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Spatially resolved, multiplexed digital characterization of
protein and RNA expression in FFPE tissue sections
Abstract
Spatially-resolved Multiplexed Profiling
Proof of Concept Example
Imaging-based techniques are powerful tools for analyzing protein and mRNA
expression and localization within the tumor microenvironment. However, they
typically suffer from a number of challenges, including lack of dynamic range,
difficult quantitation, and labor intensive workflow for very limited multiplexing.
We have developed a novel platform based on the nCounter® barcoding
technology that enables spatially resolved, digital characterization of proteins and
mRNA in a highly multiplexed (up to 800-plex) assay. The assay relies upon
probes coupled to photocleavable oligonucleotide tags which are released from
discrete regions of the tissue using focused through-objective UV (~ 365nm)
exposure. An automated prototype capable of imaging, selective illumination using
a DMD (digital mirror device), and sample collection was developed by modifying
a standard microscope. Cleaved tags are quantitated in an nCounter® assay, and
counts are mapped back to tissue location, yielding a spatially-resolved digital
profile of analyte abundance.
Assay features:
• High resolution (currently ~ 1 to 4 cells)
• All digital counting, with large dynamic range (> 105)
• High multiplex (currently 30, but scalable with no change in instrumentation to 800)
• Simple workflow
• Compatibility with FFPE
• No secondary antibodies or amplification reagents
NanoString Technologies 530 Fairview Avenue North, Seattle, WA 98109 In situ Protein Profiling on FFPE
(1) To establish overall
tissue morphology, a
Tonsil sample was
imaged using 2-color
fluorescence of Ki-67
(cell proliferation
marker) and CD3
(immune cell marker)
(2) Various regions of
interest (ROIs) (400
µm x 300 µm) were
then selected for
detailed molecular
profiling with an
oligo-antibody
cocktail already
bound to the sample
(3) UV cleavage of selected ROIs allows multiplex digital profiling
(nCounter counts)
PanCK (tumor marker) and CD45 (immune cell marker).
(4) Four representative ROIs and digital counts
obtained for CD3 and Ki-67. Regions include a
germinal center with high levels of Ki-67 (#5), a
germinal center with moderate levels of Ki-67 and
CD3 (#9, “mixed”), a non-germinal center with high
levels of CD3 (#1), and a connective tissue region
with low levels of Ki-67 and CD3 (#12, “control”).
Masking workflow for automated profiling of tumor
Tissue microarray containing Grade 3 colorectal cancer
epithelium (PanCK, magenta) and T cells (CD3, green)
ROIs in the same field of view
(5) Hierarchically clustered heatmap of
standardized data (across targets) to
identify regions and targets with
similar profiles.
Digital Spatial Profiling
Highly Multiplexed molecular profiling with Optical Barcodes
Mechanism of action of DSP microscope verified:
Spatially-resolved detection down to a single cell
Linearity and Limit of Detection (LOD)
Two ROIs (#1 and #5) contain regions that resemble tertiary lymphoid
structures (regions within dashed lines).
CCRF-CEM cell pellet
In situ RNA Profiling on FFPE
showing areas of various
ROIs illuminated.
A fluorescently conjugated
secondary antibody
targeting antibodies against
CD3 and CD4 were used to
visualize cell membranes.
Overall Workflow
(1) Process: FFPE tissue slide
incubated with a cocktail of oligo
conjugated antibodies
Counts obtained for the same ROIs
Circles with a diameter
from 650 µm to 50 µm
Her2 3+ Breast cancer sample
(2) View: Regions of interest (ROIs)
Tonsil sample
are identified with visible lightbased imaging
(3) Profile: Selected ROIs are
chosen for high-resolution multiplex
profiling, and oligos from the
selected region are released upon
exposure to UV light.
Ki-67 (cell proliferation marker) and PanCK (tumor marker)
(4) Collection: Photocleaved
oligos are then collected via a
microcapillary tube and stored in a
microplate well
(5) Digital counting:
Photocleaved oligos from the
spatially-resolved ROIs in the
microplate are hybridized to 4-color,
6-spot optical barcodes, enabling up
to ~1 million digital counts of the
protein targets (distributed across all
targets) in a single ROI using
standard NanoString nCounter®
instruments
LOD = background counts average + 2 x standard deviation
Single cell Analysis
Tonsil
Instrument Setup
(1) Sample tissue slide
(2) Microscope stage
(3) Buffer-gasket
(4) Objective
(5-6) Band pass filters
(7) LED light engine
(8) CMOS camera
(9) Digital mirror device (DMD) module
(10) DMD array
(11) UV LED
(12-13) Dichroic mirror
(14) Micro capillary
(15) Micro-well plate
(16) System fluid line
(17) Syringe pump module
(18) Reagent bottles
(19) Inlet/outlet line for washing
(20) Wash station
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
www.nanostring.com | [email protected] |
@nanostringtech
© 2016-2017 NanoString Technologies, Inc. All rights reserved. Patents pending.
NanoString, NanoString Technologies, the NanoString logo, nCounter, 3D Biology, and nSolver are registered
trademarks or trademarks of NanoString Technologies, Inc., in the United States and/or other countries.
Profiling results of Her2 and RPS6 protein and RNA obtained
on serial sections
Profiling results of CD45, CD20, and cytokeratin protein and RNA
stained with CD3 and CD4 rabbit
antibodies (T cell markers) and
CD19 and CD20 mouse antibodies
(B cell markers) and visualized with
goat anti-mouse and goat anti-rabbit
antibodies labeled with different
fluorophores
• New application of NanoString barcoding enables multiplexed, digital,
spatially-resolved protein/RNA profiling in FFPE tissue
Counts from single cell illumination of 12
T cells and 12 B cells.
• Variable ROI selection permits profiling of 650 um diameter areas down to
single cells.
PBMC cell pellet
with various 1, 2, or 4 cell
ROIs
Conclusion
• Detection of RNA from tissue permits characterization of up to 800 targets
without relying on commercial antibodies.
• Correlation between RNA and protein measurement is high validating the
performance of system
• In situ multiplexed protein/RNA profiling in tumor microenvironment may
enable novel biological insights for research and accelerate drug
development
Researchers interested in participating in NanoString's technology access program
for its Digital Spatial Profiling technology should contact us at [email protected].
AUTHORS: Dwayne Dunaway, Jaemyeong Jung, Yan Liang, Chris Merritt, Giang Ong,
Kristina Sorg, Isaac Sprague, Sarah Warren, Philippa Webster, Joseph M Beechem*
NanoString Technologies, Inc.
*Correspondence: [email protected]
AGBT, February 13th – 16th, 2017