Multispectral Imaging of Myeloid Cell Populations in Immunosuppressed and Immunocompetent Cutaneous Squamous Cell Carcinoma

File Descriptions: cSCC_ImgFiles.zip: Zipped file with all unmixed images cSCC_ClinicalInfo.xlsx: Excel file with clinical meta data cSCC_Img_Channel_Info.xlsx: Excel file indicating which marker is in each image channel Methods: Cutaneous squamous cell carcinoma tissues were obtained from immunocompetent and immunosuppressed patients and reviewed by a trained pathologist. Multiplexed immunofluorescence staining to identify immune cell populations in the TME was performed using tyramide signal amplification. All samples were stained together. Sections of tissue were deparaffinized in xylene and hydrated in ethanol prior to antigen retrieval with Citrate buffer (BioSB, BSB-0021) at 115°C for 15 minutes. The myeloid staining panel used included primary antibodies against CD40 (Bethyl, A700-056), CD11c (Abcam, ab52632), HLA-DR (Bethyl, A500-022A), CD68 (Abcam, ab213363), CD11b (Abcam, ab52478), and pan-cytokeratin (Cell Signaling Technology, 67306S). After each cycle of antigen retrieval, the slides were incubated with 5% BSA for protein blocking. Secondary antibodies conjugated with poly-horseradish peroxidase targeting rabbit (ThermoFisher, 32260) and mouse (ThermoFisher PI32230) were used for signal detection and amplification, along with tyramide-conjugated fluorescent dyes (Biotium, Fremont, CA) CF430 (96053), CF488A (92171), CF550R (96077), CF583R (96085), CF680R (92196), and CF754 (96090) in the presence of manual amplification buffer (Akoya Biosciences, Marlborough, MA). After each staining cycle, the sections were heat-treated in antigen retrieval buffer, either pH 6.0 Citrate or pH 9.0 EDTA (BioSB, BSB-0031), to disengage the antigen-antibody complex while the covalently bound, fluorescence-labeled tyramide remained on the tissue. After all rounds of staining were completed, tissues were treated with TrueBlack lipofuscin autofluorescence quencher (Cell Signaling Technology). Counterstaining of the cell nuclei and coverslipping was performed using VectaShield Antifade Mounting Media with DAPI (Vector Laboratories, Newark, CA). The slides were scanned using the PhenoImager Fusion (Akoya Biosciences) at 20x magnification. Single-stained slides were used to generate a spectral library using inForm 3.1 (Akoya Biosciences) for spectral unmixing. Exported images were analyzed using MATLAB R2022b (Mathworks). Bulk tissue was detected based on a maximum intensity projection across all channels. Nuclei were segmented using a bandpass filtered followed by difference of gaussians, then segmented by Otsu’s method. Local maxima indicative of nuclei centers were identified and used for a seeded watershed filter to separate overlapping nuclei. Nuclei were further used for a seeded watershed filter to segment individual cells, with a maximal dilation restricted to the nuclear diameter. Mean fluorescence intensity of individual cells was used to determine marker positivity. All studies were approved by the institutional review board at Cleveland Clinic.