Simultaneous quantification of multiple proteins in a user-defined region of a cross-sectional tissue section.

Abstract

A need exists for probes, compositions, methods, and kits for the simultaneous, multiplexed detection and quantification of protein expression within user-defined regions of tissue, user-defined cells, and / or user-defined subcellular structures within cells. The present invention relates, inter alia, to probes, compositions, methods, and kits for the simultaneous, multiplexed detection and quantification of protein expression within user-defined regions of tissue, user-defined cells, and / or user-defined subcellular structures within cells.

Description

[Background technology]

[0001] Cross-references relating to related applications This application claims priority and benefit to U.S. Provisional Patent Application No. 62 / 193,819 filed on 17 July 2015, U.S. Provisional Patent Application No. 62 / 261,654 filed on 1 December 2015, U.S. Provisional Patent Application No. 62 / 277,283 filed on 11 January 2016, and U.S. Provisional Patent Application No. 62 / 323,018 filed on 15 April 2016. Each of the aforementioned applications is incorporated herein by reference in its entirety.

[0002] Background of the Invention Standard immunohistochemistry typically allows for the simultaneous detection of 3-4, but at most 6-10, protein targets. There is a need for probes, compositions, methods, and kits for the simultaneous, multiplexed detection and quantification of protein expression within user-defined regions of tissue, user-defined cells, and / or user-defined subcellular structures within cells. [Overview of the Initiative]

[0003] Summary of the Invention The present invention relates to probes, compositions, methods, and kits for the simultaneous, multiplex detection and quantification of protein expression in user-defined regions of tissue, user-defined cells, and / or user-defined subcellular structures within cells.

[0004] Aspects of the present invention relate to a method comprising the steps of (1) contacting at least one protein target in or from at least one cell in a tissue sample with at least one probe comprising a target-binding domain and a signal oligonucleotide; (2) applying sufficient force to a location in the tissue sample to release the signal oligonucleotide; and (3) recovering and identifying the released signal oligonucleotide to detect at least one protein target in or from a specific location in the tissue sample to which the force was applied. The specific location is a user-defined region of tissue, a user-defined cell, and / or a user-defined subcellular structure within a cell. The target-binding domain includes protein-binding molecules, such as antibodies, peptides, aptamers, and peptoids. In some embodiments, two or more protein targets are detected. In multiple embodiments, 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, and any number in between; for example, 800 or more different targets may be detected. In multiple embodiments, detection includes quantifying the quantity of each target.

[0005] In some embodiments, the method further comprises repeating at least steps (2) and (3) for at least a second specific location of the tissue sample, wherein the second specific location comprises at least a second cell. In some embodiments, detection comprises comparing the amount of at least one protein target in or from the first specific location with that of at least a second specific location. The at least one cell and the at least second cell may be of the same cell type or different cell types. In some embodiments, detection comprises quantifying the amount of at least one protein target in or from the first cell type and that of at least a second cell type. In some embodiments, the first and second cell types are independently selected from normal and abnormal cells, e.g., pathological and cancerous cells.

[0006] In several embodiments, the at least one cell is fixed directly to the surface or indirectly to the surface via at least one other cell. The tissue sample may be a tissue section 2–1000 μm thick, obtained, for example, from a formalin-fixed paraffin-embedded (FFPE) sample or an unfixed sample. At least one cell may be fixed or unfixed. At least one cell may be stained or labeled before step (2) to allow visualization of subcellular or cellular structures of the stained or labeled cell. Alternatively, with respect to a tissue section, a section adjacent to the section in contact with the probe may be stained or labeled before step (2) to allow determination of corresponding cells or subcellular or tissue-related structures within nearby cells in the section in contact with the probe. Such staining or labeling techniques are well known in the art.

[0007] In the above embodiment, at least one probe further comprises a linker (e.g., a cleavable linker) located between the target-binding domain and the signal oligonucleotide. The cleavable linker may be photocleavable and is cleaved by light supplied by a suitable coherent light source (e.g., a laser and a UV light source) or a suitable incoherent light source (e.g., an arc lamp and a light-emitting diode (LED)). The light source can be irradiated to at least one subcellular structure of at least one cell, and the amount of at least one protein target in or from at least one subcellular structure of at least one cell can be detected. Alternatively, the light source can be irradiated first to at least one subcellular structure of at least one cell, and then to at least one subcellular structure of at least a second cell, allowing for a comparison between the amount of at least one protein target in or from at least one subcellular structure of at least one cell and that of at least one subcellular structure of at least a second cell.

[0008] In some embodiments, the signal oligonucleotide is a single-stranded nucleic acid or a partially double-stranded nucleic acid.

[0009] In several embodiments, the sample may be cultured cells or dissociated cells (fixed or unfixed) fixed on a slide. The sample may include cells (including both primary cells and cultured cell lines) and / or tissues (including cultured or explanted tissues). The sample may include dissociated cells from cultured cells, primary cells, or explants.

[0010] In several embodiments, illuminating a region of interest smaller than the field of view (e.g., a single cell or subcellular structure within a cell) involves the use of a laser scanning device (e.g., confocal) or a digital mirror device (DMD) for guiding light.

[0011] In several embodiments, the probe is prepared by a stable, site-selective cysteine ​​bioconjugation reaction, preferably against the heavy chain of the hinge region of the antibody. In several embodiments, the probe may contain multiple (i.e., two or more, e.g., two, three, four, five or more) labeled oligonucleotides for each antibody.

[0012] Detection includes polymerase reactions, reverse transcriptase reactions, hybridization to oligonucleotide microarrays, mass spectrometry, hybridization to fluorescent molecular beacons, sequencing reactions, or nCounter® molecular barcodes. In preferred embodiments, nCounter® systems and methods from NanoString Technologies® are used.

[0013] In several embodiments, the signal oligonucleotide is recovered from the tissue via a liquid layer, turbulence, or transition flow. The flow may be, for example, via a channel having a depth of 25 to 500 μm between the tissue and a fluid device or an impermeable layer placed on the tissue.

[0014] In some embodiments, the signal oligonucleotide is recovered from a proximal solution of at least one cell, for example, at least immediately above. The proximal solution may be recovered by aspirating, for example, a pipette, capillary tube, microarray pin, flow cell with holes, or another suitable aspiration system known in the art, or any combination thereof. The capillary tube may be equipped with an optical device that can transmit the force of light, for example, UV light, to at least one cell. The pipette or microarray pin may be mounted on an array comprising multiple pipettes or microarray pins. The proximal solution may contain an anionic polymer, for example, dextran sulfate, and / or salmon sperm DNA, and / or the recovered signal oligonucleotide may be added to a solution containing an anionic polymer, for example, dextran sulfate, and / or salmon sperm DNA. In addition to or instead of salmon sperm DNA, other nonspecific blocking agents known in the art may be used.

[0015] In several embodiments, the method provides simultaneous spatially resolved protein detection in tissue samples.

[0016] In some embodiments, the digital readout has a linear dynamic range of 5 logs or more.

[0017] In several embodiments, the probe is typically supplied to the sample at a lower concentration than that used for immunohistochemistry (IHC) or 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 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 25, 1 / 30, 1 / 50, 1 / 60, 1 / 70, 1 / 80, 1 / 90, 1 / 100, 1 / 200, 1 / 300, 1 / 400, 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, 1 / 1000, 1 / 2000, or less, and any value in between. In several embodiments, the probe is provided in concentrations of 100nM, 70nM, 60nM, 50nM, 40nM, 30nM, 20nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM, 0.01nM, and less than or equal to any value in between.

[0018] In several embodiments, tissue samples are attached to slides and first imaged using fluorescence (e.g., fluorescently labeled antibodies and fluorescent stains (e.g., DAPI)), and then protein expression is digitally counted from the samples.

[0019] In several embodiments, reverse purification, including affinity purification methods that involve contacting the complete probe molecule with an immobilized oligonucleotide complementary to a portion of the complete probe, or with an immobilized antibody or protein-binding motif that recognizes and binds to a portion of the complete probe, is used to isolate the complete probe molecule from the released signal oligonucleotide. In several embodiments, the target-binding domain of the complete probe includes a universal purification tag or a sequence that is partially complementary to the immobilized oligonucleotide, or that can be recognized or bound by an immobilized antibody or protein-binding motif. Any such tag or sequence known in the art may be used in these embodiments.

[0020] Any aspect or embodiment described herein can be combined with any other aspect or embodiment disclosed herein. While the disclosures are described in conjunction with their detailed description, the above description is intended to be illustrative and not to limit the scope of the disclosure as defined by the appended claims. Other aspects, advantages, and modifications are also within the scope of the following claims.

[0021] The patents and scientific literature referenced herein establish knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated herein by reference. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific papers cited herein are incorporated herein by reference.

[0022] A patent or application file must include at least one color drawing. A copy of the patent or publication of the patent application, accompanied by the color drawing, will be provided by the Patent Office upon request and payment of the required fees. [Brief explanation of the drawing]

