Methods of amplifying spatial signals of one or more macromolecules colocalized with a target analyte within a biological sample

Abstract

This disclosure relates to methods for identifying spatial signals of one or more macromolecules colocalized with one or more target analytes in a high dimensional biological sample.

Description

METHODS OF AMPLIFYING SPATIAL SIGNALS OF ONE OR MORE MACROMOLECULES COLOCALIZED WITH A TARGET ANALYTE WITHIN A BIOLOGICAL SAMPLECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 727,702, filed December 4, 2024, the entire contents of which are incorporated by reference herein.BACKGROUND OF THE INVENTION

[0002] There is a growing interest and demand for high-dimensional multiplex imaging technology to generate large-scale spatial expression and co-expression datasets for various biomarkers. This advanced imaging technique allows researchers to simultaneously observe and analyze multiple molecular targets within a single sample. It ensures accurate cell discrimination and preservation of the native architectural context. High-dimensional multiplex imaging provides valuable insights into cellular interactions, signaling pathways, and disease mechanisms, thereby enhancing the understanding of cellular processes and offering a comprehensive view of biological systems.

[0003] One platform for high-dimensional multiplex imaging is AKOYA Biosciences' PhenoCycler-Fusion (PCF) technology1. This platform combines the principles of immunofluorescence (IF) techniques with DNA barcoding to enable the simultaneous detection of numerous molecular targets. Instead of using fluorescent dyes, each antibody is labeled with a unique DNA barcode. Upon staining a sample with a mixture of barcoded antibodies, the DNA barcodes are sequentially detected using complementary fluorescent DNA oligos, known as reporters. This barcoding system allows for multiplexing capabilities via iterative cycles of staining and imaging.SUMMARY OF THE INVENTION

[0004] The present disclosure extends an insight that many existing high-dimensional platforms for imaging spatial signals of analytes of interest within a sample are incompatible with signal amplification. The present disclosure is based, in part, on the insight that tyramide-signal amplification (TSA) can provide enhanced signal detection for one or more macromolecules colocalized with one or more target analytes with increased flexibility in label selection.

[0005] In one aspect, provided are methods of amplifying spatial signals of one or more macromolecules colocalized with one or more target analytes within a biological sample. In some embodiments, the method comprises (i) contacting the biological sample with a first antibody that binds to the target analyte, (ii) contacting the biological sample with an antibody complex, wherein the antibody complex comprises a second antibody that binds to the first antibody, and an enzyme that is conjugated to the second antibody, wherein the enzyme is capable of activating tyramide, and (iii) contacting the biological sample with a label complex, wherein the label complex comprises a label that is conjugated to tyramide. In some embodiments, the enzyme conjugated to the second antibody catalyzes oxidation of the tyramide on the label complex into reactive free radicals that deposit onto the one or more macromolecules colocalized with the target analyte in the biological sample. In some embodiments, the spatial signals of the one or more macromolecules colocalized with the target analyte are identified by detecting the label.

[0006] In some embodiments, the high dimensional biological sample is a tissue section, tissue, tumor section, or tumor.

[0007] In some embodiments, the free radicals react with tyrosine or trytophan on the one or more macromolecules colocalized with the target analyte.

[0008] In some embodiments, the one or more macromolecules colocalized with the target analyte are in low abundance in the biological sample as compared to the target analyte. In some embodiments, the one or more macromolecules colocalized with the target analyte are less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the total non-target material.

[0009] In some embodiments, the enzyme is selected from the group consisting of: hydrolysases, peroxidases, oxidases, esterases, glycosidases and phosphatases. In some embodiments, the enzyme is horseradish peroxidase (HRP).

[0010] In some embodiments, the method further comprises removing the first antibody and the second antibody after step (iii). In some embodiments, removing the first and second antibodies comprises a heat-induced epitope retrieval (HIER) process.

[0011] In some embodiments, step (iii) is carried out in the presence of hydrogen peroxide.

[0012] In some embodiments, the label of step (iii) is hapten. In some embodiments, the label is selected from the group consisting of biotin, digoxigenin, fluorescein, and dinitrophenol.

[0013] In some embodiments, the biological sample is a Formalin-fixed, paraffin-embedded (FFPE) sample.

