NanoString GeoMx DSP TMA-TNP Phase 4 Protein assay
Heidi S Feiler, Koei Chin, Jinho Lee, Gabriel Zangirolani, Christopher Corless
Abstract
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
FFPE slide sample preparation
FFPE slide preparation
Bake FFPE slides at 60°C for 1h 0m 0s .
Deparaffinize by sequential incubation with Xylene (0h 3m 0s twice), 100% EtOH (0h 1m 0s), 95% EtOH (0h 1m 0s ) and 70% EtOH (0h 1m 0s ).
Briefly rinse the slides with diH2O (distilled water) and incubate with 1x Citrate antigen retrieval buffer (Epitope retrieval buffer, 6.0) for 0h 15m 0s at high pressure in a pressure cooker.
Take out the slides from the pressure cooker and let them cool down to room temperature around 0h 25m 0s.
Wash the slides 5 times with TBS-T for 0h 2m 0s each.
Antibody panel incubation
DSP protein panel and visualization marker incubation
Mark off the entire tissue section with a hydrophobic pen to create a reagent boundary.
Block the slides for 1h 0m 0s at room temperature using the Nanostring blocking buffer (Buffer W)
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 220µL 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 220µL.
Incubate slides with all antibody panels and visualization markers for at 4°C.
Post fixation and DNA staining
Post fixation
After antibody incubation, wash the slides 3 times with TBS-T for 0h 3m 0s each.
Fix the slides with 10% NBF (Neutral buffered Formalin) for 0h 30m 0s at RT.
Wash 3 times with TBS-T for 0h 3m 0s each, then stain the samples with SYTO-13, prepared by 1:10 dilution in TBS for 0h 15m 0s at RT.
Slides are briefly washed 2 times with TBS-T for 0h 3m 0s each.
Loading samples to DSP
DSP run preparation
Scrape the hydrophobic barrier off with a scalpel or a straight-edged razor.
Place slides on the GeoMx slide tray and clean the back of the slide with 70% EtOH.
Once the gasket is sealed, place 6 mL of Buffer S on slides.
Load the GeoMx slide tray onto the DSP platform.
GeoMx operation
Slide scan, ROI (Region of Interest) selection and AOI (Area of Interest) segmentation.
Log onto GeoMx software and start with “New / Continue Run”.
After loading slides with the collection plate information, DSP is ready to scan slides.
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
When the scan area for each slide has been adjusted with sensitivity setting, select Scan .
ROI selection and AOI segmentation
After scanning is done, each color channel intensity is adjusted to show visualization markers along with tissue or cell line property.
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.
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 .
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.
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.
Hybridization
Hybridization
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 1h 0m 0sin 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.
Add 10µL of DEPC-treated water to each well and resuspend the DNA oligo samples for 0h 30m 0s at RT.
(1) Add and mix well 12µL 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 108µL (= 12ul x 9 Probe A) in the Probe A tube.
(2) Add and mix well 12µL of Probe B (Universal probe) with 87µLof DEPC water in a fresh 1.5ml Eppendorf tube (labeled Probe B tube, total volume is 99µL).
(3) Calculate and add 80µLof 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 640µLof hybridization buffer was added (80 x 8 rows).
The Probe/Master Mix is made by adding 64µLfrom the Probe A tube and 64µLfrom the Probe B into the Master mix tube (64µL + 64µL + 640µL) for a total of 768µL.
The Hybridization CodeSet for each row (A-H) is taken out from -80°C and thawed at RT
84µL of Probe / Master mix is added to each hybridization Codeset and mixed well.
Prepare the hybridization plate separately (96-well plate)
8µL of hybridization CodeSet mix was added to each well per each row in the hybridization plate.
3µL of DNA oligo samples from the rehydrated plate (out of 10ul) was mixed into each hybridization Codeset (8ul + 3ul = total 11ul / well).
Seal the hybridization plate with aluminum foil using the microplate heat sealer and briefly spin it down in a centrifuge at 2000g.
Samples are hybridized at 67°C for 16h 0m 0s in the PCR Thermocycler and then kept at 4°C until loading in the nCounter MAX system.
nCounter reading
nCounter preparation and reading
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.
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.
The cartridge is transferred to nCounter to read the counts with defined CDF (CodeSet Design Form) setting downloaded from the DSP plate information.
The RCC (Reporter Code Count) file from nCounter is downloaded to your PC and imported into the DSP server.
DSP data analysis
QC DSP data and analysis
Select and queue the slides to analyze using "New Analysis" in the DSP server.
Determine the New Analysis file name and save it in the designated folder.
Open an analysis file and perform the QC with preset parameters.
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
