Ds 0001

The main clinical and pathological characteristics of LSCC patients are presented in Supplementary Table 4. Clinical staging and the anatomical site of the tumors were assessed according to the 6^th edition of the Union for International Cancer Control (UICC 2008) tumor-node-metastasis classification of malignant tumors. Detection of FoxP3 (Ab22510, IgG₁, 1:50 dilution, Abcam, Cambridge, MA, USA) and CD25 (Ab61777, IgG, 1:100 dilution, Abcam) was performed on 4 μm-thick, paraffin-embedded sections of tissue samples with the indicated antibodies using a double staining kit (DS-0001, ZSGB-BIO, Beijing, China). For chemokine receptor ligand staining, sections of tissue samples were stained with anti-MCP-1, -MDC, or -TARC antibodies using an ABC kit (PK-4001, ZSGB-BIO).

For evaluation of the percent of CD34⁺ PD-L1⁺ vessels in tumor tissues. All patient sections were double-stained with anti-CD34 antibody (ZA-0550, ZSGB-BIO) and anti-PD-L1 antibody (66248-1-lg, Proteintech) using a double-staining kit (DS-0001, ZSGB-BIO). First, the most-abundant sites of blood vessels were selected under fourfold magnification of microscope, and four sites were randomly selected under a 40-fold objective lens, and the proportion of PD-L1⁺ CD34⁺ cells in all CD34⁺ vessels was calculated and averaged. The average percentage was used as the final result for each section. To evaluate the positive expression of VEGFA (ab1316, Abcam) and hypoxia-inducible factor α (HIF-1α) (ab113642, Abcam), stained sections were digitally analyzed at ×400 resolution using an Olympus BX-UCB. HIF-1α and VEGFA expression were scored by the H-score system, respectively. H score calculation method is based on previous literature^{15 (link)}.