GeoMx
Digital Spatial Profiler®

Grant Application Package

Section 1: Information to provide
in grant application

1.

Throughput: >10 sections per day

the DSP scans the slides and presents a high quality image
for region of interest selection. The oligonucleotide tags
are released from user-selected regions of the tissue
with focused UV light based on the user region andsegmentation selections. The digital micromirror deviceof the DSP instrument tunes the UV light with 1 micron
resolution. This allows great flexibility for region selection
including irregularly shaped and non-continuous segments.
Released tags are quantitated with nCounter opticalbarcodes or an Illumina® next-generation sequencer (NGS).
Finally, counts are mapped back to the tissue location by
the software, resulting in a spatially-resolved digital profile
of protein or mRNA abundance.

2.

Multi-analyte: RNA and Protein

1.

3.

Single Cell Segment Resolution

4.

Minimum Segment Size Recommendation:• Target detection is dependent on the size andnumber of cells in segment and expression level• For small cells (e.g., T Cells or B cells): 10-20 cells
for protein, 50-200 cells for RNA• For large cells (e.g., astrocytes Neurons): 1-5 cells
for protein and 5-20 RNA

A 5-10µm thick formalin-fixed, paraffin-embedded(FFPE) or fresh frozen tissue section is stained witha cocktail of standard morphology markers (i.e.
fluorescent antibodies) combined with either oligotagged antibodies (protein) or oligo-tagged ISH probes(RNA). Oligo tags are attached to the antibody andISH probes via a UV-photocleavable linker. Staining canbe performed manually or an automated protocol isavailable on the Leica BOND RX.

2.

Regions of interest (ROI) are selected by the user on
the visualized tissue. Using fluorescent morphology
markers and our auto-segmentation algorithm, users
can further divide an ROI into individual segments, suchas specific tissue compartments or cell populations.
(Please refer to section: GeoMx DSP ROI selection).

3.

After ROI/segment selection, oligos from the selected
regions are released upon focused exposure to UV light.

4.

The photocleaved oligos are aspirated via a
microcapillary tube and stored in microplate wells.

5.

Lastly, the photocleaved oligos from the spatiallyresolved ROIs in the microplate are (1) quantified using
standard NanoString nCounter® or next-generation
sequencing Illumina® workflows.

Platform Overview
GeoMx® Digital Spatial Profiler (DSP) is the spatial biology
solution.
DSP enables morphology-driven, high-plex profiling on asingle FFPE or fresh frozen tissue section. DSP technology
combines standard immunofluorescence techniques withnCounter optical barcoding quantification. The system
specifications are as follows:

5.

6.

Multiplexing capability:• 96 targets with nCounter readout• 20,000+ targets with NGS readout
Quantitative: 6 logs of dynamic range

Technology Overview & WorkflowGeoMx® Digital Spatial Profiler (DSP) is a novel platform
developed by NanoString Technologies. DSP enables
spatially resolved, high-plex (10s -10,000s) digitalquantitation of proteins and mRNA in tissue. The assay
utilizes unique reagents (antibodies or RNA probes)
coupled to UV photocleavable oligonucleotide barcodes.
After incubation/hybridization of the GeoMx reagents alongwith visualization reagents to slide-mounted formalin fixedparaffin-embedded (FFPE) or fresh frozen tissue sections,

GeoMx DSP Workflow

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

2

GeoMx DSP Instrumentation and Assays
Spatial biology experiments with GeoMx require theDSP instrument for slide scanning, ROI selection, andoligo collection. Downstream of the DSP, there are two
instrumentation platforms for quantitative readout:
NanoString nCounter® platform or a standard library
preparation workflow for analysis on Illumina® NGS
platforms.
The GeoMx DSP platform is commercially available fromNanoString Technologies. DSP results correlate withthose from immunohistochemistry high concordance
is observed between DSP results generated using both
antibodies or RNA probes. Oligo conjugation does notinterfere with antibody or RNA probe specificity andonce conjugated ensures robust quantification of protein
and RNA abundance. Additionally, analytical validation
data has demonstrated multiplexing compatibility. Lastly,
any commercially-available antibody can be customlabeled with oligo tags and used in a DSP protocol.
(reference- internal data)

GeoMx DSP ROI Selection
The DSP software enables the user tremendous flexibility in the ROI selection strategy to profile tissue. In particular,
highlighted below are five unique profiling modalities designed to interrogate tissue samples.
The segmentation and cell-type specific approaches are achieved through the use of masks in software based on thefluorescent marker staining of the tissue (e.g. PanCK+ vs. PanCK-). UV light then specifically targets the ROI with multiple
illumination events, enabling the separate profiling of complex and interdigitated cell populations within a selected ROI (e.g.
tumor vs. stroma).

