TITLE: NanoString GeoMx DSP TMA-TNP Phase 4 Protein assay
AUTHORS: Jinho Lee, Gabriel Zangirolani, Koei Chin, Heidi S Feiler, Christopher Corless

[DESCRIPTION]

This protocol outlines the NanoString GeoMx DSP phase 4 protein assay that was applied in the Human Tumor Atlas Network (HTAN) Tissue MicroArrary (TMA)-TransNetwork Project (TNP).

The TMA-TNP evaluates various characterization and analytics methodologies on a large array of breast tumor samples representing a broad spectrum of disease state and subtype. A commercially available anonymized breast tumor TMA was purchased and serial sections were distributed.  Participating HTAN Centers characterized the FFPE specimens using various imaging platforms and generated a spatially resolved cell type/state census using each center’s method of choice. Data was recorded in a common repository to enable joint analysis.

The protocol that immediately precedes this one for TMA-TNP Phase 4 can be found at:  dx.doi.org/10.17504/protocols.io.ewov1o7wolr2/v1.  It describes FFPE block serial sectioning, slide processing and TMA sample distribution.

In this protocol, the DSP protein assay was performed with the following human marker panels:  Immune cell profiling core, Immune activation status, Immune-Oncology (IO) Drug target, Cell death, Pan-tumor, MAPK and PI3K/AKT panels including OHSU custom panel (Cell cycle and DNA damage).  A total of 85 protein targets were evaluated in the TMA samples. Two compartments (Tumor and Stroma) in each TMA core were analyzed to determine cell-to-cell interactions in the tissues.




[STEPS]
SECTION: FFPE slide sample preparation
1. FFPE slide preparation
SECTION: FFPE slide sample preparation
1.1. Bake FFPE slides at 60 °C for 60 min .

SECTION: FFPE slide sample preparation
1.2. Deparaffinize by sequential incubation with Xylene (3 min  twice), 100% EtOH (1 min), 95% EtOH (1 min ) and 70% EtOH (1 min ).

SECTION: FFPE slide sample preparation
1.3. Briefly rinse the slides with diH2O (distilled water) and incubate with 1x Citrate antigen retrieval buffer (Epitope retrieval buffer, pH 6.0) for 15 min at high pressure in a pressure cooker.

SECTION: FFPE slide sample preparation
1.4. Take out the slides from the pressure cooker and let them cool down to room temperature around 25 min. 

SECTION: FFPE slide sample preparation
1.5. Wash the slides 5 times with TBS-T for 2 min each.

SECTION: Antibody panel incubation
2. DSP protein panel and visualization marker incubation




SECTION: Antibody panel incubation
2.1. Mark off the entire tissue section with a hydrophobic pen to create a reagent boundary.
SECTION: Antibody panel incubation
2.2. Block the slides for 60 min at room temperature using the Nanostring blocking buffer (Buffer W)

SECTION: Antibody panel incubation
2.3. Freshly prepare each commercial protein panel in 1:25 dilution and each visualization marker (PanCK and CD45) in 1:40 dilution with Buffer W to a volume of 220 µL per slide.

For the custom antibody panel,  the final concentration at 250 ng/ml per antibody is calculated and adjusted to the final volume of 220 µL.

SECTION: Antibody panel incubation
2.4. Incubate slides with all antibody panels and visualization markers for   at 4 °C. 

SECTION: Post fixation and DNA staining
3. Post fixation
SECTION: Post fixation and DNA staining
3.1. After antibody incubation, wash the slides 3 times with TBS-T for 3 min each.

SECTION: Post fixation and DNA staining
3.2. Fix the slides with 10% NBF (Neutral buffered Formalin) for 30 min at RT.

SECTION: Post fixation and DNA staining
3.3. Wash 3 times with TBS-T for 3 min  each, then stain the samples with SYTO-13, prepared by 1:10 dilution in TBS for 15 min  at RT.

