Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue

Abstract

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.

Keywords: cancer diagnostics; high-content cellular analysis; image analysis; mTOR; multiplexing.

Fig. 1.

MxIF data acquisition, image processing, and data analysis scheme. (A) In the laboratory, background autofluorescence (AF) tissue images are acquired before subsequent application of fluorescent dye-conjugated primary antibodies. Stained images are then acquired, followed by dye inactivation and restaining with new directly conjugated antibodies. New images are acquired, and the cycle is repeated until all target antigens are exhausted. Times associated with each step are indicated. (B) Stained images are registered, background AF is removed from each stained image, and images are segmented into epithelial and stromal regions, followed by identification of individual cells and corresponding plasma membrane, cytoplasm, and nuclear regions. Pixel-level data are summarized in cellular features, which is subsequently queried in data analysis (C). Data analysis can consist of a variety of statistical and visual explorations. In this work, we use K-median clustering to group cells with similar mTOR activity.

Fig. 2.

Enhanced visualization of complex tissues and MxIF coupled with DNA FISH. Breast cancer tissue was stained and analyzed by the MxIF process: (A) Cytokeratin protein staining serves as epithelial cellular marker; (B) samples are imaged to confirm dye inactivation and generate background images used in the autofluorescence removal process; (C) DAPI signal is imaged every cycle and provides a reference for image registration; and (D) Na⁺/K⁺ATPase staining serves as a membrane marker for determining cell borders. Each fluorescent channel is pseudocolored, and after registration, a merged image can be generated (E). Segmentation maps are produced for subcellular specific analysis (F). An H&E-like representation of the tissue can be generated using selective color assignment of the fluorescent signals (G). Marker stains can be viewed with chromogen-like pseudocoloring (H) including hematoxylin-like nuclear counterstain from the DAPI signal. Combined staining with dye-conjugated antibodies and DNA FISH in breast cancer tissue shows pan-keratin and HER2 protein stains together with HER2 and CEP17 FISH. Pseudocolored composite images from a representative field of view at 40× magnification are shown as follows: (I) immunofluorescence for pan-keratin (green) and Her2 (red) with DAPI (blue); (J) FISH signals for HER2 (red), CEP17 (green), and DAPI (blue); (K) Her2 immunofluorescence (red) from first imaging round overlaid with HER2 FISH (purple), CEP17 FISH (green), and DAPI (blue); and (L) magnified view of the area marked in K, highlighting Her2 amplified cells (solid arrows) showing colocalization of HER2 gene amplification and Her2 protein expression, and normal cells (dashed arrow) with normal FISH pattern and undetectable Her2 protein signal.

Fig. 3.

Multiplexed immunofluorescence of signal transduction pathways in CRC. 747 FFPE stage I–III CRC specimens arrayed on three TMAs were stained for 61 protein antigens including markers of epithelial, immune and stromal cell lineage, subcellular compartments, oncogenes, tumor suppressors, and significant posttranslational protein modifications. Pseudocolored images of signaling and regulatory molecule staining in one small field of view are shown. Nuclei are counterstained with DAPI and pseudocolored blue in all images. (A, 1) TMA core depicted in virtual H&E. (A, 2) Pseudocolored overlaid immunofluorescence of epithelial cells stained positive for pancytokeratin and stroma area stained positive for α-smooth muscle actin. (A, 3) Major subcellular compartments are detected by immunofluorescence staining of ribosomal protein S6 (cytoplasm) and Na⁺K+ATPase (plasma membrane), and the nucleic acid stain DAPI (nucleus). (B) Activation of mitogenic and anabolic signaling pathways in CRC cells. Multiplexed immunofluorescence of signaling protein expression and phosphorylation shows complex activation and repression patterns of regulatory and signal transduction pathways. The white arrow indicates a single cell expressing each feature and the cyan arrow indicates a cell with differential expression of features.

Fig. 4.

Cluster analysis shows large-scale divergence of signaling to RPS6, 4E-BP1, and ERK1/2 in CRC cells. (A) K-medians clustering heat map of 4E-BP1 pThr37/46, RPS6 pSer 235/236, and ERK1/2 pT202/Y204 staining in 3.6 × 10⁵ CRC cells from 720 subjects (abbreviated as p4EBP1, pS6, and pERK). (B) K-medians clustering heat map of enrichment in clusters from A in 436 subjects with ≥50 cells positive for p4E-BP1, pS6, or pERK. (C–E) Pseudocolor overlaid images of representative subjects enriched for mutually exclusive subject-level signaling (C and D) and a subject with many cell clusters (E) are shown. Mapping of cell clusters back on to images demonstrates cases exhibiting homogeneity (F and G) or heterogeneity (H) of cluster assignment. The legends in F–H show the colors selected to represent each cluster.