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94 Orion™: 16-plex single-step stain and imaging of oral squamous cell carcinoma
  1. Eric Kaldjian,
  2. Tad Georg and
  3. Selena Larkin
  1. RarCyte, Inc., Seattle, WA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background In order to understand the subcellular nature of the tumor microenvironment (TME), high quality imaging at single-cell resolution is needed as a basis for downstream biomarker quantitation and predicting patient outcomes. Here we investigate a sample of an infiltrating squamous cell carcinoma of the tongue using whole slide, single-step stain and imaging at single-cell resolution.

Methods The images profile a whole slide section of an infiltrating squamous cell carcinoma of the tongue. The FFPE squamous cell section was stained with a 16-plex immunofluorescence (IF) panel in one staining round followed by whole slide imaging with the Orion spatial biology platform. Tissue autofluorescence was imaged and isolated as an additional fluorescence channel. The full protocol is fairly quick and simple, using standard histology tools:

  • Mount sections on glass slides

  • De-paraffinize and perform antigen retrieval

  • Quench autofluorescence

  • Stain slides with a panel of ArgoFluor™ conjugated antibodies

  • Coverslip with ArgoFluor Mounting Medium and cure overnight

  • Image whole slides at 20X magnification using the Orion instrument

  • Process to ome.TIFF and analyze

Results Multiplexed imaging uncovered the tumor cells recapitulate the organizational structure of the normal epithelium where most express E-Cadherin similar to basal mucosal epithelium. Yet in some regions, the cells differentiate into squamous cells and express Pan-CK (figure 1). In contrast to the normal epithelium, the tumor is highly proliferative and stimulates a host fibrocellular immune response that is demonstrated by a mixture of immune cells at the border of the tumor nodule.

Conclusions There is a need to show biology of the TME in a high quality, subcellular fashion as it provides a more in-depth understanding into such biology and thus, can lead to a greater spatial investigation for human disease. Here multiplexed imaging identified normal tongue mucosa, the host response to the tumor, and a variety of T cell sub-types, including regulatory T cells and activated T cells, confirming the biological differences in tissue composition and cellular interactions between these tissues and provides crucial information for future spatial quantitation.

Abstract 94 Figure 1

Whole-slide tissue section of an infiltrating squamous cell carcinoma of the tongue stained with a 16-plex biomarker panel and imaged in one scan on Orion.

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