Simultaneous quantification of gene expression in user-defined regions of cross-sectioned tissue

Abstract

The present invention relates to simultaneous quantification of gene expression in user-defined regions of cross-sectioned tissue. In particular, the present invention relates to probes, compositions, methods and kits for simultaneous, multiplexed detection and quantification of protein and / or nucleic acid expression in user-defined tissue regions, user-defined cells, and / or user-defined intracellular subcellular structures.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a divisional application of the invention patent application entitled "Simultaneous Quantification of Gene Expression in User-Defined Regions of Cross-Sectioned Tissue" with international application PCT / US2016 / 042460, which has an international application date of July 15, 2016 and enters China with application number 201680054038.4. This application claims priority and benefits to U.S. Provisional Application No. 62 / 193,809 filed on July 17, 2015; U.S. Provisional Application No. 62 / 261,657 filed on December 1, 2015; U.S. Provisional Application No. 62 / 277,289 filed on January 11, 2016; and U.S. Provisional Application No. 62 / 323,023 filed on April 15, 2016. Each of the above applications is incorporated herein by reference in its entirety.

[0003] Technical Field and Background Art

[0004] Standard immunohistochemistry and in situ hybridization methods allow for simultaneous detection of up to six to ten protein or nucleic acid targets, typically three to four targets. There is a need for probes, compositions, methods, and kits for simultaneous, multiplexed detection and quantification of protein and / or nucleic acid expression in user-defined tissue regions, user-defined cells, and / or user-defined intracellular subcellular structures. Summary of the invention

[0005] The present invention relates to probes, compositions, methods and kits for simultaneous, multiplexed detection and quantification of protein and / or nucleic acid expression in user-defined tissue regions, user-defined cells, and / or user-defined intracellular subcellular structures.

[0006] One aspect of the present invention relates to a method comprising the following steps: (1) contacting at least one target in at least one cell in a tissue sample or from at least one cell in a tissue sample with at least one probe comprising a target binding domain and a signaling oligonucleotide; (2) providing a force to a location in the tissue sample sufficient to release the signaling oligonucleotide; and (3) collecting and determining the released signaling oligonucleotide, thereby detecting at least one target in a specific location of the tissue sample to which the force is provided or from a specific location of the tissue sample to which the force is provided. The specific location is a user-defined tissue region, a user-defined cell, and / or a user-defined subcellular structure within a cell. The target can be a nucleic acid (e.g., mRNA and miRNA) and / or a protein. The target binding domain can be a single-stranded nucleic acid, a partially double-stranded nucleic acid, or a protein binding molecule, such as an antibody, a peptide, an aptamer, and a peptoid. In an embodiment, two or more targets (i.e., proteins, nucleic acids, and combinations thereof) are detected. In embodiments, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1920, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more targets, and any number therebetween, for example, 800 or more different targets can be detected. In embodiments, detection includes quantification of the abundance of each target.

[0007] In embodiments, the method further includes repeating at least steps (2) and (3) at least a second specific location of the tissue sample, wherein the second specific location comprises at least a second cell. In embodiments, detection includes comparing the abundance of the at least one target in or from the first specific site with the abundance of the at least one target in or from the second specific site. The at least one cell and at least the second cell may be the same cell type or different cell types. In some embodiments, detection includes quantifying the abundance of the at least one target in or from the first cell type, and the at least one target in or from the second cell type. In embodiments, the first and second cell types are independently selected from normal cells and abnormal cells, such as diseased and cancer cells.

[0008] In embodiments, the at least one cell is directly fixed to the surface, or is indirectly fixed to the surface by at least one other cell. The tissue sample can be a tissue section of 2 to 1000 μm thick, for example, from a paraffin-embedded (FFPE) sample fixed by formalin-or obtained from an unfixed sample. The at least one cell can be fixed or unfixed. The at least one cell can be dyed or labeled before step (2) to allow visualization of the subcellular or cellular structure in the dyed or labeled cell. Alternatively, for tissue sections, the adjacent sections of the sections contacting the probe can be dyed or labeled before step (2) to allow assessment of the structures related to the subcellular, cell or tissue in the corresponding cells or nearby cells in the sections contacted with the probe. This dyeing or labeling technique is well known in the art.

[0009] In the above aspects, at least one probe further includes a joint (e.g., a cleavable joint) located between the target binding domain and the signaling oligonucleotide. The cleavable joint can be photocleavable, which is cut by light provided by a suitable coherent light source (e.g., a laser and UV light source) or a suitable incoherent light source (e.g., an arc lamp and a light emitting diode (LED)). The light source can irradiate at least one subcellular structure of the at least one cell, and the abundance of at least one nucleic acid target in or from at least one subcellular structure of the at least one cell can be detected. Similarly, the light source can first irradiate at least one subcellular structure of the at least one cell, and then irradiate at least one subcellular structure in at least a second cell, allowing the abundance of at least one target in or from at least one subcellular structure of the at least one cell to be compared with the abundance of at least one target in or from at least one subcellular structure in at least a second cell.

[0010] In an embodiment, the signaling oligonucleotide is a single-stranded nucleic acid or a partially double-stranded nucleic acid.

[0011] In embodiments, the sample can be a cultured cell or isolated cell (fixed or unfixed) that has been fixed to a slide. The sample can include cells (including primary cells and cultured cell lines) and / or tissues (including cultured or explanted). The sample can include cultured cells, primary cells, or isolated cells from explants.

[0012] In embodiments, illuminating a target area smaller than the field of view (eg, a single cell or a subcellular structure within a cell) includes using a laser scanning device (eg, confocal) or a digital micromirror device (DMD) to direct light.

[0013] In an embodiment, the probe is prepared by a cysteine ​​bioconjugation method, which is stable and preferably site-specific for the hinge region heavy chain of an antibody. In an embodiment, the probe may comprise multiple (ie, more than one, such as 2, 3, 4, 5 or more) labeled oligonucleotides / antibodies.

[0014] Detection includes polymerase reaction, reverse transcriptase reaction, hybridization to oligonucleotide microarray, mass spectrometry, hybridization to fluorescent molecular beacons, sequencing reaction, or nCounter ® In a preferred embodiment, nCounter ® Systems and from NanoString Technologies ® method.

[0015] In an embodiment, the signaling oligonucleotide is collected from the tissue by liquid laminar flow, vortex flow or transition flow. The flow can pass through a channel having a depth of 25 to 500 μm between the tissue and a fluidic device or an impermeable barrier placed on the tissue.

[0016] In embodiments, the signal oligonucleotide is collected from the near end of the at least one cell, for example, at least the solution immediately above. The near end solution can be collected by suction, for example, by a pipette, a capillary, a microarray needle, a flow cell comprising a hole, or another suitable suction system as known in the art, or any combination thereof. The capillary can include an optical device, which can transmit optical force, for example, UV light to the at least one cell. The pipette or microarray needle can be connected to an array comprising a plurality of pipettes or microarray needles. The near end solution can include anionic polymers, for example, dextran sulfate and / or salmon sperm DNA and / or the signal oligonucleotide collected can be added to a solution comprising anionic polymers, for example dextran sulfate and / or salmon sperm DNA. In addition to salmon sperm DNA, or instead of salmon sperm DNA, other non-specific blockers as known in the art can be used.

[0017] In embodiments, the methods provide for simultaneous spatially resolved DNA, RNA and / or protein detection of tissue samples.

[0018] In an embodiment, the digital readout comprises a linear dynamic range greater than or equal to 5 logs.

[0019] In embodiments, the concentration of the probe provided to the sample is generally less than that used for immunohistochemistry (IHC) or for in situ hybridization (ISH). Alternatively, the concentration may be significantly lower than that used for IHC or ISH. For example, the probe concentration may be 2 times less, 5 times less, 10 times less, 20 times less, 25 times less, 30 times less, 50 times less, 60 times less, 70 times less, 80 times less, 90 times less, 100 times less, 200 times less, 300 times less, 400 times less, 500 times less, 600 times less, 700 times less, 800 times less, 900 times less, 1000 times less, 2000 times less, or less, and any quantity thereof. In an embodiment, the probe is provided at a concentration of 100 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM, and less, and any concentration therebetween.

[0020] In an embodiment, a tissue sample is attached to a slide and first imaged using fluorescence (eg, fluorescently labeled antibodies and fluorescent dyes such as DAPI) and then digitally enumerated for protein and / or nucleic acid expression from the sample.

[0021] In an embodiment, negative purification is used, for example, including affinity purification methods, including contacting the complete probe molecule with a fixed oligonucleotide complementary to a portion of the complete probe or a fixed antibody or protein binding motif that recognizes and binds to a portion of the complete probe to remove the complete probe molecule from the released signal oligonucleotide. In an embodiment, the target binding domain of the complete probe comprises a universal purification tag, or a sequence that is complementary to the fixed oligonucleotide portion or can be recognized or bound by a fixed antibody or protein binding motif. In these embodiments, any such tag or sequence well known in the art can be used.

[0022] Any aspect or embodiment described herein may be combined with any other aspect or embodiment disclosed herein. Although the disclosure has been described together with its detailed description, the above description is intended to illustrate and not limit the scope of the disclosure, which is limited by the scope of the appended claims. Other aspects, advantages and changes are within the scope of the claims below.

[0023] The patents and scientific literature mentioned herein constitute the knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0025] Figure 1 : Two exemplary probes are shown. The nucleic acid backbone (single-stranded DNA or single-stranded RNA) is shown as a black straight line. The probes each include a target binding domain shown in red. The upper probe includes a labeled RNA segment hybridized to the nucleic acid backbone, while the lower probe includes a labeled DNA oligonucleotide hybridized to the nucleic acid backbone. A cleavable motif (e.g., a cleavable linker, not shown) may be located between the backbone and the target binding domain, or within the backbone. The cleavable motif allows the release of the signaling oligonucleotide from the bound target nucleic acid or protein; subsequently, the signaling oligonucleotide is collected and detected.

[0026] Figure 2 : Shows the first type of probe that can directly bind to the target nucleic acid (top). In the figure at the bottom, the probe has been bound to the target nucleic acid shown as a blue curve. In this figure and the following figures, the reporter probe includes six positions (marked with red circles) that hybridize with the labeled oligonucleotide. Since the probe includes a position that can hybridize with the labeled oligonucleotide, the probe may also be referred to as a reporter probe.

[0027] Figure 3 : shows a first type of dual-probe composition of the present invention. Here, the first type of probe binds directly to the target nucleic acid, and the first type of capture probe binds directly to the target nucleic acid. The capture probe may include at least one affinity reagent, which is shown as an asterisk. The target nucleic acid in the sample is shown as a blue curve.

