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3. Mapping proteins and transcripts in the TIME
3.1 Chromogenic and fluorescent immunodetection in FFPE
As with any chemical stain, H&E is poorly suited to the analysis of molecular features of
the TIME such as immune cell cluster of differentiation (CD) markers or cytokines.
Chromogenic IHC [54] on FFPE sections provides a robust method to leverage antibody
specificity to localize an enzyme to sites where a target antigen is most abundant in a tissue
section. There, the enzyme generates an insoluble precipitate upon reaction with a chromogenic substrate. In indirect IHC, the primary antibody is chemically conjugated to
horseradish peroxidase (HRP) or alkaline phosphatase (AP). However, it is far more
common to perform indirect detection using a secondary antibody-enzyme conjugate
(Fig. 3 , left). For example, a polyclonal antibody raised in a goat to recognize the constant
region (Fc) of rabbit immunoglobulin (a typical species for monoclonal or polyclonal
primary antibodies) is chemically crosslinked to HRP or AP to be used as a secondary
antibody for chromogenic IHC. Deparaffinized and rehydrated tissue sections are treated
with heat and moisture to depolymerize formaldehyde and reverse crosslinks in a process
299Spatial mapping of the tumor immune microenvironment
Fig. 3 Overview of chromogenic vs fluorescent IHC staining. (Left) Chromogenic IHC uses an enzymatic
reaction to deposit a colored compound near the target antigen. Visualization is typically accomplished using bright-field microscopy. (Right) Fluorescent detection uses a fluorophore to provide a
signal for visualization by fluorescence microscopy.
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dubbed "antigen retrieval" [55]. Then, the tissue is treated to prevent nonspecific antibody binding, and the primary antibody is applied to detect the biomarker. After washing, the secondary antibody conjugate is applied, and the unbound conjugate is washed
away. Then, incubation with the chromogenic substrate deposits precipitate to reveal the
distribution of the antigen. The standard chromogen, DAB (3,3
0
-diaminobenzidine),
yields a dark brown precipitate when incubated with HRP and hydrogen peroxide.
The brown color contrasts with the blue of hematoxylin which is typically used (with
or without eosin) as a chemical counterstain to provide morphological context, making
bright-field microscopy sufficient to visualize the distribution of chromogen precipitate.
Despite well-understood limitations [56], chromogenic IHC is a critical tool in the
clinical laboratory and remains the most important assay for examining the TIME in
patient samples. For example, a chromogenic IHC test to assay expression levels of
PD-L1 within a tumor is approved by the United States Food and Drug Administration
as a companion assay for ICI therapy using PD-1 or PD-L1 therapeutic antibodies [7].
However, patients whose tumors test positive for PD-L1 may not be significantly more
likely to respond to treatment [57]. In turn, beyond the level of staining, the distribution
and abundance of PD-L1 are not reliable indicators of therapeutic response.
Another challenge for chromogenic IHC is that antigens that are expressed at low
levels and/or damaged by suboptimal tissue processing may produce a weak or false negative signal. This may obviate detection by standard secondary antibody-enzyme conjugates but be compatible with detection using a polymerized secondary antibody-enzyme
construct, which has the potential to increase sensitivity several fold by facilitating signal
amplification [58]. This and several other methods have been used to increase the ratio of
HRP enzymes to the antigen [59]. Multiple signal amplification strategies are commercialized. For example, a biotinylated primary or secondary antibody may be detected with
greater sensitivity using a streptavidin-HRP conjugate than via direct crosslinking. Alternatively, in the ABC method, the biotinylated antibody is detected by polymers formed
in situ from avidin and biotinylated HRP. Linking polymers of HRP to secondary antibodies is a common approach for signal amplification, with multiple options and formats
commercially available.
