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Figure S4. Multiplex immunofluorescence images showing densities of various tumor-associated immune cell phenotypes as determined by panel 1 and panel 2 markers from representative NSCLCs treated with neoaduvant chemotherapy (NCT) or not treated with NCT (non-NCT). Numbers of T lymphocytes (CD3+), helper T cells (CD3 + CD4+), tumor-associated macrophages (TAM; CD68+), activated natural killer cells (CD57 + granzyme B + CD45RO−), memory antigen experienced cells (CD45RO + PD-1+), and antigen experienced PD-1+ cells as well as PD-L1+ malignant cells were higher in the NCT group than in the non-NCT group. Images ×200. (PPTX 29362 kb)

Table S2. Median densities of tumor-associated immune cells in NSCLCs from patients who received neoadjuvant chemotherapy (NCT) or did not receive NCT (non-NCT), by tumor compartment and histology (N = 112). (DOCX 23 kb)

Figure S5. Multiplex immunofluorescence images showing densities of various tumor-associated immune cell phenotypes (TAICs) as determined by panel 1 and panel 2 markers from the stromal and epithelial compartments of representative NSCLCs treated with neoadjuvant chemotherapy (NCT) or not treated with NCT (non-NCT). In general, densities of TAICs were higher in the stromal compartments than in their respective epithelial compartments in both NCT and non-NCT tumors. Overall, density of tumor-associated macrophages (TAMs; CD68+) was higher in NCT tumors than in non-NCT tumors, and density of memory/regulatory cells (CD45RO + FOXP3+) was lower in the NCT group than in the non-NCT group. Images ×200. (PPTX 15137 kb)

Table S1. Median densities of tumor-associated immune cells in NSCLCs of patients who received neoadjuvant chemotherapy (NCT) or did not receive NCT (non-NCT), by tumor compartment (N = 112) (DOCX 21 kb)

Figure S3. Characterization of immune cell phenotyping using marker panel 2. Cell phenotypes were defined as memory cells (CD45RO, including memory/natural killer cells CD45RO + CD57 + granzyme B−, memory/regulatory cells CD45RO + FOXP3+, memory antigen experienced cells CD45RO + PD-1+, and other CD45RO+ cells), memory/regulatory cells (CD45RO + FOXP3+), memory antigen experienced cells (CD45RO + PD-1+), activated natural killer cells (CD57 + granzyme B + CD45RO−), and antigen experienced cells (PD-1, including PD-1 + CD45RO+ and other PD-1+ cells). Each type of cell is shown as individual images by marker and by a composite image. (PPTX 103717 kb)

Figure S2. Characterization of immune cell phenotyping using marker panel 1. Cell phenotypes were defined as malignant cells expressing PD-L1 (AE1/AE3 + PD-L1+), T lymphocytes (CD3; pan T-cell marker including helper T cells CD3 + CD4+, cytotoxic T cells CD3 + CD8+, and other CD3+ T cells), helper T cells (CD3 + CD4+), cytotoxic T cells (CD3 + CD8+), tumor-associated macrophages (TAMs; CD68+), and TAMs expressing PD-L1 (CD68 + PD-L1+). Each type of cell is shown as individual images by marker and by a composite image. (PPTX 102487 kb)

Figure S1. Representative multiplex immunofluorescence (mIF) workflow, showing different steps in the process of analysis. (A) Vectra 3.0 multispectral microscope used for the analysis. (B) Phenochart 1.0.4 software showing an image scanning at ×10 and selection of five intratumoral areas (669×500 μm each) for further analysis. (C) mIF image scanning at ×20 and viewed in the InForm 2.1.3 image analysis software. (D) Composed image showing the different phenotypes with panel 1. (E) Tumor segmentation (epithelial and stromal compartments) using the InForm software. (E) Cell segmentation to characterize individual cells using DAPI as a counterstaining marker, and cell phenotyping identifying different subgroups of cells supervised by a pathologist. (PPTX 6362 kb)

Methylation frequency of APC and/or RASSF1A in present study and other 5 articles. (DOCX 80 kb)

Clinical characteristics of 316 advanced lung cancer patients. (DOCX 18 kb)

Primers and probes used in duplex qMSP. (DOCX 13.7 kb)

Supplementary Materials. (PDF 1208 kb)

Supplementary Materials. (PDF 1208 kb)