Multiplex, Lung Cancer, TME (Akoya Biosciences, Inc.) Multiplex, Lung Cancer, TME (Akoya Biosciences, Inc.)

Process APP




Multiplex, Lung Cancer, TME (Akoya Biosciences, Inc.)

Developed for Akoya Biosciences, Inc.

The widely used biomarker panel for lung cancer (CD8, PD-1, PD-L1, CK, CD68, FoxP3 and CD68) was developed to understand immune infiltration in the face of a PD-L1 blockade. It can be utilized to assess (a) the change in the immune interaction across the tumor-stroma interface and (b) the composition on either side of the normal-tumor invasive edge. Phenotypes are identified using the Multiplex Phenotyping module. This APP is designed to work with amplified fluorescence signals that have been unmixed with multispectral imaging, in which each stain is spectrally isolated from both spectrally overlapping fluorophores and background autofluorescence.

Figure 1

Figure 1

Part of a raw multiplexing image

Figure 2

Figure 2

Automatic tumor detection based on the presence of CK staining

Figure 3

Figure 3

Automatic cell segmentation based on the presence of DAPI staining and automatic identification of phenotypes based on the trained phenotyping APP

Figure 4

Figure 4

Generated t-SNE plot showing the detected phenotypes


Auxiliary APPs

APP: “01 Tumor Detection” The auxiliary APP ”01 Tumor Detection” is used for automatic tumor detection

APP: “02 Find Cells” The auxiliary APP ”02 Find Cells” is used for automatic identification of cells

Quantitative Output variables

The output variables obtained from this protocol are:

  • Phenotyping: A .csv file that holds the mean pixel value in each band for each cell
  • Location: A .csv file that holds the position of each cell

The APP also outputs the total number of cells positive for different combinations of markers in both tumor and stroma. For a full list, please contact us.


Step 1: Load and run the APP “01 Tumor Detection” for tumor identification

Step 2: Load and run the APP “02 Find Cells” for cell identification

Step 3: Load and run the APP “03 Phenotyping” is used for division of the cells into identified phenotypes


The tissue is initially segmented into tumor and stroma based on the presence of cytokeratin staining. Following tissue segmentation, individual cells are identified based on the presence of DNA (DAPI) and membrane proteins where available. The unsupervised phenotyping algorithm is trained across multiple images resulting in identification of cellular phenotypes. The trained APP can be used for division of identified cells into the identified phenotypes. The phenotypes can be counted and further visualized in a Phenotypic Matrix, Phenotypic Profile and/or tSNE plots for a better understanding of the data.

Staining Protocol

Akoya Biosciences’ Opal Multiplex IHC Detection Kits [1,2] are used.


This APP was developed for Akoya Biosciences, Inc., a company dedicated to the field of multiplexed biomarker analysis and immunofluorescence tissue analysis.


1. Akoya Biosciences, Inc. PhenoImager Assays (Last updated: April 2023)

2. Lu, S. et. al., Comparison of Biomarker Modalities for Predicting Response to PD-1/PD-L1 Checkpoint Blockade: A Systematic Review and Meta-analysis, JAMA Oncology 2019, 5 (8), 1195-1204, DOI

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