Image mass cytometry staining
TMA, prostate, breast carcinoma, ovarian carcinoma, MCD
PROLIFERATIVE, ENDOMETRIOID CARCINOMA
An Immuno-Oncology (IO)-IMC panel with 36 validated markers was designed to include lymphoid (CD45RO, CD3, CD4, CD8, CD19, Granzyme B, CD94, OX40), myeloid (CD11b, CD14, CD68, MPO, CD11c, HLA-DR, CD163, CD33), immune-modulatory (CCR6, PD-L1, CD73, FOXP3, TIM3, LAG3, VISTA, B7-H3, ICOS), epithelial (pan cytokeratin, panCK), proliferative (Ki67), and leukocyte/constitutive (CD45, β2-microglobulin, GAPDH, NaKATPase) markers. The IO-IMC panel was optimized by single plex-IHC assays of BSA-free monoclonal antibodies, followed by single plex indirect immunofluorescence (IF) assays of each metal-isotope-tagged antibody (Maxpar Labeling Kits; Fluidigm), and a final multiplex staining step containing the whole cocktail with 36 metal-isotope-tagged antibodies. A control TMA (4 μm diameter) including normal and neoplastic tissues (tonsil, appendix, placenta, prostate, breast carcinoma, ovarian carcinoma, and endometrioid carcinoma) was used for each staining assay, and to select the optimal concentration per antibody showing a constant cellular subtype expression, and a stable pattern of staining in the same control tissue cores observed by single- and multiplex staining. Fifty consecutive 4 µm thickness unstained slides were obtained from the TMA block of colon biopsy specimens. To select the unstained slides for IMC staining, three slides at different levels (1, 25, and 50) were stained with H&E. The level 25 showed the highest number of cases (46) with at least one core evaluable, therefore a standardized IMC manual staining protocol was performed in three adjacent levels (22, 23, and 24). The staining protocol included one single antigen retrieval step (Tris buffer, pH 9.0, two-cycles of 10 min at 99°C), an overnight IMC panel cocktail incubation step at 4°C followed by Iridium and Ruthenium counterstaining. One field of view (FOV) per core (800 µm x 800 µm) was ablated and imaged by Hyperion Imaging System (Fluidigm) and MCD Viewer Software (v1.0.560.2, Fluidigm) for further digital image analysis.