Tumor-associated macrophages promote progression and the Warburg effect via CCL18/NF-kB/VCAM-1 pathway in pancreatic ductal adenocarcinoma

Abstract

Tumor-associated macrophages (TAMs) are frequently found near pancreatic cancer cells, but it is uncertain whether they are involved in pancreatic cancer progression and the Warburg effect. Here, we show that CCL18 secreted by TAMs facilitates malignant progression and induced a glycolytic phenotype in pancreatic cancer, partially owing to paracrine induction of VCAM-1 in pancreatic cancer cells. Reciprocally, VCAM-1-induced lactate production from pancreatic cancer cells with enhanced aerobic glycolysis activates macrophages to a TAM-like phenotype, forming a positive feedback loop. VCAM-1 was found to be highly expressed in human pancreatic ductal adenocarcinoma (PDAC) tissues and cell lines, and is associated with disease progression and predicts clinical outcome in PDAC patients. Flow cytometry analysis further demonstrated that VCAM-1 downregulation induced an accumulation of PDAC cells in G0/G1 phase, accompanied by a significant decrease in S phase. Downregulation of VCAM-1 significantly inhibited proliferation, colony formation, migration, and invasion of PDAC cells in vitro, whereas the ectopic expression of VCAM-1 had the opposite effect. VCAM-1 on pancreatic cancer cells might tethers THP-1 monocytes to cancer cells via counter-receptor interaction, providing a survival advantage to pancreatic cancer cells that infiltrate leukocyte-rich microenvironments. Furthermore, downregulation of VCAM-1 could repress tumor growth in mouse xenograft models. In particular, our results highlighted the contribution of VCAM-1 to the maintenance of the Warburg effect in PDAC cells. Finally, we investigated the clinical correlations of CCL18 and VCAM-1 in human PDAC specimens. In summary, these findings indicate that the CCL18/PITPNM3/NF-kB/VCAM-1 regulatory network might provide a potential new therapeutic strategy for PDAC.

Fig. 1. Differences and characterizations in mRNA expression profiles between pancreatic cancer cell line PANC-1-alone control groups (NPC groups) and the PANC-1-co-cultured TAMs groups (NPM groups).

a Scatter plots are used to evaluate the difference in the expression of mRNAs between the NPC groups and the NPM groups. The values plotted on X and Y axes are the averaged normalized signal values of each group (log2 scaled). The middle green line refers to no difference between the two groups, and the flanking green lines represent twofold changes. The mRNAs above the top green line and below the bottom green line indicate more than twofold changes between the two groups. b Box plots for the normalized gene expression data of the NPC groups and the NPM groups. c Volcano plots used for visualizing differential expression between two different conditions. The vertical lines correspond to twofold (log2 scaled) up and down, respectively, and the horizontal line represents a p value of 0.05 (−log10 scaled). The red points in plot represent the differentially expressed mRNAs with statistical significance. d Hierarchical cluster analysis of all target mRNAs. The mean entities of all target mRNAs, where at least three out of six samples have flags in present or marginal. Flags are attributes that denote the quality of the entities using methods from GeneSpring software. e Hierarchical cluster analysis of the top 30 up and downregulated mRNAs. Red and green colors represent up- and downregulated genes, respectively. f The top 10 upregulated mRNAs are listed by fold change, among which the cell adhesion molecule VCAM-1 was the most upregulated gene with ~ 7.07-fold change

Fig. 2. VCAM-1 is aberrantly overexpressed in PDAC tissues and cell lines.

