WCLC 2025 - Posters & ePosters-P3.03.48 Fluorescence Lifetime Imaging in Fixed NSCLC Samples Demonstrate Differences Between Tissue Compartments

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This study investigates fluorescence lifetime imaging microscopy (FLIM) applied to formalin-fixed paraffin-embedded (FFPE) non-small cell lung cancer (NSCLC) tissue microarrays, aiming to characterize metabolic and structural heterogeneity across tumor, stromal, and immune compartments at per-cell resolution. A cohort of 58 NSCLC patient tissue cores encompassing approximately 650,000 segmented cells was analyzed using a Leica Falcon X FLIM system combined with multiplex immunofluorescence (MIF) staining for markers including PanCK (tumor), CD3/CD20 (immune), αSMA, and FAP (stromal). <br /><br />Results showed significant differences in FLIM lifetimes between histological compartments: tumor regions exhibited shorter lifetimes than stroma, while tertiary lymphoid structures (TLS) demonstrated the longest lifetimes, indicating that FLIM decay properties reflect tissue-specific metabolic and microenvironmental variations. No significant lifetime differences were found between adenocarcinoma and squamous cell carcinoma subtypes, smoking status, or cancer stage, suggesting FLIM signatures are driven by cellular microenvironment more than broad clinical categories. Importantly, lower tumor FLIM lifetimes correlated with poorer recurrence-free survival, highlighting prognostic potential.<br /><br />At a single-cell level, tumor cells marked by PanCK were distinctly separated from non-tumor populations by FLIM lifetime (p < 0.0001). Immune subsets (CD3+ T-cells and CD20+ B-cells) displayed distinct FLIM profiles from other immune cells but not significantly from each other. Stromal cancer-associated fibroblast subsets showed marked heterogeneity: FAP+ fibroblasts had different lifetimes than αSMA+ fibroblasts, dual FAP/αSMA+ cells, and unclassified stroma, consistent with their spatial distribution and functional diversities in the tumor microenvironment.<br /><br />Overall, FLIM provides a powerful, label-free metabolic and structural imaging approach that distinguishes NSCLC cellular lineages and microenvironments, offering potential as a complementary tool for research and prognosis. Future work aims to expand the cohort size, validate clinical correlations, and refine FLIM profiling as a high-content biomarker in lung cancer pathology.

Asset Subtitle

Alexander Hunt

Meta Tag

Speaker Alexander Hunt

Topic Tumor Biology – Translational Biology

Keywords

fluorescence lifetime imaging microscopy

FLIM

non-small cell lung cancer

NSCLC

formalin-fixed paraffin-embedded

FFPE

tumor microenvironment

multiplex immunofluorescence

metabolic heterogeneity

prognostic biomarker