Tumor-associated neutrophils and macrophages interaction contributes to intrahepatic cholangiocarcinoma progression by activating STAT3

Abstract

Background: Tumor-associated neutrophils (TANs) and macrophages (TAMs) can each influence cancer growth and metastasis, but their combined effects in intrahepatic cholangiocarcinoma (ICC) remain unclear.

Methods: We explored the distributions of TANs and TAMs in patient-derived ICC samples by multiplex immunofluorescent staining and tested their separate and combined effects on ICC in vitro and in vivo. We then investigated the mechanistic basis of the effects using PCR array, western blot analysis and ELISA experiments. Finally, we validated our results in a tissue microarray composed of primary tumor tissues from 359 patients with ICC.

Results: The spatial distributions of TANs and TAMs were correlated with each other in patient-derived ICC samples. Interaction between TANs and TAMs enhanced the proliferation and invasion abilities of ICC cells in vitro and tumor progression in a mouse xenograft model of ICC. TANs and TAMs produced higher levels of oncostatin M and interleukin-11, respectively, in co-culture than in monoculture. Both of those cytokines activated STAT3 signaling in ICC cells. Knockdown of STAT3 abolished the protumor effect of TANs and TAMs on ICC. In tumor samples from patients with ICC, increased TAN and TAM levels were correlated with elevated p-STAT3 expression. All three of those factors were independent predictors of patient outcomes.

Conclusions: TANs and TAMs interact to promote ICC progression by activating STAT3.

Keywords: cytokines; liver neoplasms; macrophages; neutrophil infiltration; tumor microenvironment.

Figure 1

Correlation of tumor-associated neutrophils (TANs) and tumor-associated macrophages (TAMs) in intrahepatic cholangiocarcinoma (ICC). Representative immunofluorescence images of CD66b (green), CD68 (red) and DAPI (blue) staining showing (A) a small cluster and (B) a larger cluster of TANs and TAMs in ICC. Scale bars: 50 μm. (C) G(r) and (D) K(r) functions showed the theoretical distribution (blue line) and the actual distribution (red line) of distances between TANs and TAMs. (E) A scatter plot illustrated the positive correlation between the numbers of TANs and TAMs. (F) A scatter plot illustrated the positive correlation between the RNA expression levels of CD66b and CD68.

Figure 2

Biological effect of the interaction between tumor-associated neutrophils (TANs) and tumor-associated macrophages (TAMs) on intrahepatic cholangiocarcinoma (ICC) cells. (A) CCK8 cell proliferation assay showed that conditioned medium (CM) from TAN and TAM promoted ICC cell proliferation. Data are shown as the mean±SD (n=6 for each group). (B) Invasion of ICC cells cultured with CM from TAN and TAM compared with that of control ICC cells. The graphs show the number of invasive cells after 48 hours. Data are shown as the mean±SD (n=6 for each group). (C) Cultured with CM from TAN and TAM increased the colony-formation activity of ICC cells Data are shown as the mean±SD (n=6 for each group). *P<0.05, ***p<0.001.

Figure 3

Influence of the interaction between tumor-associated neutrophils (TANs) and tumor-associated macrophages (TAMs) on intrahepatic cholangiocarcinoma (ICC) growth and metastasis. (A) Schematic of the mouse xenograft experiments. Control group: 1×10⁷ ICC cells were injected into the subcutaneous space of the upper left flank region of NOD-Prkdc^scid IL2rg^tm1/Bcgen mice at day 0. TANs group: 1×10⁷ ICC cells were co-injected with 1×10⁶ TANs into the subcutaneous space of the upper left flank region of mice at day 0, and TANs were injected into the tumor at the indicated time (red arrows). TAMs group: 1×10⁷ ICC cells were co-injected with 1×10⁶ TAMs into the subcutaneous space of the upper left flank region of mice at day 0, and TAMs were injected into the tumor at the indicated time (yellow arrows). TANs+TAMs group: 1×10⁷ ICC cells were co-injected with 1×10⁶ TANs and TAMs mixture (TANs:TAMs=1:1) into the subcutaneous space of the upper left flank region of mice at day 0, and TANs and TAMs mixture were injected into the tumor at the indicated time (blue arrows). All mice were monitored once every 5 days and killed 5 weeks later. (B) Xenografts containing TANs and TAMs produced greater tumor volume and more pulmonary metastasis (C) than xenografts composed of ICC cells alone, *p<0.05, **p<0.01, ***p<0.001. Scale bars: 50 μm. Data are shown as the mean±SD (n=4 for each experiment mice group).

