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Background: Non-Small Cell Lung Cancer (NSCLC) is the leading cause of cancer-related death worldwide and is usually diagnosed in an already locally or systemically advanced state. Depending on the stage of the tumor, surgical therapy is limited and systemic therapy required. Recent developments with targeted therapies and immune checkpoint blockade resulted in improved survival for a limited number of patients but the magnitude of patients will still progress. The transforming growth factor beta signaling pathway (TGFβ) is frequently activated in lung cancer, involved in malignant progression and a possible target for therapy [1]. TGFβ signaling is known to inhibit immune responses, immune cell proliferation and to dampen effector functions in various immune cells [2]. Therefore we set out to investigate the activation of the TGFβ signaling pathway in different immune cell populations in human lung cancer to better define the cells that are affected by TGFβ.

Methods: Multiplexed immuno-fluorescence staining and multi-spectral imaging was used to investigate a cohort of > 200 early stage NSCLC specimens assembled on TMAs for activation of the TGFB signaling pathway by targeting phosphorylated SMAD3 and different immune cell markers. Image analyses were conducted to analyze spatial relationships and local abundances in the tumor as well as stroma in tissue samples from the tumor center or margin

Results: Overall, a significantly increased number of CD3, proliferating CD3 (p <0.001) and CD3-phospho SMAD3 (p <0.05) cells were observed in the stroma compared to tumor tissues, however the overall percentage of CD3pS3 remained unaltered between tumor and stroma, suggesting an equal impact on both compartments. In addition, adenocarcinomas exhibited a significantly increased abundance of CD3 in the tumor and stroma in both, the invasive margin or the central region of the tumor compared to squamous cell carcinomas.

Conclusions: Using multi-parameter tissue analyses to investigate the abundance of specific immune cell populations and pathway activation in the tumor-microenvironment of lung cancer tissues enables detailed analysis of immune signaling phenotypes. Somewhat surprisingly, these studies suggest that TGFβ impacts an equal percentage of CD3 T cells in both the stroma and tumor center.

Acknowledgements: Sebastian Marwitz is funded by the Deutsche Forschungsgemeinschaft (DFG) via MA 7800/1-1.


  1. Marwitz S, Depner S, Dvornikov D, Merkle R, Szczygiel M, Müller-Decker K, et al. Downregulation of the TGF-β pseudoreceptor BAMBI in non-small cell lung cancer enhances TGF-β signaling and invasion. Cancer Research.
  2. Li MO, Wan YY, Sanjabi S, Robertson A-KL, Flavell RA. TRANSFORMING GROWTH FACTOR-β REGULATION OF IMMUNE RESPONSES. Annual Review of Immunology. 2006; 24(1): 99–146.

Ethics Approval: This study was approved by University of Lübeck´s institutional review board number (Az. 18-026).

Publication Date


Clinical Institute





Cancer, immunotherapy, multispectral imaging, transforming growth factor beta, TGFB, non-small cell lung cancer, NSCLC, histology, T cells




Poster presented at Society for Immunotherapy of Cancer Annual Meeting, Washington, D.C., November 7 – 11, 2018.

Application of multiplexed immunofluorescence and multispectral imaging to investigate TGFβ pathway activation of immune cell populations in human lung cancer

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