Immunohistochemistry assay

CD molecules and Vimentin were detected using immunohistochemistry in lung cancer tissues obtained from 30 patients with lung cancer who received tumor resection from May 2018 to September 2020 in Harbin Medical University Cancer Hospital. This study was approved by the Ethics Committee of Harbin Medical University and written informed consent was obtained from all the participants. CD68, CD16, CD163 and CD8 were used to mark macrophages, M1 macrophages, M2 macrophages and cytotoxic T lymphocyte (CTL), respectively. Vimentin is the mesenchymal marker.

In brief, the endogenous peroxidase in sections was removed by incubating with H₂O₂ at room temperature (RT) for 10 min after routine dewaxing and hydration. Antigens were retrieved by micro-wave heating. The sections were incubated with primary antibody against CD68 (Abcam, Cambridge, MA, USA; No. ab213363; 1:100 dilutio), CD16 (Abcam; No. ab183354; 1:200 dilution), CD163 (Abcam; No. ab182422; 1:500 dilution), CD8 (Abcam; No. ab101500; 1:800 dilution) and Vimentin (Abcam; No. ab16700; 1:200 dilution) at 4°C overnight, and then incubated with the streptavidin-biotin peroxidase-labeled secondary antibody at 37 °C for 30 min. DAB substrate (ZSGB Bio, Beijing, China) was used to show the positive result.

CD-positive cells were counted in five high power fields (HPFs) through microscopy. The average value was used to reflect the number of macrophages or CTL infiltrated in cancer tissues. The expression of Vimentin was quantified by scoring the area and intensity of staining. Staining intensity was quantified as follows: negative(0), weak,¹ moderate,² or strong.³ Staining area was scored according to the percentage of positive cells: none(0), <25%,¹ 25–50%,² 50–75%,³ or >75%.⁴ The final score was the intensity score × the area score. The results were evaluated by two pathologists in a double-blind manner.