Samenvatting

INTRODUCTION
Extensive studies of the immunological and stromal lung tumor microenvironment (TME) have improved understanding of tumor progression and therapy resistance. A recent study on human lung adenocarcinoma specimens revealed that higher intra-tumoral nerve fiber density is linked to poor prognosis. Previously, we demonstrated enhanced CD8+ T cells activation in the lung TME of mice upon vagal nerve stimulation, emphasizing neuronal influence on tumor progression. However, the mechanisms underlying lung cancer-specific innervation via neurogenesis or axonogenesis remain largely unexplored.
METHODS
Orthotopic models are underrepresented in TME innervation research. To address this, we optimized a single-nodule-based orthotopic lung cancer model via intrathoracic injection of Lewis lung carcinoma (LLC) cells in C57BL/6 mice (n=12, 2E5 LLC cells, n=12, 1E6 LLC cells). Tumor growth was monitored via bioluminescence imaging (BLI) and microCT (5 min @113 mGy). As such collected lungs, engrafted with tumor nodules of 1-5mm, are a proxy for early and progressive stages of disease. Using in toto lung light sheet microscopy, we analyzed the innervation profile, staining for GAP43 (axonogenesis), VAChT (parasympathetic) and TH (sympathetic). The spatial organization will be visualized by staining blood (PECAM-1) and lymphatic (LYVE-1) vessels next to immune cells (CD68, CD56, CD3).
RESULTS/DISCUSSION
One week after intrathoracic tumor inoculation, localized tumor growth was observed via BLI and microCT imaging in the lungs of 5/24 mice. Of these, 3 mice were sacrificed to represent the early stage of disease, while all remaining mice were monitored for progressive disease. In 6/25 mice, a low signal in BLI could not be confirmed in microCT, highlighting the difference in sensitivity and specificity between both. Figure 1 shows a small, localized tumor in the left lung lobe one week post inoculation (p.i.). For subsequent imaging, tumor-bearing lungs will be isolated, perfused, fixed, and prepared for in toto imaging using an optimized label-free tissue clearing protocol. Figure 2 represents a whole lung, three weeks p.i. of intravenously challenged LLC, visualized using the LaVision light sheet microscope (488 nm excitation) at ACAM, University of Antwerp, to map autofluorescence. Three-dimensional images were attained with Imaris software, marking the tumor volumes in green.
CONCLUSIONS
We successfully engrafted single tumor nodules in the left lung lobe, confirmed by BLI and microCT imaging. In early-stage disease, BLI outcompetes microCT for its sensitivity, while microCT enables precise volumetric measurements of tumors at all disease stages. With a successful orthotopic lung cancer model and an optimized tissue clearing protocol we are ready to visualize the onset and evolution of lung TME innervation via in toto imaging.
NOVELTY (149/150)
Optimizing an intrathoracic syngeneic lung cancer model, this work enhances in-depth analysis of TME innervation via cutting-edge imaging techniques.
IMPACT(138/150)
Findings will support neuroscience and neuro-engineering research fields towards deeper understanding in neuro-immune-cancer interactions.

Conference

ConferenceEuropean Molecular Imaging Meeting
Verkorte titelEMIM 2025
Land/RegioSpain
StadBilbao
Periode11/03/2514/03/25
Internet adres

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