Immune cells in the tumor microenvironment have a critical role in lung cancer control versus progression and metastasis. In this study, we explored the role of tumor-infiltrating-lymphocyte subpopulations in lung cancer progression by performing in vitro co-culturing, in vivo mouse tumor models, and human lung cancer tissue staining. CM from the Co-culture of activated lymphocytes and lung cancer cells was able to induce an EMT transition in both human and mouse tumor cells, characterized by deregulation of epithelial and mesenchymal markers and increased migration. Co-Culture CM displayed a distinct secretome profile when compared to the supernatant of either lymphocytes or cancer cells alone. Specifically, cytokines IL-9, IL-17 A, and F were increased in Co-Culture CM.Detection of Th9 and Th17 cells in tissue microarrays from human lung cancer tissue correlated with a decrease in patient survival and these cells were frequently identified in the stromal area of tumors. Co-Culture of lung cancer cells with the specific Th9 and Th17 subtypes replicated the deregulation of EMT markers and migration induction observed with Lymphocyte CM, thereby pointing toward these cells as responsible for the observed effects on cancer cell behavior. Additionally, Co-culturing of lung cancer cells with Th9 or Th17 cells modulated the expression profile of genes implicated in EMT, metastasis, and angiogenesis, such as MMPs and the Klf family of transcription factors. Stimulation with the individual effector cytokines, IL-9 and IL-17, reproduced the results observed by exposure to CM, further supporting the involvement of Th9 and Th17 cells, and their effector cytokines, in tumor development and progression.Co-injection of Th9 and/or Th17 cells with tumor cells in mouse lung cancer models promoted primary tumor growth and metastasis. Th9 especially affected tumor size and weight after subcutaneous injection, whereas Th17 cells had a specific effect on metastasis in models of primary tumor and metastasis. Consequently, inhibition of IL-9 or IL-17 cytokines by neutralizing antibodies decreased protein expression of EMT markers and slowed lung cancer progression and metastasis. In conclusion, our results assign Th9 and Th17 lymphocytes a prominent role in the induction of lung cancer cell EMT, thereby promoting migration and metastatic spreading and could provide novel therapeutic strategies targeting some of the targets described in our work.
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