[0028] Figure 4 : Shows a second type of probe (or reporter probe) that can indirectly bind to a target nucleic acid in a sample (upper part). Here, in the lower figure, the target of the probe is an intermediate oligonucleotide shown in green, which in turn binds to a target nucleic acid in the sample shown as a blue curve. It can be said that the intermediate oligonucleotide is a probe as defined herein because it contains a nucleic acid backbone and is capable of binding to a target nucleic acid.

[0029] Figure 5: shows a second type of dual-probe composition of the invention. Here, the second type of probe binds indirectly to a target nucleic acid in a sample (via an intermediate oligonucleotide, shown in green), and the second type of capture probe binds indirectly to a target nucleic acid in a sample (via another intermediate oligonucleotide, shown in orange). The capture probe may include at least one affinity reagent, which is shown with an asterisk.

[0030] Figure 6 : Shows the second type of probes that indirectly bind to target nucleic acids in the sample ( Figure 4 The positioning of the cleavable motif in the probe (or in the reporter probe) affects which substances are included with the released signaling oligonucleotide.

[0031] Figure 7 : Shows three types of probes for detecting proteins. In the top configuration, the probe comprises a nucleic acid linked to a protein binding domain; in this configuration, a cleavable motif (e.g., a cleavable linker, not shown) can be included between the nucleic acid and the protein binding domain or within the nucleic acid itself. In the middle configuration, the protein binding domain is linked to the nucleic acid, and the probe hybridizes to the nucleic acid. The probe (comprising a target binding domain and a nucleic acid linked to the protein binding domain (shown in green)) can bind to the probe before or after the target binding domain binds to the protein target (e.g., Figure 8 ). A cleavable motif may be included in one or both of the backbone or the nucleic acid attached to the protein binding domain. In this configuration, the Figure 2 and 4 The first or second type of probe shown in the figure is used to detect proteins. In the lower configuration, the protein binding domain is attached to the nucleic acid, and the intermediate oligonucleotide (shown in red) hybridizes to both the probe and the nucleic acid attached to the protein binding domain. In this configuration, the Figure 2 and 4 The first or second type of probe shown in is used to detect the protein.

[0032] Figure 8 :show Figure 7 The two upper panels show the probe before and after it has bound to a protein. The next panel shows the probe after its cleavable motif has been cleaved; in this panel, the cleavable motif is between the nucleic acid and the target binding domain. Once the nucleic acid has been released, it can be considered a signaling oligonucleotide. In the lower panel, the signaling oligonucleotide (released probe nucleic acid) is replaced by a reporter probe (e.g., Figure 2 and 4 shown in ).

[0033] Fig. 9 : Display from Figure 7The probe in the intermediate configuration shown in Figure 8 The positioning of the cleavable motif within the probe or within the reporter probe affects those substances that are included with the released signaling oligonucleotide.

[0034] Fig.10 : Shows the steps in the method of the present invention, wherein a signaling oligonucleotide from a region of interest (ROI) is detected.

[0035] Fig.11 :Showing the steps in the method of the present invention, wherein the target area is located on the first continuous section of the tissue sample, and the probe is applied to the second continuous section of the tissue sample. Release and collect signal oligonucleotides from the probe that is bound to the target in the first target area of ​​the second continuous section. Subsequently, release and collect signal oligonucleotides from the probe that is bound to the target in the second (until the nth) target area of ​​the second continuous section.

[0036] Fig.12 : Shows multiplex detection of multiple target nucleic acids and / or proteins from a first target region, followed by multiplex detection of multiple target nucleic acids and / or proteins from a second target region.

[0037] Fig.13 : A diagram illustrating the steps in the method of the present invention. The method shown may be referred to herein as "nCounter ® Digital Multiplex Immunohistochemistry (IHC).

[0038] Fig.14 : is a flow chart showing the use of nCounter compared with standard TSA-based multiplex IHC (lower part) ® Digital multiplex IHC (top) allows for simplified workflow and higher multiplexing.

[0039] Fig.15 : A photograph showing a digital micromirror device (DMD) attached to a Ti-E microscope (top) and a bright field image of a FFPE tissue section (bottom). Illumination (white dots) on the FFPE tissue (bright field image) shows multiple ROIs of approximately ~10-20 µm in size (i.e., the size of a single cell).

[0040] Fig.16: Schematic illustration of the components and optical paths involved in the invention when the method includes the use of a digital micromirror device (DMD). Wide field illumination using the DMD is focused on the sample. The LED provides sufficient illumination to excite the entire field of view at once, and with single cell illumination such that ~80-600 DMD pixels illuminate a 10 µm diameter cell. Common grade DMDs will provide adequate single cell resolution. DS: dichroic mirror; FW: filter wheel; and DMD: digital micromirror device.

[0041] Fig.17 : Illustration of the components and optical paths involved in the present invention when the method includes the use of a laser scanning device (e.g., a confocal scanning device). In the confocal scanning configuration, the light is directed by galvo-mirrors. This method requires an inexpensive 405 nm laser. DS: dichroic mirror; FW: filter wheel; and MM: motorized micromirror.

[0042] Fig.18 : Micrographs showing the overall histomorphology of tonsil samples, which were first imaged using dual-color fluorescence of Ki-67 (cell proliferation marker; green) and CD3 (immune cell marker; red). Twelve regions (including Fig.19 ).

[0043] Fig.19 : Yes Fig.18 nCounter for Ki-67 and CD3 in the four regions shown in ® Figure of data counts. Images are obtained from serial sections (to allow for inspection of various additional controls). In general, samples can be imaged using fluorescent antibodies and then digitally counted using the same slide (by UV exposure). Multiple targets analyzed across 12 regions (including the four regions shown here) show different profiles of Ki-67 and CD3 localization. Below the figure is the magnification of the four regions.

[0044] Fig. 20 : Display from Fig.18 Example counts of a 30-plex oligo-antibody cocktail across 12 regions of interest (ROIs) of the tonsil sample shown in . Data were acquired from serial sections (to allow examination of various additional controls).

[0045] Fig.21 : A micrograph showing the overall histomorphology of T cells in a melanoma sample from a lymph node, which was first imaged using three-color fluorescence of CD3 (red), CD8 (green), and DAPI (blue). The white circle has a diameter of 25 µm and encloses three cells.

[0046] Fig. 22 : nCounter showing CD3 conjugate release from FFPE lymph node tissue sections (5 µm thick) as a function of UV irradiation area (100 µm to 1 mm in diameter). ® Data. The field of view aperture size is shown below the graph.

[0047] Fig.23 : Shown as a function of UV irradiated area (100 µm to 1 mm in diameter) and from Fig.21 and 22 nCounter of CD45 conjugates released from FFPE lymph node tissue sections (5 µm thick) from the same experiment shown in ® data.

[0048] Fig.24 : Shown as a function of UV irradiated area (100 µm to 1 mm in diameter) and from Figures 21 to 23 nCounter of PD1 conjugates released from FFPE lymph node tissue sections (5 µm thick) from the same experiment shown in ® data.

[0049] Fig.25 : Shows a tissue microarray (TMA; left panel) of breast tumor tissue containing variable levels of Her2 protein, as shown in a micrograph (middle panel) of Her2 fluorescence determined by IHC staining. The right panel shows an enlargement of a single area of ​​the middle panel.

[0050] Fig.26 : nCounter showing 48 typical regions versus Her2 status (ASCO-CAP guidelines) ® Count data.

[0051] Fig. 27 :against Fig.26 The 48 regions mentioned, nCounter ® Count the relative total pixel intensity (x10 3 )’s drawing.

[0052] Fig.28 : is a micrograph to determine the overall tissue morphology of a melanoma sample, which first uses dual-color fluorescence imaging of CD3 (red) and DAPI (blue). 10 exemplary regions are identified using white boxes.

[0053] Fig.29 : Display from Fig.28 Example counts of 30 oligo-antibody mixtures across 10 regions of interest (ROIs) for the samples shown in FIG.

[0054] Fig. 30A and Fig. 30B : Micrograph showing single cells (blue) in a tonsil tissue sample (green) illuminated by UV using a digital micromirror device (DMD).

[0055] Figures 31A to 31D are micrographs showing single cells (bright white) in tonsil tissue samples irradiated with UV using a digital micromirror device (DMD). Fig.31B The single cells mentioned in Fig. 31A are highlighted; Fig.31D The single cells mentioned in Figure 31C are highlighted.

[0056] Fig.32 : Shows the steps in a spatially resolved FFPE tissue protein assay. The steps are similar to those of a nucleic acid detection assay, but in the nucleic acid detection assay, the sample is bound to a probe comprising a nucleic acid target binding domain rather than an antibody.

[0057] Fig.33 : Showing the steps in spatially resolved FFPE tissue protein assay.

[0058] Fig.34 : Shows data from an embodiment in which the entire tissue or entire sample is irradiated, for example using a standard UV gel cassette, to release signaling oligonucleotides previously attached to the probes.

[0059] Fig.35 : shows an embodiment in which, for example, a microscope is used to illuminate parts of the tissue or sample, ie UV cutting under the microscope (time titration experiment).

[0060] Fig.36 : shows an embodiment in which, for example, a part of a tissue or a sample is irradiated using a microscope, i.e., UV cutting under a microscope (irradiated area titration test).

[0061] Fig.37 : shows an embodiment in which, for example, a portion of a tissue or sample is irradiated using a microscope, ie UV cutting under a microscope (irradiation area titration test - multiple targets).

[0062] Fig.38 : An embodiment is shown in which marker expression is first determined for a target region in a tissue (eg, a breast cancer sample), and this target region is then irradiated (eg, using UV) to release the probe oligonucleotide from the probe.

[0063] Fig.39 : Shows an embodiment where tissue is embedded in a flow cell. Shows data for multiple fractions. Fig.38Data are provided, where the expression of fluorescently labeled markers in a predetermined target region is determined. A photograph and schematic diagram showing the configuration of the instrument are also shown.

[0064] Fig.40 : shows an embodiment in which tissue is embedded in a flow cell with small holes. Also shown are photographs and schematics showing the configuration of the instrument.

[0065] Figures 41A to 41C : Shows that an embodiment using a flow cell with small holes (rather than collecting eluate from the entire tissue surface) has a significant improvement in signal to noise ratio. Also shown are photographs and schematics showing the instrument configuration.

[0066] Figures 42A to 42C : Shows data for embodiments using a flow cell with small wells (12 or 96 well format).

[0067] Fig.43A and B: Data are shown to exclude background signal from the flow cell where whole tissue elution was performed ( Fig.43A ) and the background signal from the flow cell where elution occurs directly on the target area ( Fig.43B ) for comparison.

[0068] Fig.44 : is a schematic diagram showing an aspiration embodiment for multiple target areas, using open surface eluent collection. A multi-tube array for aspiration / dispensing eluent with rotary valve selection is shown here.

[0069] Fig.45 : Includes photographs and schematics showing an embodiment in which the eluent is collected by capillaries (microaspirator).