[0014] In some embodiments, the method comprises repeating steps (i)-(iii) to identify spatial signals of one or more macromolecules colocalized with a second target analyte in the biologicalsample with a different set of: (a) a first antibody that binds to the second target analyte, (b) an antibody complex comprising a second antibody that binds to the first antibody of (a), and an enzyme that is conjugated to the second antibody of (b), wherein the enzyme is capable of activating tyramide, and (d) a label complex, wherein the label complex comprises a label that is conjugated to tyramide. In some embodiments, the enzyme conjugated to the second antibody of (b) catalyzes oxidation of the tyramide on the label complex into reactive free radicals that deposit onto the one or more macromolecules colocalized with the second target analyte within the biological sample. In some embodiments, the spatial signals of the one or more macromolecules colocalized with the second target analyte are identified by detecting the label of (d).

[0015] In some embodiments, the method comprises (iv) contacting the biological sample with a third antibody that binds to a second target analyte, (v) contacting the biological sample with a second antibody complex, wherein the second antibody complex comprises a fourth antibody that binds to the third antibody, and a second enzyme that is conjugated to the fourth antibody, wherein the enzyme is capable of activating tyramide, and (vi) contacting the biological sample with a second label complex, wherein the label complex comprises a second label that is conjugated to tyramide. In some embodiments, the second enzyme conjugated to the fourth antibody catalyzes oxidation of the tyramide on the second label complex into reactive free radicals that deposit onto one or more macromolecules colocalized with the second target analyte within the biological sample. In some embodiments, the spatial signals of the one or more macromolecules colocalized with the second target analyte are identified by detecting the second label.

[0016] In some embodiments, a method of identifying spatial signals of one or more macromolecules colocalized with one or more target analytes within a biological sample comprises the steps (i) to (iii), and step (iv) detecting the label(s). In some embodiments, step (iv) further comprises immunostaining the label(s).

[0017] In some embodiments, the spatial signals provide three-dimensional locations.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 is a schematic view of an exemplary signal amplification of a target of interest.

[0019] FIGS 2A-2F are immunohistochemical images of Pan-Cytokeratin (PanCK) in nonsmall cell lung cancer (NSCLC) samples with (shown as biotin) or without (shown as PanCK) signal amplification. The scare bars for FIGS. 2 A, 2C and 2E are 400 pm, and the scare bars for FIGS. 2B, 2D and 2F are 50 pm.26087

[0020] FIGS. 3A-3F are immunohistochemical (3A-3C) or immunofluorescent (3D-3F) images of CD56 in tonsil. FIGS. 3A-3C are images from CD56 immunohistochemistry (IHC) staining with signal amplification. FIGS. 3D-3F are images from the TSA amplification using an anti- CD56 antibody and tyramide-biotin, where biotin was used for PhenoCycler-Fusion detection instead of CD56. The scare bars for FIGS. 3 A and 3D are 400 pm. The scare bars for FIGS. 3B and 3E are 100 pm. The scare bars for FIGS. 3C and 3F are 50 pm.

[0021] FIGS. 4A-4D are immunohistochemical (4A-4B) or immunofluorescent (4C-4D) images of CD56 in gastroesophageal junction (GEJ) tumor. FIGS 4A and 4B are images from CD56 IHC staining with signal amplification. FIGS. 4C and 4D are images of biotin signals from PhenoCycler-Fusion detection, after the TSA treatment using an anti-CD56 antibody and tyramide-biotin. The scare bars for FIGS. 4A and 4C are 400 pm. The scare bars for FIGS. 4B and 4D are 100 pm.

[0022] FIGS. 5A-5D are immunofluorescent images of PanCK in NSCLC samples. After the TSA treatment, both original epitopes (PanCK in FIGS. 5 A and 5B) and the new hapten epitopes (digoxygenin in FIGS. 5C and 5D) were detected by barcoded antibodies, showing colocalization of the two signals. The scare bars for FIGS. 5A and 5C are 400 pm. The scare bars for FIGS. 5B and 5D are 50 pm.DETAILED DESCRIPTION OF THE DISCLOSURE

[0023] The present disclosure provides signal amplification that can be applied to multiplex and / or multi-dimensional imaging systems. The present disclosure is based, in part, on the insight that a low-abundant target analyte can be transformed into a new high-abundant label, improving sensitivity in signal detection. In some embodiments, such signal amplification can be used for high dimensional multiplexing platforms, e.g., for analysis of tissue sections and other similar sample types.

