Methods, compositions and devices for isolating and analyzing regions of interest from tissues for expression analysis

Abstract

Provided herein is a method for isolating cellular components from at least one region of interest in a planar tissue section.

Description

[0001] Cross-references

[0002] This application claims priority to U.S. Provisional Application No. 62 / 637,998, filed on March 2, 2018, entitled “METHODS, COMPOSITIONS, AND DEVICES FOR ISOLATION AND EXPRESSION ANALYSIS OF REGIONS OF INTEREST FROM A TISSUE SECTION,” and U.S. Provisional Application No. 62 / 691,559, filed on June 28, 2018, entitled “METHODS, COMPOSITIONS, AND DEVICES FOR ISOLATION AND EXPRESSION ANALYSIS OF REGIONS OF INTEREST FROM A TISSUE SECTION,” the entire contents of which are incorporated herein by reference. Background Art

[0003] Conventional clinical analysis of gene and protein expression in tissue samples involves extraction of nucleic acids and proteins from sections (e.g., tissue biopsies). In the absence of selection mechanisms, analyses based on these sections represent a synthesis of expression profiles and DNA / RNA sequence data from multiple cell types within the tissue (e.g., epithelial cells, connective tissue, and immune cells). Summary of the Invention

[0004] In some aspects, the present disclosure provides a method for isolating biomolecules from at least one region of interest on a planar tissue section on a solid substrate for sequencing or analysis, comprising: (a) surrounding a defined region of interest on the planar tissue section on a solid substrate with a polymer to form a non-physical fluid barrier, wherein the diameter of the region of interest is less than 1 mm; and (b) selectively destroying in situ cells within at least one of the defined regions of interest. In some aspects, the present disclosure provides a method for isolating biomolecules from at least one region of interest on a planar tissue section on a solid substrate for sequencing or analysis, comprising: applying a chemical mask to the planar tissue section, the chemical mask surrounding the at least one region of interest in the planar tissue section on the solid substrate, wherein the chemical mask forms a barrier suitable for selectively destroying cells within the at least one region of interest, wherein the chemical mask is not a physical fluid barrier. In some aspects, the present disclosure provides a method for isolating biomolecules from at least one region of interest on a planar tissue section on a solid substrate for sequencing or analysis, comprising: dispensing a solvent suitable for disrupting cells within the region of interest in the planar tissue section onto the solid substrate to form droplets containing free cellular components, wherein the droplets are isolated from fluid communication with tissue outside the region of interest in the absence of a physical barrier. In some aspects, the present disclosure provides a method for isolating and detecting biomolecules from multiple regions of interest in a tissue section, comprising: applying a hydrophobic mask to the tissue section to isolate at least 1,000 regions of interest in the planar tissue section from each other in the absence of a physical barrier, and detecting a plurality of proteins or nucleic acids released in situ from cells within the at least 1,000 regions of interest, thereby preserving spatial location information of the proteins or nucleic acids. In some embodiments, the method is capable of isolating regions of interest having a diameter of less than or equal to about 100 microns. In some embodiments, the tissue section is a FFPE tissue section. In some embodiments, the method comprises detecting a plurality of proteins or nucleic acids released in situ from the cells, and the detecting comprises sequencing the nucleic acids released in situ from the cells. In some embodiments, the sequencing comprises labeling the nucleic acids released in situ with a barcode that identifies the location of the region of interest. In some embodiments, the barcode is not attached to a surface. In some embodiments, the labeling comprises labeling RNA released in situ and involves (a) applying oligonucleotides containing unique nucleic acid sequences to at least 1000 regions of interest; and (b) performing first-strand cDNA and second-strand cDNA synthesis on the at least 1000 regions of interest to separate the regions of interest from each other.In some embodiments of the method, cross contamination of the unique nucleic acid sequence between the cDNA molecules synthesized from the region of interest is reduced. In some embodiments, the synthesis of the first chain cDNA is carried out in situ. In some embodiments, the synthesis of the first chain cDNA and the second chain cDNA is carried out in situ. In some embodiments, sequencing the nucleic acid released from the cell in situ includes sequencing mRNA and genomic DNA. In some embodiments, sequencing the nucleic acid released from the cell in situ includes sequencing mRNA. In some embodiments, sequencing mRNA includes a pre-amplification step of the cDNA produced from the mRNA. In some embodiments, the pre-amplification step is LM-PCR, PCR using random hexamer primers, PCR using polyadenylic acid (poly-A) specific primers, or any combination thereof. In some embodiments, sequencing the nucleic acid released from the cell in situ includes sequencing genomic DNA. In some embodiments, sequencing genomic DNA includes a whole genome amplification (WGA) step of the genomic DNA. In some embodiments, the WGA step is degenerate oligonucleotide primer (DOP) PCR, multiple displacement amplification (MDA), multiple annealing loop amplification (MALBAC), PicoPlex, isothermal amplification with self-degenerate primers, or a combination thereof.

