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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.

Rationale Recent studies indicate that tumor-associated macrophages (MΦ) with an M2 phenotype can influence cancer progression and metastasis, but the regulatory pathways remain poorly characterized. Objectives This study investigated the role of tumor-associated MΦ in lung cancer. Methods Coculturing of MΦ with mouse Lewis lung carcinoma (LLC1) and 10 different human lung cancer cell lines (adenocarcinoma, squamous cell carcinoma, and large cell carcinoma) caused up-regulation of CCR2/CCL2 and CX3CR1/CX3CL1 in both the cancer cells and the MΦ. Measurements and main results In the MΦ-tumor cell system, IL-10 drove CCR2 and CX3CR1 up-regulation, whereas CCL1, granulocyte colony-stimulating factor, and MIP1α were required for the up-regulation of CCL2 and CX3CL1. Downstream phenotypic effects included enhanced LLC1 proliferation and migration and MΦ M2 polarization. In vivo, MΦ depletion (clodronate, MΦ Fas-induced apoptosis mice) and genetic ablation of CCR2 and CX3CR1 all inhibited LLC1 tumor growth and metastasis, shifted tumor-associated MΦ toward M1 polarization, suppressed tumor vessel growth, and enhanced survival (metastasis model). Furthermore, mice treated with CCR2 antagonist mimicked genetic ablation of CCR2, showing reduced tumor growth and metastasis. In human lung cancer samples, tumor MΦ infiltration and CCR2 expression correlated with tumor stage and metastasis. Conclusions Tumor-associated MΦ play a central role in lung cancer growth and metastasis, with bidirectional cross-talk between MΦ and cancer cells via CCR2 and CX3CR1 signaling as a central underlying mechanism. These findings suggest that the therapeutic strategy of blocking CCR2 and CX3CR1 may prove beneficial for halting lung cancer progression.

Background: Circulating fibrocytes (CFs) are bone marrow derived, mesenchymal progenitor cells that have emerging role in many diseases. CFs was shown to participate in tissue remodeling in pulmonary hypertension and fibrosis, via secretion of different cytokines and growth factors. Nevertheless, their role in the lung cancer still has to be delineated. Thus, our aim is to identify the role of CFs in lung cancer progression and metastatic potential. Results/methods: We generated CFs by isolating human peripheral blood mononuclear cells (PBMCs) and culturing them for 10 days until they differentiated into CFs. Purity (≥95%) of the CFs population was analyzed by flow cytometry and immunofluorescence. Co-culturing of A549 (human alveolar adenocarcinoma cells) and CFs for 12 and 24 hrs, followed by collection of conditioned medium (CM) and RNA from both cells yielded interesting results. Co-cultured CM promoted proliferation (1.31±0.07 versus 1.5±0.13) and migrating capacity (64.6±26.5 versus 472.8±103.4) of A549 tumor cells. In addition, co-cultured CM caused epithelial-to-mesenchymal transition (EMT) of A549 cells; epithelial markers (E-cadherin, cytokeratin) were downregulated, and mesenchymal markers (α-smooth muscle actin, fibronectin) were upregulated. In vivo to study the CFs involvement in lung cancer, we co-injected CFs with A549 cells or A549 cells alone and measured the tumor growth after 28 days. The tumor size was significantly increased (1354.8±333.6 versus 3042.4±373.4) in co-injected group (CF+A549) compared to A549 alone. Screening for genes regulated in co-injected group tumors showed regulation of tumor microenvironment, an increase in macrophage markers (CSF1R, CD68, CD200, MMD), angiogenesis markers (EDNRB, THBS1) and ECM remodeling markers (MMP14, CTSB, CTSH) compared to A549 tumor alone. Conclusions: We believe that circulating fibrocytes may play positive role in the tumor growth and progression. The increase in EMT and migration, may suggest their involvement in invasion and metastasis. Targeting CFs and their secretory molecules can be of therapeutic importance in lung cancer. Citation Format: Alina Asafova, Vandana Nikam, Anja Schmall, Werner Seeger, Robert Voswinckel, Rajkumar Savai. Involvement of circulating fibrocytes in the progression of adenocarcinomas by modulating EMT and tumor microenvironment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2604. doi:10.1158/1538-7445.AM2013-2604