Immunofluorescence (IF), or immunodetection with fluorescent antibodies, was
demonstrated by Albert Coons decades prior to the development of chromogenic
IHC [60, 61], yet has remained a niche technique for clinical pathology. Conjugating
primary or secondary antibodies to small-molecule fluorophores allows sensitive and
high-resolution detection of antibody binding using any modern fluorescence microscope (Fig. 3, right). Fluorescence brightness is proportional to the local concentration
of fluorophores, and optical filters allow independent excitation and detection of multiple
fluorophores bound to a single sample. Thus, IF is better suited to quantitative measurement and detecting colocalized antigens on a tissue section than chromogenic IHC.
However, clinical use of IF is limited by the availability of appropriate microscopes,
the lack of practical fluorescent counterstains to help appreciate morphology, and a high
autofluorescent background observed in many tissues.
3.2 Multiplex immunodetection in FFPE
Understanding the full complexity o f the TIME requires the ability to map many
biomarkers in the same tissue sample simultaneously. Often, it is satisfactory to perform
multiple IHC experiments in parallel, for example, to independently evaluate PD-L1
staining and enumerate total CD8
same tissue block. However, this approach is not compatible with the evaluation of
multiple antigens in single cells, such as determining whether CD8
pressing perforin or PD-1, which would provide information on cytotoxic T cell activation or exhaustion, respectively. Therefore multiplexing, or detection of multiple
biomarkers together on one tissue sample, is now accept ed as a critical tool for analysis
of the TIME [62].
While most IHC is performed using a single primary antibody, there are wellestablished methods to apply IHC to map two, three, four, or more antigens at once
in a single tissue section [63–66]. Multiplex staining in chromogenic IHC requires the
use of chromogens that yield different precipitate colors, potentially allowing a series
of visually distinct chromogens to be reacted in sequence with enzyme-conjugated secondary antibodies. However, limitations of the chemistry such as masking of antigens by
precipitates can confound reliable colocalization, even in double staining IHC. As alternative enzyme substrates to chromogens, tyramides form phenoxyl radicals that covalently modify nearby protein tyrosines. Performing cycles of indirect detection and
enzyme inactivation with a different fluorescent tyramide for each primary antibody,
up to seven or eight antigens can be mapped on a single tissue section [67]. This
so-called tyramide signal amplification (TSA) method has long been commercialized,
with substrates and kits now offered by several companies compatible with standard
autostainers. TSA may also have applications for multiplexed chromogenic IHC [68].
The Opal fluorescent TSA chemistry and multispectral imaging technology, developed
by Cambridge Research & Instrumentation and most recently acquired by Akoya, offers
a well-validated tool to examine patterns of up to seven TIME biomarkers on one section
[69–71]. TSA has been used productively over the past decade to explore determinants of
immunotherapy response, leading to dozens of publications (e.g., [71–73]).
For multiplexed IF, chemical fluorophores covering the spectrum from ultraviolet
excitation to far-red emission are commercially available as N-hydroxysuccinimide esters
that are readily conjugated to antibodies, offering the potential to distinguish dozens of
individual fluorophores or combinations at one time using spectral detection [74, 75].
Thus, using direct conjugation of fluorophores with distinct excitation and emission
spectra provides a simple and practical means to apply four or more fluorescently labeled
+
cytotoxic T cells on proximate sections from the
+
cells are also ex-
301Spatial mapping of the tumor immune microenvironment
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primary antibodies simultaneously and then map them independently. Multiplexed
detection of directly labeled antibodies is standard in flow cytometry, but direct IF in thin
sections is limited to abundantly expressed biomarkers with well-defined subcellular distributions. An interesting application of direct fluorescence localization is in the detection
of specific T cell receptors in tissue [76]. Here, in place of an antibody, a multivalent
probe is used to overcome the low affinity of binding of TCRs to peptide-MHC complexes. The capability to track multiple tumor-specific TCRs in situ is an important missing tool for TIME analysis.
Toward enhancing the sensitivity of detection in IF, many alternatives to covalent
antibody labeling with chemical fluorophores have been described. Bright fluorescent
nanoparticles such as quantum dots have long been used for multiplexing IF with moderate success [77]. Fluorescent polymers [78] are similarly bright but more chemically
tractable for conjugation.
