaling, hence enhancing the CD8+ T cell response (Fig. 7).

Furthermore, the pan-cancer analyses between CCL18 and immune checkpoints expression have important implications for cancer immunotherapy. As a number of inhibitory immunoreceptors have been identified and studied, 40 known immune checkpoint genes were collected, and were correlated with the expression of CCL18 (Fig. S9). We found 26 checkpoints were highly co-expressed with CCL18 including but not limited to PD-L1 (CD274), LAG3, TIGIT and CTLA4. Compared with other 33 cancer types in The Cancer Genome Atlas (TCGA) (Fig. S10), CCL18 was co-expressed with multiple immune checkpoints in 14 cancers. This demonstrated the great potential of CCL18 to be served as a pan-cancer target for future immunotherapy.

There are major efforts to develop therapeutic strategies to overcome immune resistance by using a combination of checkpoint blockers, and to improve drug efficacies and response rate of patients. Recent reports have indicated the strategies to improve phagocytic ability and reduce tumor growth via blocking PD-1 on TAMs [58] or promoting M2 macrophage polarization [59, 60]. TME changes not only influence tumor progression, but also dramatically influence the efficacy of cancer therapy. Monocyte derived macrophages build an essential inflammatory niche sha** tumor immune microenvironment. Recent progress defined the molecular landscapes and mechanisms of macrophage differentiation [61] held the promise to uncover its heterogeneity and functional roles within tumors. TAMs secrete cytokines and chemokines that can suppress T cell recruitment and activation, thereby promoting resistance to immune checkpoint inhibition. Novel therapeutic techniques, as demonstrated here, have the potential to synergize with checkpoint inhibitors, chemotherapy, and/or radiation therapy to enhance the overall efficacy of cancer treatment.

Conclusion

In summary, our study provided a comprehensive landscape of TME in ESCC by characterizing the cell–cell interactions dynamics of ESCC TME, thus provided novel insights to understand the co-evolution of tumor and immune system in ESCC. We identified that CCL18, mainly secreted by macrophages, and may serve as a target for the diagnosis and immunotherapy of ESCC by combining bioinformatic analysis and in vitro validation of the associated signaling pathway. We designed and screened a CCL18 blockade peptide and validated its antitumor activity in vitro and in vivo. As summarized in Fig. 8, CCL18 blockade might inhibit esophageal carcinogenesis by preventing TAMs recruitment and cancer cell proliferation.

Fig. 8
figure 8

Schematic illustration of the possible antitumor mechanism of CCL18/CCL3 blockade. CCL18 and CCL3 in the ESCC TME are secreted by TAMs and other myeloid cells. Pep3 may elicit antitumor effects in ESCC by preventing infiltration of tumor-associated macrophages (TAM) and proliferation of ESCC cancer cells, via CCL3/CCR1/CCR5 and CCL18/PITPNM3 pathways

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