[0070] Fig.46A and B: including display from Fig.45 Data for an embodiment in which the eluate is collected by capillary (microaspirator).

[0071] Fig.47 : is a schematic diagram showing an embodiment of aspiration for multiple target areas or for a single target area, using an open surface for eluate collection. Shown here is a multi-tube array for aspiration / dispensing and a single tube / pipette with a fixed position using suction versus capillary action.

[0072] Fig.48 : is a schematic diagram showing illumination and fluid collection by a combined capillary and lens.

[0073] Fig.49 : is a schematic diagram showing steps in an embodiment of a spatially resolved FFPE tissue assay comprising a 96-well grid.

[0074] Fig.50 : Shows protein expression data obtained from a single cell or two cells using the methods and devices described herein.

[0075] Fig.51 : Identify target regions located on serial sections from a single tumor sample.

[0076] Fig.52 : Shows counts obtained from 6 of the 9 RNA probes included in the assay of Example 16.

[0077] Fig.53 :show Fig.52 Means and standard deviations of counts are shown in .

[0078] Fig.54 : Display simultaneous hybridization to nCounter ® Molecular barcodes and ® nCounter ® The system digitizes RNA expression data and protein data for counted probes.

[0079] Fig.55 : Shows RNA expression data obtained from single-stranded DNA probes and partially double-stranded DNA probes.

[0080] Fig.56 : Shows RNA expression data obtained from probes hybridized in the presence of salmon sperm DNA.

[0081] Fig.57 : Shows RNA expression data from a PSA (prostate specific antigen) specific probe.

[0082] Fig.58 : Shows that the specificity of the probe is improved at non-standard sub-nM concentrations. DETAILED DESCRIPTION

[0083] The present invention is based in part on probes, compositions, methods and kits for simultaneous, multiplexed detection and quantitation of protein and / or nucleic acid expression in user-defined tissue regions, user-defined cells, and / or user-defined subcellular structures within cells.

[0084] The present invention provides a comparison of the characteristics and abundance of target proteins and / or target nucleic acids present in a first target region (e.g., tissue types, cells (including normal and abnormal cells), and subcellular structures within cells) with the characteristics and abundance of target proteins and / or target nucleic acids present in a second target region. There is no predetermined upper limit to the number of target regions and comparisons that can be made; the upper limit relates to the size of the target regions relative to the size of the sample. For example, when a single cell represents the target region, then the slice may have hundreds to thousands of target regions; however, if the tissue slice includes only two cell types, then the slice may have only two target regions (each including only one cell type).

[0085] The present invention provides a higher multiple degree than using standard immunohistochemistry or in situ hybridization methods. Standard immunohistochemistry methods allow for simultaneous detection of up to 6 to 10 protein targets, more typically 3 to 4 protein targets. Similarly, in situ hybridization methods are limited to simultaneous detection of less than 10 nucleic acid targets. The present invention provides detection of large combinations of nucleic acid targets and / or protein targets from a defined area of ​​a sample. The present invention provides an objectively measured improvement through digital quantification and improved reliability and consistency, thereby enabling comparison of results between multiple centers.

[0086] The probes of the invention may have a nucleic acid backbone (single-stranded DNA or RNA) with defined positions capable of hybridizing (non-covalently binding) to at least one labeled oligonucleotide. Figure 1 . Such probes (which have a limited position capable of hybridizing (non-covalently binding) with at least one labeled oligonucleotide) are also referred to herein as reporter probes. The number of positions on the reporter probe ranges from 1 to 100 or more. In an embodiment, the number of positions ranges from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15, 20, 30, 40 or 50, or any range therebetween. In fact, there is no restriction on the number of positions on the skeleton (for detecting target nucleic acids and / or for detecting target proteins), because the engineering of such skeletons is completely within the capabilities of technicians. The number of target nucleic acids and / or proteins detectable by a group of probes depends on the number of positions included in the probe skeleton.

[0087] As used herein, a labeled oligonucleotide refers to an RNA segment that includes a detectable label, or a DNA oligonucleotide that includes a detectable label.

[0088] The position of nucleic acid backbone can be with at least one oligonucleotide hybridization of mark (non-covalent binding).Alternatively, the position can be with at least one oligonucleotide hybridization lacking detectable label.Each position can be hybridized to 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or 21 to 100 oligonucleotides of mark (or unlabeled) or more.The quantity of the labelled oligonucleotide hybridized with each position depends on the length of the position and the size of the oligonucleotide.The length of the position can be between about 300 to about 1500 nucleotides.The length of the oligonucleotide of labelling (or unlabelling) can be at about 20 to about 1500 nucleotide length variation.In embodiments, the length of the oligonucleotide of labelling (or unlabelling) can be at about 800 to about 1300 ribonucleotide variation. In other embodiments, the length of the labeled (or unlabeled) oligonucleotide can vary from about 20 to about 55 deoxyribonucleotides; such oligonucleotides are designed to have a melting / hybridization temperature between about 65 and about 85°C, such as about 80°C. For example, a position of about 1100 nucleotides in length can be hybridized to between about 25 and about 45 oligonucleotides, each of which comprises a length of about 45 to about 25 deoxyribonucleotides. In an embodiment, each position is hybridized to about 34 labeled oligonucleotides of about 33 deoxyribonucleotides in length. The labeled oligonucleotide is preferably a single-stranded DNA.

[0089] Each labeled oligonucleotide can use one or more detectable labeling monomer labels. The label can be at the end of the oligonucleotide, at a site in the oligonucleotide, or a combination thereof. The oligonucleotide can include amine-modified nucleotides that allow detectable labeling to be coupled to the nucleotide.

[0090] The labeled oligonucleotide of the present invention can use any label of a variety of labeling monomers, such as fluorescent dyes, quantum dots, dyes, enzymes, nanoparticles, chemiluminescent labels, biotin, or other monomers known in the art that can be detected directly (e.g., by light emission) or indirectly (e.g., by the combination of fluorescently labeled antibodies). The preferred embodiment of the labeling that can be utilized by the present invention is a fluorophore. A variety of fluorophores can be used as labeling monomers for labeling nucleotides, including but not limited to GFP-related proteins, cyanine dyes, fluorescein, rhodamine, ALEXA Flour , Texas Red, FAM, JOE, TAMRA and ROX. A variety of different fluorophores are known, and more fluorophores across the entire spectrum will continue to be produced.

[0091] The label that is combined with each position (by the hybridization of the position and the marker oligonucleotide) is spatially separable and can be spectrally distinguished from the label of the previous position or the subsequent position. A series of orderly spatially separable and spectrally distinguishable labels of probes are referred to as bar codes or marker codes in this article. Bar codes or marker codes allow determination of target nucleic acids or target proteins that have been combined with specific probes.

[0092] The labeled oligonucleotide is hybridized to its position under a standard hybridization reaction (e.g., 65°C, xSSPE); this allows for spontaneous assembly of the reporter probe or probes. Probes using longer RNA molecules as labeled oligonucleotides (e.g., as described in US2003 / 0013091) must be preassembled at the manufacturing site rather than by the end user, and at higher temperatures to avoid crosslinking through the multiple backbones of the longer RNA molecules; the preassembly step is followed by purification to remove excess unhybridized RNA molecules, which would increase background. The use of short single-stranded labeled oligonucleotides (e.g., comprising deoxyribonucleotides) greatly simplifies the manufacture of probes and reduces the costs associated with their manufacture.

[0093] In embodiments, the concentration of the probe provided to the sample is generally less than that used for immunohistochemistry (IHC) or for in situ hybridization (ISH). Alternatively, the concentration may be significantly lower than that used for IHC or ISH. For example, the probe concentration may be 2 times less, 5 times less, 10 times less, 20 times less, 25 times less, 30 times less, 50 times less, 60 times less, 70 times less, 80 times less, 90 times less, 100 times less, 200 times less, 300 times less, 400 times less, 500 times less, 600 times less, 700 times less, 800 times less, 900 times less, 1000 times less, 2000 times less, or less, and any quantity therebetween. In an embodiment, the probe is provided at a concentration of 100 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, 0.01 nM, and less, and any concentration therein.

[0094] Commercially available kits, software, and systems can be used, such as the nCounter ® Box nCounter ® Systems, detection and quantification probes.

[0095] Background noise during protein detection can be reduced by performing negative purification of the intact probe molecule. This can be accomplished by affinity purification of antibodies or photocleavable adapters after collecting the eluate from the target area. Typically, the released signaling oligonucleotide will not detach from the solution. For this step, a protein-G or -O method in a pipette tip, tube, or plate can be used. Such equipment and reagents are commercially available.

[0096] Background noise during nucleic acid detection can be reduced by performing negative purification of the complete probe molecule. This can be accomplished by performing affinity purification of the target binding domain or a light-cleavable linker after collecting the eluent from the target area. Typically, the released signaling oligonucleotide will not be detached from the solution. To assist in negative purification, a universal purification sequence may be included in the probe, for example in the target binding domain.

[0097] Figure 1 Two exemplary probes comprising a single-stranded nucleic acid backbone and a target binding domain are shown, shown in red. The upper probe comprises a labeled RNA segment that hybridizes to a position in the backbone, while the lower probe comprises a labeled DNA oligonucleotide that hybridizes to a position in the nucleic acid backbone. Figure 1 The colors shown elsewhere in this disclosure are non-limiting; other colored labels and other detectable labels known in the art can be used in the probes of the invention.

[0098] The probe of the present invention can be used to detect a target nucleic acid. Figure 2 and 4 This aspect is illustrated. This probe includes at least a skeleton and a target nucleic acid binding region. The target nucleic acid binding region preferably has a length of at least 15 nucleotides, and more preferably a length of at least 20 nucleotides. In a specific embodiment, the length of the target nucleic acid binding region is about 10 to 500, 20 to 400, 30 to 300, 35, 40 to 200, or 50 to 100 nucleotides. Probes and methods for combining and identifying target nucleic acids have been described in, for example, US2003 / 0013091, US2007 / 0166708, US2010 / 0015607, US2010 / 0261026, US2010 / 0262374, US2010 / 0112710, US2010 / 0047924, and US2014 / 0371088, each of which is incorporated herein by reference in its entirety. The target nucleic acid may be DNA or RNA, and is preferably cRNA, messenger RNA (mRNA) or miRNA; preferably the DNA is cDNA.

[0099] The probes of the present invention can be used to directly hybridize to a target nucleic acid. Figure 2The probe (or composition) of this embodiment is illustrated. The probe includes a target nucleic acid binding domain, shown in red. The target nucleic acid is shown as a blue curve. Figure 3 The diagram illustrates a dual probe composition comprising Figure 2 The capture probe and the capture probe are shown in Figures 1 and 2. The capture probe comprises at least one affinity reagent, shown as an asterisk. The at least one affinity portion can be attached to the capture probe in a covalent or non-covalent manner. Various affinity portions suitable for purification and / or for immobilization are known in the art. Preferably, the affinity portion is biotin, avidin or streptavidin. Other affinity tags are recognized by specific binding partners and therefore promote separation or immobilization by affinity binding with a binding partner that can be fixed on a solid support. In these figures, each probe comprises 6 positions for hybridization with a labeled oligonucleotide, each position being determined by a colored circle.