Given the limitations of direct labeling, it is more common to exploit indirect detection with fluorescent secondary antibodies in multiplexed IF. Here, using primary antibodies that are different subclasses (mouse IgG1, IgG2b, or IgG3) or isoforms (IgG vs.
IgM) and/or derived from a different species (mouse vs. rabbit) allows multiple biomarkers to be detected independently by different fluorescent Fc-specific secondary antibodies. However, with most validated high-quality antibodies having been derived in
mice or rabbits, the requirement to identify different species is a barrier that has long stymied progress in multiplexing with indirect IF. The growing popularity of rabbit monoclonal antibodies, which are all one IgG subclass, is only exacerbating this problem.
Modifying antibodies with biotin for detection with fluorescent streptavidin or with
digoxigenin or other haptens [79] for detection with antihapten antibodies can extend
multiplexing. Preassembly of antibodies from the same species with different fluorescent
streptavidins or with fluorescent secondaries (e.g., Thermo Zenon, ChromoTek NanoSecondaries, NanoTag Fluo-Tag) is another way to overcome this limitation.
3.2.1 Deep profiling by cyclic IHC and IF
As a means to achieve very high multiplexing, multiple antibodies, whether from different or the same species, can be examined in sequence by performing cycles of direct or
indirect staining and stripping/erasing on one tissue section. Multiple strategies have been
used to remove the signal of the prior cycle while preserving the remaining antigens for
subsequent detection. Though slow and cumbersome when performed manually, cyclic
staining is readily implemented as an automated iterative staining system for dozens of
markers (Fig. 4).
Applied to IHC, multiple markers can be detected by sequential chromogenic reactions
and "erasing" the precipitate along with eluting bound antibody. While DAB leads to a
Fig. 4 Overview of cyclic immunostaining using directly-labeled fluorescent antibodies. Following tissue preparation, staining is performed in multiple rounds. Each iteration includes staining with a new
set of fluorescent antibodies, followed by an inactivation step. Cycles are repeated until all biomarkers
have been imaged.
303Spatial mapping of the tumor immune microenvironment
relatively stable precipitate, the red HRP chromogen AEC (3-amino-9-ethylcarbazole)
can be removed with ethanol. This is leveraged in the sequential immunoperoxidase labeling and erasing (SIMPLE) strategy [80] to build up a composite image of multiple antigens
via cycles of primary antibodystaining, indirect detection with HRP-conjugated secondary
antibodies, imaging, and erasing. Several approaches to cyclic IF on FFPE tissue sections
that can be enabled in the laboratory have been described that chemically inactivate or
bleach the fluorophore or elute the primary and secondary antibodies [81–85].Among
these, the most practical option for "home brew" may be tissue-based cyclic immunofluorescence (t-CyCIF) [83, 86] where the signal is erased by bleaching fluorophores with
hydrogen peroxide solution at high pH in white light, limiting antigen loss in each cycle.
A simple erasing methodology developed by GE Healthcare, now Cytiva, is based on the
chemical inactivation of cyanine dyes to enable cyclic IF on tissue sections [87]. An automated staining and imaging device is currently marketed as the Cell DIVE system for sixty
marker analysis. In the toponome imaging system (TIS) [88], in each cycle, a pair of fluorescent primary antibodies are incubated with a tissue section, imaged, and then photobleached. This simple strategy has been adapted to enable 96 biomarkers to be
interrogated on the same tissue sample via the Miltenyi MACSima system, facilitating deep
profiling of the TIME.
