Multiplexed immunofluorescence delineates proteomic cancer cell states associated with metabolism
Anup Sood, … , Steven M. Larson, Ingo K. Mellinghoff
Anup Sood, … , Steven M. Larson, Ingo K. Mellinghoff
Published May 5, 2016
Citation Information: JCI Insight. 2016;1(6):e87030. https://doi.org/10.1172/jci.insight.87030.
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Resource and Technical Advance Oncology

Multiplexed immunofluorescence delineates proteomic cancer cell states associated with metabolism

Abstract

The phenotypic diversity of cancer results from genetic and nongenetic factors. Most studies of cancer heterogeneity have focused on DNA alterations, as technologies for proteomic measurements in clinical specimen are currently less advanced. Here, we used a multiplexed immunofluorescence staining platform to measure the expression of 27 proteins at the single-cell level in formalin-fixed and paraffin-embedded samples from treatment-naive stage II/III human breast cancer. Unsupervised clustering of protein expression data from 638,577 tumor cells in 26 breast cancers identified 8 clusters of protein coexpression. In about one-third of breast cancers, over 95% of all neoplastic cells expressed a single protein coexpression cluster. The remaining tumors harbored tumor cells representing multiple protein coexpression clusters, either in a regional distribution or intermingled throughout the tumor. Tumor uptake of the radiotracer 18F-fluorodeoxyglucose was associated with protein expression clusters characterized by hormone receptor loss, PTEN alteration, and HER2 gene amplification. Our study demonstrates an approach to generate cellular heterogeneity metrics in routinely collected solid tumor specimens and integrate them with in vivo cancer phenotypes.

Authors

Anup Sood, Alexandra M. Miller, Edi Brogi, Yunxia Sui, Joshua Armenia, Elizabeth McDonough, Alberto Santamaria-Pang, Sean Carlin, Aleksandra Stamper, Carl Campos, Zhengyu Pang, Qing Li, Elisa Port, Thomas G. Graeber, Nikolaus Schultz, Fiona Ginty, Steven M. Larson, Ingo K. Mellinghoff

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Figure 1

Experimental design.

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Experimental design. Immunofluorescence approach. A single 3- to 5-μm un...
Immunofluorescence approach. A single 3- to 5-μm unstained section from a routinely collected formalin-fixed and paraffin-embedded (FFPE) tumor tissue block was used from each tumor for the multiplex iterative imaging cycles (n = 20). Background autofluorescence (AF) tissue images were acquired before subsequent application of fluorescent dye-conjugated primary antibodies. Stained images were then acquired, followed by dye inactivation and restaining with new directly conjugated antibodies. New images were acquired, and the cycle was repeated until all target antigens were exhausted. Stained images were registered. Background AF was removed from each stained image. Images were segmented into epithelial and stromal regions using boundaries of cytokeratin staining, followed by identification of individual cells and corresponding plasma membrane (as determined by Na+K+ATPase staining), cytoplasm (S6 staining), and nuclear regions (DAPI). Biomarker pixel-level intensity data, which were subsequently queried in data analysis, were quantified at cell level. Three different metrics per marker (mean, standard deviation, and 90% hot spot) were used, amounting to about 155 million measurements. Data analysis included K-median clustering to groups of cells based on similar biomarker intensity levels. Each field of view (FOV) was manually reviewed. Only FOVs with >90% IDC cells on histopathological review were included in the analysis.