Cancer immunophenotyping by seven‐colour multispectral imaging without tyramide signal amplification

Abstract

Checkpoint blockade immunotherapies have revolutionised cancer treatment in the last decade. Nevertheless, these are only beneficial for a small proportion of cancer patients. Important prognosticators for response to immunotherapy are the mutation burden of tumours as well as the quality and quantity of tumour-infiltrating immune cells. High-throughput multiplex immunophenotyping technologies have a central role in deciphering the complexity of anti-tumour immune responses. Current techniques for the immunophenotyping of solid tumours are held back by the lack of spatial context, limitations in the number of targets that can be visualised simultaneously, and/or cumbersome protocols. We developed a tyramide signal amplification-free method for the simultaneous detection of seven cellular targets by immunofluorescence. This method overcomes limitations posed by most widespread techniques and provides a unique tool for extensive phenotyping by multispectral fluorescence microscopy. Furthermore, it can be easily implemented as a high-throughput technology for validation of discovery sets generated by RNA sequencing or mass cytometry and may serve in the future as a complementary diagnostic tool.

Pre-existing inflammatory immune microenvironment predicts the clinical response of vulvar high-grade squamous intraepithelial lesions to therapeutic HPV16 vaccination

“…Dim markers were tyramide signal amplified with Opal to enable their detection by fluorescence microscopy, and primary antibodies with clashing species and isotypes in the panel were directly labeled with fluorochromes or detected with species/isotype-specific antibodies. 28 A complete overview of the design of the T cell panel and myeloid cell panel is presented in online supplementary files 1 and 2, respectively. In the seven color immunofluorescent procedure, 4 μm FFPE tissue sections were deparaffinized, endogenous peroxidase was blocked with hydrogen peroxide, and heat-induced epitope retrieval was performed with citrate (10 mM, pH 6.0) in the T cell panel and with tris-EDTA (10 mM/1 mM, pH 9.0) in the myeloid cell panel.…”

“…On the second day, after detection of the previous with respective fluorescently labeled secondary antibodies, the directly labeled primary antibodies were incubated for 5 hours and, lastly, DAPI was applied as nuclear counterstain. 28 Quantification of immune cells in the tMe Immunofluorescence images were acquired with the Vectra V.3.0.5 multispectral imaging microscope (Perki-nElmer) at 20× magnification and exposure times were set to avoid spectral overlap. Immune cells in the TME were automatically phenotyped and counted with inForm V. Open access software was trained to segment epithelial and stromal fractions, segment DAPI +nucleated cells, and to assign a phenotype to each cell (online supplementary file 3a).…”

High-dimensional cytometric analysis of colorectal cancer reveals novel mediators of antitumour immunity

“…A six-marker immunofluorescence panel was applied to 5 µm frozen tissue sections of 4 MMR-deficient and 4 MMR-proficient colorectal tumours, as described previously 21. Details on immunofluorescence antibody staining are available in online supplementary methods and online supplementary table S4.…”

CD163+cytokine-producing cDC2 stimulate intratumoral type 1 T cell responses in HPV16-induced oropharyngeal cancer

“…Immunofluorescent four color staining was performed for 10 selected OPSCC tumors as described previously. 16 17 Antibody specificity and optimal antigen retrieval were assessed by single-plex IHC using tonsils. Following incubation with superblock buffer (Thermo Fisher Scientific, Waltham, Massachusetts, USA), the primary antibodies and isotype/species-specific secondary fluorescent antibodies were applied ( online supplementary table 2 ).…”