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3.2.2 Barcode-based multiplexing methods
The practical value of being able to probe with multiple mouse or rabbit monoclonal
antibodies at one time and the limitations of direct labeling have led to multiple antibody
barcoding strategies. Stimulated by immuno-PCR [89], a large class of promising technologies for multiplexed immunodetection have been reported in the academic and patent literature in the last two decades that leverage antibody-oligonucleotide conjugates to
localize multiple fluorophores to the antibody-antigen complex. Here, multiplexing can
be readily achieved via barcoding each antibody with a different oligonucleotide and then
color-coding the detection chemistry. Nearly the full range of nucleic acid detection and
amplification technologies have been applied to fluorescent immunolocalization including annealing complementary probes, strand displacement, gel, and nanostructure assembly, hybridization chain reaction, isothermal amplification, rolling circle amplification
(RCA), ligation, primer extension, and in situ sequencing by synthesis (partly reviewed
here [90]). Detecting pairs of antibody-oligonucleotide conjugates by in situ proximity
ligation assay [91] can enhance both sensitivity and specificity. While most of these
methods have yet to be applied to the analysis of the TIME in FFPE tissue sections,
Ultivue has fully commercialized a DNA-barcoding, amplification, and detection technology, InSituPlex [92] (Fig. 5, top), where each of four primary antibodies is conjugated
to a unique oligonucleotide, the antibody barcode amplified by primer-exchange reaction [93], and then detected by annealing of probes labeled with one of four fluorescent
dyes. This four-plex IF assay has been implemented on autostainers and recently applied
Fig. 5 Overview of new commercial multiplexing methods. InSituPlex uses primary antibodies conjugated to DNA barcodes, which are amplified before fluorescence-labeled probes are hybridized to the
DNA. Amplified DNA is shown. UltraPlex uses peptide tag-conjugated primary antibodies and antipeptide tag fluorophore-conjugated secondary antibodies.
to explore determinants of ICI response [94, 95]. For higher multiplexing, a cocktail of
multiple primary antibodies is applied and all their barcodes are amplified. Deeper profiling is achieved by cycles of annealing four detection probes, imaging, and elution. This
enables interrogation of the TIME with a large panel of antibodies. Along the same lines,
using the Akoya Biosciences CODEX (codetection by indexing) instrument, panels of
antibody-oligonucleotide conjugates are detected in parallel via cycles of annealing and
eluting of fluorescently labeled oligonucleotide probes, providing comparatively rapid
deep profiling by cyclic IF.
As an alternative to DNA barcoding, in the Cell IDx UltraPlex strategy, primary antibodies are conjugated with individual peptide tags (Fig. 5, bottom). Here, much like using
secondary antibodies to recognize the species of primary antibodies by their distinct Fc
domains, each primary antibody can be detected by a different monoclonal antibody raised
to recognize one of the peptide tags. As with DNA barcoded antibodies, the peptidetagged primary antibodies can be applied as a cocktail. Then the antitag "secondary" antibodies are applied, which are provided as fluorescent or enzyme conjugates, enabling multiplexing of several antibodies of the same species in IHC or IF. UltraPlex is rapid, readily
adaptedto autostainers, and, using panels of four primary antibodies, validated for analysis of
patterns in the TIME potentially linked to immunotherapy response [96, 97].Afterimaging, sections can be stained with H&E to align fluorescence to morphology. To increase
multiplexing, additional panels of antibodies can be examined on adjacent thin sections.
Cyclic staining for deep profiling with this technology appears straightforward.
305Spatial mapping of the tumor immune microenvironment
3.3 In situ hybridization to detect transcripts
As a complementary approach to protein biomarker analysis by IHC or IF, detection of
RNA transcripts by in situ hybridization (ISH) enables detection of the expression patterns of RNA biomarkers in the TIME with labeled oligonucleotide probes. Of particular relevance, patterns of expression of small secreted proteins such as TNFα, TGFβ, and
IFNγ are important indicators of immune cell differentiation, activation, and suppression.
