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. 2016 Jan 1;7(1):42-9.
doi: 10.7150/jca.12923. eCollection 2016.

Increasing the Inflammatory Competence of Macrophages with IL-6 or with Combination of IL-4 and LPS Restrains the Invasiveness of Pancreatic Cancer Cells

Aino N E Salmiheimo  1 , Harri K Mustonen  1 , Sanna A A Vainionpää  1 , Zhanlong Shen  2 , Esko A J Kemppainen  1 , Hanna E Seppänen  1 , Pauli A Puolakkainen  1
Affiliations

Increasing the Inflammatory Competence of Macrophages with IL-6 or with Combination of IL-4 and LPS Restrains the Invasiveness of Pancreatic Cancer Cells

Aino N E Salmiheimo et al. J Cancer. .

Abstract

Recent studies suggest that pro-inflammatory type M1 macrophages inhibit tumor progression and that anti-inflammatory M2 macrophages enhance it. The aim of this study was to examine the interaction of type M1 and M2 macrophages with pancreatic cancer cells. We studied the migration rate of fluorescein stained pancreatic cancer cells on Matrigel cultured alone or with Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) differentiated macrophages or with Macrophage Colony Stimulating Factor (M-CSF) differentiated macrophages, skewing the phenotype towards pro- and anti-inflammatory direction, respectively. Macrophage differentiation was assessed with flow cytometry and the cytokine secretion in cell cultures with cytokine array. Both GM-CSF and M-CSF differentiated macrophages increased the migration rate of primary pancreatic adenocarcinoma cell line (MiaPaCa-2) and metastatic cell line (HPAF-II). Stimulation with IL6 or IL4+LPS reversed the macrophages' increasing effect on the migration rate of MiaPaCa-2 completely and partly of HPAF-II. Co-culture with MiaPaCa-2 reduced the inflammatory cytokine secretion of GM-CSF differentiated macrophages. Co-culture of macrophages with pancreatic cancer cells seem to change the inflammatory cytokine profile of GM-CSF differentiated macrophages and this might explain why also GM-CSF differentiated macrophages promoted the invasion. Adding IL6 or IL4+LPS to the cell culture with MiaPaCa-2 and GM-CSF or M-CSF differentiated macrophages increased the secretion of inflammatory cytokines and this could contribute to the reversion of the macrophage induced increase of cancer cell migration rate.

Keywords: M1; M2; invasion; macrophages; pancreatic adenocarcinoma.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
GM-CSF (skewed towards M1) and M-CSF (skewed towards M2) differentiated macrophages expressed different surface protein distribution in flow cytometry. Comparing GM-CSF and M-CSF macrophages proportion of cells positive to M2 markers CD209, CD163 and CD14 was significantly higher in M-CSF differentiated macrophages but M2 marker CD206 was less positive and also M1 marker CD80 was higher (#p<0.05). This shows a partly incomplete polarization with these commonly used stimulants. MiaPaCa-2 cells increased only the CD16 positivity of GM-CSF macrophages but had no significant effect on M-CSF differentiated macrophages. IL6 induced no significant changes to the surface expression of GM-CSF differentiated macrophages. Adding IL4 and LPS to M-CSF differentiated macrophages increased the proportion of cells positive to M2 markers CD209 and CD206 and to M1 marker CD16 but M2 markers CD163 and CD14 decreased (*p<0.05). Error bars show the standard error of mean.
Figure 2
Figure 2
Both GM-CSF and M-CSF differentiated macrophages increased the migration rate of pancreatic cancer cells. This could be reversed by adding IL6 to GM-CSF or IL4 and LPS to M-CSF differentiated cell culture. MiaPaCa-2 and HPAF-II cell invasion rate was measured in Matrigel cultured alone and with macrophages when they were cultured in medium containing (A) GM-CSF with and without IL6 and (B) M-CSF with and without IL4 and LPS. Error bars show the standard error of mean. *p<0.05.
Figure 3
Figure 3
Cytokine secretion of macrophages and pancreatic cancer cells was studied with cytokine array. These figures show only the cytokines where significant changes observed on the array panel. (A) When GM-CSF differentiated macrophages were co-cultured with pancreatic cancer cells their inflammatory cytokine secretion decreased significantly. When IL6 was added to the cell culture the inflammatory cytokine secretion was restored. (B) In M-CSF differentiated macrophages co-cultured with pancreatic cancer cells stimulation with IL4 and LPS activated the secretion of inflammatory cytokines. Error bars show the standard error of mean. *p<0.05
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