High Dimensional Imaging Mass Cytometry Panel to Visualize the Tumor Immune Microenvironment Contexture

Abstract

The integrative analysis of tumor immune microenvironment (TiME) components, their interactions and their microanatomical distribution is mandatory to better understand tumor progression. Imaging Mass Cytometry (IMC) is a high dimensional tissue imaging system which allows the comprehensive and multiparametric in situ exploration of tumor microenvironments at a single cell level. We describe here the design of a 39-antibody IMC panel for the staining of formalin-fixed paraffin-embedded human tumor sections. We also provide an optimized staining procedure and details of the experimental workflow. This panel deciphers the nature of immune cells, their functions and their interactions with tumor cells and cancer-associated fibroblasts as well as with other TiME structural components known to be associated with tumor progression like nerve fibers and tumor extracellular matrix proteins. This panel represents a valuable innovative and powerful tool for fundamental and clinical studies that could be used for the identification of prognostic biomarkers and mechanisms of resistance to current immunotherapies.

Keywords: biomarkers; imaging mass cytometry; immune therapies; panel design; tumor immune microenvironment.

Figure 1

Stepwise procedure for immunodetection by IMC.

Figure 2

IMC staining condition optimization. Two antibody incubation conditions and three antibody dilutions were tested for each antibody of the IMC panel. (A) The markers CD8 and CD206 are representative of the variations induced by incubation time and temperature (1h30 at +4°C and overnight at room temperature (RT)). (B) The markers CD14 and tenascin C are representative of the variations induced by antibody dilution (1/100, 1/200 and 1/400). Scale bar = 100 μm.

Figure 3

Visualization by IMC of structural and cellular TiME components in a single region of cSCC section. Overlaid and single antibody signals representing lymphatic vessels (podoplanin), blood vessels and CAFs (aSMA), nerve fibers (pan-neurofilament), tumor cells (pan-cytokeratin), ECM (fibronectin) and immune cells (CD45) compared to nuclei and HES staining of the same region of cSCC-1 section. Scale bar = 100 μm.

Figure 4

Visualization by IMC of tumor cell heterogeneity in a single region of cSCC section. Overlaid and single antibody signals targeting pan-cytokeratin, EGFR, β-Catenin, podoplanin and Ki67 markers compared to nuclei and HES staining of the same region of cSCC-2 section. Scale bar = 100 μm.

Figure 5

Visualization by IMC of immune cell diversity in a single region of cSCC section. Representative cSCC region, from cSCC-3 section, showing the detection of Langerhans cells (Langerin), neutrophils (CD15), T cells (CD3), macrophages (CD68), B cells (CD20), tumor cells (pan-cytokeratin) and CAFs, blood and lymphatic vessels (α-SMA and podoplanin). (AA–CC) Identification of immune cell colocalizations. (A) Identification of T cell subset distribution: cytotoxic T cells (CD8⁺Ganzyme B⁺) (A-1), TIGIT-expressing CD4⁺ and CD8⁺ T cells (A-2), regulatory T cells (CD4⁺FOXP3⁺) (A-3) and proliferating T cells (CD3⁺Ki67⁺) (A-4). (B) Overlaid and single antibody signals targeting CD15, myeloperoxidase, and granzyme B neutrophil markers within tumor islet (pan-cytokeratin). (C) Identification of macrophage subsets. Overlaid and single antibody signals targeting CD14, CD206, CD204, CD68, CD163 and pan-cytokeratin. Scale bar = 100 μm.

Figure 6

Visualization by IMC of immune cell colocalization with TiME components in a single region of cSCC section. Representative cSCC region, from cSCC-4 section, showing the detection of macrophages (CD68), T cells (CD3), blood vessels and CAFs (α-SMA) ECM (fibronectin and tenascin C) and nerve fibers (pan-neurofilament and Tubulin-β-III) with overlaid and single antibody signals images. (A) Immune cells invading a nervous sheath zone. (B) Immune cell egress from blood vessels. (C) leukocyte-infiltrated tumor islet. (D) ECM protein-rich area. (E) Immune cell-rich area. Scale bar = 100 μm.