While determining which cells in the TIME are releasing secreted proteins is poorly matched to IHC or IF, ISH to detect messenger RNA (mRNA) in the cytoplasm is ideal for
this purpose. Synthesis of biotinylated or digoxigenin-labeled probes, detection with
streptavidin conjugated to HRP, or indirect detection with antidigoxigenin antibody,
and chromogenic detection with DAB provides a direct equivalent to IHC. Fluorescent
streptavidin or antidigoxigenin enables fluorescent ISH (FISH) as an equivalent to indirect immunofluorescence. Beyond simple annealing, a wide range of nucleic acid-based
amplification methods have been applied to ISH. Among these, in situ branched DNA
signal amplification (bDNA) [98] has been successfully commercialized as ACD
RNAscope and Thermo Fisher ViewRNA using detection via proximity of pairs of
bound hybridization probes to achieve single-molecule specificity and sensitivity.
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This bDNA method enables both chromogenic and fluorescent single-molecule mRNA
detection in FFPE tissue sections with automated stainers. Further, bDNA detection is
readily combined with IF to allow simultaneous interrogation of proteins and transcripts
in FFPE tissue sections [99]. While synthesizing a single probe labeled with one or a few
fluorophores may not be sufficiently sensitive, assembling a panel of fluorescent probes to
tile across a transcript can concentrate enough in one spot to allow detection and counting of single mRNAs, enabling single-molecule FISH (smFISH) [100]. Given the
pointillist labeling, this approach is ideally suited to multiplexing. The smFISH approach
has been commercialized as Stellaris by BioSearch who offers off-the-shelf sets of genespecific probes in multiple fluorescent colors. smFISH is compatible with cryosections
[101] but displays unsatisfactory signal-to-noise in FFPE sections. This has been partly
addressed by method optimization [102] and rolling circle amplification [103]. Among
alternative probe designs that have been proposed for smFISH, split-FISH appears to
offer significant advantages for speed, sensitivity and specificity [104].
As with deep profiling by IF, sequential staining strategies have been implemented in
FISH to maximize multiplexing. In sequential barcoding FISH (seqFISH), probes that
detect each mRNA target are synthesized with four fluorophores. In each cycle, mixtures
of probes are applied in which each probe is only represented by one color and the changing fluorescence at each position reflects which probe is binding [105] (Fig. 6). Similar to
Fig. 6 SeqFISH barcoding. SeqFISH restains each biomarker multiple times using the same oligonucleotide probe, switching the fluorophore each cycle. The sequence of the fluorophore is used as a
barcode to identify the biomarker, allowing the same set of two to four dyes to be used to label thousands of RNA molecules in the same tissue section.
seqFISH, multiplexed error-robust FISH (MERFISH) [106] uses sequential imaging to
encode a unique fluorescent identifier for each mRNA. First, a series of encoding probes
are applied to the sample, each containing a targeting sequence in the center and two
readout sequences on either side. Following the initial hybridization, the first set of fluorescently labeled readout probes are conjugated to the readout sequences and imaged.
This process is repeated with each set of readout probes. Not all RNA molecules will
have a corresponding readout probe in every round of hybridization, thus, the presence
or absence of a signal can be used to generate a binary barcode for each unique transcript.
Although these iterative ISH methods may not be compatible with FFPE tissue sections,
they can be successfully implemented in properly cleared cryosections [107].
3.4 High multiplexing with Digital Spatial Profiling
NanoString nCounter is a distinct oligonucleotide barcoding technology that leverages a
dedicated flow-cell reader to distinguish the 4096 possible codes on Reporter Tags formed
by ordering four fluorescent colors along six positions along a nucleic acid backbone. In
Digital Spatial Profiling (DSP) [108,109], sets of Reporter Tags are linked to ISH probes or
conjugated to antibodies via a UV cleavable linker. The probes are applied in a cocktail to
an FFPE tissue section or cryosection along with up to four fluorescently labeled antibodies
to help identify morphology or immune subsets. Then, using conventional IF as a guide,
one or more regions of interest (ROIs) are selected. The ROI can be a segment of the
tumor or multiple spots to capture cells with specific markers. Then, each ROI is illuminated by UV light and the released Reporter Tags are eluted and captured for detection in
the nCounter reader. The DSP platform is particularly powerful for mRNA profiling on
slides but has also enabled relatively deep profiling of immune subsets and immune response
to immunotherapy based on detecting CD markers and other antigens in the TIME
[110–112], albeit without the single-cell resolution of other strategies. This is less of a lim-
itationwhen interrogating the immune cell make-up of tertiary lymphoid structures, where
dense clusters of innate and adaptive immune cells form in or near tumors and serve a role in
potentiating immune response [113,114].