[0005] In some aspects, the present disclosure provides a tissue slice comprising at least 1000 water droplets isolated from each other in the absence of a physical barrier and unable to communicate with fluid. In some embodiments, the tissue slice comprises a hydrophobic mask that isolates the 1000 water droplets from each other and unable to communicate with fluid. In some embodiments, the at least 1000 water droplets contain at least 1000 unique oligonucleotides. In some embodiments, the hydrophobic mask comprises at least one layer of cyanoacrylate polymer. In some embodiments, the hydrophobic mask comprises at least one non-silylated fluoroalkyl layer. In some embodiments, the non-silylated fluoroalkyl layer comprises fluoroacrylate or non-silylated perfluoroalkane compound. In some embodiments, the tissue slice comprises at least one perfluoroalkylsilane layer. In some embodiments, the perfluoroalkylsilane layer comprises perfluoroalkyltrichlorosilane or perfluorotris(dimethylamino)silane. In some embodiments, the perfluoroalkyl trichlorosilane is FOTS (tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane). In some embodiments, the perfluoroalkyl tris(dimethylamino)silane is PF 10TAS (perfluorodecyl tris(dimethylamino)silane). In some embodiments, the oligonucleotide is not attached to a surface.

[0006] In some aspects, the present disclosure provides a method for isolating cellular components from at least one region of interest on a planar tissue slice on a solid substrate, comprising: (a) applying a chemical mask to the planar tissue slice, the chemical mask surrounding the at least one region of interest in the planar tissue slice on the solid substrate; and (b) dissolving the cellular components within the at least one region of interest by dispensing a solution comprising one or more extractants onto the region of interest, thereby selectively lysing cells surrounding the at least one region of interest. In some embodiments, dispensing the solution onto the region of interest comprises: (a) dispensing the solution comprising one or more extractants onto a reagent transfer device comprising at least one reagent transfer array element located on a surface of a second solid substrate; (b) contacting the at least one reagent transfer array element located on the surface of the second solid substrate with the planar tissue slice such that the at least one reagent transfer array element spatially corresponds to the at least one region of interest in the planar tissue slice, wherein the contacting causes the solution comprising the one or more extractants to be transferred to the at least one region of interest in the planar tissue slice, thereby dissolving the cellular components within the at least one region of interest in the solution comprising the one or more extractants. In some embodiments, the method further comprises isolating at least one dissolved cellular component from the at least one region of interest in the planar tissue section on the solid substrate. In some embodiments, the method comprises sequencing nucleic acids in the cellular component dissolved from the at least one region of interest. In some embodiments, the method comprises performing tandem mass spectrometry analysis on proteins in the cellular component dissolved from the at least one region of interest. In some embodiments, the solution in (b) further comprises a soluble tag, wherein the soluble tag corresponds to the at least one region of interest. In some embodiments, the soluble tag is an oligonucleotide. In some embodiments, the oligonucleotide comprises a sample index sequence corresponding to the region of interest and, optionally, a specific molecular tag sequence. In some embodiments, the oligonucleotide comprises a charge tag corresponding to the region of interest. In some embodiments, the oligonucleotide is double-stranded. In some embodiments, the oligonucleotide is conjugated to beads. In some embodiments, the soluble tag is a tandem mass spectrometry tag. In some embodiments, the one or more extractants comprise one or more surfactants, proteases, tonicity modifiers, chaotropic agents, nucleases, buffers, protease inhibitors, phosphatase inhibitors, or nuclease inhibitors. In some embodiments, the chemical mask has a contact angle of 60-155 degrees. In some embodiments, the chemical mask is applied with a hydrophobic masking solution comprising a fluoroalkane or fluoroacrylic polymer.In some embodiments, the chemical mask solution is applied with the aid of a solution comprising an acrylate or a cyanoacrylate. In some embodiments, the chemical mask solution further comprises a solvent. In some embodiments, the solvent is a propylene glycol derivative, a fluorocarbon, or an alcohol. In some embodiments, the solvent is perfluorooctane, perfluoro-2-methylpentane, perfluoro-1,3-dimethylcyclohexane, perfluorodecalin, or 1,3-difluoropropane. In some embodiments, the at least one region of interest is circular and the diameter of the region of interest is 1 mm or less, 500 microns or less, 250 microns or less, 125 microns or less, 100 microns or less, 80 microns or less, 50 microns or less, 25 microns or less, or 15 microns or less. In some embodiments, the area of ​​at least one region of interest is less than about 7.8×10. 5 square micrometers. In some embodiments, the at least one reagent transfer array element located on the surface of the second solid substrate comprises at least one hydrophilic region defined by a hydrophobic region. In some embodiments, the at least one reagent transfer array element located on the surface of the second solid substrate is capable of delivering a solution volume of 10,000 picoliters or less, 1,000 picoliters or less, 500 picoliters or less, 250 picoliters or less, 100 picoliters or less, 50 picoliters or less, 10 picoliters or less, or 2 picoliters or less. In some embodiments, the hydrophobic region surrounding the at least one hydrophilic region has a contact angle of 60-155 degrees. In some embodiments, the method comprises lysing cellular components from one or more regions of interest in the planar tissue section. In some embodiments, the method comprises lysing cellular components from at least 10, at least 100, at least 1000, or at least 10,000 regions of interest in the planar tissue section. In some embodiments, the hydrophobic mask surrounding the at least one region of interest in the planar tissue section comprises a grid pattern. In some embodiments, the hydrophobic mask surrounding the at least one region of interest in the planar tissue section comprises an elliptical shape. In some embodiments, the hydrophobic mask surrounding the at least one region of interest in the planar tissue section on a solid substrate is applied to the planar tissue section using a piezoelectric inkjet delivery device. In some embodiments, the thickness of the planar tissue section is about 2 to about 50 μm. In some embodiments, the thickness of the planar tissue section is about 1 to about 15 μm. In some embodiments, the planar tissue section is a formalin-fixed, paraffin-embedded (FFPE) tissue section. In some embodiments, the planar tissue section is a non-fixed tissue section.

