High-dimensional multiplexed immunohistochemical characterization of immune contexture in human cancers

Grace Banik, Courtney B. Betts, Shannon M. Liudahl, Shamilene Sivagnanam, Rie Kawashima, Tiziana Cotechini, William Larson, Jeremy Goecks, Sara I. Pai, Daniel R. Clayburgh, Takahiro Tsujikawa, Lisa M. Coussens

Research output: Chapter in Book/Report/Conference proceedingChapter

29 Scopus citations


Biomarker assessments of tumor specimens is widely used in cancer research to audit tumor cell intrinsic as well as tumor cell extrinsic features including the diversity of immune, stromal, and mesenchymal cells. To comprehensively and quantitatively audit the tumor-immune microenvironment (TiME), we developed a novel multiplex immunohistochemistry (mIHC) platform and computational image processing workflow using a single formalin-fixed paraffin-embedded (FFPE) tissue section. Herein, we validated this platform using nine matched primary newly diagnosed and recurrent head and neck squamous cell carcinoma (HNSCC) sections sequentially subjected to immunodetection with a panel of 29 antibodies identifying malignant tumor cells, and 17 distinct leukocyte lineages and their functional states. Image cytometric analysis was applied to interpret chromogenic signals from digitally scanned and coregistered light microscopy-based images enabling identification and quantification of individual tumor cells, structural features, immune cell phenotypes and their functional state. In agreement with our previous study via a 12-plex imaging mIHC platform, myeloid-inflamed status in newly diagnosed primary tumors associated with significantly short progression free survival, independent of lymphoid-inflamed status. Spatial distribution of tumor and immune cell lineages in TiME was also examined and revealed statistically significant CD8+ T cell exclusion from tumor nests, whereas regulatory T cells and myeloid cells, when present in close proximity to tumor cells, highly associated with rapid cancer recurrence. These findings indicate presence of differential immune-spatial profiles in newly diagnosed and recurrent HNSCC, and establish the robustness of the 29-plex mIHC platform and associated analytics for quantitative analysis of single tissue sections revealing longitudinal TiME changes.

Original languageEnglish (US)
Title of host publicationTumor Immunology and Immunotherapy - Integrated Methods Part A
EditorsLorenzo Galluzzi, Nils-Petter Rudqvist
PublisherAcademic Press Inc.
Number of pages20
ISBN (Print)9780128186770
StatePublished - 2020

Publication series

NameMethods in Enzymology
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988


  • Cancer immunology
  • Histopathology
  • Immunohistochemistry
  • Multiplex imaging

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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