Despite its potential, the current DSP platform has several weak points. The reliance
on four-plex immunofluorescence to identify ROIs and the small yield of Reporter Tags
from each cell makes many kinds of measurements impractical. While collecting
Reporter Tags from multiple cells in a single section based on shared cell subtype markers
offers a practical alternative, this will obscure spatial and cell-to-cell heterogeneity.
Although a larger region of tissue can be mapped by examining a grid of ROI’s, each
spot is simply a sample of the local cells. Further, although DSP has the potential to interrogate hundreds to thousands of antigens at once, the current strategy is to offer panels of
tagged antibody probes that do not provide the profiling depth or customization of the
iterative staining methods.
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As a way to increase multiplexing with the DSP strategy, NanoString Reporter Tags
can also be detected and quantified using massively parallel sequencing, which has been
applied to increase the range of detection of RNAs [109]. This approach can also be
applied to codetection of proteins with oligonucleotide-tagged antibodies as is already
practical in multiomic single-cell analysis (CITE-seq) [115–117], potentially enabling
simultaneous interrogation of tens of thousands of antigens and transcripts at a time in
each DSP region of interest.
3.5 Multiplexing in FFPE tissue beyond fluorescence
While fluorescence detection has proven more than satisfactory for a wide range of multiplexed assay formats, fluorophores can display low signal-to-background, limited spatial
resolution, spectral overlap across optical channels, and problematic quenching and photobleaching. Further, results are subject to the varying performance of scanners and
microscopes and lack of standards for image processing software. Among the alternative
antibody labeling strategies compatible with multiplexed optical detection at cellular resolution, molecular or nanoparticle tags that can be detected by vibrational Raman spectroscopy are particularly promising [118–120]. By leveraging dedicated microscopes and
the narrow emission bands that can be achieved with small-molecule labels such as the
Carbow probes [121], Raman spectroscopy offers the potential to independently image
dozens of antigens at subcellular resolution in thin tissue sections.
In mass spectrometry immunohistochemistry (MSIHC) [122], labeling antibodies
with elemental mass tags offers a powerful approach for multiplexing but creates a challenge for scanning mass spectrometry at cellular resolution. This has been addressed by
two similar strategies, imaging mass cytometry (IMC, commercialized by Fluidigm) [123]
and multiplexed ion beam imaging (MIBI, recently commercialized by IONpath) [124].
Here, primary antibodies conjugated to carriers loaded with one of over a hundred available heavy metals as monoisotopic tags [125] are applied to the tissue as a cocktail, and
then the tissue is scanned with a laser (IMC) or ion beam (MIBI) to vaporize the tags for
detection in a simple mass spectrometer. The spectra obtained at each scan position allow
independent detection of the ion count for each mass tag and thus quantitative mapping
of each antibody. Although primarily applied to protein detection, IMC has also been
adapted to enable highly multiplexed detection of RNAs using bDNA amplification
and mass-tagged probes, a strategy compatible with dual detection along with masstagged antibodies on the same tissue section [126].
Given the very high multiplexing and excellent quantitation but the limited spatial
resolution of this methodology, a practical way to examine the data is to assign the protein
or RNA expression to individual cells in the sample [127]. Toward these ends, metal
counterstaining as with ruthenium tetroxide [128] may enhance cell segmentation. Once
cells are defined, dissection of the TIME using MSIHC is facilitated by compatibility with
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