[0023] [Figure 1] Shows two representative probes. The nucleic acid backbone (either single-stranded DNA or single-stranded RNA) is shown as a straight black line. Each probe contains a target-binding domain shown in red. The upper probe contains a labeled RNA segment hybridized to the nucleic acid backbone, whereas the lower probe contains a labeled DNA oligonucleotide hybridized to the nucleic acid backbone. A cleavable motif (e.g., a cleavable linker, not shown) can be placed between the backbone and the target-binding domain or within the backbone. The cleavable motif enables the release of signal oligonucleotides from the bound target nucleic acid or protein; thereafter, the signal oligonucleotides are recovered and detected. [Figure 2] Shows a first type of probe that can bind directly to a target nucleic acid (top). In the following image, the probe is shown bound to the target nucleic acid as a blue curve. In this drawing and subsequent drawings, the reporter probe contains six locations for hybridizing to a labeled oligonucleotide (indicated by colored circles). Since the probe contains locations for hybridizing to the labeled oligonucleotide, the probe can also be referred to as a reporter probe. [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 contain at least one affinity reagent, which is shown as a star. The target nucleic acid in the sample is shown as a blue curve. [Figure 4] Shows a second type of probe (or reporter probe) that can bind indirectly to a target nucleic acid in a sample (top). Here, the target of the probe is a mediator oligonucleotide shown in green, and it binds sequentially to the target nucleic acid in the sample shown as a blue curve in the lower image. The mediator oligonucleotide may be said to be a probe as defined herein because it contains a nucleic acid backbone and can thus bind to the target nucleic acid. [Figure 5]The second type of dual probe composition of the present invention is shown. Here, the second type probe indirectly binds to the target nucleic acid in the sample (via a mediating oligonucleotide shown in green), and the second type capture probe indirectly binds to the target nucleic acid in the sample (via another mediating oligonucleotide shown in orange). The capture probe may contain at least one affinity reagent, which is indicated by an asterisk. [Figure 6] This shows the release of signal oligonucleotides from a second type of probe (illustrated in Figure 4) indirectly bound to the target nucleic acid in the sample. The location of the cleavable motif within the probe (or reporter probe) influences which material is included in the released signal oligonucleotide. [Figure 7] Three types of probes used to detect proteins are shown. In the top form, the probe comprises a nucleic acid attached to a protein-binding domain; in this form, a cleavable motif (e.g., a cleavable linker, not shown) may be contained between the nucleic acid and the protein-binding domain, or within the nucleic acid itself. In the middle form, the protein-binding domain is attached to a nucleic acid, and the probe hybridizes to that nucleic acid. The probe (with a target-binding domain and a nucleic acid attached to the protein-binding domain (shown in green)) can be bound by the probe before and after its target-binding domain binds to a protein target (as shown in Figure 8). The cleavable motif may be contained in either the backbone or the nucleic acid attached to the protein-binding domain, or both. The first or second type of probe shown in Figures 2 and 4 may be used in this form to detect proteins. In the bottom form, the protein-binding domain is attached to a nucleic acid, and a mediating oligonucleotide (shown in red) hybridizes to both the probe and the nucleic acid attached to the protein-binding domain. The first or second type of probe shown in Figures 2 and 4 may be used in this form to detect proteins. [Figure 8]Figure 7 shows the probe in the middle and bottom. The two images at the top show the probe before and after it binds to a protein. The next image shows the probe after its cleavable motif has been cleaved; in this image, the cleavable motif is between the nucleic acid and the target binding domain. Once the nucleic acid is released, it can be considered a signal oligonucleotide. In the bottom image, the reporter probe binds to the signal oligonucleotide (the nucleic acid from which the probe was released) (as shown, for example, in Figures 2 and 4). [Figure 9] Figure 7 shows the middle morphology of the probe, and Figure 8 shows the release of signal oligonucleotides from the probe. The location of the cleavable motif within the probe (or reporter probe) influences which materials are included in the released signal oligonucleotide. [Figure 10] The present invention provides steps for detecting a signal oligonucleotide from a region of interest (ROI). [Figure 11] The steps of the present invention are shown, wherein the region of interest is located in the first serial section of the tissue sample, and the probe is applied to the second serial section of the tissue sample. The signal oligonucleotide is released from the probe bound to the target in the first region of interest of the second serial section and recovered. Next, the signal oligonucleotide is released from the probe bound to the target in the second (up to the nth) region of interest of the second serial section and recovered. [Figure 12] Multiple target nucleic acids and / or proteins are detected from the first region of interest, followed by multiple target nucleic acids and / or proteins from the second region of interest. [Figure 13] The steps of the present invention are illustrated. The method shown may also be referred to herein as “nCounter® Digital Multiple Immunohistochemistry (IHC)”. [Figure 14] This flowchart demonstrates the simplified workflow and more advanced multiplexing possible with nCounter® Digital Multiplexed IHC (top) compared to a standard TSA-based multiplexed IHC (bottom). [Figure 15] This photograph shows bright-field images of a digital mirror device (DMD) (top) and an FFPE tissue section (bottom) attached to a Ti-E microscope. Light-irradiated areas (white dots) on the FFPE tissue (bright-field image) indicate multiple ROIs approximately 10-20 μm in size, i.e., single-cell size. [Figure 16] The components and optical paths related to the present invention when the method involves the use of a digital mirror device (DMD) are illustrated. Wide-field illumination using a DMD is focused on the sample. The LED provides illumination sufficient to excite the entire field of view at once and provides single-cell illumination such that ~80-600 DMD pixels illuminate cells with a diameter of 10 μm. A standard-grade DMD will provide sufficient single-cell resolution. DS: Dichroic mirror, FW: Filter wheel, and DMD: Digital mirror device. [Figure 17] The components and optical paths related to the present invention when the method involves the use of a laser scanning device (e.g., a confocal scanning device) are illustrated below. In a confocal scanning configuration, a galvanized mirror guides the light. This method requires an inexpensive 405 nm laser. DS: dichroic mirror, FW: filter wheel, and MM: motorized mirror. [Figure 18] Micrographs establishing the overall histological morphology of the tonsil sample, initially imaged using two-color fluorescence: Ki-67 (a green cell proliferation marker) and CD3 (a red immune cell marker), are shown. Twelve regions (including the four regions magnified in Figure 19) are identified by white frames. [Figure 19]Figure 18 shows graphs illustrating nCounter® data counts for Ki-67 and CD3 across four regions. Images were obtained from serial sections (to allow for various additional controls to be investigated). Generally, samples can be imaged using fluorescent antibodies and then digitally counted (by UV exposure) using the same slides. Multiple targets analyzed across 12 regions (including the four regions shown here) exhibit different profiles of Ki-67 and CD3 localization. The graphs below show magnifications for the four regions. [Figure 20] Figure 18 shows representative counts of 12 regions of interest (ROIs) from tonsil samples using a 30-plex oligo antibody cocktail. Data were obtained from serial sections (to allow for various additional controls to be investigated). [Figure 21] Micrographs establishing the overall histological morphology of T cells in a melanoma sample from a lymph node, initially imaged using three colors of fluorescence: CD3 (red), CD8 (green), and DAPI (blue). The white circles, 25 μm in diameter, surround three cells. [Figure 22] The nCounter® data regarding the CD3 complex released from FFPE lymph node tissue sections (5 μm thick) as an effect of UV irradiation (diameter 100 μm to 1 mm) is shown. The field of view aperture size is shown below the figure. [Figure 23] The nCounter® data regarding the CD45 complex released from FFPE lymph node tissue sections (5 μm thick) and from the same experiments shown in Figures 21 and 22, as an example of the effect of UV irradiation (diameter 100 μm to 1 mm), are shown. [Figure 24] The effects of UV irradiation (diameter 100 μm to 1 mm) are shown in nCounter® data regarding PD1 complexes released from FFPE lymph node tissue sections (5 μm thick) and from the same experiments shown in Figures 21-23. [Figure 25]The left panel shows a tissue microarray (TMA) of breast tumor tissue, including variable levels of Her2 protein, as shown in the micrograph (center panel) with Her2 fluorescence identified by IHC staining. The right panel shows a magnified view of a single region from the center panel. [Figure 26] This shows nCounter® count data for 48 representative regions against Her2 status (ASCO-CAP guidelines). [Figure 27] Plots of nCounter® count versus total pixel intensity (×10³) for the 48 regions mentioned in Figure 26. [Figure 28] This is a micrograph establishing the overall histological morphology of a melanoma sample initially imaged using two colors of fluorescence: CD3 (red) and DAPI (blue). Ten representative regions are identified by white frames. [Figure 29] Figure 28 shows representative counts of 10 regions of interest (ROIs) from the sample using a 30-plex oligo antibody cocktail. [Figure 30A] This is a microscopic image showing UV irradiation using a digital mirror device (DMD) on a single cell (blue) in a tonsil tissue sample (green). [Figure 30B] This is a microscopic image showing UV irradiation using a digital mirror device (DMD) on a single cell (blue) in a tonsil tissue sample (green). [Figure 31A-31D] This is a micrograph showing UV irradiation of a single cell (bright white) in a tonsil tissue sample using a digital mirror device (DMD). This is a micrograph showing UV irradiation of a single cell (bright white) in a tonsil tissue sample using a digital mirror device (DMD). Figure 31B highlights the single cell mentioned in Figure 31A. This is a micrograph showing UV irradiation of a single cell (bright white) in a tonsil tissue sample using a digital mirror device (DMD). This is a micrograph showing UV irradiation of a single cell (bright white) in a tonsil tissue sample using a digital mirror device (DMD). Figure 31D highlights the single cell mentioned in Figure 31C. [Figure 32]The steps of a spatially resolved FFPE tissue protein assay are shown. These steps are the same as those of a nucleic acid detection assay, except that the sample is bound to a probe containing a nucleic acid target binding domain rather than an antibody. [Figure 33] The steps for the spatially degraded FFPE tissue protein assay are shown. [Figure 34] The data shown is from embodiments in which the entire tissue or sample is irradiated, for example, using a standard UV gel box, to release signal oligonucleotides before being attached to a probe. [Figure 35] Embodiments in which a portion of the tissue or sample is irradiated, for example using a microscope, i.e., UV cutting under a microscope (time titration experiment). [Figure 36] This describes an embodiment in which a portion of the tissue or sample is irradiated, for example, using a microscope, i.e., UV sectioning under a microscope (irradiation-area titration experiment). [Figure 37] This example shows an embodiment in which a portion of the tissue or sample is irradiated, for example, using a microscope, i.e., UV sectioning under a microscope (irradiation area titration experiment - multiple targets). [Figure 38] This embodiment describes a region of interest within a tissue (e.g., a breast cancer sample) that is first identified in relation to the expression of a marker, and then this region of interest is irradiated (e.g., with UV) to release a signal oligonucleotide from a probe. [Figure 39] An embodiment in which tissue is embedded in a flow cell is shown. Data for multiple fractions are presented. Similar to the data in Figure 38, the region of interest is pre-identified with respect to the expression of a fluorescently labeled marker. Photographs and illustrations showing the configuration of the apparatus may also be presented. [Figure 40] An embodiment is shown in which tissue is embedded in a flow cell with small holes. Photographs and diagrams illustrating the configuration of the apparatus are also provided. [Figure 41A]Embodiments using a flow cell with small holes demonstrate a significant signal-to-noise improvement over the recovery of eluate from the entire tissue surface. Photographs and diagrams illustrating the configuration of the apparatus are also provided. [Figure 41B] Embodiments using a flow cell with small holes demonstrate a significant signal-to-noise improvement over the recovery of eluate from the entire tissue surface. Photographs and diagrams illustrating the configuration of the apparatus are also provided. [Figure 41C] Embodiments using a flow cell with small holes demonstrate a significant signal-to-noise improvement over the recovery of eluate from the entire tissue surface. Photographs and diagrams illustrating the configuration of the apparatus are also provided. [Figure 42A] The data for embodiments using flow cells with small holes (12 or 96-hole format) are shown. [Figure 42B] The data for embodiments using flow cells with small holes (12 or 96-hole format) are shown. [Figure 42C] The data for embodiments using flow cells with small holes (12 or 96-hole format) are shown. [Figure 43A] Figure 43A shows the background signal from a flow cell in which whole tissue elution was performed, compared to the background signal from a flow cell in which elution occurred directly over the region of interest (Figure 43B). [Figure 43B] Figure 43A shows the background signal from a flow cell in which whole tissue elution was performed, compared to the background signal from a flow cell in which elution occurred directly over the region of interest (Figure 43B). [Figure 44] This diagram illustrates eluate recovery at an open surface for an embodiment of aspiration of multiple regions of interest. Here, a multi-tube array for eluate aspiration / distribution using a rotary valve switch is shown. [Figure 45] Includes photographs and diagrams illustrating embodiments in which the eluate is recovered through a capillary tube (microaspirator). [Figure 46A]The data from the embodiment shown in Figure 45, in which the eluate is recovered through a capillary tube (microaspirator), is presented. [Figure 46B] The data from the embodiment shown in Figure 45, in which the eluate is recovered through a capillary tube (microaspirator), is presented. [Figure 47] This diagram illustrates embodiments of aspiration of multiple regions of interest or eluate recovery on an open surface for a single region of interest. Here, a multi-tube array using pipetting versus capillary action for aspiration / distribution, or a single tube / pipette in a fixed position, is shown. [Figure 48] This diagram illustrates irradiation and fluid recovery using a combination of capillary tubes and lenses. [Figure 49] This is a diagram illustrating the steps of an embodiment of a spatially degraded FFPE tissue assay with a 96-well grid. [Figure 50] Protein expression data obtained from a single cell or two cells using the methods and apparatus described herein are shown. [Figure 51] Identify regions of interest located on serial sections from a single tumor sample. [Figure 52] The counts obtained for six of the nine RNA probes included in the assay of Example 16 are shown. [Figure 53] Figure 52 shows the mean and standard deviation of the counts. [Figure 54] The image shows RNA expression data and protein data for probes that were simultaneously hybridized with nCounter® molecular barcodes and digitally counted by an nCounter® system from NanoString Technologies®. [Figure 55] RNA expression data obtained from single-stranded DNA probes and partially double-stranded DNA probes are shown. [Figure 56] The RNA expression data obtained from probes hybridized in the presence of salmon sperm DNA is shown. [Figure 57]This shows RNA expression data obtained using a probe specific to PSA (prostate-specific antigen). [Figure 58] This demonstrates that the probe's specificity is enhanced at non-standard, sub-nM concentrations. [Modes for carrying out the invention]