[0024] When an original target analyte (e.g., comprising a target epitope) of interest is low in amount in a sample, existing technologies (e.g., PCF technology) utilizing a barcoded primary antibody and the complimentary reporter provide weak signals. The present disclosure provides methods for amplifying spatial signals of one or more macromolecules colocalized with one or more target analytes within a biological sample using a TSA treatment.Definitions

[0025] So that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all26087 other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0026] Reference to “or” indicates either or both possibilities unless the context clearly dictates one of the indicated possibilities. In some cases, “and / or” was employed to highlight either or both possibilities.

[0027] As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0028] The term “about”, when modifying the quantity (e.g., mg) of a substance or composition, or the value of a parameter characterizing a step in a method, or the like, refers to variation in the numerical quantity that can occur, for example, through typical measuring, handling and sampling procedures involved in the preparation, characterization and / or use of the substance or composition; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make or use the compositions or carry out the procedures; and the like. In certain embodiments, “about” can mean a variation of ± 10%.

[0029] As used herein, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of’ and “consisting essentially of’ the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0030] “ Consists essentially of,” and variations such as “consist essentially of’ or “consisting essentially of,” as used throughout the specification and claims, indicate the inclusion of any recited elements or group of elements, and the optional inclusion of other elements, of similar or different nature than the recited elements, that do not materially change the basic or novel properties of the specified dosage regimen, method, or composition.

[0031] As used herein, the term “colocalized” as used in reference to macromolecules and / or analytes, is defined to mean that two macromolecules and / or analytes have spatial overlap within a given biological sample. In specific embodiments, “colocalized” means “in the vicinity of’.

[0032] As used herein, the term “in the vicinity” in this context may encompass, and in specific embodiments, is a shortest distance between the objects of from 0 to 50 pm, from 0 to 20 pm, from 0 to 10 pm, from 0 to 5 pm, from 0 to 2 pm, from 0 to 1 pm, 0 to 500 nm, from 0 to 200 nm, from 0 to 100 nm, from 0 to 50 nm, from 0 to 20 nm, from 0 to 10 nm, from 0.01 to 50 pm, from 0.01 to 20 pm, from 0.01 to 10 pm, from 0.01 to 5 pm, from 0.01 to 2 pm, from 0.01 to 1 pm, 0.01 to 500 nm, from 0.01 to 200 nm, from 0.01 to 100 nm, from 0.01 to 50 nm, from 0.01 to 20 nm, or from 0.01 to 10 nm, for example.

[0033] As used herein, the term “high dimensional biological sample” is meant to refer to a biological sample that allows for the imaging within the tissue microenvironment. In some embodiments, high dimensional biological samples include tissue sections, tissues, tumor sections, or tumors.

[0034] As used herein, the term “target analyte” is meant to refer to a target epitope, protein, or nucleic acid.

[0035] As used herein the term “target macromolecules” is meant to refer to one or more macromolecules within a biological sample having a target analyte. In specific embodiments, the one or more macromolecules are in the vicinity of, or in close proximity with, the target analyte.

[0036] As used herein, the term “low abundant target” is meant to refer to a target having a substantially lower copy number or concentration as compared to the copy number or concentration of macromolecules in the biological sample. By “non-target material”, it is meant macromolecules in the biological sample other than the target analyte. In some embodiments, the non-target material is of a similar chemical nature as the target (e.g., nucleic acids or proteins). In some embodiments, the non-target material is genomic background. In some embodiments, the low abundant target is present in a sample at a lower copy number than the limit of detection. In some embodiments, the low abundant target is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the total non-target background (e.g., genomic background, protein background, cellular component background, and / or non-human metagenomics background) in the sample.Signal Amplification

[0037] In some embodiments, the method for amplifying spatial signals of one or more macromolecules colocalized with one or more target analytes within a biological sample comprises (i) contacting the biological sample with a first antibody that binds to an epitope of a target analyte; (ii) contacting the biological sample with an antibody complex, wherein the antibody complex comprises a second antibody that binds to the first antibody, and an enzymethat is conjugated to the second antibody; and (iii) contacting the biological sample with a label complex, wherein the label complex comprises a label that is conjugated to tyramide, the biological sample has more copies of the label complex than the target analyte, and the label spatially overlaps with the target analyte.

