Nrf2 activation drive macrophages polarization and cancer cell epithelial-mesenchymal transition during interaction

Background The M2 phenotype of tumor-associated macrophages (TAM) inhibits the anti-tumor inflammation, increases angiogenesis and promotes tumor progression. The transcription factor Nuclear Factor (erythroid-derived 2)-Like 2 (Nrf2) not only modulates the angiogenesis but also plays the anti-inflammatory role through inhibiting pro-inflammatory cytokines expression; however, the role of Nrf2 in the cancer cell and macrophages interaction is not clear. Methods Hepatocellular carcinoma cells (Hep G2 and Huh 7) and pancreatic cancer cells (SUIT2 and Panc-1) were co-cultured with monocytes cells (THP-1) or peripheral blood monocytes derived macrophages, then the phenotype changes of macrophages and epithelial-mesenchymal transition of cancer cells were detected. Also, the role of Nrf2 in cancer cells and macrophages interaction were investigated. Results In this study, we found that cancer cells could induce an M2-like macrophage characterized by up-regulation of CD163 and Arg1, and down-regulation of IL-1b and IL-6 through Nrf2 activation. Also, Nrf2 activation of macrophages promoted VEGF expression. The Nrf2 activation of macrophages correlated with the reactive oxygen species induced by cancer cells derived lactate. Cancer cells educated macrophages could activate Nrf2 of the cancer cells, in turn, to increase cancer cells epithelial-mesenchymal transition (EMT) through paracrine VEGF. These findings suggested that Nrf2 played the important role in the cancer cells and macrophages interaction. Conclusions Macrophage Nrf2 activation by cancer cell-derived lactate skews macrophages polarization towards an M2-like phenotype and educated macrophages activate Nrf2 of the cancer cells to promote EMT of cancer cells. This study provides a new understanding of the role of Nrf2 in the cancer cell and TAM interaction and suggests a potential therapeutic target. Electronic supplementary material The online version of this article (10.1186/s12964-018-0262-x) contains supplementary material, which is available to authorized users.

0.5% BSA containing HBSS-). Then a 1.068 g/ml Optiperp-HBSS (1 part Optiprep and 4 part 0.5% BSA containing HBSS-) was overlaid on. On the top, there was 1 ml HBSS-. The buffy coat /Optiperp mixture was centrifuged at 600g for 25 min at room temperature. The layer which was between HBSSand 1.068 Optiperp-HBSS was collected as monocytes. The isolated monocytes were cultured in RPMI1640 containing 10%FBS.
For the macrophage differentiation, the isolated PMBC were stimulated with 20nM M-CSF (461-ML, R&D, USA) for six days.

Flow cytometry
The detached cells were blocked the non-specific Fc-mediated by mouse serum with 15 minutes.
Then the cells were incubated with the PE-conjugated anti-CD163 antibody (eBioscience, Thermo Fisher Scientific, USA) 30 minutes on ice. After three times washing, the cells were analyzed by flow cytometry. The data of flow cytometry were analyzed with FlowJo V10 (FlowJo, LLC, USA).

Immunohistochemistry
Briefly, patients' tumor tissue sections were incubated with ice-cold neutral buffer containing 10% formalin and fixed overnight at 4˚C. Fixed sections were paraffin embedded. For immunohistochemistry, 4-μm sections were deparaffinized in xylene and rehydrated sequentially in ethanol. For antigen retrieval, slides were boiled in 0.01 M sodium citrate (pH 6.0) for 30 min. Sections were blocked by incubation with 5% goat serum in PBS for 60 min. After blocking, the sections were firstly incubated with CD163 (1:500) antibody overnight 4 °C. The sections were washed and incubated with the HRP-conjugated second antibody (Dako Japan, Tokyo, Japan) for 60 min. After washing, the sections were incubated with diamino-benzidine. Antigen retrieval and blocking as before was performed again. The sections were then incubated with the anti-Nrf2 antibody (1:250) and visualized using HistoGreen (Linaris, Dossenheim, Germany). Before mounted, nucleuses were stained with nuclear fast red (N3020, Sigma, USA).    HO-1 and Nqo-1 expression of cancer cells were measured by PCR (n=4). Graphs show the data as mean ± SD. *, P < 0.05, compared between two groups. E, cancer cells stimulated with conditioned medium of PBMC derived macrophage (p-M0) or PBMC derived TEM (p-TEM) and Nrf2-knockde down cancer cells were stimulated with conditioned medium of PBMC derived TEM (p-TEM+si-Nrf2), the migrated cancer cell were observed and counted (scale bar 200 µm) (n=3). Graphs show the data as mean ± SD. *, P < 0.05, compared between two groups. F, cancer cells stimulated with conditioned medium of PBMC derived macrophage (p-M0) or PBMC derived TEM (p-TEM) and Nrf2-knocked-down cancer cells were stimulated with conditioned medium of PBMC derived TEM (p-TEM+si-Nrf2), the morphology of cancer cells was observed (scale bar 50 µm) (n=3). G, cancer cells of which Nrf2 were knocked down or not were stimulated with conditioned medium of PBMC derived TEM, E-cadherin and N-cadherin expression of cancer cells were detected by western blot.

Figure S5
A, conditioned medium of PBMC derived TEM in which the VEGF was neutralized (p-TEM-CM+anti-VEGF) or not (p-TEM-CM+IgG) were used to stimulate cancer cells during migration assay, the migrated cancer cells were observed and counted (scale bar 100 µm) (n=3). Graphs show the data as mean ± SD. *, P < 0.05, compared between two groups. B, the morphology of cancer cells which stimulated with VEGF neutralized conditioned medium of PBMC derived TEM (scale bar 50 µm). C, the E-cadherin and N-cadherin expression of cancer cells which stimulated with VEGF neutralized conditioned medium of PBMC derived TEM were detected by western blot. D, the nuclear Nrf2 levels of cancer cells which stimulated with VEGF neutralized conditioned medium of PBMC derived TEM or not were evaluated by western blot. E, HO-1 and Nqo-1 expression of cancer cells which stimulated with VEGF neutralized conditioned medium of PBMC derived TEM were assessed by PCR (n=4).
Graphs show the data as mean ± SD. *, P < 0.05, compared between two groups.