[0100] Any probe of the invention may comprise an affinity moiety.

[0101] The probes of the present invention can be used to hybridize indirectly (via an intermediary oligonucleotide) to a target nucleic acid present in a sample. Figure 4 The probe (or composition) of this embodiment is illustrated. The probe includes a target nucleic acid binding domain shown as red, which is bound to a synthetic oligonucleotide (intermediate oligonucleotide; shown as green), which in turn binds to the target nucleic acid in the biological sample. It can be said that the intermediate oligonucleotide is a probe as defined herein, because it includes a nucleic acid backbone and can bind to the target nucleic acid. The target nucleic acid present in the biological sample is shown as a blue curve. Figure 5 The diagram illustrates a dual probe composition comprising Figure 4 Probe and capture probe. In these embodiments, the target nucleic acid binding region of probe is different from the regional hybridization of the intermediate oligonucleotide (that is, synthetic oligonucleotide) of the target nucleic acid present in the sample. Therefore, the target binding region of probe is independent of the final target nucleic acid in the sample. This allows the economic and fast flexibility in the determination design, because the target (present in the sample)-specific component of the determination is included in the cheap and widely available synthetic DNA oligonucleotide, rather than in the more expensive probe. This synthetic oligonucleotide is simply designed by including the region with the target nucleic acid hybridization present in the sample and the region with the probe hybridization. Therefore, it is possible to detect infinitely diverse target nucleic acids (present in the sample) using a single indirect binding probe group in different experiments by simply replacing the target-specific (synthetic) oligonucleotide part of the determination.

[0102] Probe or the probe of the present invention may include the following region, which allows the release of signal oligonucleotide after applying a suitable force. In a non-limiting example, the region is a cleavable motif (e.g., a restriction enzyme site or a cleavable joint). The cleavable motif allows the release of signal oligonucleotide from the target nucleic acid or protein of combination, and then collects and detects the signal oligonucleotide. As used herein, the signal oligonucleotide is a probe region currently having a position hybridized with at least one labeled oligonucleotide, or a probe region (e.g., a nucleic acid molecule) that can be released from the target binding domain of the probe. When it can be separated from other parts of the probe (i.e., cutting and releasing), the signal oligonucleotide is referred to as releasable. The example of a cleavable part includes but is not limited to a light-cleavable joint.

[0103] In the probes of the invention (as described herein), the cleavable motif may be located between the nucleic acid and the target binding domain, between the backbone and the target binding domain, or within the backbone. Figure 6 In the present invention, a non-limiting choice of the position of the cleavable motif can be deduced from a gap in the probe or a gap in the intermediate oligonucleotide.

[0104] The probe of the present invention can be used to detect a target protein. Figure 7 The probe (or composition) of this embodiment is illustrated. This probe includes at least a backbone and a target protein binding region. In the protein targeting probe of the present invention, the signaling oligonucleotide can be a nucleic acid attached to the protein binding domain. In these probes, the signaling oligonucleotide is targeted and bound by a probe containing a position for hybridizing a labeling oligonucleotide. This probe is shown in Figure 7 In the middle panel of . Here, the signaling oligonucleotide is visible as a green line. The probe can be bound by the probe before the probe (through its protein binding domain) binds to the protein or after it binds to the protein. The signaling oligonucleotide does not need to be bound by the probe until it has been released from the target binding domain (this embodiment is not shown).

[0105] The probe region capable of binding to the target protein includes a molecule or assembly, which is designed to bind at least one target protein, at least one target protein surrogate or both, and can form a molecular complex comprising a protein probe and a target protein under appropriate conditions. The region capable of binding to the target protein includes an antibody, a peptide, an aptamer or a peptoid. Antibodies can be obtained from a variety of sources, including but not limited to polyclonal antibodies, monoclonal antibodies, monospecific antibodies, recombinantly expressed antibodies, humanized antibodies, plant antibodies, etc. In this article, the terms protein, polypeptide, peptide and amino acid sequence are used interchangeably to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids or synthetic amino acids. These terms also include amino acid polymers that have been modified, such as by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation, such as conjugation using a labeling component. The term amino acid as used herein refers to natural and / or non-natural or synthetic amino acids, including but not limited to glycine, and both D or L optical isomers, and amino acid analogs and peptoids. Probes and methods for binding and identifying target proteins have been described, for example, in US 2011 / 0086774, the contents of which are incorporated herein by reference in their entirety.

[0106] In an embodiment, the probe is prepared by a cysteine ​​bioconjugation method that is stable, preferably specific to the heavy chain site of the hinge region of the antibody. This preparation method provides a relatively controllable stoichiometric ratio of the labeled oligonucleotide to the antibody. The probe can include multiple (i.e., more than 1, such as 2, 3, 4, 5 or more) labeled oligonucleotides / antibodies. Typically, the sensitivity of a "heavier" probe comprising 3 or 4 labeled oligonucleotides / antibodies is significantly lower than that of an antibody lacking a labeled oligonucleotide, or a "lighter" probe comprising 1 or 2 labeled oligonucleotides / antibodies.

[0107] Protein targeting probes and nucleic acid targeting probes can be used simultaneously as long as conditions allow binding of both protein targets and nucleic acid targets. Alternatively, when conditions allowing binding of both protein targets and nucleic acid targets are not possible, protein targeting probes and nucleic acid targeting probes can be used sequentially.

[0108] Probe group is synonymous with probe composition. Probe group includes at least one kind of probe, i.e., points to a target. Probe group preferably includes at least two, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more kinds of probes. Probe group can include one or more copies of each kind of probe.

[0109] Only the first probe set may be applied to the sample. Alternatively, a second (or higher number) of probe sets may be subsequently applied to the sample. The first set and the second (or higher number) may target nucleic acids only, proteins only, or a combination thereof.

[0110] In the present invention, two or more targets (i.e., proteins, nucleic acids, or a combination thereof) are detected; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more targets are detected, and any number of targets in between.

[0111] The cell type or tissue type that can be targeted, the probe group is predetermined. For example, if the tissue is breast cancer, the probe group will include probes pointing to proteins associated with breast cancer cells (such as Her2, EGFR and PR) and / or probes pointing to proteins associated with normal breast tissue. In addition, the probe group can be predetermined based on the developmental state of the targeted cells or tissues. Alternatively, the probe group can be predetermined based on the subcellular localization of the target, such as the nucleus, cytoplasm and cell membrane. For example, antibodies pointing to Foxp3, histone H3 or P-S6 mark the nucleus, antibodies pointing to CD3, CD4, PD-1 or CD45RO mark the cytoplasm, and antibodies pointing to PD-L1 mark the cell membrane.

[0112] Probes can be chemically synthesized or can be produced biologically using a vector into which a nucleic acid encoding the probe has been cloned.

[0113] Any probe or set of probes described herein can be used in the methods and kits of the invention.

[0114] For the probes described herein, the association of a label code with a target nucleic acid or target protein is not fixed.

[0115] The probe of the present invention can be used to detect target nucleic acid or protein present in any sample (e.g., biological sample). As will be appreciated by those skilled in the art, the sample can contain any number of things, including but not limited to: cells (including primary cells and cultured cell lines) and tissues (including cultured or explanted). In an embodiment, the tissue sample (fixed or unfixed) is embedded, serially sliced, and fixed to a microscope slide. As is well known, a pair of serial sections will include at least one cell present in two serial sections. The structure and cell type located on the first serial section will have similar positioning on adjacent serial sections. The sample can be a cultured cell or a discrete cell (fixed or unfixed) that has been fixed to a slide.

[0116] In embodiments, the tissue sample is a biopsied tumor or part thereof, i.e., a clinically relevant tissue sample. For example, the tumor may be from breast cancer and the sample may be a resected lymph node.

[0117] The sample may be obtained from almost any organism, including multicellular organisms, such as plants, fungi and samples of the animal kingdom; preferably the sample is obtained from an animal, such as a mammal. Human samples are particularly preferred.

[0118] In some embodiments, the probes, compositions and kits described herein are used for diagnosis of a condition. The term diagnosis or diagnosis of a condition as used herein includes predicting or diagnosing a condition, determining a constitution susceptible to a condition, monitoring treatment of a condition, diagnosing a therapeutic response to a disease, and prognosis of a condition, progression of a condition, and response to a specific treatment of a condition. For example, a tissue sample can be assayed according to any of the probes, methods or kits described herein to determine the presence and / or amount of a marker of a disease or malignant cell type in a sample (relative to a non-diseased condition), thereby diagnosing or grading a disease or cancer.

[0119] Typically, samples attached to a slide can first be imaged using fluorescence (e.g., fluorescent antibodies or fluorescent dyes such as DAPI) to identify morphology, target regions, target cell types, and single cells, and then protein and / or nucleic acid expression can be digitally counted from the sample on the same slide.

[0120] The compositions and kits of the present invention may include probes and other reagents, such as buffers and other reagents known in the art, to facilitate the binding of proteins and / or nucleic acids in a sample, ie, for performing a hybridization reaction.

[0121] The kit will also include instructions for using the components of the kit, including but not limited to information necessary for hybridizing the labeled oligonucleotide to the probe, the probe to the target-specific oligonucleotide, the target-specific oligonucleotide to the target nucleic acid, and / or the probe to the target protein.

[0122] Exemplary protocols for detecting target nucleic acids and / or target proteins are described below and are shown in Figures 10 to 14 (upper) middle.

[0123] Cells (live or fixed) or tissue sections (e.g., formalin-fixed paraffin embedded (FFPE)) prepared in accordance with multiplex immunohistochemistry methods and / or nucleic acid in situ hybridization methods are prepared and fixed to glass slides or suitable solid supports. Access to the surface of the cell or tissue section is maintained to allow fluid exchange; this can be achieved by using a fluid chamber reagent exchange system (e.g., Grace™ Bio-Labs, Bend OR). On a serial section or on an adjacent serial section, a target region (ROI) is determined where the probe will be provided. In the first example, a full "macro-feature" imaging method is performed on the target cell / tissue, such as DAPI staining, membrane staining, mitochondrial staining, specific epitope staining, and specific transcript staining to determine the overall macro-features of the target cell / tissue. Optionally, on a serial section adjacent to the serial section, a target region (ROI) where the probe will be provided is determined; here, on the first serial section ( Fig.11 and 12 This procedure will roughly define the target area on adjacent serial slices ( Fig.10 The red line in the small and medium panels B, and Fig.11 and 12 The green oval and green triangle of slice #2 in Figure 1, where the signaling oligonucleotide will be released from the probe after applying an appropriate and directed force. Continuous slices can be approximately 5µm to 15µm apart from each other.

