PhenoCycler (formerly known as CODEX)

Services

CPTR offers highly multiplexed imaging using the PhenoCycler platform. The technology allows for detection of a maximum of 40 protein targets in the same tissue section at single cell resolution, with quantitative data for each protein. Since 2018 CPTR has been part of the PhenoCycler early access program. Now, the technology is available as a service as collaborative support with cost recovery for the consumables.

Tissue Types

CPTR has extensive experience with imaging fresh frozen tissues, as well as working knowledge and experience with FFPE and TMAs.

• Mouse Fresh Frozen
• Human Fresh Frozen
• Human FFPE
• TMAs (tumor microarrays)

Tissue Preperation & Sectioning

Users are responsible for harvesting and preparing tissue sections onto coated coverslips for PhenoCycler experiments. Use poly- L-Lysine coated coverslips for fresh frozen and Vectabond or APES treated coverslips for FFPE tissues. The coated coverslips are provided by CPTR.

For best quality sections, special care should be taken both for collection and fixation of tissues. For these steps follow general histology procedures (Akoya’s manual for freezing specimens and manual for frozen tissue sectioning).

The following websites have technical notes for best practices for tissue processing:

• http://www.ihcworld.com/_protocols/histology/frozen_section_technique_1.htm
• http://www.ihcworld.com/_protocols/histology/frozen_section_technique_2.htm
• http://www.ihcworld.com/_protocols/histology/frozen_section_technique_3.htm
• http://www.ihcworld.com/_protocols/histology/frozen_section_technique_4.htm

Tissue size: The recommended tissue thickness for fresh frozen is 8 um (max 10 um) and for FFPE the recommendation is 4-5 um. The tissue should not exceed 1.6 x 1.6 cm. The tissues should be placed in the middle of the coverslip as much as possible and free of folds and wrinkles.

Molecular Histopathology and Histoserv have experience with this type of tissue sectioning.

Number of sections: CPTR requests 3-5 tissues sections for each sample and recommends having H&E staining on adjacent slides. If you need help with slide scanning, CPTR coordinates with LGCP for automated scanning and images are uploaded into HALO image analysis platform for viewing and collaboration.

Detected targets by PhenoCycler: The maximum number of targets that can currently be detected is 40 – driven by the number of commercially available barcodes. The antibody panel comprises of commercial and custom conjugated antibodies.

The commercial antibody panel is different based on the species and tissue type: mouse FF, human FF, human FFPE (CPTR Target List)

Custom antibody conjugation:  Depending on the project desired targets, antibodies can be custom conjugated and added to the PhenoCycler panel. Users are responsible for selecting and validating the specific antibody clone for the target of interest using PhenoCycler staining and fixation conditions (Protocol).  After selection of the antibody clone and identifying the barcode that fits the panel, CPTR performs the antibody conjugation and validation on PhenoCycler. The minimum amount of antibody required is ~ 70 ug, in a carrier protein- and glycerol-free format. Extra tissue sections with known positive and negative staining should be provided for testing custom antibodies.

Note: custom antibody development and thorough validation takes time and can delay a project.

Tissue staining, imaging, and processing: After finalizing the antibody panel, CPTR staff performs tissue staining and imaging following recommended best practices from Akoya Biosciences, using commercial PhenoCycler microfluidics connected to a Keyence microscope. The whole tissue section or different regions are imaged using a 20X objective acquiring multiple Z-stacks. Imaging time depends on the area of the tissue and number of targets and can take 1-2 days per sample. Raw images are processed to generate aligned and improved 2D stitched 16-bit tiff images.

Results delivered: A summary power point slide deck generated from PhenoCycler processor with experimental details. Stitched tiff images for each individual signal, as well as composite images, are uploaded into HALO for viewing, qualitative and single cell level quantitative analysis. Tile by tile raw or processed images are delivered upon request.

Data Analysis: For basic analysis, CPTR recommends HALO (IndicaLabs) image analysis software for area, single cell level and spatial analysis. The cloud version of the software is available for investigators through CBIIT and includes AI enabled tools for tissue classifications and cellular/nuclear segmentation. CPTR staff provides guidelines and can direct users to training resources for PhenoCycler image analysis. Single cell level quantitative data can be exported as csv files for further analysis in any third-party software and/or NIDAP (link) for further analysis.

Price: The reagent cost for staining a PhenoCycler slide is $300 + n*19, where n is the number of commercial or custom antibodies used. For example, the cost for using a 20-ab panel is $680, using a 30-ab panel is $870. The price of PhenoCycler staining is eligible for 50% OSTR subsidy up to $10,000 per lab per fiscal year. The cost for CPTR custom conjugated antibody is the same as commercial. Custom antibody development is a shared cost between the investigator and CPTR. The user buys the antibody and performs the pre- and post-conjugation validation by conventional immunofluorescence (IF), CPTR performs the conjugation and validation on PhenoCycler.

If interested in PhenoCycler-Fusion 2.0 technology for a project:  The first step is to contact the facility at kedein@mail.nih.gov and briefly describe the project. Next, we will schedule a meeting to discuss your needs, clarifying the sample type, number and antibody panel needed and ask you to submit a project proposal at https://cptr.ccr.cancer.gov/projm/. Upon project approval you will be asked to fill out the sample submission form and prepare the samples.