SECTION: Post fixation and DNA staining
3.4. Slides are briefly washed 2 times with TBS-T for 3 min  each.

SECTION: Loading samples to DSP
4. DSP run preparation
SECTION: Loading samples to DSP
4.1. Scrape the hydrophobic barrier off with a scalpel or a straight-edged razor.
SECTION: Loading samples to DSP
4.2. Place slides  on the GeoMx slide tray and clean the back of the slide with 70% EtOH.
SECTION: Loading samples to DSP
4.3. Once the gasket is sealed, place 6 mL of Buffer S on slides.
SECTION: Loading samples to DSP
4.4. Load the GeoMx slide tray onto the DSP platform.
SECTION: GeoMx operation
5. Slide scan, ROI (Region of Interest) selection and AOI (Area of Interest) segmentation.
SECTION: GeoMx operation
5.1. Log onto GeoMx software and start with “New / Continue Run”. 
SECTION: GeoMx operation
6. ROI selection and AOI segmentation 
SECTION: GeoMx operation
6.1. After scanning is done, each color channel intensity is adjusted to show visualization markers along with tissue or cell line property. 
SECTION: GeoMx operation
5.2. After loading slides with the collection plate information, DSP is ready to scan slides.
SECTION: GeoMx operation
5.3. A slide scan name is created and panel/visualization marker information is selected as below:
 In the Probe Reagent Kit field, in any order select Human Immune Cell Profiling Protein Core, Human Immune Activation Status Protein, Human IO Drug Target Protein, Human Cell Death Protein, Human MAPK Signaling Protein, Human Pan-Tumor Protein, Human PI3K/AKT Signaling Protein, and SQ-35430 OHSU Custom

Select the FITC/525 nm, Cy5/568 nm and Texas Red/615 nm channels.
For FITC/525 nm, select SYTO 13 as fluorophore, DNA as biological target and enter 50 as exposure time.
For Cy5/568 nm, select Alexa 532 as fluorophore, PanCK as biological target and enter 300 as exposure time.
For Texas Red/615 nm, select Alexa 594 as fluorophore, CD45 as biological target and enter 300 as exposure time. 
Select FITC/525 nm as focus channel 
SECTION: GeoMx operation
5.4.  When the scan area for each slide has been adjusted with sensitivity setting, select Scan.
SECTION: GeoMx operation
6.2. Each ROI is determined and selected by pathologist's guide, and drawn with circle (maximum 660um radius), rectangle (maximum 660x785um) or polygonal shape (maximum 660x785um).

    TNP TMA slide contains total 88 cores. 
    Due to the limitation of scan area in the slide loading slot,  only 44 cores per slide can be scanned and collected.
    Two TMA slides (88 cores were embedded and slightly shifted to either left or right side of slide to cover half of 88 
    cores in each slide)  are required to collect all cores.
SECTION: GeoMx operation
6.3. In the segment menu, 2 segmentation classes (Tumor, stromal and others) are added and  parameters are set in the following order:

For Tumor segmentation (Segment 1), Alexa 532 (PanCK) is set to positive ("+") and the others set to ignore ("0") for the tumor collection

For stromal segmentation (Segment 2), Alexa 532 (PanCK), Alexa 594 (CD45) and FITC 525 (SYTO 13) were set to ignore ("0") to collect all other regions  

Then click Generate Segments.
SECTION: GeoMx operation
6.4. Once all segments are automatically generated, each channel parameter needs to be manually re-adjusted with pathologist's input to confirm if the segmentation is correctly done.

Caution: less than 20 cells in each segment is removed from collection due to threshold for low signal. 
SECTION: GeoMx operation
6.5. Once all AOI segmentation is complete, the "Exit Scan Workspace" button icon is clicked to approve ROI selection and samples are collected in a 96-well plate.
SECTION: Hybridization
7. Hybridization 
SECTION: Hybridization
7.1. After completion of sample collection, the 96-well plate is finalized, removed from the DSP and transferred to the PCR thermocycler.