[0124] Fig.13 and 14 (Top) The steps of the present invention are further illustrated. Fig.13 The steps shown in the figure include the following. (1) Processing: FFPE slide-encapsulated tissue is incubated with a cocktail of primary antibodies conjugated to DNA oligomers via photocleavable linkers, along with a limited number of visible wavelength imaging agents. (2) Inspection: Regions of interest (ROIs) are identified at low-plex using visible light-based imaging agents to map out the overall “architecture” of the tumor slide (e.g., imaging cell nuclei and / or using one or two key tumor biomarkers). (3) Profiling: Selected ROIs are selected for high-resolution multiplex profiling, and oligomers are released from the selected regions after exposure to UV light. (4) Plating: Free photocleaved oligomers are then collected, e.g., by a microcapillary-based “pipette,” and stored in microplate wells for subsequent quantification. (5) Digital counting: During the digital counting step, photocleaved oligomers from spatially resolved ROIs in the microplate are hybridized to a 4-color, 6-spot optical barcode, allowing for the use of a standard NanoStringnCounter ®Readout instruments (e.g., SPRINT, Flex, and MAX), digitized counts of up to ~1 million protein targets (dispersed across up to 800 markers) in a single ROI.

[0125] The target region can be a tissue type, a cell type, a cell, or a subcellular structure within a cell present in a cell.

[0126] A composition comprising a set of probes is applied to the serial sections, each probe comprising a releasable signal oligonucleotide. The probe group may alternatively comprise a probe targeting a protein, a targeting nucleic acid or both. The composition may comprise a capture probe. When the probe is indirectly bound to the target (protein and / or nucleic acid), the composition applied comprises an intermediate oligonucleotide. The composition will comprise other reagents known in the art to promote the binding of proteins and / or nucleic acids in the sample.

[0127] A blocking step is performed before and / or after application of the composition.

[0128] For probes including a photocleavable linker, a solid support (e.g., a microscope slide) is placed in a microscope capable of providing excitation light at a wavelength capable of cleaving the photocleavable linker. The light is used to excite the first target region ( Fig.10 The red line in the small and medium-sized figure B and Fig.11 and 12 ROI i ), thereby cleaving the photocleavable linker and releasing the signaling oligonucleotide. Figure 6 and 9 As shown in , the signal oligonucleotide includes at least one probe region, which currently has a position to bind to at least one labeled oligonucleotide, or includes a nucleic acid from a probe, which binds or can bind to a reporter probe. By directing excitation light only to ROIi, the signal oligonucleotide is released only from the probe in ROIi, and not from the probe outside ROIi, and the probe outside ROIi retains its signal oligonucleotide. Therefore, only the probe bound to the target in ROIi is collected, thereby allowing the characteristics and amount of the target (protein and / or nucleic acid) in ROIi to be detected.

[0129] Wash the surface of the slide with a small amount of buffer (~5 to 30 µl) and collect the eluate (containing the released signaling oligonucleotide) into the first sample container ( Fig.12 The slice surface was further washed to remove any released signaling oligonucleotide omitted from the eluate.

[0130] Excite the second target region with light ( Fig.11 and 12 ROI j), thereby cutting the photocleavable linker and releasing the signaling oligonucleotide from the second target region. Again, by directing the excitation light only to ROIj, the signaling oligonucleotide is released only from the probes within ROIj, and not from the probes outside ROIj, which retain their signaling oligonucleotides. Therefore, only for the probes bound to the target within ROIj, the signaling oligonucleotide is collected, thereby allowing the characteristics and amount of the target (protein and / or nucleic acid) located within ROIj to be detected.

[0131] Wash the surface of the slide with a small amount of buffer (~5 to 30 µl) and collect the eluate (containing the released signaling oligonucleotide) into the first sample container ( Fig.12 The slice surface was further washed to remove any released signaling oligonucleotide omitted from the eluate.

[0132] Repeat the extraction step, washing step, and cleaning step until all target regions (until the ROI n ) Collect signal oligonucleotides.

[0133] Other advantages, features and embodiments of the present invention are described in the appendix submitted with this article. Various methods and equipment for collecting signal oligonucleotides and various ways for providing force are shown. In addition, the appendix also provides unexpected improved results obtained by specific embodiments of the present invention that exceed other embodiments. Data confirming an improvement in signal-to-noise ratio of about 7 times to about 200 times are shown.

[0134] Detection can use any microscope type device or system known in the art. Devices or systems may include wide field illumination and digital micromirror devices (DMD; see Fig.15 and 16 ); advantages include reduced cost, since the DMA and controller can also drive the LED (which will photocleave the probe) and add substantially no additional cost, provide convenient operation, allow small feature sizes of ~1 mm, which will include cells of 10-40 mm, and take advantage of available consumer electronics (such as projectors). The device or system may include a laser scanning device, such as a confocal, see Fig.16 The advantage of this is that smaller morphological features can be illuminated and imaged; however, the use of these devices involves additional costs.

[0135] Use polymerase reactions, reverse transcriptase reactions, hybridization to oligonucleotide microarrays, mass spectrometry, hybridization to fluorescent molecular beacons, sequencing reactions, or nCounter ®Molecular barcodes, in each eluate sample, identify multiple target proteins and / or target nucleic acids present in each target region in the sample. nCounter as described in US2003 / 0013091, US2007 / 0166708, US2010 / 0015607, US2010 / 0261026, US2010 / 0262374, US2010 / 0112710, US2010 / 0047924, US2014 / 0371088, and US2011 / 0086774 ® Systems and from NanoString Technologies ® The method is the preferred method for determining target proteins and / or target nucleic acids. ® Systems and from NanoString Technologies ® The method allows for the simultaneous multiplex determination of multiple (800 or more) different target proteins and / or target nucleic acids.

[0136] Collectively, the characteristics and abundance of target proteins and / or target nucleic acids present in a first target region (e.g., tissue types, cells (including normal and abnormal cells), and subcellular structures within cells) can be compared to the characteristics and abundance of target proteins and / or target nucleic acids present in a second target region or more target regions.

[0137] The present invention provides for multiplexed detection and comparison of up to 800 target proteins from, for example, a tumor and its adjacent normal tissue; thus, enabling systematic exploration of a tumor and its microenvironment.

[0138] The invention can be used in ongoing clinical studies to illustrate new responses to immunotherapy and other targeted therapies.

[0139] The present invention also enables the discovery of immune biomarkers, for example in tumors, which can be used in the development of companion diagnostics.

[0140] Immunohistochemistry is a powerful technique for analyzing protein expression and localization in FFPE tissue sections. However, it suffers from multiple challenges, including lack of dynamic range, difficulty in quantification, and labor-intensive workflows for very limited multiplexing. ® A novel platform of barcoding technology that enables highly multiplexed (up to 800-assay) assays (i.e., nCounter ®In digital multiplex immunohistochemistry (IHC) assays), spatially resolved digital characterization of proteins is performed. The assay relies on antibodies coupled to photocleavable oligonucleotide tags that are released from discrete areas of tissue using focused UV (e.g., ~365 nm) exposure through an objective lens. ® The label of quantitative cutting in the determination, and the count is plotted back to the tissue location, to produce a spatially resolved digital overview of protein abundance. Protein detection can be carried out together or separately with nucleic acid detection determination, and the nucleic acid detection determination uses a nucleic acid probe comprising a light-cleavable oligonucleotide label. Therefore, the present invention can provide a spatially resolved digital overview of protein abundance, a spatially resolved digital overview of protein and nucleic acid abundance, or a spatially resolved digital overview of nucleic acid abundance.

[0141] Advantages of the assay include, but are not limited to, high sensitivity (e.g., ~1 to 4 cells), fully digital counting, and a large dynamic range (>10 5 ), high multiplexing (e.g., 30 targets and expandable to 800 targets without changing instruments), simple workflow, compatibility with FFPE, no secondary antibodies (for protein detection) or amplification reagents, and the prospect of use in clinical assays.

[0142] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0143] Unless otherwise stated or obvious from the context, the term "or" as used herein should be construed as inclusive and encompasses both "or" and "and."

[0144] Unless otherwise indicated or apparent from the context, the term "about" as used herein should be understood to be within the normal tolerance range in the art, such as within 2 standard deviations of the mean. Approximately can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value. Unless otherwise apparent from the context, all numerical values ​​provided herein are modified by the term "about".

[0145] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Preferred materials and methods are described herein, but other probes, compositions, methods and kits similar or equivalent to those described herein can also be used in the practice of the present invention. It should be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting. Example

[0146] Example 1: The present invention provides a "barcoding landscape" for quantifying multiple targets in FFPE tissue sections.

[0147] Heterogeneity within tumors has become a major challenge to the implementation of targeted therapeutics. Historically, immunohistochemistry (IHC) has been used to assess the spatial heterogeneity of proteins; however, it is difficult to use to quantify protein abundance with high multiplexity and a wide dynamic range.

[0148] In this example, proteins in formalin-fixed paraffin-embedded (FFPE) tissue sections are labeled using antibody-containing probes that include a photocleavable linker and a fluorescent barcode. Subsequently, the probes in the defined ROI of the FFPE tissue section are exposed to focused UV light, thereby releasing the signaling oligonucleotide (including the fluorescent barcode) from the ROI. The released signaling oligonucleotide is washed out and collected from the FFPE sample. Subsequently, the signaling oligonucleotide is purified by a PCR product from NanoString Technologies. ® nCounter ® The system identifies and digitally counts the fluorescent barcodes of the signaling oligonucleotides released, thereby quantifying the abundance of each targeted protein in the user-defined spatial region of the tissue section. After releasing and collecting the signaling oligonucleotides from the first ROI, the focused UV light is exposed to the second ROI of the FFPE tissue section to define the ROI, thereby releasing the signaling oligonucleotides from the second ROI. In this non-limiting embodiment, it is observed that the numerical value of the observed count is highly linear relative to the UV irradiation area (0.97 < R 2 <0.99), and has a detection spatial resolution of about 100 µm x 100 µm, or about 100 cells. Unexpectedly, the present invention provides a "barcoding landscape" for quantifying up to 800 targets in a single FFPE tissue section with a dynamic range of 5.5 logs (base 10).

[0149] Example 2: The present invention provides a practical and feasible method for quantifying protein expression without signal amplification and for achieving a higher order of magnitude of target antigen multiplexing in FFPE tissue sections.