[0024] Detailed description of the invention The present invention is in part based on probes, compositions, methods, and kits for the simultaneous, multiplex detection and quantification of protein and / or nucleic acid expression within user-defined regions of tissue, user-defined cells, and / or user-defined subcellular structures within cells.

[0025] The present invention provides a comparison of the identity and quantity of target proteins and / or target nucleic acids present in a first region of interest (e.g., tissue type, cells (including normal and abnormal cells), and intracellular subcellular structures) with the identity and quantity of target proteins and / or target nucleic acids present in a second region of interest. There is no predefined upper limit on the number of regions of interest and the number of comparisons that can be performed; the upper limit is related to the size of the region of interest relative to the sample size. For example, when a single cell is the sample of the region of interest, the section may have hundreds or thousands of regions of interest; however, if a tissue section contains only two cell types, then the section may have only two regions of interest (each containing only one cell type).

[0026] This invention provides a higher degree of multiplicity than is possible with standard immunohistochemistry or in situ hybridization. Standard immunohistochemistry allows for the simultaneous detection of up to 6–10 protein targets, although 3–4 protein targets are more typical. Similarly, in situ hybridization is limited to the simultaneous detection of fewer than 10 nucleic acid targets. This invention enables the detection of numerous combinations of nucleic acid targets and / or protein targets from a defined area of ​​a sample. This invention achieves increased objective measurement through digital quantification, as well as enhanced reliability and consistency, thereby enabling the comparison of results across multiple centers.

[0027] The probes of the present invention may have a nucleic acid backbone (single-stranded DNA or RNA) having a predetermined position that can hybridize (non-covalently bond) with at least one labeled oligonucleotide. See Figure 1. Such probes (having a predetermined position that can hybridize with at least one labeled oligonucleotide) are also referred to herein as reporter probes. The number of positions on the reporter probe backbone ranges from 1 to 100 or more. In some embodiments, the number of positions ranges from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-15, 20, 30, 40, or 50, or any range in between. In practice, there is no limit to the number of positions on the backbone (for detecting target nucleic acids and / or target proteins), as the design of such backbones is well within the capabilities of those skilled in the art. The number of target nucleic acids and / or proteins detectable by a set of probes depends on the number of positions included in the probe backbone.

[0028] As used herein, labeled oligonucleotides refer to RNA segments containing a detectable label or DNA oligonucleotides containing a detectable label.

[0029] A position in the nucleic acid skeleton can hybridize (non-covalently bond) with at least one labeled oligonucleotide. Alternatively, a position can hybridize with at least one oligonucleotide lacking a detectable label. Each position can hybridize with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21-100 or more labeled (or unlabeled) oligonucleotides. The number of labeled oligonucleotides that hybridize to each position depends on the length of that position and the size of the oligonucleotides. Positions can be about 300-1500 nucleotides in length. The length of the labeled (or unlabeled) oligonucleotides varies from about 20-1500 nucleotides. In some embodiments, the length of the labeled (or unlabeled) oligonucleotides varies between about 800-1300 ribonucleotides. In several other embodiments, the length of the labeled (or unlabeled) oligonucleotides varies by about 20 to about 55 deoxyribonucleotides; such oligonucleotides are designed to have a melting / hybridization temperature of about 65 to about 85°C, for example, about 80°C. For example, a position of about 1100 nucleotides in length may hybridize to about 25 to 45 oligonucleotides, each oligonucleotide having a length of about 45 to 25 deoxyribonucleotides. In several embodiments, each position is hybridized to about 34 labeled oligonucleotides with a length of about 33 deoxyribonucleotides. The labeled oligonucleotides are preferably single-stranded DNA.

[0030] Each labeled oligonucleotide may be labeled with one or more detectable label monomers. The label may be at the end of the oligonucleotide, at a dot within the oligonucleotide, or a combination thereof. The oligonucleotide may contain amine-modified nucleotides, which enable the coupling of the detectable label to the nucleotide.

[0031] The labeled oligonucleotides of the present invention can be labeled with any of the various labeling monomers known in the art, such as fluorescent dyes, quantum dots, pigments, enzymes, nanoparticles, chemiluminescent markers, biotin, or other monomers that can be detected directly (e.g., by emission) or indirectly (e.g., by binding to a fluorescently labeled antibody). A preferred example of labeling available in the present invention is fluorescent dye molecules. Several fluorescent dye molecules can be used as labeling monomers for labeling nucleotides, including, but are not limited to, GFP-related proteins, cyanine dyes, fluorescein, rhodamine, Alexa Flour®, Texas Red, Fam, JOE, TAMRA, and ROX. Several different fluorescent dye molecules covering the full spectrum are known and continue to be produced for longer periods.

[0032] The label associated with each position (by hybridization of the labeled oligonucleotide and the position) is spatially separable and spectrally degradable from the label at the preceding or subsequent position. The spatially separable and spectrally degradable sequence of probe labels, also referred herein as a barcode or label code, enables the identification of a target nucleic acid or target protein conjugated by a particular probe.

[0033] Labeled oligonucleotides hybridize to their positions under standard hybridization conditions, e.g., 65°C, 5xSSPE; this allows for the self-assembly of reporter probes or probes. Probes using longer RNA molecules as labeled oligonucleotides (as described, e.g., in US2003 / 0013091) The probes must be pre-assembled at the manufacturing site rather than by the end user, and at a higher temperature to avoid crosslinking of multiple backbones via longer RNA molecules; this pre-assembly step is followed by purification to remove excess non-hybridized RNA molecules (including deoxyribonucleotides) that increase the background of the RNA molecules. The use of short, single-stranded labeled oligonucleotides (including deoxyribonucleotides, for example) greatly simplifies the production of the probes and further reduces the costs associated with their manufacturing method.

[0034] In several embodiments, the probe is typically supplied to the sample at a lower concentration than that used for immunohistochemistry (IHC) or 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 1 / 2, 1 / 5, 1 / 10, 1 / 20, 1 / 25, 1 / 30, 1 / 50, 1 / 60, 1 / 70, 1 / 80, 1 / 90, 1 / 100, 1 / 200, 1 / 300, 1 / 400, 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, 1 / 1000, 1 / 2000, or less, and any value in between. In several embodiments, the probe is provided in concentrations of 100nM, 70nM, 60nM, 50nM, 40nM, 30nM, 20nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.9nM, 0.8nM, 0.7nM, 0.6nM, 0.5nM, 0.4nM, 0.3nM, 0.2nM, 0.1nM, 0.09nM, 0.08nM, 0.07nM, 0.06nM, 0.05nM, 0.04nM, 0.03nM, 0.02nM, 0.01nM, and less than or equal to any value in between.

[0035] The probes can be detected and quantified using commercially available cartridges, software, and systems, such as the nCounter® system using nCounter® cartridges.

[0036] Background noise during protein detection can be reduced by performing complete back-purification of the probe molecule. This can be done by performing affinity purification of the antibody or photocleavable linker after recovering the eluate from the region of interest. Typically, the released signal oligonucleotide is not removed from the solution. Protein G or O mechanisms in pipette tips, tubes, or plates can be used for this step. Such devices and reagents are commercially available.

[0037] Background noise during nucleic acid detection can be reduced by performing complete back-purification of the probe molecule. This can be done by performing affinity purification of the target-binding domain or photocleavable linker after recovering the eluate from the region of interest. Typically, the released signal oligonucleotide is not removed from the solution. For assistance in back-purification, a universal purification sequence may be included in the probe, e.g., the target-binding domain.

