Abstract 1635: Multiplexed analysis of fixed tumor tissues using imaging mass cytometry
Authors: Villarroel-Espindola Franz , Carvajal-Hausdorf Daniel , Datar Ila , Esch Amanda , Rashidi Narges , Nassar Ala , Ren Shelly , Montgomery Ruth R. , Herbst Roy S. , Rimm David L. , Schalper Kurt A. 
Published: July 2017

Abstract
Introduction: Comprehensive evaluation of the tumor microenvironment with preservation of spatial context requires simultaneous in situ detection of multiple targets. Current fluorescence-based methods can accommodate up to 5-6 distinct markers and can be applied to formalin fixed, paraffin embedded tissue (FFPE). Imaging mass cytometry uses metal-conjugated antibodies and mass spectrometry to perform highly multiparametric, quantitative measurement of protein targets within tissue sections providing unprecedented detail of quantitative tissue labeling. To date, the IMC technology has not been widely available and its performance has not yet been compared with other validated methods.
Methods: Using multiplexed quantitative immunofluorescence (QIF, AQUA/Genoptix) and imaging mass cytometry (IMC, Fluidigm) we validated and optimized metal-conjugated assays to detect HER2 (29D8-CST, 176Yb), CD3 (DAKO, 170Er), Ki-67 (B56-BD biosciences, 168Er), Histone H3 (D1H2-CST, 176Yb), pancytokeratin (C11-Biorad, 162Dy), Vimentin (RV202-BD Pharmingen, 156Gd), LipoR (Fluidigm, 115In) and DNA/nuclei (DNA intercalator, Fluidigm, 191/193Ir). The markers were applied to FFPE samples from tumor cell lines, human tonsil, lymph node; and breast and lung carcinomas represented in tissue microarray format. Data was visualized using MCD viewer and ImageJ software and results from QIF and IMC were compared.
Results: Metal conjugation did not affect the performance of the primary antibodies studied here. The staining patterns of the epithelial/tumor and stromal compartments evaluated using the IMC platform and QIF were comparable. HER2 signal was higher in tumors and cell lines harboring HER2 amplification by FISH/IHC. The CD3 signal was higher in T-cell lymphoma cells and in the inter-follicular areas of human tonsil and lymph nodes. Ki-67 signal was nuclear, co-localized with Histone H3 and showed positivity in lymphoid germinal centers, cytokeratin-positive carcinoma cells and CD3+ tumor infiltrating lymphocytes. Results obtained using the IMC at different time points in serial tissue sections were comparable (P&lt;0.05).
Conclusion: Quantitative and reproducible measurement of immune and non-immune targets with spatial resolution using the IMC platform is feasible in FFPE breast and lung carcinomas and the results are comparable to multiplexed QIF. Expansion of the current panel to include &gt;30 markers for immune cell phenotypes/function and tumor markers is ongoing.
Citation Format: Franz Villarroel-Espindola, Daniel Carvajal-Hausdorf, Ila Datar, Amanda Esch, Narges Rashidi, Ala Nassar, Shelly Ren, Ruth R. Montgomery, Roy S. Herbst, David L. Rimm, Kurt A. Schalper. Multiplexed analysis of fixed tumor tissues using imaging mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1635. doi:10.1158/1538-7445.AM2017-1635