Different In Situ Immune Patterns between Primary Tumor and Lymph Node in Non-Small-Cell Lung Cancer: Potential Impact on Neoadjuvant Immunotherapy

Figure 1

Multiplex immunofluorescence staining and multispectral imaging to visualize the expression of CD3, CD8, PD-L1, and cytokeratin (CK) in primary tumors (PTs) and metastatic lymph nodes (mLNs). (a) Fields of view (FOVs) of the center of tumor (CT) and invasive margin (IM) regions were identified at low magnification (×10). (b) Images of representative FOVs that are categorized as CT in sections with tumor tissue being too small. (c, d) FOVs of CT region in PT (c) and mLN (d). Staining was performed for CD3 for T lymphocytes (yellow), CD8 for cytotoxic T cells (green), type II CK for epithelial cells (purple), 4-6-diamidino-2-phenylindole (DPAI) for nuclei (blue), and PD-L1 (red). (e, f) FOVs of IM region in PT (e) and mLN (f). Hand-drawn training regions allowed images to be segmented into tumor compartment (presence of cytokeratin positivity (red)) and stroma compartment (absence of cytokeratin positivity (green)). (g, h) Densities of CD3+ and CD8+ tumor-infiltrating lymphocytes (TILs) in tumor and stroma compartments of PT (g) and mLN (h) were assessed. Higher densities of CD3+ and CD8+ were observed in stroma compartment compared to tumor compartment in both PT and mLN. Significance was evaluated using a Wilcoxon signed-rank test. .

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