The DSP so_ware enables the user tremendous ﬂexibility in the ROI selec0on strategy to proﬁle 0ssue. In
par0cular, highlighted below are ﬁve unique proﬁling modali0es designed to interrogate 0ssue samples:
Geometric

Segmentation

Cell-Type SpecificContour

GriddedWhat is the heterogeneityof expression in different
regions of my tissue?

What is the expression profile ofdistinct biological compartments(e.g., Tumor-TME)?

What is the expression profile
of a specific cell population
in my tissue?

How does the immune
environment change on eitherside of an infiltrate boundary?

What novel targets areuncovered with deep mappingof a specific tissue region?

The segmenta0on and cell-type speciﬁc approaches are achieved through the making of masks in so_ware
based on the ﬂuorescent marker staining of the 0ssue (e.g. PanCK+ vs. PanCK-). UV light then speciﬁcallytargets the ROI with mul0ple illumina0on events, enabling the separate proﬁling of complex andFOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

3

GeoMx DSP Data Analysis
The GeoMx DSP comes with built-in data analysis software that enables users to easily visualize and interpret their data. The
intuitive interface displays high-resolution images side-by-side with quantitative profiling data. The connection between theimage and profiling data is preserved allowing users to interact with either dataset in real time. For example, users can selecta data point within a data visualization and the software will automatically highlight the ROI or segment from which theexpression data was generated. The data analysis workflow includes QC and normalization operations, as well as several datavisualization methods for various types of scientific and statistical inquiries.
Users can auto-generate figures such as heatmaps, box plots, and bar graphs, to visualize the distribution of protein orgene expression across multiple tissue compartments (e.g., tumor versus stroma); discover statistical changes in expression
between experimental groups (e.g., t-tests, linear mixed models) that can also be visualized in volcano and PCA plots;
visualize large data matrices using dendrograms and clustering analyses to discover biomarkers; and, perform pathway
analyses to understand disease mechanisms of action and identify novel targets.

GeoMx DSP Consumables
UV Photocleavable
linkerTargetComplimentary
Sequence

UV Photocleavable
linkerIndexing Oligo
Sequamce

Indexing Oligo
Sequence

Target RNA

The GeoMx DSP is a multi-analyte platform that canspatially resolve both protein and RNA from two serial
sections. The available and highly validated content covers
Immuno-Oncology, Immunology, and Neuroscience.
Protein content is designed for flexibility with a ~20-plex
core panel that includes additional positive and negativecontrols. Additional ~10-plex modules are available and canbe mixed and matched with the core based on an individual
researcher’s needs (up to 96-plex total). Users can also add
up to 5 custom targets through Abcam (>6000+ antibodiesto select from) or up to 10 custom targets of interest using
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

NanoString’s Protein Barcoding Service (more details forcustom antibodies below). A Core panel must always be run.
The following content is commercially available onnCounter readout:
1.

Human IO Protein – Immune Cell Profiling Core, IO
Drug Target Module, Immune Activation Status Module,Immune Cell Subtyping Module, Pan-Tumor Module,
Cell Death Module, AKT/PI3K Signaling Module, andMAPK Signaling Module
42.

Mouse IO Protein - Immune Cell Profiling Core, IO Drug
Target Module, Immune Activation Status Module,Immune Cell Subtyping Module, and Pan-Tumor Module3.

Human Neuroscience Protein – Neural Cell Profiling
Core, Alzheimer’s Pathology Module, Parkinson’sPathology Module, Alzheimer’s Pathology ExtendedModule, Autophagy Module, and Glial Cell Subtyping
Modulegene targets of interest. CTA launch expected 2020
H2.
3.

Human Whole Transcriptome Atlas (HuWTA) for NGS:18,000+ protein encoding mRNA targets encompasses
the whole human transcriptome and is relevant forany type of research. The HuWTA takes a targeted
approach to quantify transcripts in every biologicalpathway and across all gene expression levels. It has
been pruned of uninformative high expressors likeribosomal proteins from the target list, so that reads arecomprised of informative protein coding genes. Users
can add up to 30 custom targets of interest to countunique transcript variants such as isoforms, lnRNA andexogenous transcripts. HuWTA launch expected 2021
H1.

4.