[0038] In some embodiments, the methods provided herein create a new epitope that is different from the original epitope in the target analyte of interest. In some embodiments, a concentration and / or number of molecules having the new epitope is higher than that of the original epitope. In some embodiments, detection of the new epitope provides higher signals than the original epitope. In some embodiments, the new epitope is created within the vicinity of the original epitope. In some embodiments, detection of the new epitope provides similar spatial signals to those of the original epitope.

[0039] In some embodiments, the methods provided herein utilize TSA. TSA is a technique used in immunohistochemistry, immunocytochemistry, immunofluorescence, and in situ hybridization to amplify weak signals and improve the sensitivity of detection2 4. It is useful for detecting low-abundance targets or targets with low antibody affinity.

[0040] The TSA process involves several steps. First, a first antibody specific to the target of interest is bound to the epitope of the target in the sample. Then, a second antibody conjugated with an enzyme (e.g., horseradish peroxidase (HRP)) is added, binding to the first antibody. Next, a tyramide reagent solution containing a label (e.g., tyramide conjugated label (e.g., phenolic compound, e.g., tyramide-hapten molecules, e.g., tyramide-biotin or tyramide- digoxygenin)) is introduced. The enzyme attached to the second antibody activates tyramide which catalyzes the covalent deposition of tyramide radicals onto nucleophilic residues (e.g., tyrosine on proteins near the enzyme). This leads to the formation of highly dense tyramide-label deposition around the target site. The tyramide-label molecules, which spatially overlap with the original epitope, become the new epitope for detection. Since the new epitope are more abundant than the original epitope, the fluorescence signal is amplified.

[0041] The deposited tyramide-label molecules are then detected using a reporter molecule that specifically binds to the label (e.g., hapten), generating an amplified fluorescent signal. One of the advantages of TSA is the catalytic deposition of numerous tyramide-label molecules near each enzyme, providing signal amplification up to 1000-fold over conventional immunolabeling methods. This increased sensitivity allows detection of low-copy number targets.

[0042] In some embodiments, the enzyme converts the label complex into a free radical(s) that covalently reacts with a macromolecule in the vicinity of the target analyte. In some embodiments, the enzyme is selected from the group consisting of hydrolysases, peroxidases,oxidases, esterases, glycosidases and phosphatases. In some embodiments, the enzyme is horseradish peroxidase (HRP).

[0043] In some embodiments, tyramide of the label complex reacts with tyrosine or trytophan. In some embodiments, tyramide of the label complex reacts with tyrosine or trytophan on proteins near the enzyme.

[0044] In some embodiments, the target analyte is low abundant in the biological sample. In some embodiments, the target is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the total nontarget material.

[0045] In some embodiments, the methods further comprise removing the first antibody and the second antibody. In some embodiments, the removing step comprises a heat-induced epitope retrieval (HIER) process.

[0046] In some embodiments, the label is hapten. In some embodiments, the label is selected from the group consisting of biotin, digoxigenin, fluorescein, and dinitrophenol. In some embodiments, the label is biotin, or digoxigenin. In some embodiments, the label is biotin. In some embodiments, the label is digoxigenin.

[0047] In some embodiments, the label complex is tyramide-hapten. In some embodiments, the label complex is selected from the group consisting of tyramide-biotin tyramide-digoxigenin, tyramide- fluorescein, and tyramide- dinitrophenol. In some embodiments, the label complex is tyramide-biotin or tyramide-digoxigenin. In some embodiments, the label complex is tyramide- biotin. In some embodiments, the label complex is tyramide-digoxigenin.

[0048] In some embodiments, the label complex is contacted with the biological sample in the presence of peroxide (e.g., hydrogen peroxide). In some embodiments, the enzyme converts the label complex into a free radical that covalently reacts with a macromolecule in the vicinity of the target analyte in the presence of peroxide (e.g., hydrogen peroxide).

[0049] In some embodiments, the methods further comprise repeating steps (i)-(iii) to identify spatial locations of one or more different target analytes in the biological sample with a different set of (a) a first antibody that binds to an epitope of the second target analyte, (b) an antibody complex comprising a second antibody that binds to the first antibody, and an enzyme that is conjugated to the second antibody, and (d) a label complex.

[0050] The present disclosure also provides an insight into the challenges faced by many existing platforms that allow high-dimensional imaging, as they often encounter difficulties in identifying a suitable antibody clone for such platform. The reason behind this challenge is thatmany existing platforms require antibody modification that can alter the antibody’s performance. However, the methods provided herein offer additional flexibility in antibody choices.