[0150] Quantitative multiplex immunohistochemistry has emerged as an area of ​​interest in oncology because of its unique ability to determine spatiotemporal organization and interdependencies, which further defines how checkpoint blockade affects the tumor microenvironment. This example describes a one-step, amplification-free staining method using a photocleavable oligo-tagged primary antibody that interacts with a target antigen within FFPE tissue sections. Ultraviolet (UV) light irradiation is applied to release the oligomers from the antibody, and the eluate is then collected, quantified, and digitally counted, which corresponds to antigen abundance.

[0151] Various conjugation methods were first investigated; this identified a cysteine ​​bioconjugation method that was stable, specific primarily to the hinge region heavy chain site, and relatively controllable in terms of the stoichiometric ratio of oligonucleotide to antibody.

[0152] Linear regression analysis was then performed to determine the relationship between the UV-induced cleavage area and the measured digitized protein counts; a high degree of linearity was observed (0.97 < R 2 < 0.99), confirming the basic mechanism / premise associated with this multiplex protein counting approach for FFPE tissues.

[0153] In order to determine the influence of the presence of conjugated oligonucleotides on antibody-antigen interaction, the performance of the following aspects of the labeled oligonucleotide-conjugated antibody and the unmodified antibody under the same conditions in FFPE tissue sections is compared: sensitivity, specificity and signal intensity. The antibody targeting the antigen located in the nucleus, cytoplasm or membrane is selected to determine the relationship between the subcellular localization of the antibody performance and the target antigen. The selected antibody targets Foxp3, histone H3, P-S6 (nuclear antigen), CD3, CD4, PD-1, CD45RO (cellular antigen), and PD-L1 (membrane antigen). In terms of sensitivity, generally, it is found that the "heavier" oligonucleotide conjugated antibody (with 3 or 4 labeled oligonucleotides / antibody) is not conjugated to the antibody or "lighter" oligonucleotide conjugated antibody (with 1 or 2 labeled oligonucleotides / antibody) when compared, the sensitivity is significantly lower. Between the unconjugated or "lighter" oligonucleotide conjugated antibodies, no significant differences in sensitivity, specificity or intensity are observed between the target antigens of the nucleus, cytoplasm and membrane.

[0154] The present invention provides highly multiplexed protein profiling that measures absolute protein expression levels using a practical and feasible approach to comprehensively define the immune landscape in tumors before and during immunotherapeutic intervention.

[0155] Example 3: The present invention provides spatially resolved, multiplexed protein detection from FFPE tissues.

[0156] method

[0157] Antibodies: Antibodies used in this example and Examples 4-6 may include: "Target (clone ID, supplier)")": H3 (D1H2, CST), CD8 (OTI3H6, Origene), CD4 (SP35, Spring Bio), FOXP3 (D2W8E, CST), B7-H3 (D9M2L, CST), S6 (54D2, CST), B7-H4 (D1M8I, CST), Granzyme B (OTI4E4, Origene), Ki67 (8D5, CST), PD-1 (Nat105, Cell Marque), CD3 (MRQ-39, Cell Marque), Vista (D1L2G, CST), Her2 (29D8, CST), PR (D8Q2J, CST), ER (SP1, Spring Bio), EGFR (D38B1, CST), CD56 (MRQ-42, Cell Marque), PD-L1 (E1L3N, CST), CD45 (2B11&PD7 / 26, Cell Marque), TIM-3 (D5D5R, CST), and Pan-keratin (C11, CST), CD45RO (UCHL1, Cell Marque).

[0158] Microscopic observation of tonsils: 5 µm sections of tonsil FFPE blocks (Amsbio) were mounted on slides. IHC was performed using standard methods. Antigen retrieval was performed using a pressure cooker. Tonsil sections were stained using the primary CD3 antibody MRQ-39 (Rabbit mAb, CellMarque) and the primary Ki-67 antibody 8D5 (Mouse mAb, CST). Secondary incubations were performed using Alexa594-labeled goat α rabbit (Life Tech.) and Alexa488-labeled goat α mouse (Life Tech.).

[0159] Here, a sample attached to a slide is first imaged using fluorescent antibodies, and then the expression of the protein is digitally counted from the sample.

[0160] Use something like Figures 10 to 14 (top) The UV cut-out of the selected ROI allowed the digital profiling of all 30 species (nCounter ® count).

[0161] result

[0162] Fig.18Shown are photomicrographs to determine the overall tissue morphology of tonsil samples, which were first imaged using dual-color fluorescence of Ki-67 (cell proliferation marker; green) and CD3 (immune cell marker; red). Fig.19 Multiple targets analyzed (four areas magnified in ), showing three different profiles of Ki-67 and CD3 localization. Fig.19 Shows Fig.18 nCounter for Ki-67 and CD3 in the four regions shown in ® Data count. Fig. 20 Shows the Fig.18 30 kinds of example counts of oligo-antibody mixtures on 12 target regions (ROI) of tonsil sample shown in . Data are obtained from serial sections (to allow examination of various additional controls). As shown, the regions of tissue samples can be classified based on the intensity and characteristics of the markers expressed. Exemplary classification is shown as: "CD3-enriched", "Ki67-enriched", "mixed", and "connective tissue".

[0163] These data show that the present invention provides spatially resolved detection of multiple (here at least 30) protein markers. By expanding the number of protein probes (antibodies) used, up to 800 different protein markers can be detected with similar resolution.

[0164] Example 4: The present invention provides multiplex protein detection from FFPE and achieves single-cell resolution.

[0165] method

[0166] Melanoma microscopy: 5 µm sections of melanoma (lymph node derived) FFPE blocks (Asterand) were mounted on slides. IHC was performed using standard methods. Antigen retrieval was performed using a pressure cooker.

[0167] Here, the sample is first imaged using fluorescence, and then the expression of the protein is digitally counted from the sample.

[0168] Use something like Figures 10 to 14 (top) The steps are as shown in (top).

[0169] result

[0170] Fig.21 : A micrograph showing the overall histomorphology of T cells in a melanoma sample from a lymph node, which was first imaged using three-color fluorescence of CD3 (red), CD8 (green), and DAPI (blue). The white circle has a diameter of 25 µm and encloses three cells.

[0171] Fig. 22 : nCounter showing CD3 conjugate release from FFPE lymph node tissue sections (5 µm thick) as a function of UV irradiation area (100 µm to 1 mm in diameter). ® Data. The limit of detection counts (LOD = background counts + 2x standard deviation) corresponds to a spatial resolution of 26 µm in diameter. The field of view aperture size is indicated below the graph. Fig.23 and Fig.24 Data for CD45 and PD1 (each from the same experiment) are shown.

[0172] The data show that the spatial detection capability of the present invention corresponds to about 1 to 4 cells.

[0173] Example 5: The present invention provides quantitative performance of clinically relevant assays

[0174] method

[0175] Use something like Figures 10 to 14 (top) The steps are as shown in (top).

[0176] Breast cancer tissue microarray (TMA): TMA BR1504a was obtained from US Biomax, Inc., and H&E staining images were obtained from the US Biomax webpage (World Wide Web (www) biomax.us / tissue-arrays / Breast / BR1504a). Fig.25 The slices of the same block as the slices shown in the left side of the small figure were stained with Her2 primary antibody 29D8 (Rabbit mAb, CST) and goat α rabbit (Life Tech.) labeled with Alexa594. The counts of histone H3, ribosomal protein S6, estrogen receptor, progesterone receptor, mouse IgG isotype control and rabbit IgG isotype control were also obtained (data not shown). The Her2 pathological score for TMABR1504a is provided by US Biomax, Inc. (World Wide Web ( www ) biomax.us / tissue-arrays / Breast / BR1504a). Use Her2 primary antibody 29D8 (Rabbit mAb, CST) and Alexa594 labeled goat α rabbit (Life Tech.) for staining. Although other rabbit primary antibodies were used in the first mixture, the fluorescence from these antibodies was negligible compared to Her2 fluorescence. ImageJ software was used to obtain total pixel intensity (at λ =594). For this reason, the background value was set to intensity = 0, and the highest intensity was set to intensity = 255. The sum of all pixel intensities for each ROI is shown.

[0177] Here, the sample is first imaged using fluorescence, and then the expression of the protein is digitally counted from the sample.

[0178] result

[0179] Fig.25 (Left panel) shows a tissue microarray (TMA) of breast tumor tissue containing variable levels of Her2 protein, as shown in a micrograph (middle panel) of Her2 fluorescence determined by IHC staining. The right panel shows an enlargement of a single area of ​​the middle panel; such areas are stained using a multiplex antibody cocktail.

[0180] Fig.26 nCounter showing 48 representative regions versus Her2 status (ASCO-CAP guidelines) ® Count data. Fig. 27 For the 48 regions mentioned above, nCounter ® Count the relative total pixel intensity (x10 3 ) icon.

[0181] These mathematical count data showed significant correlation with visual Her2 status according to ASCO-CAP guidelines (R2 = 0.51, Fig.26 ) compared with the fluorescence intensity (R2 = 0.92, Fig. 27 ) are highly correlated.

[0182] Example 6: The present invention reveals the abundance of specific cell types in tissue samples.

[0183] Use something like Figures 10 to 14 (top); first the melanoma sample attached to the slide is imaged using fluorescence, and then the expression of the protein is digitally counted from the sample.

[0184] Fig.28 Shown is a micrograph of the overall tissue morphology of a melanoma sample using dual-color fluorescence of CD3 (immune cell marker; red) and DAPI (nuclei; blue). Expression data were obtained from 10 regions identified by white boxes using a cocktail of 30 antibodies. Fig.29 Shows the Fig.28 Example nCounter® counts of 30 oligo-antibody mixtures on 10 regions of interest (ROIs) of the melanoma sample shown in . Counts are shown for 13 markers, each with expression counts above background. Regions 5, 6, 7, identified as "immune infiltrate enriched," have the highest expression of T-cell markers and T-cell regulatory markers.

[0185] These data show that the present invention provides spatially resolved detection of multiple (here at least 30) protein markers. By expanding the number of protein probes (antibodies) used, up to 800 different protein markers can be detected with similar resolution.

[0186] Example 7: A digital micromirror device (DMD) is capable of illuminating single cells.

[0187] 30 and 31 are photomicrographs showing that UV irradiation using a digital micromirror device (DMD) can irradiate single cells in tonsil tissue samples.

[0188] These data show that the present invention is capable of single cell resolution when using DMD.

[0189] Example 8: The gel cassette is capable of irradiating the entire sample and releasing signaling oligonucleotides from probes bound to the entire sample.

[0190] Fig.34 The following embodiment is shown, wherein the whole tissue or sample is irradiated, for example, using a standard laboratory UV gel box. Here, FFPE tissue slides are placed on a light plate, a wax pencil holding buffer solution (TBS) is used to cover the FFPE tissue, and UV light exposure (276 - 362nm, e.g., 302nm; ~5mW / cm2) is applied to the tissue through a glass slide (1 mm thick). 2 ). The data show that within about 1 minute of UV exposure, most of the signaling oligonucleotides were released from the FFPE-bound antibodies. Counts were normalized to the positive control.