Mouse Whole Transcriptome Atlas (MuWTA) for NGS:
18,000+ protein encoding mRNA targets encompasses
the whole mouse transcriptome and is relevant forany type of research. The MuWTA takes a targeted
approach to quantify transcripts in every biologicalpathway and across all gene expression levels. The
probes have been designed toward BalbC, C57B6, andNOD strains and has considerable cross-reactivity withother strains. It has been pruned of uninformative highexpressors like ribosomal proteins from the target list,
so that reads are comprised of informative protein
coding genes. Users can add up to 30 custom targets
of interest to count unique transcript variants such as
isoforms, miRNA and exogenous transcripts. MuWTA
launch expected 2021 H1.

All NanoString antibody panels are validated in a pre- andpost-conjugation screen, a sensitivity/specificity screen
using FFPE cell pellets, an interaction screen, and a TMA
screen using normal and tumor TMAs (see SupportingData).
Custom antibodies:
Custom Antibodies from Abcam: Abcam provides 1-5 custom
antibodies to extend GeoMx protein assays. To add custom
antibodies to a GeoMx protein assay, users place theorder directly through Abcam, using one of Abcam’s IHCvalidated recombinant rabbit monoclonal antibodies, andthe form found at: www.abcam.com/dsp. See the tablebelow for order requirements. Abcam custom antibodies aretested in a pre- and post-conjugation screen.
Custom Antibodies from NanoString: NanoString provides

1-10 custom antibodies through the NanoString Protein
Barcoding Services (PBS). To add custom antibodies ofinterest to a GeoMx protein assay, users submit a PBS
Antibody Submission Form found at: www.nanostring.
com/pbs Users purchase IHC-validated antibodies directlyfrom a vendor and ship them to NanoString for barcoding.
NanoString custom antibodies are tested for the presenceof the conjugated tag, but are not tested with IHC or furthervalidated.

All NanoString RNA probes are validated in a bioinformatic
screen and undergo technical QC(see Supporting Data).

Training and support
Guidance on further validating custom antibodies can beprovided upon request.
RNA Content:
1. Human IO RNA for nCounter– Immune Cell Pathways
Panel with 84 targets (including controls) and room toadd up to 10 custom targets (+2 additional controls)of interest for a total of up to 96 targets. Fixed panel
content covers characterization of the global immune
response, microenvironment immune activity, tumor
reactivity, and the Tumor Inflammation Signature.
2.

Human Cancer Transcriptome Atlas (CTA) for NGS:
1,800+ mRNA gene targets that cover important
aspects of immune response, tumor biology and themicroenvironment. The CTA curated content is deeply
annotated with 55 major pathways, immune cellsubsets, and clinically-derived gene sets such as theTumor Inflammation Signature (TIS) and PAM50 tumor
subtype geneset. Users can add up to 30 custom mRNA

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

Upon purchase and acquisition of the GeoMx DSP
instrument, NanoString will provide on-site installation andcalibration of the instrument by a trained field engineer.
Once the instrument has been qualified, a ProductApplication Scientist (PAS) will be on site to provide acomprehensive introduction to the GeoMx DSP platform.
The introduction will cover routine use of the instrument,
experimental design, project consultations, and data
analysis. In addition, the PAS will train the lab scientist toensure that they have hands-on experience in operating
the instrument, implementing the selected GeoMx protocol,and running an experiment from end-to-end . Lastly, thePAS will provide data analysis training with the GeoMx Data
Analysis software so that the customer is familiar with alldata analysis methods to process, analyze and interpret
their data. Upon successful completion of the training,
the customer can access support through the NanoString
GeoMx Customer Experience Team regarding experimentaldesign, instrumentation, consumables, and software.
5Tips to maximize success with the grant application process
Emphasize Collaboration
The GeoMx DSP software enables collaboration and secured remote-access. The GeoMx instrument makes it possible tosecurely access your data from anywhere and at anytime through a web browser. The software also provides administrators
the ability to assign roles and permissions to their team members and collaborators. The platform allows multiple users withinlarge labs, core facilities, or contract research organizations to securely manage and share their data.
Enabling the DSP workflow in your research environment will facilitate deep and meaningful collaborations between
scientists, pathologists, and biostatisticians. Hypothesis-driven study design, pathology-driven ROI selection, and data-driven
analyses will enable translational discoveries and contributions to the fields of science and medicine. Acknowledging thesecollaborative opportunities and providing details on how the working relationships will be fostered can increase the successof your grant application.
PATHOLOGIST:ROI Selection &Approval

LAB TECHNICIAN:

Instrument Operation

BIOSTATISTICIAN:

Analyzes Data

PRINCIPALSCIENTISTS:

Reviews Results

Benefits to local research environment
The digital spatial profiler (DSP) quantifies biology with spatial context. Any tissue that can be sectioned including FormalinFixed Paraffin Embedded (FFPE) and fresh frozen tissues can benefit from the platform. Our reagents are designed towards
human and mouse, but our reagents have appreciable and useful cross-reactivity with non-human primate and rattus
respectively. Information on cross-reactivity is available upon request. The DSP is relevant to any individual using tissue slides
to study biology, making the technology a valuable addition to the local research ecosystem.