[0051] For example, if no suitable antibody clone can be identified for a particular target on a multiplexing platform, a TSA reaction can be performed using a clone that works for regular IHC. The resulting novel hapten epitope can then be detected using a validated antibody clone against this hapten. This method potentially allows for the detection of any target, even if antibody modification renders the preferred clone completely incompatible with the platform.

[0052] FIG. 1 demonstrates an exemplary method for amplifying spatial signals of one or more macromolecules colocalized with one or more target analytes within a biological sample. This method involves a pre-detection treatment procedure that transforms the original epitope of the target analyte into a new and more abundant epitope using TSA. Before the multiplex detection, a first antibody is applied to bind to the original epitope, followed by the binding of an HRP- conjugated second antibody to the first antibody. The sample is then incubated with hydrogen peroxide and a tyramide-conjugated hapten (e.g., tyramide-biotin or tyramide digoxigenin). HRP, which is only present at the location of the target analytes, converts the tyramide-hapten conjugates into free radicals that covalently react with macromolecules (e.g., nucleophilic residues (e.g., tyrosine, tryptophan)) in the vicinity of the epitope, resulting in the deposition of a large quantities of haptens at the site of the original epitopes. Afterward, both the first and second antibodies are removed from the tissue with heat-induced epitope retrieval (HIER) process. Since the binding of tyramide to the tissue is covalent, only the antibodies are removed, leaving the tyramide-hapten still on the tissue. As the hapten is also an immunogen, it can be used as an epitope for immunostaining. Additionally, because tyramide-haptens are colorless and non- fluorescent, they do not interfere with downstream imaging. The TSA process, applied in this manner, transforms the original epitope into a new and much more abundant hapten-based epitopes at the same location as the original epitopes. During the multiplex detection process, instead of detecting the original low-abundant epitope, these haptens are used as the new epitope for detecting the target of interest. Therefore, instead of using a first antibody against the original target, a primary antibody against the hapten (novel epitope) is used, which makes the detection much easier due to its significantly greater abundance. As a result, low-expressing targets that were difficult to detect and previously had to be excluded from high-dimensional multiplexing can now be included in panels using this method.26087EXAMPLESExample 1: Experimental methods

[0053] The present example explains methods of experiments that are included in Examples 2- 3.Materials and Methods

[0054] The details of the tissues used herein are listed in Table 1.Table 1. Tissues (FFPE)

[0055] The details of the antibodies used herein are listed in Tables 1 and 2.Table 2. Barcoded antibodiesTable 3. Unconjugated antibodies

[0056] The details of other reagents used herein are listed in Table 4.Table 4. Other Reagents26087

[0057] The details of the sample slides used herein are described in Tables 5 and 6.26087Table 5. PCF stainingTable 6. DAB stainingTissue Preparation

[0058] Formalin-fixed, paraffin-embedded (FFPE) tissue blocks were sectioned at a 5 pm thickness and allowed to air dry overnight. Slides were baked at 60 °C for 1 hour before use and deparaffinized. Following the deparaffinization, the FFPE slides were subjected to heat-induced epitope retrieval (HIER) in citrate buffer at pH 6.0 at 120°C for 4 minutes. After the HIER process, the slides were allowed to cool down. Subsequently, the slides were immersed in deionized water, followed by two immersions in TBST.Slides pre-treatment with TSA

[0059] Pre-treatment with TSA was performed on the Leica Bond RX Autostainer. For tyramide-biotin only, slides were blocked in the Avidin Blocking System for 10 minutes, washed three times in TBST, and then blocked in the Biotin Blocking System for 10 minutes. They were washed three times in TBST again. Endogenous peroxidase was blocked by incubating in 3% hydrogen peroxide for 10 minutes, followed by three washes in TBST. Then, the slides were blocked in the PKI blocking buffer for 5 minutes. The slides were then incubated in the primary antibody for 60 minutes, washed three times in TBST, incubated in the Opal Polymer HRP for 15 minutes, and washed three times in TBST. The slides then treated with either tyramide- digioxigenin or tyramide-biotin for 10 minutes and washed three times in TBST. After the TSA step, the slides are treated with Bond ER1 solution for 20 minutes at 100 °C and washed three times in TBST. Once the autostainer program was complete, the slides were removed from machine and incubated in deionized water for 2 minutes, repeated twice.26087Phenocycler-fusion staining of TSA-treated slides