Figure 4

Tumor-associated neutrophils (TANs) and tumor-associated macrophages (TAMs) produced oncostatin M (OSM) and interleukin (IL)-11, respectively, in co-culture. (A) A PCR array showed altered expression of cytokines and chemokines by TANs after co-culture with TAMs. (B) A PCR array showed altered expression of cytokines and chemokines by TAMs after co-culture with TANs. (C) quantitative reverse transcription (qRT)-PCR and western blot analysis validated the messenger RNA (mRNA) and protein levels of OSM in TANs after co-culture with TAMs. (D) qRT-PCR and western blot analysis validated the mRNA and protein levels of IL-11 in TAMs after co-culture with TANs. (E) ELISA confirmed the presence of OSM and IL-11 in conditioned media obtained from co-cultured TANs and TAMs. (F) Representative images of OSM expression (magenta) in TANs (CD66b, green cells), and IL-11 expression (cyan) in macrophages (CD68, red cells) obtained by opal multiplex immunofluorescent staining of intrahepatic cholangiocarcinoma (ICC) samples. Scale bars: 50 μm.

Figure 5

Biological effect and mechanism of oncostatin M (OSM) and interleukin (IL)-11 in intrahepatic cholangiocarcinoma (ICC) cells. (A) Western blot analysis showed the phosphorylation state of STAT3 and downstream STAT3 targets including vascular endothelial growth factor (VEGF), cyclin D1 and BCL-2 in ICC cells after OSM and IL-11 treatment. (B) CCK8 cell proliferation assay showed ICC cell proliferation in the indicated treatment groups. Data are shown as the mean±SD (n=6 for each group). (C) Matrigel-invasion assay showed the invasion of ICC cells in the indicated treatment groups. Data are shown as the mean±SD (n=6 for each group). (D) Colony-formation activity of ICC cells in the different treatment groups. Data are shown as the mean±SD (n=6 for each group). *P<0.05, **p<0.01, ***p<0.001.

Figure 6

The role of STAT3 signaling in the protumor effect of the interaction between tumor-associated neutrophils (TANs) and tumor-associated macrophages (TAMs) in intrahepatic cholangiocarcinoma (ICC). (A) CCK8 cell proliferation assay showed ICC cell proliferation after treatment with TANs and TAMs, shRNA-STAT3 or the STAT3 inhibitor S3I-201 (200 μM). Data are shown as the mean±SD (n=6 for each group). (B) Matrigel-invasion assay showed ICC cell invasion after treatment with TANs and TAMs, shRNA-STAT3 or the STAT3 inhibitor S3I-201. Data are shown as the mean±SD (n=6 for each group). (C) Colony-formation activity of ICC cells after treatment with TANs and TAMs, shRNA-STAT3 or the STAT3 inhibitor S3I-201, ***p<0.001. Data are shown as the mean±SD (n=6 for each group). (D) Tumor volume and (E) pulmonary metastasis of mouse xenografts composed of ICC cells treated with TANs and TAMs with or without shRNA-STAT3 or the STAT3 inhibitor S3I-201. *P<0.05, **p<0.01, ***p<0.001. Scale bars: 100 μm. Data are shown as the mean±SD (n=4 for each experiment mice group).

Figure 7

Tumor-associated neutrophil (TAN) levels were correlated with tumor-associated macrophage (TAM) infiltration, p-STAT3 expression and prognosis in patients with intrahepatic cholangiocarcinoma (ICC). (A and B) Expression of CD66b, CD68 and p-STAT3 in representative ICC tumor samples, Scale bars: 100 μm. (C) A scatter plot illustrated the correlation between the numbers of TANs and TAMs and p-STAT3 expression. (D) The numbers of TANs and TAMs, p-STAT3 expression and the combination of those factors (TANs/TAMs) was predictive of prognosis in patients with ICC.