Multiplex Immunohistochemistry for Immune Cell Profiling

For immune cell profiling we conducted mfIHC. Details of the procedure have been described previously [28 (link)]. Tyramide signal amplification was used employing an Opal IHC kit (PerkinElmer, Waltham, MA) according to the manufacturer's instructions. We previously constructed a T-cell panel, comprising CD3, CD4, CD8, FOXP3 and T-bet. This panel also included cytokeratin to differentiate tumor and stromal areas, and DAPI for staining nuclei [28 (link)]. Primary antibodies used were: CD3 (clone SP7, Abcam, Tokyo, Japan), CD4 (clone 4B12, Leica Microsystems, Tokyo, Japan), CD8 (clone 4B11, Leica Microsystems), FOXP3 (D6O8R, Cell Signaling Technology, Danvers, MA), T-bet (clone 4B10, Santa Cruz Biotechnology, Dallas, CA) and cytokeratin (clone AE1/AE3, Dako Agilent Technologies, Santa Clara, CA). Employing an automated imaging system (Vectra ver. 3.0, PerkinElmer), an average of 20 areas at × 200 were captured for each patient. An image analyzing software program (InForm, PerkinElmer) segmented cancer tissue into cancer cell nests (intratumoral) and the framework (stromal) region, and detected immune cells with specific phenotypes. Representative images of tissue segmentation and cell phenotype recognition are shown in Additional file 2. Infiltrating immune cells were quantified using an analytic software program (Spotfire, TIBCO, Palo Alto, CA) and then calculated per area.

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Onagi H., Horimoto Y., Sakaguchi A., Ikarashi D., Yanagisawa N., Nakayama T., Nakatsura T., Ishizuka Y., Sasaki R., Watanabe J., Saito M., Saeki H., Hayashi T., Arakawa A., Yao T, & Kitano S. (2022). High platelet-to-lymphocyte ratios in triple-negative breast cancer associates with immunosuppressive status of TILs. Breast Cancer Research : BCR, 24, 67.

Publication 2022

CD3 (clone SP7, Vectra ver. 3.0

Antibodies Cancer Cells Clone Cytokeratin Dapi Nuclei Opal Patient Phenotype T cell Tissue Tumor Vectra

Corresponding Organization :

Other organizations : Juntendo University, The Cancer Institute Hospital, Japanese Foundation For Cancer Research

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Infiltrating immune cell counts (CD3, CD4, CD8, FOXP3, T-bet) in cancer cell nests (intratumoral) and stromal regions

control variables

Cytokeratin to differentiate tumor and stromal areas
DAPI for staining nuclei

controls

No positive or negative controls were explicitly mentioned in the input.

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