[0060] The slides were transferred from deionized water to a hydration buffer, and incubated for 2 minutes twice. They were then incubated in a staining buffer for 30 minutes. An antibody cocktail was prepared by diluting barcoded antibody in the staining buffer, with 2.375% (v / v) of each of the N Blocker, G Blocker, J Blocker and S Blocker. Approximately 150-200 pL of the antibody cocktail was deposited on each sample to completely cover the entire tissue. The slides were kept in a humidity chamber and incubated for 3 hours at room temperature or overnight at 4 °C. Following the antibody incubation, the sample slides were washed with the staining buffer for 2 minutes twice.

[0061] Next, the tissue post fixation was performed by incubating the sample slides with the Post-Staining Fixing Solution (1.6% PF A) in a Coplin jar for 10 minutes. They were then washed with PBS, three times for 30 seconds each. Subsequently, the slides were incubated with ice-cold methanol in the Coplin jar for 5 minutes, followed by another wash with PBS, three times for 30 seconds each. The Final Fixative Solution was prepared by diluting 20 pL of the Fixative Reagent from the Staining kit in 1 mL of lx PBS. The slides were placed back to the humidity chamber, and 200 pL of the diluted Final Fixative Solution was deposited on the sample and incubated for 20 minutes. After that, the slides were washed with PBS, three times for 30 seconds each, and stored in a Coplin jar with 40 ml of a storage buffer at 4 °C before imaging.

[0062] Fluorescent multiplexed images were acquired using AKO YA’ s Phenocycler-Fusion instruments. The phenocycler reporter plate was prepared and the Phenocycler-Fusion run was set up following the manufacturer’s instructions.DAB staining with counterstaining

[0063] The slides were stained with a Thermo autostainer after baking, deparaffinization and HIER. Endogenous peroxidase was blocked by incubating the slides in 3% hydrogen peroxide for 10 minutes. They were then washed once with TBST. Next, the slides were incubated with the primary anti-human CD56 antibody at a concentration of 5 pg / mL for 60 minutes. After incubation, the slides were washed twice with TBST. Subsequently, the slides were incubated with Envision mouse linker for 15 minutes, followed by two washes with TBST. The Envision Flex HRP was then added to the slides and incubated for 20 minutes, followed by two washes with TBST. The slides were then incubated with DAB for 10 minutes and washed once with TBST. After that, the slides were incubated in DAB enhancer for 7 minutes and washed once with deionized water.26087

[0064] Once the autostainer program was complete, the slides were removed from machine and immersed in deionized water. They were counterstained with Mayer’s Hematoxylin using the Leica Stainer XL. The staining program for the Leica Stainer XL was as follows:1) Mayer's Hematoxylin (Poly Scientific Cat# S216) for 30 seconds;2) Deionized Water for 2 minutes;3) Richard Allan's Bluing Reagent (Fisher Cat# 7301) for 20 seconds;4) Deionized Water for 2minutes.

[0065] Following the counterstaining, the slides were dried in a 60°C oven and then coverslipped with mounting medium (Dako Cat# CS703; Lot# 157257) before being viewed under a microscope.Slide scanning

[0066] Brightfield immunohistochemical images were acquired using the Leica Aperio AT2 scanner with a 40x magnification.Example 2: Detecting Pan-Cytokeratin (PanCK)

[0067] The present example demonstrates the detection of Pan-Cytokeratin (PanCK) using the methods described herein.

[0068] Pan-Cytokeratin (PanCK) was selected, since it is known to be highly expressed in tumor samples. Non-small cell lung cancer (NSCLC) FFPE samples were treated with TSA using tyramide-biotin after staining the samples with an anti-PanCK antibody, and an HRP-conjugated second antibody. This process allowed for the deposition of biotin in the areas where PanCK is distributed. Subsequently, all the antibodies were removed, leaving only the original tissue along with the deposited tyramide-biotin. The samples then underwent PhenoCycler-Fusion staining with an anti-PanCK and anti-biotin antibody cocktail, each labeled with a distinct barcode, and were detected using corresponding reporters. The results show colocalization between the PanCK signals (see FIGS. 2A and 2B) and the Biotin signals (see FIGS. 2C and 2D). The biotin recapitulated the original distribution of the PanCK epitopes. Thus, instead of directly detecting PanCK, the detection of biotin can be used to reveal the distribution of PanCK.Example 3: Signal amplification of a low-abundant target