[0038] Figure 1 shows two representative probes containing a single-stranded nucleic acid backbone and a target-binding domain shown in red. The upper probe contains a labeled RNA segment hybridized to a position within the backbone, while the lower probe contains a labeled DNA oligonucleotide hybridized to a position within the nucleic acid backbone. The colors shown in Figure 1 and other parts of this disclosure are not limiting; other colored labels and other detectable labels known in the art may be used with the probes of the present invention.

[0039] The probe of the present invention can be used to detect a target nucleic acid. Figures 2 and 4 illustrate this embodiment. Such a probe includes at least a backbone and a target nucleic acid-binding region. The target nucleic acid-binding region is preferably at least 15 nucleotides in length, and more preferably at least 20 nucleotides in length. In certain embodiments, the target nucleic acid-binding region is about 10–500, 20–400, 25, 30–300, 35, 40–200, or 50–100 nucleotides in length. Probes and methods for binding to and identifying target nucleic acids are described, for example, in 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.

[0040] The protein target may be a complete protein, multiple polypeptides, polypeptides, or peptides.

[0041] The probes of the present invention may be used to directly hybridize to a target nucleic acid. Figure 2 illustrates a probe (or composition) of this embodiment. The probe includes a target nucleic acid binding domain, shown in red. The target nucleic acid is shown as a blue curve. Figure 3 illustrates a dual probe composition comprising the probe and capture probe of Figure 2. The capture probe includes at least one affinity reagent, indicated by an asterisk. The at least one affinity moiety can be attached to the capture probe by covalent or non-covalent bond. Various affinity moieties suitable for purification and / or immobilization are known in the art. Preferably, the affinity moiety is biotin, avidin, or streptavidin. Other affinity tags are recognized by a specific binding partner, thereby facilitating separation and immobilization by affinity binding to the binding partner (which can be immobilized on a solid support). In these drawings, each probe includes six positions for hybridizing to a labeled oligonucleotide, each position identified by a colored circle.

[0042] Any probe of the present invention may include an affinity portion.

[0043] The probe of the present invention can be used to indirectly hybridize to a target nucleic acid present in a sample (via a mediating oligonucleotide). Figure 4 illustrates a probe (or composition) of this embodiment. The probe includes a target nucleic acid binding domain, shown in red, which binds to a synthetic oligonucleotide (mediating oligonucleotide; shown in green) that sequentially binds to the target nucleic acid in the biological sample. The mediating oligonucleotide may be said to be the probe as defined herein, because it contains 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 illustrates a dual probe composition including the probe and capture probe of Figure 4. In these embodiments, the target nucleic acid binding region of the probe hybridizes to a region of a different mediating oligonucleotide (i.e., synthetic oligonucleotide) than the target nucleic acid present in the sample. Thus, the target binding region of the probe is independent of the final target nucleic acid in the sample. This allows for economical and rapid flexibility in assay design, as assays for target-specific components (present in the sample) are contained in synthetic DNA oligonucleotides, which are less expensive and more widely available than more expensive probes. Such synthetic oligonucleotides are simply designed to contain a region that hybridizes to the target nucleic acid present in the sample and a region that hybridizes to the probe. Therefore, a single set of indirectly binding probes can be used to detect an infinite variety of target nucleic acids (present in the sample) in different experiments simply by replacing the target-specific (synthetic) oligonucleotide portion of the assay.

[0044] The probe or probe of the present invention includes a region that enables the release of a signal oligonucleotide after the application of a suitable force. In one, but not limited to, example, the region is a cleavable motif (e.g., restriction enzyme site or cleavable linker). The cleavable motif enables the release of a signal oligonucleotide from the bound target nucleic acid or protein, which is then recovered and detected. As used herein, a signal oligonucleotide is a region of the probe that has a position to hybridize to at least one labeled oligonucleotide or a region of the probe (e.g., a nucleic acid molecule) that can be released from the target binding domain of the probe. A signal oligonucleotide is said to be releaseable when it is cleaved from the rest of the probe (i.e., cleaved and released). Examples of cleavable motifs include, but are not limited to, photocleavable linkers.

[0045] In the probes of the present 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. In Figure 6, a non-limiting selection of the location of the cleavable motif may be inferred from gaps in the probe or gaps in the mediating oligonucleotide.

[0046] The probes of the present invention can be used to detect target proteins. Figure 7 illustrates a probe (or composition) of this embodiment. Such a probe includes at least a backbone and a target protein-binding region. In the protein-targeting probes of the present invention, the signal oligonucleotide may be a nucleic acid attached to the protein-binding domain. In these probes, the signal oligonucleotide is targeted and bound by the probe, which includes a site for hybridizing to a labeled oligonucleotide. Such a probe is shown in the center image of Figure 7, where the signal oligonucleotide is seen as a green line. The probe may be bound by the probe before it binds to a protein (via its protein-binding domain) or after it has bound to a protein. The signal oligonucleotide does not need to be bound by the probe until it has already been released from its target-binding domain (not shown in this embodiment).

[0047] Examples of probe regions capable of binding to target proteins include molecules or assemblies designed to bind to at least one protein target protein, at least one protein target protein substitute, or both, and to form a molecular complex containing the protein probe and the target protein under appropriate conditions. Examples of regions capable of binding to target proteins include antibodies, peptides, aptamers, or peptoids. Antibodies can be obtained from a variety of sources, including, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, recombinant antibodies, humanized antibodies, and plant antibodies (plantibodies). The terms protein, polypeptide, peptide, and amino acid sequence are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be fragmented by non-amino acid or synthetic amino acids. The term also includes amino acid polymers modified by, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other operations such as binding with a labeling component. As used herein, the term amino acid refers to any of the following: natural and / or non-natural, or synthetic amino acids, including, but not limited to, glycine and both D or L optical isomers, amino acid analogs, and peptide mimetic compounds. Probes and methods for binding to and identifying target proteins are described, for example, in US2011 / 0086774, which is incorporated herein by reference in its entirety.

[0048] In several embodiments, the probe is prepared by a site-selective cysteine ​​bioconjugation reaction, preferably stable to the hinge region heavy chain of the antibody. This method of preparation results in a relatively controllable stoichiometric ratio of labeled oligonucleotide to antibody. The probe contains multiple (i.e., two or more, e.g., 2, 3, 4, 5 or more) labeled oligonucleotides per antibody. Generally, "heavier" probes (containing three or four labeled oligonucleotides per antibody) are significantly less sensitive than antibodies lacking labeled oligonucleotides or "lighter" probes (containing one or two labeled oligonucleotides per antibody).

[0049] Protein-targeting probes and nucleic acid-targeting probes can be applied simultaneously, provided that the conditions allow for binding to both protein and nucleic acid targets. Alternatively, protein-targeting probes and nucleic acid-targeting probes can be applied sequentially when the conditions that allow for binding to both protein and nucleic acid targets are not available.

[0050] A set of probes is synonymous with a composition of probes. A set of probes includes at least one probe species, i.e., one targeting one target. Preferably, a set of probes includes at least two, for example, 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 probe species. A set of probes may include one or more copies of each probe species.

[0051] Only the first set of probes may be applied to the sample. Alternatively, a second set (or a larger number) of probes may be applied to the sample afterward. The first and second (or larger number) sets may target nucleic acids only, proteins only, or a combination thereof.

[0052] In this invention, two or more targets (i.e., proteins, nucleic acids, or combinations 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, and any number in between.

[0053] A set of probes may be pre-defined based on the target cell or tissue type. For example, if the tissue is breast cancer, the set of probes would include probes targeting proteins associated with breast cancer cells (e.g., Her2, EGFR, and PR) and / or probes targeting proteins associated with normal breast tissue. Furthermore, the set of probes may be pre-defined based on the differentiation state of the target cell or tissue. Alternatively, the set of probes may be pre-defined based on, for example, the subcellular localization of interest, e.g., nucleus, cytoplasm, and membrane. For example, antibodies targeting Foxp3, histone H3, or P-S6 label the nucleus, antibodies targeting CD3, CD4, PD-1, or CD45RO label the cytoplasm, and antibodies targeting PD-L1 label the membrane.

[0054] The probe may be chemically synthesized or biologically produced using a vector from which the nucleic acid encoding the probe has been cloned.

[0055] Any probe or probe set described herein may be used in the methods and kits of the present invention.

[0056] The labeling codes for the probes described herein have not yet been determined for use with respect to target nucleic acids or target proteins.

[0057] The probe of the present invention can be used to detect any sample, for example, a biological sample, in which a target nucleic acid or protein is present. As will be understood by those skilled in the art, the sample may include, but is not limited to, a variety of things, including cells (including both primary cells and cultured cell lines) and tissues (including cultured or explanted tissues). In some embodiments, a tissue sample (fixed or unfixed) is embedded, serially sectioned, and fixed onto a microscope slide. As is well known, a set of serial sections contains at least one cell present in both serial sections. The structure and cell type located in the first serial section have similar positions in adjacent serial sections. The sample may be cultured cells fixed on a slide, or dissociated cells (fixed or unfixed).

[0058] In several embodiments, the tissue sample is a biopsy tumor or a portion thereof, i.e., a clinically relevant tissue sample. For example, the tumor may originate from breast cancer. The sample may be an excised lymph node.

[0059] The sample can be obtained from virtually any organism, including multicellular organisms, in the plant, fungi, and animal kingdoms; preferably, the sample is obtained from an animal, such as a mammal. Human samples are particularly preferred.

[0060] In several embodiments, the probes, compositions, methods, and kits described herein are used for the diagnosis of disease. Where used herein, the terms diagnosis or diagnosis of disease include the prediction or diagnosis of disease, the determination of predisposition to disease, the observation of treatment for disease, the diagnosis of the response to treatment of disease, and the prognosis of disease, the progression of disease, and the response of disease to specific treatment. For example, a tissue sample can be assayed according to any of the probes, methods, or kits described herein to measure the presence and / or quantity of markers of disease or malignant cell types in the sample (against a non-pathological state), thereby to diagnose or stage disease or cancer.

[0061] Generally, samples attached to a slide are first imaged using fluorescence (e.g., fluorescent antibodies or fluorescent stains (e.g., DAPI)) to identify morphology, regions of interest, cell types of interest, and single cells, and then protein and / or nucleic acid expression can be digitally counted from the samples on the same slide.

[0062] 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, i.e., for carrying out hybridization reactions.