Publications
The utility of the GeoMx DSP system is demonstrated already in over a dozen peer-reviewed publications, many in leading
journals. Please click here to view current GeoMx DSP Publications.

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

6Supporting Data
Reproducibilityof GeoMx DSP

Reproducibility of GeoMx DSPAll GeoMx antibodies and RNA Probes go through a robust validation process to ensure they will perform as expected andresearcherscan use
GeoMx reagents
off the highshelf without
having tospend
validating panels.
Twoserial tonsil
sections
demonstrateconcordance
with24 time
co-registered
ROIs. Additionally, serial sections
Reproducibilityof GeoMx
DSP
analyzed on the GeoMx DSP for protein (Figure 1) or RNA (Figure 2) expression demonstrate high reproducibility. For
additional reproducibility data see: Gupta et al., 2020 (Rimm); Merritt et al., 2020

Two serial tonsil sections demonstrate high concordance with 24 co-registered ROIs.

GeoMx® DSP High-Plex RNA was performed on 5 µm FFPE sections. High reproducibility wasdemonstrated between experimental replicates with serial sections with a pre-commercial panel of 4,998
FIGURE 1: Two serial tonsil sections demonstrate high concordance with 24 co-registered ROIs
probes.

GeoMx® DSP High-Plex RNA was performed on 5 µm FFPE sections. High reproducibility wasdemonstrated between experimental replicates with serial sections with a pre-commercial panel of 4,998
probes.

FIGURE 2: GeoMx® DSP High-Plex RNA was performed on 5 µm FFPE sections. High reproducibility was demonstrated between experimental replicates with
serial sections with a pre-commercial panel of 4,998 probes.

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

7Ordering Information
Ancillary Products
Product

Description

Catalog Number

GeoMx™ Digital Spatial Profiler Slide Tray

Additional 4-slide holder

GMX-DSP-TRAY

GeoMx Digital Spatial Profiler
Product

Description

Catalog Number

GeoMx Digital
Spatial Profiler

GeoMx™ Digital Spatial Profiler Analysis Instrument. Includes 1 year manufacturers warranty.

GMX-DSP-1Y

GeoMx™ Digital Spatial Profiler Analysis Instrument. Includes 1 year manufacturers warrantyand 1 year service contract.

GMX-DSP-2Y

GeoMx™ Digital Spatial Profiler Analysis Instrument. Includes 1 year manufacturers warranty
and 2 year service contract.

GMX-DSP-3Y

GeoMx™ Digital Spatial Profiler Analysis Instrument. Includes 1 year manufacturers warranty
and 3 year service contract.

GMX-DSP-4Y

GeoMx™ Digital Spatial Profiler Analysis Instrument. Includes 1 year manufacturers warranty
and 4 year service contract.

GMX-DSP-5Y

Materials Included
Item

Quantity

Logitech MK540 Advanced Keyboard, Mouse set

1LED Monitor 4K 8MP Color 27”

1Slide Holder Assembly

196 Well Plate Adapter

1Uninterruptible Power Supply

1Request a quote: GeoMx@nanostring.com

References
1Gupta S, Zugazagoitia J, Martinez-morilla S, Fuhrman K, Rimm DL. Digital quantitative assessment of PD-L1 using digital spatial
profiling. Lab Invest. 2020; epub ahead of print.

2

Merritt CR, Ong GT, Church SE et al. Multiplex digital spatial profiling of proteins and RNA in fixed tissue. Nat Biotechnol
202;38:586â€“599.

Terms and Conditions
Winning submissions will be selected by NanoString at its sole discretion. Award will be shipped in 2020. By submitting your application, you agree that NanoString may use your name, abstract title, abstract
and your submitted quote for marketing purposes. Additional Terms and Conditions apply, see grant application website for details.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
©2020 NanoString Technologies, Inc. All rights reserved. NanoString, NanoString Technologies, nCounter, and the NanoString logo are trademarks or registered trademarks of NanoString Technologies, Inc.,in the United States and/or other countries.
MAY 2020 MK0481

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