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Additional file 1: FigureS1. Expression,distribution, and prognostic significance of CCL3, CCL18 and their receptors inESCC TME. (A) Bubble heatmap showing expression levels of CCL3,CCL18 and their receptors in different cell types and locations based on ESCC scRNA-seqdataset. Dot size indicates percentage of expressing cells, colored based onnormalized expression levels. (B) Kaplan-Meier curves of overall survival ofESCC microarray cohort stratified by the expression of CCL3 and its receptorsCCR1 and CCR5. (C) Kaplan-Meier curves of overall survival of ESCC microarraycohort stratified by the expression of CCL18 receptors ACKR1, ERG28 and CCR8. FigureS2. Cell-cell communication network of ESCC. Differential number of possible interactions and interaction strength(edge weight) between the five major cell types between peri- and tumor sites(A and B). Number of possible interactions between the 11 cell types in peri-and tumor sites (C and D). Figure S3. Cell-cellcommunication in 4-nitroquinoline 1-oxide (4NQO) inducedmouse ESCC model. (A) UMAP visualization of 19,829 cells from 3 normal mice, 3 carcinoma in situ(CIS) mice and 3 invasive carcinoma (ICA) mice (CRA002118). (B) Differential numberof possible interactions between any two cell populations. Red (positivevalues) and blue (negative values) in the color bar indicate higher number ofpredicted interactions in normal and tumor mice samples, respectively. (C)A circle plotshowing the number of possible interactions in normal mice estimated byCellChat. (D) A circle plot showing the number of possible interactions in CIS and ICAmice estimated by CellChat. (E)A circle plot showing the cell-cell communicationnetwork of CCL pathway estimated by CellChat. (F)The significantly related ligand–receptor interactions of CCL pathway in theESCC mouse model inferred by CellChat analysis. Figure S4. The expression of CCL3 or its receptor inthe ESCC tissues or macrophages. (A)The mRNA expression level of CCL3, CCR1 and CCR5 in the ESCC tissues of 4-NQOinduced mice with ESCC by qPCR. (B) The expression of CCL3 and co-localizationwith macrophages (F4/80) in the mouse ESCC tissues. Representative views were shown. (C) The expression of CCL3 in BMDM polarized M1 or M2 macrophages inmRNA and protein levels. (*P < 0.05, ***P < 0.001). Figure S5.The cell cycle and apoptosis of EC-109, EC-109 Vector and EC-109 shPITPNM3cells. (A) Cell cycles of EC-109, EC-109 Vectorand EC-109 shPITPNM3 cells treated with or without 40ng/mL CCL18 for 72 h. *P< 0.05. (B) Flow cytometry analysis of the apoptosis of EC-109, EC-109Vector and EC-109 shPITPNM3 cells treated with or without 40ng/mL CCL18 for 72h. n.s., no significance. Figure S6. The proliferation, colony formation and migration analysis ofEC-109, EC-109 Vector and EC-109 shPITPNM3 cells. (A) The cell growth curve and cell viability of EC-109, EC-109 Vector andEC-109 shPITPNM3 cells determined by the cell number counting and MTT assay. (B)The colony formation of EC-109, EC-109 Vector and EC-109 shPITPNM3 cells toform clones. (C) Representative results of the Transwell assay of EC-109,EC-109 Vector and EC-109 shPITPNM3 cells at 48 h. Bar = 200 μm. (D) Representativeimages of the wound-healing assay of EC-109, EC-109 Vector and EC-109 shPITPNM3cells after 48 h. n.s., no significance. Figure S7. (A)Representative results of the Transwell assay of EC-109 cells in the presenceor absence of rhCCL18 at increasing concentrations (10–40 ng/mL) for 48 h. (B)Representative images of the wound-healing assay of EC-109 cells in thepresence or absence of rhCCL18 at increasing concentrations (10–40 ng/mL) at 0h and 48 h. n.s., no significance. Figure S8. The expression of PITPNM3 inthe ESCC tissues with or without Pep3 treatment. (A) The mRNA expression level of PITPNM3 in the ESCC tissues of 4-NQOinduced mouse model by qPCR. (B) Representative images of PITPNM3 staining inthe ESCC tissues, bar = 50 μm. ***P < 0.001. Figure S9. Correlationof CCL18 with immune scores, CD8 T cells and 40 known immune checkpoints in ESCC. The correlationmap between 40 common immune checkpoint genes andthe expression of CCL18 is as follows: *** P < 0.001, ** P< 0.01, * P < 0.05. Most correlated immune checkpoint genes arehighlighted in red. Figure S10. Correlation of CCL18 with known immunecheckpoints. Calculated from RNA-seqexpression data from The Cancer Genome Atlas (TCGA)datasets across 33 different cancer types. *** P < 0.001, ** P< 0.01, * P < 0.05.

MPT64 is one of the secreted proteins from Mycobacterium tuberculosis. Little is known about its role in infection by Mycobacterium tuberculosis. In this study, we demonstrated that MPT64 could dose-dependently inhibit the apoptosis of RAW264.7 macrophages induced by PPD-BCG. Quantitative real-time PCR results showed that the expression of bcl-2 increased in macrophages treated with MPT64 compared with PPD-treated cells. Furthermore, the results provided strong evidence that bcl-2 up-regulation was positively controlled by miRNA-21. Finally, NF-κB was identified as the transcription factor for miRNA-21 using a ChIP assay. It can be concluded from our study that MPT64 could inhibit the apoptosis of RAW264.7 macrophages through the NF-κB-miRNA21-Bcl-2 pathway.