[0063] The kit also includes, but is not limited to, instructions for using the kit's components, including information necessary for hybridizing labeled oligonucleotides to probes, hybridizing probes to target-specific oligonucleotides, hybridizing target-specific nucleotides to target nucleic acids, and / or hybridizing probes to target proteins.

[0064] Typical protocols for detecting target nucleic acids and / or target proteins are described below and as shown in Figures 10–14 (top).

[0065] Cells (live or fixed) or tissue sections (e.g., formalin-fixed paraffin-embedded (FFPE)) prepared according to multiple immunohistochemistry and / or nucleic acid in situ hybridization methods are prepared and fixed onto glass slides or suitable solid supports. Access to the surface of the cell or tissue section is maintained and fluid exchange is permitted; this can be achieved by using a fluid chamber reagent exchange system (e.g., Grace® Bio-Labs, Bend OR). A region of interest (ROI) is identified on a serial section to which a probe is provided, or on an adjacent serial section. Firstly, to measure the overall macroscopic features of the cells / tissue of interest, a complete “macroscopic feature” imaging methodology for the cells / tissue of interest, e.g., DAPI staining, membrane staining, mitochondrial staining, specific epitope staining, and specific transcript staining, is performed. Alternatively, the region of interest (ROI) is identified on a serial section adjacent to the serial section providing the probe; here, imaging of the complete "macroscopic features" (as previously described) is performed on the first serial section (section number 1 in Figures 11 and 12). Generally, this imaging identifies the region on the adjacent serial section (red line in panel B of Figure 10, and the green ellipse and green triangle in section number 2 in Figures 11 and 12) from which the signal oligonucleotide is released from the probe, by applying a suitable and indicated force. The serial sections are separated from each other by approximately 5 μm to 15 μm.

[0066] Figures 13 and 14 (top) further illustrate the steps of the present invention. The steps shown in Figure 13 include: (1) Process: FFPE slide-mounted tissue is incubated with a primary antibody cocktail conjugated to DNA oligos via a photocleavable linker, along with a limited number of visible wavelength imaging reagents. (2) Overview: Regions of interest (ROIs) are identified using low-plex visible light-based imaging reagents to establish the overall "structure" of the tumor section (e.g., imaging of the nuclei and / or imaging with one or two major tumor biomarkers). (3) Profile: Extracted ROIs are selected for high-resolution multiplex profiling, and oligos are released from the selected region after exposure to UV light. (4) Plating: The free, photocleaved oligos are then collected, for example, via a microcapillary-based "sipper" and stored in a microplate well for subsequent quantification. (5) Digital counting: During the digital counting step, photosection oligos from spatially resolved ROIs in a microplate are hybridized into four-color, six-spot optical barcodes, enabling up to 1 million digital counts of protein targets (distributed within up to 800 plex markers) in a single ROI using standard NanoString nCounter® readout instruments (e.g., SPRINT, Flex, and MAX).

[0067] The region of interest may be the tissue type, cell type, cells, or intracellular structures present in the sample.

[0068] A composition comprising a set of probes (each probe containing a releaseable signal oligonucleotide) is applied to serial sections. The set of probes may include probes targeting proteins, probes targeting nucleic acids, or probes targeting both. The composition may also include a capture probe. When the probes indirectly bind to targets (proteins and / or nucleic acids), the applied composition includes a mediating oligonucleotide. The composition may also include other reagents known in the art to facilitate the binding of proteins and / or nucleic acids in the sample.

[0069] The blocking step is performed before and after the composition is applied.

[0070] With respect to a probe containing a photocleavable linker, the solid support (e.g., a microscope slide) is placed in a microscope capable of supplying excitation light at a wavelength that can cleave the photocleavable linker. The first region of interest (red line in panel B of Figure 10, and ROI in Figures 11 and 12) is shown. i The probe is excited by light, thereby cleaving a photocleavable linker and releasing a signal oligonucleotide. As illustrated in Figures 6 and 9, the signal oligonucleotide comprises at least one region of the probe that currently has a site to bind to at least one labeled oligonucleotide, or nucleic acid from a probe that is bound to or can be bound to by a reporter probe. By directing the excitation light only to the ROIi, signal oligonucleotides are released only from probes within the ROIi and not from probes located outside the ROIi, and their signal oligonucleotides remain intact. Thus, signal oligonucleotides are recovered only with respect to probes bound to targets within the ROIi, thereby enabling the detection of the identity and quantity of targets (proteins and / or nucleic acids) located within the ROIi.

[0071] The surface of the section is washed with a small amount of buffer, and the eluate (~5-30 μl) containing the released signal oligonucleotides is collected in the first sample container (indicated as sample "i" in Figure 12). The surface of the section is further rinsed to remove any released signal oligonucleotides that did not enter the eluate.

[0072] The second region of interest (ROIj in Figures 11 and 12) is excited by light, thereby cleaving the photocleavable linker and releasing a signal oligonucleotide from the second region of interest. Similarly, by directing the excitation light only to the ROIj, the signal oligonucleotide is released only from probes within the ROIj and not from probes located outside the ROIj, leaving those signal oligonucleotides intact. Thus, the signal oligonucleotide is recovered only with respect to probes bound to targets within the ROIj, thereby enabling the detection of the identity and quantity of targets (proteins and / or nucleic acids) located within the ROIj.

[0073] The surface of the section is washed with a small amount of buffer, and the eluate (~5-30 μl) containing the released signal oligonucleotides is collected in the first sample tube (indicated as sample "j" in Figure 12). The surface of the section is further rinsed to remove any released signal oligonucleotides that did not enter the eluate.

[0074] The excitation step, washing step, and rinsing step encompass all regions of interest (maximum ROI). n This process is repeated until the signal oligonucleotide is recovered.

[0075] Further advantages, features, and embodiments of the present invention are illustrated in the appendix filed together. Examples include various methods and devices for recovering signal oligonucleotides, as well as various methods for supplying force. Furthermore, surprisingly, the appendix yielded improved results obtained from specific embodiments of the invention over other embodiments. Data demonstrating signal-to-noise improvements of approximately 7 to 200 times are shown.

[0076] Detection can be performed using any type of microscope device or system known in the art. The device or system may include wide-field illumination along with a digital mirror device (DMD; see Figures 15 and 16). The advantages of this include reduced costs, as the DMD and controller can also drive the LED (which optically cuts the probe), enabling small feature sizes of ~1 mm, which would include cells of 10–40 mm, with virtually no additional cost, and further, ease of implementation by utilizing readily available household appliances (such as projectors). The device or system may include, for example, a confocal, laser scanning device; see Figure 16. The advantage of this is that smaller morphological features can be illuminated and imaged; however, additional costs are associated with these devices.

[0077] Multiple target proteins and / or target nucleic acids present in each region of interest of a sample are identified in each eluate sample using polymerase reactions, reverse transcriptase reactions, hybridization to oligonucleotide microarrays, mass spectrometry, hybridization to fluorescent molecular beacons, sequencing reactions, or nCounter® molecular barcodes. The nCounter® systems and methods from NanoString Technologies® described in US2003 / 0013091, US2007 / 0166708, US2010 / 0015607, US2010 / 0261026, US2010 / 0262374, US2010 / 0112710, US2010 / 0047924, US2014 / 0371088, and US2011 / 0086774 are preferred means for identifying target proteins and / or target nucleic acids. The nCounter® system and method from NanoString Technologies® enables simultaneous multiple identification of multiple (more than 800) different target proteins and / or target nucleic acids.

[0078] Simultaneously, it is possible to compare the identity and quantity of target proteins and / or target nucleic acids present in the first region of interest (e.g., tissue type, cell type (including normal and abnormal cells)) with the identity and quantity of target proteins and / or target nucleic acids present in the second region of interest, or even more regions of interest.

[0079] This invention enables the multiple detection and comparison of up to 800 proteins of interest from discontinuous regions within tumors (e.g.) and adjacent normal tissues, thereby allowing for a systematic investigation of tumors and their microenvironment.

[0080] This invention can be used to advance clinical trials to elucidate novel responses to immunotherapy and other targeted therapies.

[0081] The present invention also enables the discovery of tumor immunobiomarkers, which can be used (for example) in the development of companion diagnostics.

[0082] Immunohistochemistry is an effective technique for analyzing protein expression and localization in FFPE tissue sections. However, it suffers from many challenges, including a labor-intensive workflow due to limited dynamic range, difficult quantification, and very limited multiplexing. Disclosed here is a novel platform based on nCounter® barcoding technology that enables spatially resolved, digital characterization of proteins in highly multiplexed (up to 800 plexes) assays, i.e., nCounter® digital multiplex immunohistochemistry (IHC) assays. The assay relies on antibodies linked to photocleavable oligonucleotide tags emitted from discontinuous regions of tissue using focused, target-transmitted UV (e.g., ~365 nm) exposure. The cleavage tags are quantified in the nCounter® assay, the counts are mapped back to the tissue distribution, and a spatially resolved digital profile of protein quantity is obtained. Protein detection can be performed in conjunction with, or separately from, a nucleic acid detection assay using nucleic acid probes containing photocleavable oligonucleotide tags. Therefore, the present invention may provide a spatially resolved digital profile of protein quantity, a spatially resolved digital profile of protein and nucleic acid quantity, or a spatially resolved digital profile of nucleic acid quantity.

[0083] The advantages of the aforementioned assay, though not limited to these, include: high sensitivity (e.g., ~1-4 cells), all counts are digital, and it has a wide dynamic range (>10). 5 ), it offers high multiplexing (30 targets, and scalability to, for example, up to 800 targets without changing instruments), a simple workflow, compatibility with FFPE, no need for secondary antibodies or amplification reagents (for protein detection), and clinical assay potential.

[0084] As used in this specification and the attached claims, the singular forms “a,” “an,” and “the” also include plural nouns unless otherwise explicitly indicated by context.

[0085] Unless otherwise specifically stated or evident from the context, the word “or” as used herein is understood to be inclusive, and therefore encompasses both “or” and “and.”

[0086] Unless otherwise stated or evident from the context, the term “about” as used herein is understood to mean, for example, within the range of standard tolerance in the art, e.g., within two standard deviations of the mean. “About” may be understood to mean 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 evident from the context, all numerical values ​​provided herein are modified by the term “about.”

