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222-D Automated hyperplex staining and imaging system on various cancer tissues using off the shelf antibodies
  1. Li Yen Chong1,
  2. Felicia Wee1,
  3. Jeffrey Lim1,
  4. Craig Joseph1,
  5. Zhen Wei Neo1,
  6. Jia Ying Joey Lee2,
  7. Lit-Hsin Loo2 and
  8. Joe Yeong1,3
  1. 1Institute of Molecular and Cell Biology (IMCB), Agency of Science, Technology and Research (A*STAR), Singapore, Singapore
  2. 2Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore, Singapore
  3. 3Singapore General Hospital, Singapore, Singapore


Background Interactions between tumor and neighboring immune cells are dynamic and complex within the tumor microenvironment (TME), requiring the use of spatial biology techniques to characterize them, and thereby identify potential targets for immunotherapy. While technologies supporting deep dives into spatial biology have rapidly evolved across modalities, including multiplex immunohistochemistry,1 2 a limited number of protein markers can be stained sequentially. Therefore, as the demand for studying different cell phenotypes increases, the number of protein markers required for staining grows exponentially too. Here, we demonstrate the use of an automated hyperplex staining and imaging system to stain 40-plex on various cancer tissues using off the shelf antibodies. A comparison of current available platforms can be found at (table 1).

Methods Heat-mediated antigen retrieval was performed on formalin-fixed paraffin embedded (FFPE) patient tissues: colorectal cancer (CRC), nasopharyngeal cancer (NPC), lung cancer and gastric cancer tissue microarray (GC TMA). Fresh frozen patient NPC, lung cancer and oropharyngeal squamous cell carcinoma (OSCC) tissues were post-fixed and permeabilized. Some of these cancers were immuno-oncotherapy (IO)-treated. Tonsil tissue was used for antibodies optimization and validation. Sequential immunofluorescence protocol was performed using the COMET platform (Lunaphore Technologies SA, Switzerland) with microfluidic chips for the spatial detection of 40 markers with off the shelf primary antibodies (table 2) and fluorophore-conjugated secondary antibodies.3

Results The 40 markers were optimized on human tonsil sections within 5 weeks (figure 1). Two 40-plex slides were concurrently stained and imaged under 24 hours at a time. 9mm x 9mm high resolution whole slide images acquired then provide an overview of the TME including tumor nest and stroma. The display of several immune cell and tumor markers allows for easy visualization of the spatial proximity of lymphoid aggregates to the tumor (figures 2 and 3). Furthermore, the expression of selected markers in the GC TMA can be viewed and cores of interest can be easily identified (figure 4). The images were hosted on ImmunoAtlas ( for interactive visualization (table 3).

Conclusions Using this technology, complex cellular phenotypes can be studied with higher plexes. Coupled with spatial data, in-depth analyses can be conducted to stratify patients’ status of treatment response and disease progression. Moreover, preserved tissue integrity allows for downstream stains like immunohistochemistry to be conducted on the same tissue section. Work in progress includes downstream analysis of the images on the VisioPharm platform and hosting of more 40-plex images in the ImmunoAtlas site.

Acknowledgements We would like to thank our institutions (IMCB, BII, A*STAR) for their continuous support in our work. We would like to extend our gratitude to Lunaphore Technologies SA for their support.


  1. Tan WCC, Nerurkar SN, Cai HY, Ng HHM, Wu D, Wee YTF, et al. Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy. Cancer Commun (Lond). 2020 Apr;40(4):135–153.

  2. Boisson A, Noel G, Saiselet M, Rodrigues-Vitoria J, Thomas N, Fontsa ML, et al. Fluorescent Multiplex Immunohistochemistry Coupled With Other State-Of-The-Art Techniques to Systematically Characterize the Tumor Immune Microenvironment. Front. Mol. Biosci. 2021 Sep;8.

  3. Migliozzi D, Pelz B, Dupouy DG, Leblond A-L, Soltermann A, Gijs MAM. Microfluidics-assisted multiplexed biomarker detection for in situ mapping of immune cells in tumor sections. Microsyst Nanoeng 2019 Nov;5(59).

Ethics Approval This study was approved by the Agency of Science, Technology and Research (A*STAR) Human Biomedical Research Office (A*STAR IRB: 2021-161, 2021-188, 2021-112).

Abstract 222-D Table 1

Comparison of current automated hyperplex protein staining and imaging platforms

Abstract 222-D Table 2

40 markers optimised on COMET

Abstract 222-D Table 3

40-plex images of different tissue types on ImmunoAtlas website

Abstract 222-D Figure 1

40 markers optimised on tonsil sample

Abstract 222-D Figure 2

40-plex CRC sample whole slide image (left); zoom-in image (right) depicting spatial organisation of lymphoid aggregate at the invasive front

Abstract 222-D Figure 3

40-plex lung cancer sample whole slide image (left); zoom-in image (right) depicting spatial organisation of lymphoid aggregate at the invasive front

Abstract 222-D Figure 4

40-plex GC TMA whole slide image (left) and zoom-in images (right) of selected cores

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