Qian Yao

Supplementary Figure S7. (A-D) Density of CD3+CD4+ and CD3+CD8+ T cells in intraepithelial and lamina propria propria/stromal regions across normal mucosa, non-serrated and serrated precursors, and carcinoma stratified by American Joint Committee on Cancer (AJCC) stage and mismatch repair (MMR) status. P values were calculated with Mann-Whitney U test (Wilcoxon rank-sum test) as compared to colorectal normal mucosa near precursor lesions (normal), carcinoma with AJCC stage II-IV (CA stage II-IV), and carcinoma with proficient mismatch repair (CA pMMR). ***: P <0.0005, **: P <0.005, *: P <0.05. Abbreviations: AJCC, American Joint Committee on Cancer; CA, colorectal invasive carcinoma; Carcinoma stage I, colorectal carcinoma with AJCC stage I; Carcinoma stage II-IV, colorectal carcinoma with AJCC stage II-IV; dMMR, deficient mismatch repair; Non-serrated, non-serrated adenomas, Normal, colorectal normal mucosa near precursor lesions; pMMR, proficient mismatch repair; Serrated, serrated lesions.

Antibodies and Staining Conditions for Multiplex Identification of T-cell Subsets and Epithelial Cells

Supplementary Figure S4. T-cell densities (/mm2) in the overall region (intraepithelial + lamina propria/stromal regions) based on the subcellular-localized CD3+ (total T-cell marker) fluorescence signals across 15 precursor and 10 carcinoma tissue microarray slides from the Health Professionals Follow-up Study, Nurses’ Health Study, and Nurses’ Health Study II.

Supplementary Figure S9. The combined T-cell status of precursor and carcinoma tissue categories in relation to anatomic location. (A-B) CD3+CD4+ and CD3+CD8+ T-cell densities in overall tissue regions stratified by anatomic location. (C-D) CD3+CD4+FOXP3+ and CD3+CD8+FOXP3+ T-cell densities in overall tissue regions stratified by anatomic location. P values were calculated with the Mann-Whitney U test (Wilcoxon rank-sum test) as compared to colorectal normal mucosa near precursor lesions (Normal mucosa) and carcinoma with proficient mismatch repair (pMMR). ***: P <0.0005, **: P <0.005, *: P <0.05. Abbreviations: CA, colorectal invasive carcinoma; dMMR, deficient mismatch repair; Non-serrated, non-serrated adenomas, Normal, colorectal normal mucosa near the precursor lesions; pMMR, proficient mismatch repair; Serrated without SSL, serrated lesions including hyperplastic polyp and traditional serrated adenoma; SSL, sessile serrated lesions.

Supplementary Figure S6. Intraepithelial and lamina propria/stromal CD3+CD4+ and CD3+CD8+ T-cell density (/mm2) stratified by tissue dysplasia grade [low-grade dysplasia (LGD) and high-grade dysplasia (HGD)] and tissue categories, which include colorectal normal mucosa, non-serrated lesions with LGD or HGD, and colorectal carcinoma. (A-F) Intraepithelial CD3+CD4+ and CD3+CD8+ T-cell densities were classified by T-cell differentiation status [CD45RA+CD45RO- (naive) and CD45RA-CD45RO+ (memory)] and functional status [FOXP3+ (regulatory)]. (G-L) Lamina propria/stromal CD3+CD4+ and CD3+CD8+ T-cell densities were classified by T-cell differentiation status (naive and memory) and functional status (regulatory). P values were calculated with the Mann-Whitney U test (Wilcoxon rank-sum test) as compared to colorectal normal mucosa near precursor lesions. ***: P <0.0005, **: P <0.005, *: P <0.05. Abbreviations: Carcinoma, colorectal invasive carcinoma; HGD, high-grade dysplasia; LGD, low-grade dysplasia; Normal, colorectal normal mucosa near the precursor lesions.

Supplementary Figure S2. Protocol for multiplex immunofluorescence staining for T cells and epithelial cells. Steps 1-4 were manually conducted. Steps 5-9 were performed with a Leica Bond RX Research Stainer (Leica Biosystems, Buffalo, Illinois, U.S.), regarding the condition of primary antibodies and fluorescence dyes (see the Supplementary Table S2). A. Deparaffinization in xylene (Fisher Chemical, Hampton, New Hampshire, U.S.) and rehydration through a graded dehydrated ethyl alcohol series (100% x3 + 95% x1 + 80% x1 + diluted water x5). B. Antigen retrieval with 2100-Retriever (62700-10, Electron Microscopy Science, Hatfield, Pennsylvania, U.S.) in ethylenediaminetetraacetic acid (EDTA) buffer pH 9.0 (S2367, Dako, Copenhagen, Denmark). C. Opal Antibody Diluent/Block (ARD1001EA, Akoya Biosciences, Marlborough, Massachusetts, U.S.). D. Opal polymer HRP Mouse + Rabbit (ARH1001EA, Akoya Biosciences). E. BOND epitope retrieval solution 1&2 (Leica Biosystems). F. Spectral DAPI (FP1490, Akoya Biosciences, Marlborough, Massachusetts, U.S.). G. Cover slips 24x50 mm No.1 thickness (12450S, Epredia, Portsmouth, New Hampshire, U.S.), prolong gold antifade reagent (P36930, Life Technologies Corporation, Eugene, Oregon, U.S.). Research Resource Identifiers (RRIDs) for primary antibodies were listed at Supplementary Table S2, but RRIDs for Opal dyes, staining solutions, and Akoya Biosciences instrumentation had not been assigned.

Supplementary Figure S1. Flow diagram for T-cell population and distribution analyses. Abbreviations: HPFS, Health Professionals Follow-up Study; NHS, Nurses’ Health Study; NHS II, Nurses’ Health Study II.

Supplementary Figure S3. Identification of distinct T-cell subsets via the combinational expression of T-cell markers (membrane CD3, membrane CD4, membrane CD8, membrane CD45RA, membrane CD45RO, nucleus FOXP3), cell cycle marker (nucleus MKI67), epithelial marker (cytoplasm KRT), and DNA marker (nucleus DAPI) at single-cell resolution. Scale bar: 2.5 (μm).

Tissue Categories in Relation to Characteristics of Colorectal Normal and Neoplasm Cases

Supplementary Figure S8. The combined T-cell status of precursor tissue categories in relation to lesion size, age of resection, and anatomic location. (A-D) CD3+CD4+ and CD3+CD8+ T-cells’ densities stratified by precursor lesions’ size, which includes small (lesion size: <5 mm), medium (5-9 mm), and large (≥10 mm) in non-serrated and serrated lesions. (E-H) CD3+CD4+ and CD3+CD8+ T-cells’ densities stratified by age of resection, which includes early-onset (age <50 years), intermediate-onset (age 50-54 years), and later-onset (age 55-69 and ≥70 years). P values were calculated with the Mann-Whitney U test (Wilcoxon rank-sum test) as compared to each adenoma category in (A-D) and colorectal normal mucosa near precursor lesions (Normal mucosa) in (E-H). ***: P <0.0005, **: P <0.005, *: P <0.05. Abbreviations: Non-serrated, non-serrated adenomas, Normal, colorectal normal mucosa near precursor lesions; Serrated, serrated lesions.