Abstract 5103: Bidirectional EMT-dependent modulation of cancer cell and M1/M2 macrophage differentiation
Authors: Astorgues-Xerri Lucile , Martinet Matthieu , Raymond Eric , Faivre Sandrine , Tijeras-Raballand Annemilaï 
Published: September 2020

Background. Head and neck (H&N) and liver carcinomas are considered as the most deadly cancers in the world. Despite advances in diagnosis and treatment, survival rates remain low mainly due to locoregional relapse, possibly triggered by the activation of epithelial to mesenchymal transition (EMT). EMT is a critical step for tumor progression, increasing motility and invasiveness of tumor cells. M1 and M2 macrophages, major components of the tumor microenvironment, were recently described as involved in EMT. Moreover, proteins expressed by cancer cells, such as CCL2 and CSF1, and promoting M2 differentiation, have been correlated with poor prognosis, and tumor aggressiveness. The aim of this study is to understand the interactions between M1/M2 macrophages and H&N or liver cancer cells considering their EMT status.

Materials and Methods. M1 and M2 macrophages were obtained from THP-1 cell after exposure to PMA followed by LPS/IFNg for M1 or IL4/IL13 for M2. Differentiation was validated by immunofluorescence (IF) using CD14 for monocyte, CD68 for macrophage, CD80 for M1, and CD163 for M2. H&N, cholangiocarcinoma (CK), and hepatocellular carcinoma (HCC) cells were characterized for their EMT status (E-cadherin/vimentin expressions) by western blot. In each tumor type, 1 epithelial and 1 mesenchymal cells were selected to analyze the effects of conditioned medium (CM) from cancer cells on macrophages differentiation, using IF. Furthermore, CCL2 and CSF1 mRNA levels were evaluated in the selected cancer cell lines. In addition, the effect of M1 and M2 CM on cancer cell proliferation, migration, and invasion were also analyzed by MTT, wound-healing, and Boyden chamber assays.

Results. Using our two steps methods, monocytes were first differentiated into macrophages (decrease of CD14 and increase of CD68 expression), and then into M1 and M2 (increase of CD80 or CD163 expression, respectively). Based on cell lines EMT status, SQ20B, SNU1196, and C3A (epithelial) and Hep2, SNU1079, and SKHep1 (mesenchymal) cells were selected. Macrophages exposed to CM from epithelial cells displayed a M1 phenotype, while macrophages exposed to CM from mesenchymal cells exhibited a M2 phenotype. Interestingly, higher mRNA expression of CCL2 and CSF1 mRNA were detected in mesenchymal cells compared to epithelial cells. In H&N and CK cell lines, M1 CM displayed increased antiproliferative effects on epithelial cells, whereas M1 CM displayed antiproliferative effects only on mesenchymal HCC cells. M1 and M2 CM increased migration and invasion in all selected cell lines, except for migration in the epithelial HCC cell line. Further characterization of M1 and M2 macrophages will be displayed at the conference.

Conclusions. This study showed that EMT status of cancer cells modulates macrophages differentiation. In fact, mesenchymal cells showed elevated levels of CCL2 and CSF1 expression, promoting M2 differentiation. Conversely, M1 and M2 displayed differential effects on H&N and liver carcinomas cell lines. These results open up new perspectives on the role of M1/M2 macrophages in EMT-dependent tumors, with the aim of developing new therapeutic approaches for patients with EMT-dependent tumors.

Citation Format: Lucile Astorgues-Xerri, Matthieu Martinet, Eric Raymond, Sandrine Faivre, Annemilaï Tijeras-Raballand. Bidirectional EMT-dependent modulation of cancer cell and M1/M2 macrophage differentiation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5103.