[0069] The present example demonstrates exemplary signal amplification of a low-abundant target.26087

[0070] The methods described herein were applied to a low-abundant target such as CD56. CD56 is weakly expressed, and amplification is needed for immunohistochemistry (IHC). Consequently, direct detection of CD56 on the PhenoCycler-Fusion platform with a barcoded antibody is challenging. For this example, human tonsil FFPE tissues were treated with TSA using an anti-CD56 antibody and an HRP-conjugated secondary antibody to deposit biotin at the sites of CD56. The distribution of biotins was then detected using an anti-biotin antibody labeled with a barcode (see FIGS. 3D-3F). As a control, CD56 IHC staining with signal amplification was performed on a neighboring section of the same tissue (see FIGS. 3A-3C).

[0071] The CD56 distributions from the control and the anti-biotin detection are very similar. The present methods were successful in detecting low-abundant proteins that were otherwise difficult to be detected on the PCF platform.

[0072] CD56 on a GEJ tumor sample was also tested, with the expression of CD56 even lower than in tonsils. Similar to the tonsil sample, TSA pretreatment was performed to generate biotin epitopes at the sites of CD56, and biotin was detected instead of CD56 using the PCF platform (see FIGS. 4C and 4D). The results were compared with CD56 IHC staining on a neighboring section of the same GEJ tumor tissue sample (FIGS. 4A and 4B). The results demonstrate a very good match in both the signal distribution and signal intensities.

[0073] This Example utilized the TSA chemistry to transform the original low-abundant epitopes into the new high-abundant hapten epitopes. By using the transformed epitopes instead of the original epitopes for subsequent detection, the sensitivity in signal detection was significantly improved. This Example demonstrates the potential fulfill the unmet need of applying signal amplification on high dimensional multiplexing platforms for analysis of tissue sections and other similar sample types.Example 4: Versatility in label selection

[0074] This Example demonstrates another exemplary label.

[0075] To demonstrate the flexibility in the choice of haptens, a different tyramide-hapten molecule, tyramide-digoxygenin (dig) was tested. PanCK was used as the target for NSCLC samples. The experiments were performed similarly to Example 2, with the substitution of tyramide-biotin with tyrmide-dig during the anti-PanCK TSA pretreatment, followed by detection using anti-dig. The results with dig demonstrate colocalization between the original PanCK epitopes (FIGS. 5A and 5B) and the new dig epitopes (FIGS. 5C and 5D), highlighting the versatile of this approach in hapten selection.26087

[0076] This Example shows the compatibility of amplifying multiple weak / difficult-to-detect targets, each corresponding to a different hapten. This can be achieved through sequential TSA processes, where each time one target is amplified and all antibodies are removed before the next round of TSA process for the next target.REFERENCES1. PhenoCycler Fusion 2 Brochure, www.akoyabio.com / wp- content / uploads / 2023 / 10 / PhenoCycler_Fusion_2_Brochure.pdf.2. Bobrow, M. N., Harris, T. D., Shaughnessy, K. J. & Litt, G. J. Catalyzed reporter deposition, a novel method of signal amplification application to immunoassays. J. Immunol. Methods 125, (1989).3. Bobrow, M. N., Shaughnessy, K. J. & Litt, G. J. Catalyzed reporter deposition, a novel method of signal amplification. II. Application to membrane immunoassays. J. Immunol. Methods 137, (1991).4. Van Gijlswijk, R. P. M. et al. Fluorochrome-labeled tyramides: Use in immunocytochemistry and fluorescence in situ hybridization, in Journal of Histochemistry and Cytochemistry vol. 45 (1997).

Claims

WHAT IS CLAIMED IS:

1. A method of identifying spatial signals of one or more macromolecules colocalized with a target analyte within a high dimensional biological sample having the target analyte, the method comprising the following steps:(i) contacting the biological sample with a first antibody that binds to the target analyte,(ii) contacting the biological sample with an antibody complex, wherein the antibody complex comprises a second antibody that binds to the first antibody, and an enzyme that is conjugated to the second antibody, wherein the enzyme is capable of activating tyramide, and(iii) contacting the biological sample with a label complex, wherein the label complex comprises a label that is conjugated to tyramide; wherein the enzyme conjugated to the second antibody catalyzes oxidation of the tyramide on the label complex into reactive free radicals that deposit onto the one or more macromolecules colocalized with the target analyte in the biological sample, and wherein the spatial signals of the one or more macromolecules are identified by detecting the label.

