Vectra polaris quantitative pathology imaging system

Optimized fluorescent multiplexed immunohistochemistry was performed using tyramide signal amplification in the Leica Bond Rx Automated Stainer (Leica Biosystems, Newcastle, UK) as previously described^{42 (link)}. Cells were stained with antibodies against CD68 (M0876, DAKO, Glostrup, Denmark), CD206 (NBP1-90020, Novus Biological, Littleton, CO, USA), PD-L1 (13684S, Abcam, Cambridge, UK) and cytokeratin (NBP2-29429, Novus, Littleton, CO, USA), and the fluorescence signals were captured with the following fluorophores: Opal 570, Opal 620, Opal 690, and Opal 780. Multiplex-stained slides were obtained using the Vectra Polaris Quantitative Pathology Imaging System (Akoya Biosciences, Marlborough, MA/Menlo Park, CA, USA). Regions of interest (ROIs) focusing on the invasive tumor margin or the active tumor-stromal interface were carefully chosen by an experienced pathologist (JK) based on the hematoxylin and eosin slides and cytokeratin expression. The images were analyzed using inForm 2.4.11 image analysis software (Akoya Biosciences, Marlborough, MA/Menlo Park, CA, US) and Spotfire software (TIBCO Software Inc., Palo Alto, CA). The data were expressed as the mean number of cells/mm² for each cell population.

Fluorescent and chromogen labeled slides were imaged at 40X using a Vectra Polaris^™ Quantitative Pathology Imaging System (Akoya Biosciences). For fluorescently labeled slides, exposures for all Opal dyes were set based upon regions of interest with strong signal intensities to minimize exposure times and maximize the specificity of signal detected. A brightfield acquisition protocol at 40X was used for chromogenically labeled slides.

IHC analysis was performed to assess the expression level of PF4 (ab303494; Abcam) in clear cell renal cell carcinoma (ccRCC) samples from Fudan University Shanghai Cancer Center (FUSCC) following manufacturers’ protocols as previously described [46 (link)]. The mIHC staining assay was conducted to investigate the abundance and distribution of PF4 (ab303494; Abcam), CD68 (ab125212; Abcam), and CD163 (25121; CST) in ccRCC adjacent normal kidney tissues following manufacturers’ protocols. Tissue slides that were bound with primary and secondary antibodies but not fluorophores were included as negative controls to assess autofluorescence. Multiplex stained slides were scanned using a Vectra Polaris Quantitative Pathology Imaging System (Akoya Biosciences) at 20 nm wavelength intervals from 440 to 780 nm with a fixed exposure time and an absolute magnification of 200×. All scans for each slide were then superimposed to obtain a single image. Multilayer images were imported to inForm v.2.4.8 (Akoya Biosciences) for quantitative image analysis.

Thirty FFPE samples were subjected to assessment of mIHC using the Akoya OPAL Polaris 7-Color Automation IHC kit (NEL871001KT). FFPE tissue samples were deparaffinized in a BOND RX system (Leica Biosystems) and then incubated sequentially with primary antibodies targeting CD68 (Abcam, ab213363, 1:1000), PD-L1 (CST, E1L3N, 13684S, 1:400), PD-1 (CST, D4W2J, 86163S, 1:200), CD163 (Abcam, ab182422, 1:500), CD3 (Dako, A0452), CD8 (Abcam, ab178089, 1:100), CD4 (Abcam, ab133616, 1:100), CD20 (Dako, L26, IR604), CD56 (Abcam, ab75813, 1:100), FOXP3 (Abcam, ab20034, 1:100) and pan-CK (Abcam, ab7753, 1:100) (Akoya Biosciences). Next, secondary antibodies and reactive Opal fluorophores were incubated. DAPI was used to stain the nucleic acids. Multiplex stained slides were scanned at 20 nm wavelength intervals from 440 nm to 780 nm with a fixed exposure time and an absolute magnification of 200 utilizing an Akoya Biosciences Vectra Polaris Quantitative Pathology Imaging System. All scans for each slide were then superimposed to create a single image. Multilayer images were imported into APTIME software (3D Medicines Inc.) for quantitative image analysis. Pan-CK staining was used to distinguish the tumor parenchyma and stroma. The numbers of stained cells per square millimeter in all nucleated cells were used to express the amounts of distinct cell types.