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.

Keywords: T-cells; biomarkers; cancer microenvironment; immune infiltration; immunotherapy; multispectral immunofluorescence; myeloid cells.

Figure 1

Workflow of seven‐target IF detection starting with tissue preparation (A), followed by detection with primary antibodies (B), which are subsequently detected with secondary antibodies (C). Afterwards, the tissue is incubated with directly conjugated antibodies (D), and incubated with DAPI as nuclear counterstain (E).

Figure 2

Immunofluorescent detection of T‐cell markers in MMR‐proficient (A) and MMR‐deficient (B) colorectal cancer tissues, namely CD3 (red), CD8 (blue), FOXP3 (cyan), CD45RO (orange), GZMB (green), keratin (white) to distinguish tumour tissue, and DAPI (grey) as nuclear counterstain. Panels C–I show DAPI, keratin, CD8, CD3, FOXP3, CD45RO, and GZMB, respectively, detected in the MMR‐deficient tumour presented in panel B.

Figure 3

Prevalence of T‐cell immunophenotypes in colorectal cancer (CRC) tissue. Phenotypes are defined as T‐helper cells (CD3⁺CD8⁻), cytotoxic T‐cells (CD3⁺CD8⁺), regulatory T‐cells (CD3⁺FOXP3⁺), memory T‐cells (CD3⁺CD45RO⁺), and activated T‐cells (CD3⁺GZMB⁺).

Figure 4

(A) Immunofluorescent detection of myeloid‐related markers in colorectal cancer tissue in which PD‐L1 was enhanced by TSA. Markers are PD‐L1 (magenta), HLA‐DR (orange), CD11c (blue), CD163 (green), CD68 (cyan), and keratin (white). (B) TSA‐enhanced PD‐L1 only, in the same tissue.