[0087] Unless otherwise specified, all academic and scientific terms used herein have the same meaning as those generally understood by those skilled in the art relating to this invention. Other probes, compositions, methods, and kits similar or equivalent to those described herein may be used in the practice of this invention, but preferred materials and methods are described herein. It should be understood that the technical terms used herein are for the purpose of describing only specific embodiments and are not intended to limit them. [Examples]

[0088] Example 1: The present invention offers the possibility of "barcoding" for quantifying multiple targets within FFPE tissue sections.

[0089] Tumor heterogeneity presents a significant challenge to the implementation of targeted therapies. Historically, immunohistochemistry (IHC) has been used to assess the spatial heterogeneity of proteins; however, quantifying protein quantities with high levels of multiplexing and a wide dynamic range remains challenging.

[0090] In this example, proteins in formalin-fixed paraffin-embedded (FFPE) tissue sections were labeled with an antibody-containing probe containing a photocleavable linker and a fluorescent barcode. The probe was then directed at a user-defined ROI of the FFPE tissue section, followed by focused UV light. The ROI was exposed to UV light, thereby releasing signal oligonucleotides (including fluorescent barcodes). The released signal oligonucleotides were washed away from the FFPE sample and recovered. Next, the fluorescent barcodes from the released signal oligonucleotides were recognized and digitally counted using an nCounter® system from NanoString Technologies®, thereby quantifying the amount of each target protein within a user-defined spatial region of the tissue section. After releasing and recovering the signal oligonucleotides from the first ROI, focused UV light was applied to a second user-defined ROI of the FFPE tissue section, thereby releasing signal oligonucleotides from the second ROI. In this unrestricted example, a high degree of linearity (0.97) was observed between the number of counts and the UV-irradiated area. <R 2 A resolution of <0.99) was observed, and the detection spatial resolution was approximately 100 μm × 100 μm, or approximately 100 cells. Surprisingly, the present invention provides the "potential for barcoding" to quantify up to 800 targets in a single FFPE tissue section with a logarithmic 5.5 (cardinal 10) dynamic range.

[0091] Example 2: The present invention provides a practical and feasible approach for quantifying protein expression without signal amplification and for achieving multiplexing of higher-order target antigens in FFPE tissue sections.

[0092] Quantitative, multi-stage immunohistochemistry has emerged as a highly intriguing field within oncology because it possesses a unique ability to identify spatial-temporal tissue and interdependencies, further defining how checkpoint blockade affects the tumor microenvironment. This example describes a one-step, amplification-free staining method using a primary antibody affixed with a photocleavable oligotag that interacts with a target antigen in FFPE tissue sections. Irradiation with ultraviolet (UV) light to release the oligo is applied, followed by eluate collection, quantification, and a digital count corresponding to the amount of antigen.

[0093] First, various binding methods were investigated; this led to the establishment of a cysteine ​​bioconjugation reaction method that is stable, site-selective, primarily for hinge region heavy chains, and relatively controllable with respect to the oligonucleotide-to-antibody stoichiometric ratio.

[0094] Next, linear regression analysis was performed to determine the correlation between UV-induced cleavage area and measured digital protein counts; from this, a high degree of linearity (0.97) was obtained. <R 2 A count of <0.99 was observed, confirming the fundamental mechanism / premise associated with this multiple protein counting method for FFPE tissue.

[0095] To determine the effect of the presence of bound oligonucleotides on antibody-antigen interactions, the performance of labeled oligonucleotide-conjugated antibodies against unmodified antibodies under identical conditions in FFPE tissue sections was compared in terms of sensitivity, specificity, and signal intensity. To determine the correlation between antibody performance and the subcellular location of target antigens, antibodies targeting antigens localized in the nucleus, cytoplasm, or membrane were selected. The selected antibodies targeted Foxp3, histone H3, P-S6 (nuclear antigen), CD3, CD4, PD-1, CD45RO (cytoplasmic antigen), and PD-L1 (membrane antigen). In terms of sensitivity, "heavier" oligonucleotide-conjugated antibodies (containing 3 or 4 labeled oligonucleotides per antibody) were generally found to be significantly less sensitive compared to unconjugated antibodies or "lighter" oligonucleotide-conjugated antibodies (containing 1 or 2 labeled oligonucleotides per antibody). No significant differences in sensitivity, specificity, or intensity were observed between unconjugated antibodies and "lighter" oligonucleotide-conjugated antibodies across nuclear, cytoplasmic, and membrane target antigens.

[0096] This invention provides highly multiplexed protein profiling for measuring absolute protein expression levels using a practical and feasible method for comprehensively defining the immune status of tumors before and during immunotherapy intervention.

[0097] Example 3: The present invention provides spatially resolved multiprotein detection from FFPE tissue.

[0098] method

[0099] Antibodies - The antibodies used in this example and Examples 4-6 are: "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 Examples include 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), and CD45RO (UCHL1, Cell Marque).

[0100] Microscopic examination of tonsils - 5 μm sections of tonsil FFPE blocks (Amsbio) were mounted on slides. Intravascular coagulation (IHC) was performed using a standard protocol. Antigen recovery was performed using a pressure cooker. Tonsil sections were stained with CD3 primary antibody MRQ-39 (rabbit mAb, Cell Marque) and Ki-67 primary antibody 8D5 (mouse mAb, CST). Secondary incubation was performed with Alexa594-labeled goat α rabbits (Life Tech) and Alexa488-labeled goat α mice (Life Tech).

[0101] Here, the samples attached to the slides were first imaged using fluorescent antibodies, and then protein expression was digitally counted from the samples.

[0102] The same steps as illustrated in Figures 10-14 (top) were used. UV cutting of the selected ROI enabled digital profiling of all 30 plexes (nCounter® count).

[0103] result

[0104] Figure 18 shows a micrograph establishing the overall histological morphology of a tonsil sample initially imaged using two-color fluorescence: Ki-67 (green cell proliferation marker) and CD3 (red immune cell marker). Multitarget analysis across 12 regions (including the four regions magnified in Figure 19) reveals three distinct profiles regarding the localization of Ki-67 and CD3. Figure 19 shows the nCounter® counts of Ki-67 and CD3 for the four regions shown in Figure 18. Figure 20 shows representative counts using a 30-plex oligo antibody cocktail for 12 regions of interest (ROIs) from the tonsil sample shown in Figure 18. Data were obtained from serial sections (to allow for various additional controls to be investigated). As shown, regions of the tissue sample were classified based on the intensity and identity of the expressed markers. Representative classifications shown: “CD3-rich”, “Ki67-rich”, “mixed”, and “connective tissue”.

[0105] These data demonstrate that the present invention provides spatially resolved detection of multiple (in this case, at least 30) protein labels. By scaling up the number of protein probes (antibodies) used, up to 800 different protein labels can be detected with similar resolution.

[0106] Example 4: The present invention provides a method for detecting multiple proteins from FFPE tissue and a single-cell resolution approach.

[0107] method

[0108] Microscopic examination of melanoma - 5 μm sections of FFPE blocks (Asterand) from melanoma (lymph node origin) were mounted on slides. IHC was performed using a standard protocol. Antigen recovery was performed using a pressure cooker.

[0109] Here, the samples were first imaged using fluorescence, and then protein expression was digitally counted from the samples.

[0110] The same steps as those illustrated in Figures 10 to 14 (top) were used.

[0111] result

[0112] Figure 21 shows a micrograph establishing the overall histological morphology of T cells in a melanoma lymph node sample, initially imaged using three colors of fluorescence: CD3 (red), CD8 (green), and DAPI (blue). The white circle, 25 μm in diameter, surrounds three cells.

[0113] Figure 22 shows nCounter® data for CD3 complexes released from FFPE lymph node tissue sections (5 μm thick) as an effect of UV irradiation (diameter 100 μm to 1 mm). The detection count limit (LOD = background count + 2 × standard deviation) corresponds to a spatial resolution of 26 μm in diameter. The field of view aperture size is shown at the bottom of the figure. Figures 23 and 24 show data for CD45 and PD1 (from the same experiment, respectively).

[0114] The aforementioned data indicates that the spatial detection capability of the present invention corresponds to approximately 1 to 4 cells.

[0115] Example 5: The present invention provides quantitative performance in clinically relevant assays.

[0116] method

[0117] The same steps as those illustrated in Figures 10 to 14 (top) were used.

[0118] Breast cancer tissue microarrays (TMA): TMA BR1504a was obtained from US Biomax, Inc., and H&E stained images were obtained from the US Biomax website (World Wide Web (www) biomax.us / tissue-arrays / Breast / BR1504a). Sections from the same block as those shown in the left panel of Figure 25 were stained with Her2 primary antibody 29D8 (rabbit mAb, CST) and Alexa594-labeled goat α rabbit (Life Tech). Counts were also obtained for histone H3, ribosomal protein S6, estrogen receptor, progesterone receptor, mouse IgG isotype control, and rabbit IgG isotype control (data not shown). Her2 pathologist scores for TMA BR1504a were provided by US Biomax, Inc. (World Wide Web (www) biomax.us / tissue-arrays / Breast / BR1504a). Staining was performed using the Her2 primary antibody 29D8 (rabbit mAb, CST) and Alexa594-labeled goat α rabbit (Life Tech). Other rabbit primary antibodies were used in the primary cocktail, but the fluorescence from those antibodies was negligible compared to the Her2 fluorescence. Total pixel intensity (at λ=594) was obtained using ImageJ software. For this, the background value was set to intensity=0, and the highest intensity was set to intensity=255. The sum of all pixel intensities per ROI is shown.

[0119] Here, the samples were first imaged using fluorescence, and then protein expression was digitally counted from the samples.

[0120] result

[0121] Figure 25 (left panel) shows a tissue microarray (TMA) of breast tumor tissue, including variable levels of Her2 protein as shown in the micrograph (center panel) with Her2 fluorescence identified by IHC staining. The right panel shows a magnified view of a single region from the center panel: this region was stained with a multi-antibody cocktail.

[0122] Figure 26 shows nCounter® count data for 48 representative regions against Her2 status (ASCO-CAP guidelines). Figure 27 shows nCounter® counts against total pixel intensity (×10) for the 48 regions mentioned in Figure 26. 3 Plot the points.

[0123] These digital count data are used for visual scoring of Her2 status according to the ASCO-CAP guidelines (R 2 Compared to =0.51 (Figure 26), the fluorescence intensity (R 2 It shows a high correlation with =0.92 (Figure 27).

