Fig 2: Interaction between BMECs and HCC cells enhances tumor growth in the spine. (A) si-CX3CL1 was transfected into BMECs to construct CX3CL1low BMECs. Transwell assays were used to examine recruitment of BMECs co-cultured with or without HMCC97H cells. The number of the recruited cells was quantified. n=3. Cell recruitment were analyzed using ANOVA test. (B) Cell vitality of BMECs was assessed using CCK-8 assay; n=3. Data were analyzed using ANOVA followed by Tukey's test. (C) Tumor size was measured based on the volume. n=5. Scale bar, 5 mm. Data were analyzed using an unpaired t-test. (D) Samples from 2 groups were examined using immunofluorescence. Magnification, ×20. The result was analyzed using an unpaired t-test. Gray bars represent the CX3CL1nor BMECs group and black bars represent the CX3CL1low BMECs group (E) Flow cytometry revealed the percentage and number of F4/80+CD11b+ cells; n=5. (F) Flow cytometry was used for detecting expression of M2 macrophage surface markers, CX3CR1, MRC1 and CD163; n=5. Cells were selected based on higher expression of CD45 for macrophages. Subsequently, CD11b and F4/80 expression were used for further characterization of macrophages. FACS (fluorescent-activated cell sorting) data were analyzed using a Kruskal Wallis test followed by Dunn's non-parametric post hoc test. HCC, hepatocellular carcinoma; BMECs, bone marrow endothelial cells; si, small interfering; CCK-8, Cell Counting kit-8; CX3CL1, C-X3-C motif chemokine ligand 1.

Fig 3: Activation of the ERK1/2 signaling pathway was positively correlated with the epithelial to mesenchymal transition of tumor cells induced by tumor associated macrophages in HNSCC tissues. Hierarchical clustering analysis of CD68, CD163, Vimentin, EGFR, Slug and p-ERK1/2 in HNSCC and normal tissues. ERK1/2, extracellular signal-regulated protein kinase 1/2; CD, cluster of differentiation; EGFR, epidermal growth factor receptor; p-, phosphorylated; HNSCC, head and neck squamous cell carcinoma; HM-SCC, HNSCC with high macrophages; LM-SCC, HNSCC with low macrophages; NM, normal mucosa.

Fig 4: Induction and acquirement of macrophage polarization. Macrophages with M0, M1 and M2 functional profiles were acquired from THP1 cells following treatment with PMA, PMA/LPS/IFN-? and PMA/IL-4/IL-13, respectively. (A) Expression of classical M0, M1 and M2 polarized macrophage surface markers (CD68 and CD163) was evaluated by flow cytometry. (B) mRNA expression levels of TNF-a, IL-1ß, TGF-ß and IL-10 in differentiated cells following induction were detected by reverse transcription-quantitative polymerase chain reaction. Data are expressed as the mean ± standard error of the mean. *P<0.05 and ***P<0.001 vs. THP1. PMA, phorbol-12-myristate-13-acetate; LPS, lipopolysaccharide; CD, cluster of differentiation; IFN-?, interferon-?; IL, interleukin; TNF-a, tumor necrosis factor-a; TGF-ß, transforming growth factor-ß.

Fig 5: Differentiation of THP-1 cells into macrophages. (A) Cell morphology and protocols used to obtain the MF and M2 macrophages. Magnification: 10×; (B,C) immunofluorescence images. CD68, CD163 and CD206 are stained in red. Nuclei are stained blue with Hoechst. Magnification: 20×. A.U. arbitrary units; (D) mRNA levels of IL8, IL10, CCL2, CCL5 and VEGF. Data are reported as mean ± SD of three independent experiments performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. THP-1 cells.