Microenvironmental Th9 and Th17 lymphocytes induce metastatic spreading in lung cancer
Ylia Salazar, … , Magdalena Huber, Rajkumar Savai
Ylia Salazar, … , Magdalena Huber, Rajkumar Savai
Published March 31, 2020
Citation Information: J Clin Invest. 2020;130(7):3560-3575. https://doi.org/10.1172/JCI124037.
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Research Article Inflammation Oncology

Microenvironmental Th9 and Th17 lymphocytes induce metastatic spreading in lung cancer

Abstract

Immune microenvironment plays a critical role in lung cancer control versus progression and metastasis. In this investigation, we explored the effect of tumor-infiltrating lymphocyte subpopulations on lung cancer biology by studying in vitro cocultures, in vivo mouse models, and human lung cancer tissue. Lymphocyte conditioned media (CM) induced epithelial-mesenchymal transition (EMT) and migration in both primary human lung cancer cells and cell lines. Correspondingly, major accumulation of Th9 and Th17 cells was detected in human lung cancer tissue and correlated with poor survival. Coculturing lung cancer cells with Th9/Th17 cells or exposing them to the respective CM induced EMT in cancer cells and modulated the expression profile of genes implicated in EMT and metastasis. These features were reproduced by the signatory cytokines IL-9 and IL-17, with gene regulatory profiles evoked by these cytokines partly overlapping and partly complementary. Coinjection of Th9/Th17 cells with tumor cells in WT, Rag1–/–, Il9r–/–, and Il17ra–/– mice altered tumor growth and metastasis. Accordingly, inhibition of IL-9 or IL-17 cytokines by neutralizing antibodies decreased EMT and slowed lung cancer progression and metastasis. In conclusion, Th9 and Th17 lymphocytes induce lung cancer cell EMT, thereby promoting migration and metastatic spreading and offering potentially novel therapeutic strategies.

Authors

Ylia Salazar, Xiang Zheng, David Brunn, Hartmann Raifer, Felix Picard, Yajuan Zhang, Hauke Winter, Stefan Guenther, Andreas Weigert, Benno Weigmann, Laure Dumoutier, Jean-Christophe Renauld, Ari Waisman, Anja Schmall, Amanda Tufman, Ludger Fink, Bernhard Brüne, Tobias Bopp, Friedrich Grimminger, Werner Seeger, Soni Savai Pullamsetti, Magdalena Huber, Rajkumar Savai

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Figure 3

CD4+ T cell subtype–specific CM induces EMT and migration in mouse cancer cells.

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CD4+ T cell subtype–specific CM induces EMT and migration in mouse cance...
(A) Naive CD44−CD62L+CD4+ T cells were isolated from C57BL/6 mice and treated with specific cytokines for 2–days to differentiate into Th9 (TGF-β and IL-4), Th17 (TGF-β and IL-6), or Th0 (without skewing cytokines) cells. Differentiation was confirmed by performing analysis for production of cell-specific cytokines, such as IL-2, IL-9, IL-17, and IFN-γ by flow cytometry. (B) Schematic experimental design showing CM from Th0, Th9, or Th17 cells or from their coculture with cancer cells used to stimulate LLC1 cells and evaluate EMT, proliferation, and migration. (C) Western blot analysis of EMT markers (N-cadherin, fibronectin, vimentin, ZO2, and cytokeratin 18) from LLC1 cell lysate after 48 hours of stimulation with Th0, Th9, or Th17 CM. (D and E) Proliferation and migration of LLC1 cells after 12-hour and 24-hour stimulation, respectively, with CM. (F) Western blot analysis of EMT markers (N-cadherin, fibronectin, vimentin, ZO2, and cytokeratin 18) from LLC1 cell lysate after 48-hour stimulation with respective coculture CMs. (G and H) Migration and proliferation of LLC1 cells after 12-hour and 24-hour stimulation, respectively, with coculture CMs. (n = 3 donors) *P < 0.05; ***P < 0.001; ****P < 0.0001 compared with LLC1 using 1-way ANOVA Dunnett’s test. Scale bars: 500 μm.