Box 2: Multi-omic analysis
methods
Tissue spatial
transcriptomics
Tissue spatial transcriptomics allows the characterization of gene
expression profiles keeping the tissue’s spatial architecture intact.
Multiple techniques have been developed for spatial transcriptomics,
mainly based on in situ hybridization, in situ capturing, in situ sequencing or microdissection (78).
Fluorescent in situ hybridization (FISH)-based methods exploit
the hybridization of fluorescent-labeled RNA-targeting probes with
pre-defined transcripts of interest, followed by imaging, visualization,
and quantification, which however is limited to the simultaneous
detection of a small number of transcripts. Higher efficiency in mRNA
detection has been reached with the usage of array-based in situ capturing methods. These arrays have attached barcoded oligonucleotides
that capture, through complementarity, the mRNAs present in the sample.
Capture is followed by reverse transcription to cDNA and NGS, allowing
the detection of more than 10,000 targets (79, 80). The widely used
Visium technology is an example of this approach (Visium spatial gene
expression, 10X Genomics) (81).
Recent technologies allow to explore the transcriptome of specific
regions of interest in FFPE samples through microdissection. The GeoMx
Digital Spatial Profiler by Nanostring allows in situ capture of
mRNAs using fluorescent-tagged RNA probes, which are linked to
UV-photocleavable DNA oligonucleotides of known sequence. The
fluorescent-tagged RNA probes are also known as imaging reagents since
they will generate a fluorescent image that allows tissue visualization
of regions where a specific mRNA is expressed. Once the investigator
selects their regions of interest, these areas are exposed to UV light
that cleaves the DNA tags in a region-specific manner. This releases
indexing oligos that are collected via microcapillary aspiration and
dispensed into a microplate and subject to Nanostring mediated counting,
or NGS (82). The RNA from FFPE fixed samples very commonly suffers
degradation. However, Visium and GeoMx technologies can retrieve good
amounts of information from these tissue samples.
Tissue spatial proteomics
The most popular methods of tissue spatial proteomics have the advantage
that FFPE samples can be used and, therefore, precious pathological
archives can be studied. Strategies for exploring spatial proteomics are
based on (i) immunofluorescence, (ii) imaging mass cytometry by time of
flight and (iii) sequencing (79). Tissue cyclic immunofluorescence
(tCycIF) is an immunofluorescence-based strategy. tCycIF uses FFPE tumor
and tissue specimens mounted on glass slides that undergo staining
cycles. In every cycle, the specimens are stained with
fluorochrome-conjugated antibodies and imaged, followed by chemical
inactivation of fluorochromes after each round of immunofluorescence
(83). Conventional wide-field, confocal or super-resolution microscopes
can be used for image acquisition. After multiple rounds of imaging, a
final high-dimensional representation of all the images is assembled
into a unique image using computational strategies. The final
high-dimension image can be segmented into all individual cells
composing the tissue to give single-cell resolution. Neighborhood
analysis can also be performed to quantify cell-cell interactions. Of
note, tCycIF does not require proprietary reagents, is robust and is a
more economical option compared to other spatial proteomics strategies.
CyTOF is a mass spectrometry-based method. In this technology, cellular
proteins are detected using antibodies that are conjugated to isotopes
from the lanthanide series of rare metals. The sample is imaged using
the Hyperion Imaging SystemTM, where these
metal-tagged antibodies are laser ablated from regions of interest in
the tissue and each ionized metal tag is detected based on differences
in their mass instead of the wavelength emitted by a fluorochrome. This
technology eliminates the autofluorescence inherent to biological
specimens, since the rare metal tags with which the antibodies are
conjugated are not present in cells. Also, compensation or background
elimination is not needed, since there is no overlap among the signal
produced by the ionized metals. In this technology, FFPE samples can be
stained with an entire panel of multiple antibodies in a single scanning
round without the need for multiple staining and washing cycles. The
image is analyzed using a proprietary software package (84).
Finally, GeoMx Digital Spatial Profiler by Nanostring can be adapted for
detection of proteins instead of transcripts (described above). In this
setting, the FFPE tissues are immunostained with UV-photocleavable
oligonucleotide-labeled antibodies. The spatial location of proteins is
again achieved by exposure of the region of interest to UV light that
photocleaves the oligos, followed by retrieval of the oligos and
sequencing. This provides an average count of oligonucleotides in every
region of interest (82, 85).
Tissue spatial genomics
Technologies for spatial resolution of the genome that can preserve
tissue architecture are less well developed. Nonetheless, using
spatially resolved DNA sequencing will finally deliver information on
the process of clonal evolution of solid tumors and provide a timeframe
for when a specific mutation appeared. FFPE samples are especially
problematic since DNA is very commonly degraded in these specimens (86).
Slide-DNA-seq is one new technology that works with cryosectioned intact
tissues. Slide-DNA-seq uses cover slip arrays coated with 10 μm
DNA-barcoded polystyrene beads, each containing a unique DNA barcode
corresponding to its spatial location in the cover slip. This is meant
to provide spatial indexing. Then, a 10-μm-thick fresh-frozen tissue
section is placed onto the barcoded bead array, treated with HCl for
histones removal and treated with the transposase Tn5 to generate DNA
fragments that will be flanked with sequencing Illumina adapters. The
barcodes are photocleaved from the beads and the resulting DNA
sequencing library is amplified by PCR (86, 87).