[0191] Example 9: Illumination from a microscope can illuminate a target area in a sample and release the signaling oligonucleotide from the probe bound to the target area.

[0192] Fig.35 The following embodiment is shown, in which for example a portion of the tissue or sample is irradiated using a microscope, i.e. UV cutting under the microscope (time titration experiment). This is in contrast to the experiment in Example 8 in which the entire sample was irradiated. Here, the UV radiation was irradiated at about 150 mW / cm 2 and 20x objective lens, applying UV LED (at 365nm). UV irradiation scans the entire tissue area determined by previous fluorescent (~590nm excitation) bright field imaging. Within about 1 second of UV exposure per field of view (FOV), most signal oligonucleotides are released from FFPE bound probes. Counts are normalized to positive control ratios. Blue: microscope data with variable exposure time length; Red: gel box 2.5 minute exposure data. Photos and schematics showing microscope instrument configuration are also shown.

[0193] Fig.36Release of signaling oligonucleotide from a uniformly distributed anti-histone (H3) antibody bound to a lung tissue sample is shown. The tissue was exposed to UV (365 nm, ~150 mW / cm 2 and 20x objective) / approx. 450µm x 330µm = 0.15 mm 2 The field of view (FOV) of the image was 100 μm. The "macro volume" used to collect the effluent was about 70 μl. This reduced the limit of detection to (FOV / 5) ~99 μm x 99 μm, and about 5 μl of effluent was collected. Therefore, in this example, the limit of detection was a niche of about 10 cells x 10 cells. These data show that the antibody signal is proportional to the spatially resolved irradiated area FOV, and the "macro fluid" limit of detection (LOD) was inferred.

[0194] Fig.37 The following embodiments are shown, wherein for example a microscopically illuminated portion of a tissue or sample is used, i.e. UV cutting under a microscope (irradiated area titration test - multiple targets) and for multiple targets. UV cutting of multiple targets in tissue is shown: two positive targets (histone H3 and ribosomal S6), and eight (8) negative targets. Only one negative target (Ox40) showed high background. Data from 0, 1, 4, 9 and 16 fields of view are shown.

[0195] Example 10: The target area can be predetermined by labeling technology, and then the target area can be irradiated to release the signaling oligonucleotide from the probe bound to the predetermined target area.

[0196] Fig.38 The following embodiment is shown, wherein the marker (here Her2) expression of a target region in a tissue (e.g., a breast cancer sample) is first determined, and then the target region is irradiated (e.g., using UV) to release the probe oligonucleotide from the bound probe. The data shown compares the amount of signaling nucleotides released from two locations: a target region predetermined to be Her2+ and a target region predetermined to be Her2- for two targets (here Her2 and histone H3).

[0197] Example 11: Samples embedded in a flow cell provide for collection of eluate from the entire sample and not just from the target area that is illuminated and from which the signaling oligonucleotide is released.

[0198] Fig.39An embodiment is shown in which tissue is embedded in a flow cell. Here, the tissue embedded in the microfluidic flow cell (9 mm annular chamber, with a volume of 100 µm height and a volume of approximately 25 µl [approximate volume is 75 µl when the flow cell has a 300 µm height]) is controlled by a syringe pump. UV cuts inside the flow cell showing the elution profile, irradiating one area (9 FOV) and eluting, followed by irradiating another area (9 FOV) and eluting. Data for multiple fractions are shown. As for the data for Example 10, the expression of fluorescently labeled markers in the target area is predetermined here.

[0199] Example 12: Samples embedded in a flow cell containing a small hole on the target area provide efficient collection of eluate from the illuminated target area and release signaling oligonucleotides from the target area rather than from the entire sample.

[0200] Fig.40 An embodiment is shown in which the tissue is embedded in a flow cell with small holes. Here, the elution is sent directly above the target area. The holes with a diameter of 0.4-1mm on the fluid chamber allow the collection of the eluate (e.g., 5μl collection volume). Tested are 9-well, 96-well formats, and 12-well formats (for tissue microarrays (TMA)). Fluorescent images are generated by combining multiple fields of view. Photos and schematics showing the instrument configuration are also shown.

[0201] Figures 41A to 41C The embodiment using a flow cell with small holes (rather than collecting eluate from the entire tissue surface) is shown to have a significant improvement in signal to noise ratio. The data show that the signal to noise ratio is improved by about 7 times by collecting the eluate with a small hole above the target area. In this embodiment, the eluate (5μl fractions) was collected using a 1mm diameter hole above the fluid flow cell (25μl chamber). Data for multiple fractions are shown.

[0202] Figures 42A to 42C Data are shown using a flow cell with small wells (12 or 96 well format). The data show an approximately 7-fold improvement in signal to noise ratio by collecting eluate in a small well above the target area. In this embodiment, the field of view illumination was focused at the center of the well; 5 µl elution volume / well.

[0203] Fig.43A A and B show data to exclude background signal from the flow cell where whole tissue elution was performed ( Fig.43A ; as in Example 11) and the background signal from the flow cell where elution occurs directly above the target area ( Fig.43B ) for comparison. Fig.43A As can be seen in Fig.43B There is a higher background for whole tissue washout. Fig.43B No differences were shown between incubations in flow cells and non-flow cells.

[0204] Example 13: Released signaling oligonucleotides can be selected by a single tube / pipette, multiple tubes / pipettes, or a multi-tube / pipette array.

[0205] Fig.44 is a schematic diagram showing an embodiment of aspiration for multiple target areas, using open surface eluent collection. A multi-tube array with rotary valve selection for aspiration / dispensing of eluent is shown here. See also Fig.47 .

[0206] Fig.45 Included are photographs and schematics showing embodiments in which the eluent is collected via capillaries (microaspirator). Fig.47 . Fig.46A and B shows the Fig.45 Data for an embodiment in which the eluate is collected by a capillary (microaspirator). This embodiment has a significant improvement in signal to noise ratio: about 10 times compared to a flow cell eluting through a hole, and about 200 times compared to whole tissue elution. Here, the LOD area is approximately 60 µm x 60 µm.

[0207] Example 14: A device comprising both illumination and elution capabilities can efficiently and accurately obtain nucleic acid and / or protein expression data from a defined target area.

[0208] Fig.48 is a schematic diagram showing illumination and fluid collection by a combined capillary and lens.

[0209] Example 15: Protein expression from single cells can be detected and quantified.

[0210] Fig.50 Protein expression data obtained from a single cell or two cells using the methods and devices described herein are shown. In the upper panel, S6 protein from at least one cell was detected and quantified, and in the lower panel, CD45 protein from at least one cell was detected and quantified.

[0211] Example 16: The methods and devices described herein provide accurate and efficient detection and quantification of spatially resolved expression of multiple RNA targets and / or protein targets.

[0212] In situ hybridization (ISH) was performed to hybridize DNA oligomer-based probes ("RNA probes"), each of which contained a target binding domain, a signaling oligonucleotide, and a photocleavable linker, to endogenous RNA. 5 μm FFPE HER2 3+ breast tissue sections were dewaxed in xylene, partially rehydrated in graded ethanol, and incubated in 70% ethanol for 1 hour at room temperature. The sections were then incubated in 40 μg / ml proteinase K for 25 minutes at 37°C. The tissues were then incubated in 50% formamide / 2X SSC for 15 minutes at room temperature and hybridized overnight at 37°C in a solution of 1 nM probe, 40% formamide, 1 mg / ml yeast tRNA, 10% dextran sulfate, and 0.2% BSA in 2X SSC. After hybridization, two stringent washes were performed in 50% formamide / 2X SSC, each for 25 minutes at 37°C. Using TO-PRO ® The sections were stained with a fluorescent nucleic acid dye, TU-3 (Thermo Fisher Scientific), to visualize tissue morphology. Subsequently, focused UV light guided by a digital micromirror device was used to cleave DNA signaling oligonucleotides from probes in user-defined regions of interest (ROIs). For each tissue section, two ROIs contained tumor tissue, two ROIs contained normal tissue, and two ROIs contained no tissue at all (the histology slide itself). After cleavage, the signaling oligonucleotides were collected and compared with the nCounter ® Molecular barcode hybridization and by NanoString Technologies ® nCounter ® Systematic digital counting. H&E was performed on tissue sections to verify tumor and normal tissue ROIs.

[0213] On serial sections, standard immunohistochemistry (IHC) was performed using "protein probes" each containing an antibody as a target binding domain, a DNA signaling oligonucleotide, and a photocleavable linker. Subsequently, anti-rabbit Alexa594 secondary antibody and TO-PRO ®-3 (Thermo Fisher Scientific) fluorescent nucleic acid dyes are stained with sections to make tissue morphology visible. Subsequently, using the focused UV light guided by a digital micromirror device, the probe cutting DNA signal oligonucleotides in the user-defined ROI are used. For each tissue section, two ROIs contain tumor tissue, one ROI contains normal tissue, and two ROIs are completely free of tissue (histology slides themselves). ROI matches the ROI selected for ISH probe cutting. After cutting, the signal oligonucleotides from the protein target are mixed with the signal oligonucleotides from the RNA target, and all are quantitatively described above. H&E is performed on the tissue section to verify tumor and normal tissue ROIs, and to verify the correct matching between ISH and IHC tissues.

[0214] Fig.51 ROIs sampled from serial sections of the same tumor sample are shown. Regions 1-4 are not shown in this image and are instead taken from tissue sections that do not contain tissue (negative control - "no tissue"). Regions 5-8 contain a small amount of tumor cells ("normal tissue"). Regions 9-12 contain a large amount of tumor cells ("tumor").

[0215] Fig.52 The counts obtained from 6 of the 9 RNA probes included in this assay are shown. For each ROI, before applying UV irradiation ("-UV" data set), and before collecting UV samples from the same region, samples are collected ("+UV" data set). When UV is not applied to the sample, background count levels are obtained; The UV dependence of the signal obtained is thus shown. Background counts are given for +UV but not for the ROI (i.e. ROI 1-4 - "no tissue") that points to tissue. Low counts (orange bars on the figure) are given to the HER2 probe in the region that is mainly normal tissue (i.e. ROI 5-8 - "normal tissue"). Higher counts are given to the HER2 probe in the region that is mainly tumor tissue (i.e. ROI 9-12 - "tumor") . Similar but lower significantly, the increase of ribosomal S6 probe (green bar in the figure) is seen. Other control probes that target RNA that is not expected to be highly expressed in this tissue type give consistent counts, which do not show the level of difference between normal and tumor tissues. These control probes are designed to target CD45, PSA (prostate-specific antigen), and two unique ERCC sequences. For clarity of consideration, Fig.53 Shows Fig.52 The mean and standard deviation of the data are shown in .

