Robust Cell Detection and Segmentation for Image Cytometry Reveal Th17 Cell Heterogeneity

Takahiro Tsujikawa, Guillaume Thibault, Vahid Azimi, Sam Sivagnanam, Grace Banik, Casey Means, Rie Kawashima, Daniel R. Clayburgh, Joe W. Gray, Lisa M. Coussens, Young Hwan Chang

Research output: Contribution to journalArticlepeer-review

13 Scopus citations


Image cytometry enables quantitative cell characterization with preserved tissue architecture; thus, it has been highlighted in the advancement of multiplex immunohistochemistry (IHC) and digital image analysis in the context of immune-based biomarker monitoring associated with cancer immunotherapy. However, one of the challenges in the current image cytometry methodology is a technical limitation in the segmentation of nuclei and cellular components particularly in heterogeneously stained cancer tissue images. To improve the detection and specificity of single-cell segmentation in hematoxylin-stained images (which can be utilized for recently reported 12-biomarker chromogenic sequential multiplex IHC), we adapted a segmentation algorithm previously developed for hematoxlin and eosin-stained images, where morphological features are extracted based on Gabor-filtering, followed by stacking of image pixels into n-dimensional feature space and unsupervised clustering of individual pixels. Our proposed method showed improved sensitivity and specificity in comparison with standard segmentation methods. Replacing previously proposed methods with our method in multiplex IHC/image cytometry analysis, we observed higher detection of cell lineages including relatively rare T H 17 cells, further enabling sub-population analysis into T H 1-like and T H 2-like phenotypes based on T-bet and GATA3 expression. Interestingly, predominance of T H 2-like T H 17 cells was associated with human papilloma virus (HPV)-negative status of oropharyngeal squamous cell carcinoma of head and neck, known as a poor-prognostic subtype in comparison with HPV-positive status. Furthermore, T H 2-like T H 17 cells in HPV-negative head and neck cancer tissues were spatiotemporally correlated with CD66b + granulocytes, presumably associated with an immunosuppressive microenvironment. Our cell segmentation method for multiplex IHC/image cytometry potentially contributes to in-depth immune profiling and spatial association, leading to further tissue-based biomarker exploration.

Original languageEnglish (US)
Pages (from-to)389-398
Number of pages10
JournalCytometry Part A
Issue number4
StatePublished - Apr 2019


  • T 17 cell phenotypes
  • cell segmentation
  • tumor immune microenvironment

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology


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