a, b The mRNA and protein levels of VCAM-1 in PANC-1 and Capan-2 cells were measured by qRT-PCR and western blotting analysis. The PANC-1 and Capan-2 cells were cultured alone, or co-cultured with TAMs for 4 days. c, d The mRNA level of VCAM-1 in 134 paired PDAC tissues and corresponding adjacent non-tumorous tissues by qRT-PCR. VCAM-1 expression levels from all tissues were normalized to β-actin expression (∆CT) and then compared with a non-tumorous tissue and converted to fold change (2^−∆∆CT). e The protein level of VCAM-1 in 4 paired PDAC tissues and their corresponding adjacent non-tumorous tissues by western blotting analysis. f, g The mRNA and protein levels of VCAM-1 were evaluated in five pancreatic cancer cell lines compared with the pancreatic ductal epithelium cell line HPDE6-C7 by qRT-PCR and western blotting analysis. ***: p < 0.001

Fig. 3. VCAM-1 mediates proliferation and G1-S checkpoint of PDAC cells in vitro.

a The expression of VCAM-1 was suppressed by specific siRNAs in PANC-1 and Capan-2 cells. b The expression of VCAM-1 was overexpressed by transfecting pcDNA3.1-VCAM-1 into PANC-1 and Capan-2 cells. c The cell viability of si-NC or si-VCAM-1 transfected PANC-1 and Capan-2 cells, as determined by CCK-8 assay. d The cell viability of pcDNA3.1-NC or pcDNA3.1-VCAM-1 transfected PANC-1 and Capan-2 cells, as determined by CCK-8 assay. (e) Top: The proliferation of si-NC or si-VCAM-1 transfected PANC-1 and Capan-2 cells by colony formation assay. Bottom: The proliferation of pcDNA3.1-NC or pcDNA3.1-VCAM-1 transfected PANC-1 and Capan-2 cells by colony formation assay. f, g The cell cycle distribution of si-NC or si-VCAM-1 transfected PANC-1 and Capan-2 cells by flow cytometry analysis. h, i The cell cycle distribution of pcDNA3.1-NC or pcDNA3.1-VCAM-1 transfected PANC-1 and Capan-2 cells by flow cytometry analysis. Values represented the mean ± SD from three independent experiments. *, ** or ***: significantly different from the corresponding control, p < 0.05, p < 0.01 or p < 0.001, respectively, by Student’s t-test

Fig. 4. VCAM-1 mediates migration and invasion of PDAC cells in vitro.

PDAC Cell line PANC-1 and Capan-2 were treated as in described in the Materials and methods. a The motility of PANC-1 and Capan-2 cells transfected with si-VCAM-1 when compared with the controls by wound-healing assay. b The motility of PANC-1 and Capan-2 cells transfected with pcDNA3.1-VCAM-1 when compared with the controls by wound-healing assay. c The migration and invasion of PANC-1 and Capan-2 cells transfected with si-VCAM-1 when compared with the controls by transwell assay. d The migration and invasion of PANC-1 and Capan-2 cells transfected with pcDNA3.1-VCAM-1 when compared with the controls by transwell assay. Values represented the mean ± SD from three independent experiments. *, ** or ***: significantly different from the corresponding control, p < 0.05, p < 0.01 or p < 0.001, respectively, by Student’s t-test

Fig. 5. VCAM-1 modulates the Warburg effect of PDAC cells in vitro.

a, b Silencing VCAM-1 expression abrogated the glycolytic capacity of PANC-1 and Capan-2 cells, as reflected by ECAR analysis. c, d Silencing VCAM-1 expression inhibited the glucose uptake and lactate production of PANC-1 and Capan-2 cells. Intracellular glucose levels were measured and normalized based on protein concentration. e, f Ectopic VCAM-1 expression facilitated the glycolytic capacity of PANC-1 and Capan-2 cells, as reflected by ECAR analysis. g, h Ectopic VCAM-1 expression promoted the glucose uptake and lactate production of PANC-1 and Capan-2 cells. i PANC-1 and Capan-2 cells expressing si-NC or si-VCAM-1 were cultured under normoxic conditions for 24 h. Acidification of the culture medium was evaluated by visually inspecting the color of the medium. j PANC-1 and Capan-2 cells expressing pcDNA3.1-NC or pcDNA3.1-VCAM-1 were cultured under normoxic conditions for 24 h. Acidification of the culture medium was evaluated by visually inspecting the color of the medium. ** or ***: significantly different from the corresponding control, p < 0.01 or p < 0.001, respectively, by Student’s t-test