DNA oligo samples in 96-well plate are completely dehydrated at 60 °C for 60 minin a PCR thermocycler. 

Caution:  the plate lid of the PCR thermocycler should be opened completely to avoid the contamination of DNA oligo from evaporation during this drying step.

SECTION: Hybridization
7.2. Add  10 µL  of DEPC-treated water to each well and resuspend the DNA oligo samples for 30 min  at RT.

SECTION: Hybridization
7.3. (1) Add and mix well 12 µL  of each Probe A per panel to a 1.5ml Eppendorf tube (labeled Probe A tube)
Since  7 commercial panels and 1 custom panel (consists of 2 Probe A ) are used, the total volume is 108 µL  (= 12ul x 9 Probe A) in the Probe A tube.
(2)  Add and mix well 12 µL  of Probe B (Universal probe) with 87 µLof DEPC water in a fresh 1.5ml Eppendorf tube (labeled Probe B tube, total volume is 99 µL).
(3) Calculate and add  80 µLof hybridization buffer per row into a fresh 1.5ml Eppendorf tube (labeled Master mix tube).  For 44 core samples, we collected a total of 8 rows in the collection plate so a total of    640 µLof hybridization buffer was added (80 x 8 rows).

The Probe/Master Mix is made by adding 64 µLfrom  the Probe A tube  and 64 µLfrom the Probe B  into the  Master mix tube (64 µL + 64 µL + 640 µL) for a total of 768 µL.   
 
SECTION: Hybridization
7.4. The Hybridization CodeSet for each row (A-H) is taken out from -80 °C  and thawed at RT
84 µL  of Probe / Master mix  is added to each hybridization Codeset and mixed well.

SECTION: Hybridization
7.5. Prepare the hybridization plate separately (96-well plate)
8 µL  of hybridization CodeSet mix was added to each well per each row in the hybridization plate.
3 µL  of DNA oligo samples from the rehydrated plate (out of 10ul) was mixed into each hybridization Codeset (8ul + 3ul = total 11ul / well).

SECTION: Hybridization
7.6. Seal the hybridization plate with aluminum foil using the microplate heat sealer and briefly spin it down in a centrifuge at 2000g.
SECTION: Hybridization
7.7. Samples are hybridized at 67 °C for 960 min  in the PCR Thermocycler and then kept at 4 °C until loading in the nCounter MAX system.
SECTION: nCounter reading
8. nCounter preparation and reading
SECTION: nCounter reading
8.1. In the DSP server, the collection plate is finalized and the library preparation file for nCounter loading is downloaded.
The sample loading volume is determined (it will vary) according to the area size calculation of total ROI/AOI in each column.
The Samples from each row (A-H) are collected and transferred into a 12-well strip PCR tube using a 12-channel multi-pipette.
SECTION: nCounter reading
8.2. Briefly, the samples are spun down in the 12-well PCR strip and then transferred to Nanostring's MAX Prep-station to load samples onto the cartridge with the standard sensitivity setting.
SECTION: nCounter reading
8.3. The cartridge is transferred to nCounter to read the counts with defined CDF (CodeSet Design Form) setting downloaded from the DSP plate information.
SECTION: nCounter reading
8.4.  The RCC (Reporter Code Count) file from nCounter is downloaded to your PC and imported into the DSP server.
SECTION: DSP data analysis
9. QC DSP data and analysis 
SECTION: DSP data analysis
9.1. Select and queue the slides to analyze  using "New Analysis"  in the DSP server.
SECTION: DSP data analysis
9.2. Determine the New Analysis file name and save it  in the designated folder.
SECTION: DSP data analysis
9.3. Open an analysis file and perform the QC  with preset parameters.
SECTION: DSP data analysis
9.4. QC passed samples are processed and the QC file (CSV) is downloaded to a PC for further analysis.


Comment: All documents related with GeoMx DSP run can be found at https://university.nanostring.com/page/document-library