2. The method of claim 1, wherein the high dimensional biological sample is a tissue section, tissue, tumor section, or tumor.

3. The method of claim 1 or claim 2, wherein free radicals react with tyrosine or trytophan on the one or more macromolecules.

4. The method of any preceding claim, wherein the one or more macromolecules are in low abundance in the biological sample as compared to the target analyte.

5. The method of claim 4, wherein the one or more macromolecules are less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the total non-target material.

6. The method of any preceding claim, wherein the enzyme is selected from the group consisting of: hydrolysases, peroxidases, oxidases, esterases, glycosidases and phosphatases.

7. The method of claim 6, wherein the enzyme is horseradish peroxidase (HRP).

8. The method of any preceding claim, further comprising removing the first antibody and the second antibody after step (iii).

9. The method of claim 8, wherein removing the first and second antibodies comprises a heat-induced epitope retrieval (HIER) process.

10. The method of any preceding claim, wherein step (iii) is carried out in the presence of hydrogen peroxide.

11. The method of any preceding claim, wherein the label of step (iii) is hapten.

12. The method of claim 11, wherein the label is selected from the group consisting of biotin, digoxigenin, fluorescein, and dinitrophenol.

13. The method of any preceding claim, wherein the biological sample is a Formalin-fixed, paraffin-embedded (FFPE) sample.

14. The method of any preceding claim, further comprising repeating steps (i)-(iii) to identify spatial signals of one or more macromolecules colocalized with a second target analyte in the biological sample with a different set of:(a) a first antibody that binds to the second target analyte,(b) an antibody complex comprising a second antibody that binds to the first antibody of (a), and an enzyme that is conjugated to the second antibody of (b), wherein the enzyme is capable of activating tyramide, and(d) a label complex, wherein the label complex comprises a label that is conjugated to tyramide, wherein the enzyme conjugated to the second antibody of (b) catalyzes oxidation of the tyramide on the label complex into reactive free radicals that deposit onto the one or more macromolecules colocalized with the second target analyte within the biological sample, and wherein the spatial signals of the one or more macromolecules colocalized with the second target analyte are identified by detecting the label of (d).

15. The method of any of claims 1-13, further comprising:(iv) contacting the biological sample with a third antibody that binds to a second target analyte,(v) contacting the biological sample with a second antibody complex, wherein the second antibody complex comprises a fourth antibody that binds to the third antibody, and a second enzyme that is conjugated to the fourth antibody, wherein the enzyme is capable of activating ty rami de, and(vi) contacting the biological sample with a second label complex, wherein the label complex comprises a second label that is conjugated to tyramide; wherein the second enzyme conjugated to the fourth antibody catalyzes oxidation of the tyramide on the second label complex into reactive free radicals that deposit onto one or more macromolecules colocalized with the second target analyte within the biological sample, and wherein the spatial signals of the one or more macromolecules colocalized with the second target analyte are identified by detecting the second label.

16. The method of claim 15, wherein the second enzyme is selected from the group consisting of: hydrolysases, peroxidases, oxidases, esterases, glycosidases and phosphatases.

17. The method of claim 16, wherein the second enzyme is horseradish peroxidase (HRP).

18. The method of claims 15-17, further comprising removing the third antibody and the fourth antibody after step (vi).

19. The method of claim 18, wherein removing the third antibody and the fourth antibody comprises a heat-induced epitope retrieval (HIER) process.

20. The method of claims 15-19, wherein step (vi) is carried out in the presence of hydrogen peroxide.

21. The method of claims 15-20, wherein the second label of step (vi) is hapten.

22. The method of claim 21, wherein the second label is selected from the group consisting of biotin, digoxigenin, fluorescein, and dinitrophenol.2608723. A method of identifying spatial signals of one or more macromolecules colocalized with one or more target analytes within a biological sample, the method comprising the steps (i) to (iii) of claims 1-22, and step (iv) detecting the label(s).

24. The method of claim 23, wherein step (iv) further comprises immunostaining the label(s).

25. The method of any preceding claim, wherein the spatial signals provide three-dimensional locations.

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