Phenoimager

Eight p16-positive oropharyngeal cancer and eight normal tonsils were used for mIHC. Formalin-fixed, paraffin-embedded block tissue microarrays with 4 μm-thick tissue sections were used. Tissue slides were deparaffinized, subjected to heat-induced epitope retrieval, and stained with the Leica Bond RX (Lecia Biosystems, Singapore) Autostainer. A multiplex IHC panel was performed on the tissue slides using the following antibodies: anti-CD68, anti-CD163, anti-CD16, and anti-cytokeratin (Table 1). Tissue slides were incubated with DAPI as a counterstain and cover-slipped with VectaShield mounting medium (Vector Labs). Control tissue samples were stained separately for each marker. Hematoxylin and eosin staining was performed on each sample and reviewed by a pathologist to ensure the representativity of the tissue sample. mIHC tissue slides were scanned on a PhenoImager (Akoya Biosciences, Marlborough, MA, USA) in qptiff format. Images were captured at a 0.5 µm pixel resolution using the 20× objective with saturation protection as a whole-slide overview.

Multispectral imaging was performed using PhenoImager at 20× magnification (Akoya Biosciences, Marlborough, MA) according to manufacturer instructions. Later, the multispectral acquired images were loaded into InForm software for unmixing and background subtraction (inForm v.3.0; Akoya Biosciences). In the inForm software, an unstained slide (without DAPI and OPAL staining) was loaded and regions with high autofluorescence signals were marked for processing. After processing, image tiles were stitched using QuPath software with the “merge multiple TIFF fields” script.

Placenta tissues (from 3 SPE, 3 control, and 1 SPE with sIUGR donors) were processed with standard paraffin section protocol to 10–30-µm-thick slides. Immunostaining with primary antibodies, including Anti-PEG10 (1:1500, Abcam, ab215035), Anti-CK7 (1:1500, Abcam, ab9021) and Anti-CD31 (1:1000, Abcam, ab9498), and DAPI followed the instruction of Opal 7 color manual Kit (AKOYA Biosciences, NEL811001KT). Fluorescent slides were scanned using the PhenoImager (Akoya Biosciences) using × 20 objective with the following exposure times: DAPI MSI, 0.38 ms; Cy5 MSI (PEG10), 17.33 ms; Cy3 (CD31), 15.76 ms; FITC (CK7), 6.58 ms. Images were generated using inForm software (Akoya Biosciences).