[0216] These RNA probe samples were also run simultaneously with protein probes from sample regions of the tumor sample. To do this, RNA and protein probes were hybridized simultaneously to the nCounter ®Molecular barcoding and ® nCounter ® The system digitizes the count. The count for this determination is displayed on Fig.54 Figure 2. Increased HER2 RNA probe counts (red bars in the upper panel) and protein probe counts (red and orange bars in the lower panel) are seen in tumor areas compared to normal areas. Only + UV samples are shown. As mentioned above, - UV control samples are not shown in this figure because they give background counts (similar to "no tissue" counts). ROI 6 and ROI 8 were removed from this analysis because no matching protein probe samples were obtained. Therefore, the signaling oligonucleotide from the protein probe and the signaling oligonucleotide from the RNA probe can be detected and quantified together.

[0217] Example 17: Partially double-stranded probes have higher signal-to-noise ratios when compared to single-stranded probes.

[0218] As described in Example 16, DNA probes (recognizing and binding to mRNA) were hybridized in situ with RNA in 5 μm FFPE tissue. Whole tissue sections mounted on separate slides were UV cut for 3 minutes in 2X SSC + 0.1% Tween 20 using a UV light box (gel box). After cutting and releasing the signaling oligonucleotide, the signaling oligonucleotide was collected by pipette and detected as described in Example 16. Fig.55 Counts for single-stranded DNA probe, partial double-stranded DNA probe, and no-probe control of HER2 3+ breast and tonsil tissue are shown in (upper panel). The signal-to-noise ratio was determined by dividing the counts by the average background counts (average ERCC counts); see Fig.55 , lower small picture.

[0219] Fig.18 : Addition of salmon sperm DNA improves probe hybridization.

[0220] As described above, DNA probes (recognizing and binding to mRNA) were hybridized in situ with RNA in 5 μm FFPE tissues. During hybridization, 1 mg / ml ultrasonically denatured salmon sperm DNA was used instead of yeast tRNA. The slides were hybridized using 1 nM probe, 40% formamide, 1 mg / ml ultrasonically denatured salmon sperm DNA, 10% dextran sulfate, and 0.2% BSA in 2X SSC. UV cutting and signal oligonucleotide collection and detection were performed as described in Example 17. In HER2 3+ breast and tonsil ( Fig.56 ) The single-stranded DNA probe is shown in . The signal-to-noise ratio was determined by dividing the counts by the average background counts (average ERCC counts).

[0221] Example 19: PSA (prostate specific antigen) RNA probes are highly specific.

[0222] As described above, DNA probes (recognizing and binding to mRNA) were hybridized in situ with RNA in 5 μm sections of FFPE prostate. A 10-minute incubation in MES at 97°C was used instead of a 1-hour ethanol incubation. UV cutting, signal oligonucleotide collection and detection, and signal-to-noise ratio calculation were performed as described in Example 17. Counts and ratios are displayed in Fig.57 middle.

[0223] Fig. 20 : Probe specificity is improved at non-standard sub-nM concentrations.

[0224] Typically, in situ hybridization (ISH) probes for recognizing RNA hybridize at 5-200 nM. Unexpectedly, the nucleic acid recognition probes of the present invention perform best at or below 0.2 nM, which is 25-1000 times lower than the standard ISH probe concentration.

[0225] DNA probes were hybridized in situ with RNA in 5 μm sections of FFPE HER2 3+ breast samples as described above. Probes were used at 5, 1, 0.2, and 0.4 nM. UV cleavage, signal oligonucleotide collection and detection, and fold change calculations were performed as described in Example 17.

[0226] Fig.58 Counts are shown to decrease as probe concentration decreases (upper panels). However, unexpectedly, when probes hybridize at sub-nM concentrations, there is a significant gain in signal ratio when comparing positive probe counts to negative control probes.

Claims

1. A method for non-diagnostic purposes, comprising: (1) contacting at least one nucleic acid target in a tissue sample with at least one probe, the probe comprising a target binding domain, a signaling oligonucleotide, and a photocleavable motif located between the target binding domain and the signaling oligonucleotide; (2) irradiating a specific location of the tissue sample with light sufficient to separate the signaling oligonucleotide from the probe, thereby generating released signaling oligonucleotide; (3) eluting the solution containing the released signaling oligonucleotide at the irradiated position; (4) collecting an eluted solution containing the released signaling oligonucleotide, wherein the released signaling oligonucleotide is collected from a solution proximal to the tissue sample; as well as (5) determining the released signaling oligonucleotide, thereby detecting at least one nucleic acid target in a specific location of the irradiated tissue sample.

2. The non-diagnostic method of claim 1, wherein the detection comprises determining the characteristics and abundance of the at least one nucleic acid target.

3. The method of claim 2 for non-diagnostic purposes, wherein the at least one nucleic acid target comprises at least two different nucleic acid targets or at least two copies of the same nucleic acid target.

4. The non-diagnostic method of claim 3, wherein the detecting comprises comparing the abundance of each of the different nucleic acid targets.

5. The non-diagnostic method of claim 1, further comprising repeating at least steps (2) to (5) at at least a second specific location in the tissue sample.

6. The non-diagnostic method of claim 5, wherein detecting comprises comparing the abundance of the at least one nucleic acid target in the specific location with the abundance of the at least one target in at least a second specific location.

7. The method of claim 1 for non-diagnostic purposes, wherein the tissue sample is fixed directly to a surface, or is fixed indirectly to the surface.

8. The method for non-diagnostic purposes according to claim 7, wherein the tissue sample is a tissue section with a thickness of 2 to 1000 μm.

9. The method of claim 8 for non-diagnostic purposes, wherein the tissue sections are obtained from formalin-fixed paraffin embedded (FFPE) samples.

10. The method for non-diagnostic purposes of claim 1, wherein the tissue sample is fixed or unfixed.

11. The non-diagnostic method of any one of claims 1 to 10, wherein the tissue sample is stained or labeled prior to step (2), thereby allowing visualization of subcellular, cellular or tissue-related structures in the stained or labeled tissue sample.

12. The method for non-diagnostic purposes according to any one of claims 1 to 10, wherein the signaling oligonucleotide is a single-stranded nucleic acid or a partially double-stranded nucleic acid.

13. The method for non-diagnostic purposes according to any one of claims 1 to 10, wherein negative purification is used to remove intact probe molecules from the released signaling oligonucleotides.

14. The method of claim 13 for non-diagnostic purposes, wherein the negative purification comprises affinity purification, which comprises contacting the intact probe with an immobilized oligonucleotide complementary to a portion of the intact probe, or with an immobilized protein binding motif that recognizes and binds to a portion of the intact probe.

15. The method of claim 13 for non-diagnostic purposes, wherein the negative purification comprises affinity purification, which comprises contacting the intact probe with an immobilized antibody that recognizes and binds to a portion of the intact probe.

16. The method of claim 14 for non-diagnostic purposes, wherein the intact probe comprises a target binding domain, the target binding domain comprising a universal purification tag, or a sequence that is partially complementary to the immobilized oligonucleotide or capable of being recognized or bound by an immobilized protein binding motif.

17. The method of non-diagnostic purpose of claim 15, wherein the complete probe comprises a target binding domain, and the target binding domain comprises a universal purification tag, or a sequence that can be recognized or bound by an immobilized antibody.

18. The non-diagnostic method of claim 1, wherein the light is provided by a light source selected from the group consisting of an arc lamp, a laser, a focused ultraviolet light source, and a light emitting diode (LED).

19. The non-diagnostic method of any one of claims 1-10 and 14-18, wherein the target binding domain comprises a single-stranded nucleic acid or a partially double-stranded nucleic acid.

20. The non-diagnostic method of any one of claims 1-10 and 14-18, wherein the nucleic acid target is DNA or RNA.

21. The method of claim 20 for non-diagnostic purposes, wherein the RNA is cRNA, mRNA or miRNA.

22. The method for non-diagnostic purposes of claim 20, wherein the DNA is cDNA.

23. A method for non-diagnostic purposes according to any one of claims 1-10, 14-18 and 21-22, wherein the signaling oligonucleotide is collected from the sample by liquid laminar flow, vortex flow or transition flow in a channel with a depth of 25 to 500 μm between the sample and a fluid device placed on the tissue sample.

24. The non-diagnostic method of claim 1, wherein the solution proximal to the tissue sample is at least immediately above the tissue sample.

25. The non-diagnostic method of claim 24, wherein the solution proximal to the tissue sample is collected by suction.

26. The method of claim 25 for non-diagnostic purposes, wherein the aspirating is by a pipette, a capillary, a microarray needle and / or a flow cell comprising a well.

27. The non-diagnostic method of claim 26, wherein the capillary tube comprises an optical device capable of transmitting ultraviolet light to the tissue sample.

28. The method of claim 26 for non-diagnostic purposes, wherein the pipette or microarray needle is connected to an array comprising a plurality of pipettes or microarray needles.

29. The non-diagnostic method of any one of claims 1-10, 14-18, 21-22, and 24-28, wherein the solution proximal to the tissue sample comprises an anionic polymer.

30. The method of claim 29 for non-diagnostic purposes, wherein the anionic polymer comprises dextran sulfate, salmon sperm DNA, or a combination thereof.

31. The method for non-diagnostic purposes of any one of claims 1-10, 14-18, 21-22, 24-28 and 30, wherein the collected signaling oligonucleotides are added to a solution comprising an anionic polymer.

32. The method for non-diagnostic purposes of claim 31, wherein the anionic polymer comprises dextran sulfate, salmon sperm DNA, or a combination thereof.

33. The non-diagnostic method of any one of claims 1-10, 14-18, 21-22, 24-28, 30 and 32, wherein the method further comprises irradiating the target area using a laser scanning device.

34. The non-diagnostic method of any one of claims 1-10, 14-18, 21-22, 24-28, 30 and 32, wherein the method further comprises illuminating the target area using a digital micromirror device.

35. The non-diagnostic method of any one of claims 1-10, 14-18, 21-22, 24-28, 30 and 32, wherein the method provides for simultaneous spatially resolved DNA and / or RNA detection of a sample.

36. The non-diagnostic method of any one of claims 1-10, 14-18, 21-22, 24-28, 30 and 32, wherein the sample is attached to a slide and the expression of nucleic acids from the sample is first imaged using fluorescence and then digitally counted.

37. The non-diagnostic method of any one of claims 1-10, 14-18, 21-22, 24-28, 30 and 32, wherein the probe is provided at a concentration of 5 nM or less.

38. The method of claim 37 for non-diagnostic purposes, wherein the probe is provided at a concentration of 1 nM or less.

39. The method of claim 38 for non-diagnostic purposes, wherein the probe is provided at a concentration of 0.4 nM or less.

40. The method of claim 39 for non-diagnostic purposes, wherein the probe is provided at a concentration of 0.2 nM or less.

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