Fig. 6. Lactate regulates the functional polarization of macrophages in vitro.

a M0 macrophages incubated with conditioned medium (CM) derived from si-VCAM-1 PDAC cells or treated with quercetin alone were less spindle-shaped and were rounder in appearance compared with M0 macrophages incubated with conditioned medium derived from si-NC PDAC cells. b–e Compared with M0 macrophages incubated with conditioned medium derived from si-NC PDAC cells, M0 macrophages incubated with conditioned medium derived from si-VCAM-1 PDAC cells or treated with quercetin alone had significantly lower mRNA levels of CD206, CD163, fibronectin, CCL18, CCL22, and IL-10 b, c and showed markedly lower protein levels of CCL18, CCL22, and IL-10 d, e, as determined by qRT-PCR and ELISA assay, respectively. f–h VCAM-1-derived lactate facilitates the M2-like polarization of macrophages in a dose-dependent manner. f Lactate stimulation induced the alternative activated M2 phenotype macrophages as characterized by a morphological change from an epithelial-like, cobblestone phenotype to a more spindle-shaped mesenchymal phenotype. g, h Compared with M0 macrophages incubated with 0 mmol/l lactate, M0 macrophages treated with increasing concentrations (5, 15, 25 mmol/l) of lactate had significantly higher mRNA levels of CD206, CD163, fibronectin, CCL18, CCL22, and IL-10 and showed prominently higher protein levels of CCL18, CCL22 and IL-10, as determined by qRT-PCR and ELISA assay, respectively

Fig. 7. Knockdown of VCAM-1 significantly inhibits tumor growth in mouse xenograft models.

a, b A subcutaneous injection of PANC-1 cells with stable knockdown of VCAM-1 or mock cells was administered into the subcutaneous bilateral hind leg of nude mice. At 28 days after subcutaneous injection, PANC-1 cells transfected with sh-VCAM-1 (white arrow) and mock cells (black arrow) produced primary tumors and a representative figure of the tumors formed is shown. c, d The qRT-PCR and western blot analyzed the mRNA and protein levels of VCAM-1 in tumor tissues from sh- VCAM-1 PANC-1 cells compared with sh-NC PANC-1 cells. e Representative images (magnification: × 200) of IHC staining of the tumor. The IHC staining showed that VCAM-1 knockdown decreased the proliferation index Ki-67. f Tumor growth curve. The points indicate mean (n = 5), and the bars indicate the SD. g Tumor weights are shown as the means of tumor weights ± SD when the tumors were harvested. ns.: not significantly different. *, ** or ***: significantly different from the corresponding control, p < 0.05, p < 0.01 or p < 0.001, respectively, by Student’s t-test

Fig. 8. CCL18 and VCAM-1 overexpression is associated with clinicopathological characteristics and poor prognosis of PDAC patients.

The expression levels of VCAM-1 and CCL18 were scored semiquantitatively based on staining intensity and distribution using the immunoreactive score (IRS) as described in the Supplementary Methods. a Representative images of CCL18 staining in PDAC tissues (categorization: low, moderate, and high). b Representative images of VCAM-1 staining in PDAC tissues (categorization: low, moderate, and high). c The Kaplan–Meier analysis of overall survival stratified by the immunoreactive score of CCL18 expression. d The Kaplan–Meier analysis of overall survival stratified by the immunoreactive score of VCAM-1 expression. The log-rank test was used to compare differences between groups. e The survival analysis showed that a higher combined CCL18 and VCAM-1 immunoreactive score correlated with poorer prognosis in PDAC patients (log-rank test, p < 0.001)