[0124] Example 6: The present invention reveals the amount of a specific cell type in a tissue sample.

[0125] The same steps as those illustrated in Figures 10 to 14 (top) were used; First, melanoma samples attached to slides were imaged using fluorescence, and then protein expression was digitally counted from the samples.

[0126] Figure 28 shows a micrograph establishing the overall histological morphology of a melanoma sample using two-color fluorescence: CD3 (immune cell marker, red) and DAPI (cell nucleus, blue). Expression data using a cocktail of 30 antibodies were obtained from 10 regions identified by white frames. Figure 29 shows representative nCounter® counts for 10 regions of interest (ROIs) from the melanoma sample shown in Figure 28 using a 30-plex oligo antibody cocktail. Counts are shown for 13 markers, each with expression counts above background. Regions 5, 6, and 7, confirmed to be "rich in immune infiltration," had the highest expression of T-cell markers and T-cell regulatory markers.

[0127] These data demonstrate that the present invention enables spatially resolved detection of multiple (in this case, at least 30) protein markers. By scaling up the number of protein probes (antibodies) used, up to 800 different protein labels can be detected with similar resolution.

[0128] Example 7: A digital mirror device (DMD) enables irradiation of single cells.

[0129] Figures 30A and 30B are micrographs showing that UV irradiation using a digital mirror device (DMD) is possible when irradiating single cells in a tonsil tissue sample.

[0130] These data demonstrate that the present invention is single-cell degradable when using DMD.

[0131] Example 8: The gel box can irradiate the entire sample and release signal oligonucleotides from the probe bound to the entire sample.

[0132] Figure 34: Data from an embodiment in which the entire tissue or sample is irradiated, for example, using a standard laboratory UV gel box. Here, the FFPE tissue slide is placed on the light panel, and a wax pen is used to hold the buffer solution (TBS), coat the FFPE tissue, and then UV light exposure (276 - 362 nm, for example, 302 nm; ~5 mW / cm 2 ) is applied to the tissue through a glass slide (1 mm thick). The data shows that most of the signal oligonucleotides were released from the FFPE-bound antibody with UV exposure within about 1 minute. Counts are normalized against a positive control.

[0133] Example 9: Irradiation from a microscope enables irradiation of the region of interest of the sample and release of signal oligonucleotides from the probes bound to the region of interest.

[0134] Figure 35 shows an embodiment in which a part of the tissue or sample is irradiated, for example, using a microscope, i.e., UV cleavage under a microscope (time titration experiment). This is in contrast to the experiment of Example 8 in which the entire sample is irradiated. Here, a UV LED (at 365 nm) is applied at ~150 mW / cm 2 using a 20× objective lens. The UV irradiation scanned the entire tissue area identified by previous fluorescence (~590 nm excitation) brightfield imaging. With UV exposure within about 1 second per field of view (FOV), most of the signal oligonucleotides are released from the FFPE-bound probes. The gel box experiment of Example 8 was used as a non-spatially resolved 100% release control. Counts are normalized against a positive control. Blue: Microscope data using variable exposure times; Red: Data for 2.5-minute exposure in the gel box. Photographs and illustrations showing the form of the microscope apparatus are also shown.

[0135] Figure 36 shows that signal oligonucleotides are released from uniformly distributed anti-histone (H3) antibodies bound to a lung tissue sample. The tissue was about 450 μm × 330 μm = 0.15 mm 2UV (365nm, ~150mW / cm²) per second per field of view (FOV) using a 20x objective lens 2 The irradiation was directed to the area. The "macrovolume" used to collect the effluent was approximately 70 μl. This reduced the detection limit to (FOV / 5) ~99 μm × 99 μm using approximately 5 μl of collected effluent. Therefore, in this example, the detection limit is approximately 10 cells × 10 cells niche. These data demonstrate that the antibody signal is proportional to the spatially resolved irradiation area FOV and evaluates the "macrofluid" detection limit (LOD).

[0136] Figure 37 shows embodiments of irradiation of a tissue or sample, for example, using a microscope, i.e., UV sectioning under a microscope and for multiple targets (irradiated area titration experiment). UV sectioning of multiple targets in the tissue is shown: two positive targets (histone H3 and ribosome S6) and eight negative targets. Only one negative target (Ox40) showed high background. Data from zero, one, four, nine, and sixteen fields of view are shown.

[0137] Example 10: The region of interest may be pre-identified by labeling techniques, and then the region of interest is irradiated, and a signal oligonucleotide is released from a probe bound to the pre-identified region of interest.

[0138] Figure 38 illustrates an embodiment in which a region of interest in a tissue (e.g., a breast cancer sample) is first identified in relation to the expression of a marker (here, Her2), and then this region of interest is irradiated (e.g., with UV) to release a signal oligonucleotide from a binding probe. The data shown compare the amounts of signal oligonucleotides released from two locations: one region of interest pre-identified as Her2+ and one region of interest pre-identified as Her2-, with respect to two targets (here, Her2 and histone H3).

[0139] Example 11: The embedded sample embedded in the flow cell allows for elution recovery from the entire sample, not only from the irradiated region of interest but also from the point where the signal oligonucleotide is released.

[0140] Figure 39 shows an embodiment in which tissue is embedded in a flow cell. Here, the FFPE tissue embedded in the microfluidic flow cell (a 9 mm circular chamber with a height of 100 μm and a volume of approximately 25 μl (when the flow cell is 300 μm high, the volume is approximately 75 μl)) is controlled by a syringe pump. UV light cut through the inside of the flow cell and showed elution profiles of irradiation and elution of one region (9 FOVs), then irradiation and elution of another region (9 FOVs). Data for multiple fractions are shown. Similar to the data in Example 10, the region of interest here was pre-identified with respect to the expression of a fluorescently labeled marker.

[0141] Example 12: A sample embedded in a flow cell containing small holes on one side of the region of interest resulted in effective elution recovery from the irradiated region of interest and from the site where the signal oligonucleotide was released, but not from the entire sample.

[0142] Figure 40 shows an embodiment in which tissue is embedded in a flow cell with small holes, where elution occurs directly on the region of interest. Holes with a diameter of 0.4–1 mm on the fluid chamber allow for the collection of eluate (e.g., a recovery volume of 5 μl). 9-well, 96-well, and 12-well configurations (for tissue microarrays (TMAs)) were tested. Fluorescence images were created by combining multiple fields of view. Photographs and illustrations illustrating the configuration of the apparatus are also shown.

[0143] Figures 41A–41C show that embodiments using a flow cell with a small hole have a significant signal-to-noise improvement over recovering eluate from the entire tissue surface. The data show that recovering eluate through a hole in the region of interest enhances the signal-to-noise ratio by approximately sevenfold. In this embodiment, a 1 mm diameter hole (25 μl chamber) on the fluid flow cell was used to recover eluate (5 μl fraction). Data for multiple fractions are shown.

[0144] Figures 42A–42C show data using a flow cell with small holes (12 or 96-well format). The data show that collecting eluate through the holes on the region of interest enhances the signal-to-noise ratio by approximately 7-fold. In this embodiment, field illumination was concentrated on the center of the holes; elution volume was 5 μl per hole.

[0145] Figures 43A and 43B show data comparing the background signal from a flow cell in which whole tissue elution was performed (Figure 43A; as shown in Example 11) with the background signal from a flow cell in which elution occurred directly over the region of interest (Figure 43B). As seen in Figure 43A, the background for whole tissue elution is higher than the background seen in Figure 43B. Furthermore, Figure 43B does not show any difference between in-flow-cell incubation and non-flow-cell incubation.

[0146] Example 13: The released signal oligonucleotides can be selected by a single tube / pipette, multiple tubes / pipettes, or a multi-tube / pipette array.

[0147] Figure 44 illustrates eluate recovery at an open surface for an embodiment of aspiration of multiple regions of interest. Here, a multi-tube array for eluate aspiration / distribution using a rotary valve switch is shown. See also Figure 47.

[0148] Figure 45 includes photographs and illustrations showing an embodiment in which eluate recovery is through a capillary tube (microaspirator). See also Figure 47. Figures 46A and 46B show data from the embodiment of Figure 45 in which eluate recovery is through a capillary tube (microaspirator). This embodiment showed a dramatic improvement in signal-to-noise ratio: the signal-to-noise ratio was increased by approximately 10 times compared to flow cell elution by hole elution, and the signal-to-noise ratio was increased by approximately 200 times compared to total tissue elution. Here, the LOD area is approximately 60 μm × 60 μm.

[0149] Example 14: A device with both irradiation and elution capabilities can efficiently and accurately obtain nucleic acid and / or protein expression data from a defined region of interest.

[0150] Figure 48 illustrates irradiation and fluid recovery using a combined capillary tube and lens.

[0151] Example 15: Protein expression can be detected and quantified from single cells.

[0152] Figure 50 shows protein expression data obtained from a single cell or two cells using the methods and apparatus described herein. In the upper panel, the S6 protein is detected and quantified from at least one cell, and in the lower panel, the CD45 protein is detected and quantified from at least one cell.

[0153] Example 16: The methods and devices described herein enable accurate and effective detection and quantification of spatially dispersed, multi-RNA target, and / or protein target expression.

[0154] In situ hybridization (ISH) was performed to hybridize DNA-based oligoprobes ("RNA probes"), each comprising a target-binding domain, a signal oligonucleotide, and a photocleavable linker, to endogenous RNA. 5 μm FFPE HER2 3+ mammary tissue sections were deparaffinized in xylene, partially rehydrated in stepwise ethanol, and incubated in 70% ethanol at room temperature for 1 hour. The sections were then incubated in 40 μg / ml proteinase K at 37°C for 25 minutes. The tissue was then incubated in 50% formamide / 2×SSC at room temperature for 15 minutes 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 2×SSC. Following hybridization, two stringent washes were performed in 50% formamide / 2×SSC at 37°C for 25 minutes each. Tissue morphology was visualized by staining the sections with TO-PRO®-3 (Thermo Fisher Scientific) fluorescent nucleic acid stain. Next, DNA signal oligonucleotides from user-defined regions of interest (ROIs) were cleaved from the probe using focused UV light induced by a semiconductor reflective device. For each tissue section, two ROIs contained tumor tissue, two contained normal tissue, and two ROIs did not contain tissue (the tissue slide itself). After cleavage, the signal oligonucleotides were recovered, hybridized to nCounter® molecular barcodes, and digitally counted using an nCounter® system from NanoString Technologies®. H&E was performed on the tissue sections to confirm tumor and normal tissue ROIs.

