Vectra polaris quantitative slide scanner

Multiplexed IF staining was performed using the Opal 7 Solid Tumor Immunology Kit (PerkinElmer). According to the manufacturer’s protocol, formalin-fixed paraffin-embedded tissue slides were deparaffinized with xylene and rehydrated through an ethanol gradient ending with a distilled water wash. Antigen retrieval was performed using AR9 (for PD-L1, CD4, and CD8) or AR6 (for CD45RO, CD3, and pan-cytokeratin) buffer and microwave treatment. The first antibody PD-L1 (clone E1L3N) was incubated, followed by secondary antibody incubation using Opal Polymer HRP. Opal-620 dye was then applied for visualization of PD-L1, and microwave treatment was performed to remove primary and secondary antibodies. The process was repeated with antibodies/fluorescent dye in the following order: CD4 (clone EP204)/Opal-520, CD8 (clone 4B11)/Opal-570, CD45RO (clone UCHL1)/Opal-650, CD3/Opal-540, pan-cytokeratin (clone AE1/AE3)/Opal-690. After applying DAPI for visualization of nuclei, slides were mounted, and cover slipped. Multiplexed slides were imaged using PerkinElmer Vectra Polaris quantitative slide scanner, and images were analyzed using Inform software (PerkinElmer, Ver. 2.4.1). Immunohistochemistry for CD8 was conducted in FFPE tissues using a CONFIRM-anti-CD8 (SP57) rabbit monoclonal primary antibody without dilution with Ventana BenchMark XT via the OptiView DAB IHC Detection Kit.

Primary human prostate specimens were collected from 118 PCa patients from Renji Hospital. Tumor differentiation was defined according to the Gleason grading system. Tumor staging was defined according to the 7th edition of the American Joint Committee on Cancer TNM staging manual [20 ].
Tissue slides were stained with primary antibody against AMACR (ab219309), CXCL1 (ab86436), LCN2 (ab125075) from Abcam (Cambridge, UK), Keratin18 (#4548, Cell Signaling Technology, Massachusetts, USA) and CD177 (LS-B1953, LifeSpan BioSciences, Washington, USA). IHC results were evaluated independently by two pathologists who were blinded to the patients’ details. The staining intensity was scored on the following scale: negative (0), weak (1), moderate (2), or strong (3). The staining extent score was on a scale of 0–3, corresponding to the percentage of immunoreactive cells (< 5, 5–25%, 26–50%, 50–100%, respectively). An aggregate score ranging from 0 to 6 was calculated by adding the intensity score and staining extent score together, resulting in a negative (0–1), low (2), moderate (3–4), and high (5–6) expression for each specimen. PerkinElmer Opal™ Tyramide Signal Amplification system was employed for multiplex protein co-staining and images were captured by VectraPolaris™ Quantitative Slide Scanner (PerkinElmer, Massachusetts, USA).