[0155] Standard immunohistochemistry (IHC) was performed on serial sections using "protein probes" equipped with antibodies, DNA signaling oligonucleotides, and photocleavable linkers, respectively, as target-binding domains. Next, the sections were stained with anti-rabbit Alexa594 secondary antibody and TO-PRO®-3 (Thermo Fisher Scientific) fluorescent nucleic acid stain to visualize tissue morphology. Then, DNA signaling oligonucleotides from user-defined regions of interest (ROIs) were cleaved from the probes using focused UV light induced by a semiconductor reflective device (DMD). For each tissue section, two ROIs contained tumor tissue, one ROI contained normal tissue, and two ROIs contained no tissue (the tissue slide itself). The ROIs were combined with the ROIs selected for ISH probe cleavage. After cleavage, signaling oligonucleotides from protein targets were mixed with signaling oligonucleotides from RNA targets, and all were quantified as previously described. H&E was performed on tissue sections to confirm the ROIs of tumor and normal tissue, and to further confirm that the ROIs were correctly matched between ISH and IHC tissues.

[0156] Figure 51 shows ROIs extracted from serial sections of the same tumor sample. Regions 1–4 are not shown in this image, but were taken instead from a portion of tissue that did not contain any tissue (negative control – “no tissue”). Regions 5–8 contained a small number of tumor cells (“normal tissue”). Regions 9–12 contained a large number of tumor cells (“tumor”).

[0157] Figure 52 shows the counts obtained for six of the nine RNA probes included in this assay. For each ROI, samples were collected before applying UV irradiation ("-UV" dataset) and before collecting positive UV samples from the same region ("+UV" dataset). Background levels of counts were obtained when UV was not applied to the samples; thereby indicating the UV dependence of the obtained signals. ROIs that were +UV but not directed towards tissue resulted in background counts (i.e., ROI1-4 - "no tissue"). Regions that were originally normal tissue (i.e., ROI5-8 - "normal tissue") showed low counts for the HER2 probe (orange bars in the graph). Regions that were originally tumor tissue (i.e., ROI9-12 - "tumor") showed higher counts for HER2. A similar, but less dramatic, increase was observed for the ribosome S6 probe (green bars in the graph). Additional control probes targeting RNAs not expected to be highly expressed in this tissue type showed consistent counts without differing levels between normal and tumor tissues. These control probes were designed to target CD45, PSA (prostate-specific antigen), and two unique ERCC sequences. For clarity, Figure 53 shows the mean and standard deviation of the data shown in Figure 52.

[0158] These RNA probe samples were also analyzed simultaneously with the protein probes used to analyze sample regions of tumor samples. For this analysis, the RNA and protein probes were simultaneously hybridized to NanoString Technologies® nCounter® molecular barcodes and digitally counted using the nCounter® system. The counts for this assay are shown in Figure 54. Increases in HER2 RNA probe counts (red bars in the upper graph) and protein probe counts (red and orange bars in the lower graph) are observed in the tumor region compared to the normal region. Only +UV samples are shown. As previously mentioned, -UV control samples are not included in this graph because they lack background counts (similar to "no tissue" counts). ROI6 and ROI8 were excluded from this analysis because matching protein probe samples were unavailable. Therefore, signal oligonucleotides from protein probes and RNA probes can be detected and quantified together.

[0159] Example 17: A partially double-stranded probe has a higher signal-to-noise ratio compared to a single-stranded probe.

[0160] A DNA probe (which recognizes and binds to mRNA) was in situ hybridized to RNA in 5 μm FFPE tissue as described in Example 16. UV cleavage was performed on the entire tissue section, mounted on separate slides, for 3 minutes using a UV light box (gel box) in 2 × SSC + 0.1% Tween 20. After cleavage and release of the signal oligonucleotide, the signal oligonucleotide was collected by pipetting and detected as in Example 16. Single-stranded DNA probe, partially double-stranded DNA probe, and unprobe-controlled counts are shown in Figure 55 (upper graph) for HER2 3+ breast and tonsil tissue. The signal-to-noise ratio was determined by dividing the count by the mean background count (mean ERCC count). See Figure 55, lower graph.

[0161] Example 18: Adding salmon sperm DNA improves probe hybridization.

[0162] A DNA probe (which recognizes and binds to mRNA) was hybridized in situ to RNA in 5 μm FFPE tissue as previously described. During hybridization, 1 mg / ml sonicated denatured salmon sperm DNA was used instead of yeast tRNA. Slides were hybridized with a solution of 1 nM probe, 40% formamide, 1 mg / ml sonicated denatured salmon sperm DNA, 10% dextran sulfate, and 0.2% BSA in 2 × SSC. UV cleavage, signal oligonucleotide recovery, and detection were performed as described in Example 17. Single-stranded DNA probes are shown in HER2 3+ breast and tonsil tissue (Figure 56). The signal-to-noise ratio was determined by dividing the count by the mean background count (mean ERCC count).

[0163] Example 19: The PSA (prostate-specific antigen) RNA probe is highly specific.

[0164] A DNA probe (which recognizes and binds to mRNA) was hybridized in situ to RNA in 5 μm sections of FFPE prostate, as previously described. Incubation in MES at 97°C for 10 minutes was used instead of 1 hour of ethanol incubation. UV cleavage, recovery and detection of signal oligonucleotides, and calculation of the signal-to-noise ratio were performed as described in Example 17. Counts and ratios are shown in Figure 57.

[0165] Example 20: The specificity of the probe is enhanced at non-standard, sub-nM concentrations.

[0166] Typically, in situ hybridization (ISH) probes used to recognize RNA hybridize at concentrations of 5–200 nM. Surprisingly, the nucleic acid recognition probe of the present invention worked best at 0.2 nM or less, which was 1 / 25–1 / 1000 lower than standard ISH probe concentrations.

[0167] The DNA probe was hybridized in situ to RNA in 5 μm sections of FFPE HER2 3+ breast samples, as previously described. The probe was used at concentrations of 5, 1, 0.2, and 0.4 nM. UV cleavage, recovery and detection of signal oligonucleotides, and calculation of magnification changes were performed as described in Example 17.

[0168] Figure 58 shows that the count decreases as the probe concentration decreases (upper graph). However, surprisingly, when the probe was hybridized at concentrations below nM, there was a significant increase in the signal-to-noise ratio of the positive probe count compared to the negative control probe.

Claims

1. (1) Contact at least one protein target in a tissue sample with at least one probe comprising a target-binding domain, a signal oligonucleotide, and a photocleavage motif located between the target-binding domain and the signal oligonucleotide; (2) Irradiate a specific location in the tissue sample with light sufficient to detach the signal oligonucleotide from the probe; (3) Elute the solution containing the detached signal oligonucleotide at the irradiated site; (4) Recover the elution solution containing the cleaved signal oligonucleotide; and (5) Identify the detached signal oligonucleotide and thereby detect at least one protein target at a specific location in the irradiated tissue sample. Methods that include...

2. The method according to claim 1, wherein the detection includes determining the identity and quantity of at least one protein target.

3. The aforementioned at least one protein target, The method according to claim 2, comprising at least two different protein targets or at least two copies of the same protein target.

4. The method according to claim 3, wherein the detection includes comparing the respective amounts of different protein targets.

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

6. The method according to claim 5, wherein the detection comprises comparing the amount of at least one protein target at a specific location with that of at least a second specific location.

7. The method according to any one of claims 1 to 6, wherein the tissue sample is fixed directly to the surface or indirectly to the surface.

8. The method according to any one of claims 1 to 7, wherein the tissue sample is a tissue section with a thickness of 2 to 1000 μm.

9. The method according to claim 8, wherein the tissue section is obtained from a paraffin-embedded (FFPE) sample fixed in formalin.

10. The method according to any one of claims 1 to 8, wherein the tissue sample is fixed or not fixed.

11. The method according to any one of claims 1 to 10, wherein the tissue sample is stained or labeled, thereby enabling visualization of subcellular structures, cellular structures, or tissue-related structures of the stained or labeled tissue sample.

12. The method according to any one of claims 1 to 11, wherein the signal oligonucleotide is a single-stranded nucleic acid or a partially double-stranded nucleic acid.

13. The method according to any one of claims 1 to 12, wherein reverse purification is used to isolate the complete probe molecule from the released signal oligonucleotide.

14. The method according to any one of claims 1 to 13, wherein the irradiation is supplied by a light source selected from the group consisting of an arc lamp, a laser, a focused UV light source, and a light-emitting diode (LED).

15. The method according to any one of claims 1 to 14, wherein the target-binding domain is selected from the group consisting of antibodies, peptides, aptamers, and peptoids.

16. The method according to any one of claims 1 to 15, wherein the detection comprises a polymerase reaction, a reverse transcriptase reaction, hybridization to an oligonucleotide microarray, mass spectrometry, hybridization to a fluorescent molecular beacon, a sequencing reaction, or molecular barcoding.

17. The method according to any one of claims 1 to 16, wherein the protein target is a complete protein, a plurality of polypeptides, polypeptides, or a peptide.

18. The method according to any one of claims 1 to 17, wherein the solution comprises an anionic polymer or salmon sperm DNA.

19. The method according to any one of claims 1 to 18, wherein the recovered signal oligonucleotide is added to a solution containing an anionic polymer and / or salmon sperm DNA.

20. The method according to claim 18 or 19, wherein the anionic polymer is dextran sulfate.

21. The method according to any one of claims 1 to 20, further comprising irradiating a region of interest using a laser scanning device or a digital mirror device (DMD).

22. The method according to any one of claims 1 to 21, wherein the method provides a spatial resolution profile of a protein in a sample.

23. The method according to any one of claims 1 to 22, wherein the detection includes providing a digital readout value that includes a linear dynamic range of > 5 logs.

24. The method according to any one of claims 1 to 23, wherein the tissue sample is attached to a slide, first imaged using fluorescence, and then nucleic acid expression is digitally counted from the tissue sample.

25. The aforementioned probe, (a) 0.01 nM to 5 nM, (b) 0.01 nM to 1 nM, (c) 0.01 nM to 0.4 nM, or (d) 0.01nM to 0.2nM The method according to any one of claims 1 to 24, prepared at the concentration of [a certain value].

Images