[0007] In some aspects, the present disclosure provides a system for isolating cellular components from at least one region of interest in a planar tissue slice on a first solid support, comprising: (a) a planar tissue slice on a first solid support, the planar tissue slice comprising at least one region of interest surrounded by a hydrophobic mask; (b) a second solid support comprising at least one reagent transfer array element on a solid substrate surface aligned with the at least one region of interest in the planar tissue slice, the at least one reagent transfer array element comprising a solution comprising one or more extractants; and (c) a motorized stage coupled to the solid support comprising the at least one reagent transfer array element and capable of moving the solid support such that the at least one reagent transfer array element on the solid substrate surface can contact the at least one region of interest in the planar tissue slice surrounded by the hydrophobic mask. In some embodiments, the system comprises a computer system configured to control the position of the motorized stage such that the at least one reagent transfer array element on the surface of the second solid substrate contacts the at least one region of interest in the tissue slice. In some embodiments, the hydrophobic masking solution is applied by a system comprising: (i) a piezoelectric inkjet delivery device capable of delivering the hydrophobic masking solution to the tissue section on the solid support, and (ii) a computer system configured to control the piezoelectric inkjet delivery device to surround at least one region of interest in the planar tissue section with the hydrophobic masking solution to produce a hydrophobic mask. In some embodiments, the at least one reagent transfer array element located on the surface of the second solid substrate comprises at least one hydrophilic region surrounded by a hydrophobic region. In some embodiments, the hydrophobic masking solution comprises C7F 15At least one of CH2OCOC(CH3)=CH2, FC-722, PerFluoroCoat or FluoroPel. In some embodiments, the hydrophobic masking solution comprises at least one of an acrylate or a cyanoacrylate. In some embodiments, the at least one reagent transfer array element located on the surface of the second solid substrate is capable of delivering a volume of 10,000 picoliters or less, 1,000 picoliters or less, 500 picoliters or less, 250 picoliters or less, 100 picoliters or less, 50 picoliters or less, 10 picoliters or less or 2 picoliters or less of solution. In some embodiments, the contact angle of the hydrophobic area surrounding the at least one hydrophilic area is 60-155 degrees. In some embodiments, the system for applying the hydrophobic masking solution comprises a UV light source capable of polymerizing the hydrophobic masking solution after application to the planar tissue section. In some embodiments, the system for applying the hydrophobic masking solution comprises equipment for chemical vapor deposition of perfluoroalkyltrichlorosilane or perfluoroalkylsilane.

[0008] In some aspects, the present disclosure provides a kit for isolating cellular components from at least one region of interest in a planar tissue section, comprising any element of any aspect of any method, system, or tissue section embodiment described herein.

[0009] In some aspects, the present disclosure provides a tissue slice comprising, from bottom to top, at least one cyanoacrylate layer followed by at least one perfluoroalkylsilane layer. In some embodiments, the perfluoroalkylsilane layer comprises perfluoroalkyltrichlorosilane or perfluoroalkyltris(dimethylamino)silane. In some embodiments, the perfluoroalkyltrichlorosilane is FOTS. In some embodiments, the tissue slice comprises at least one non-silylated fluoroalkyl layer beneath the at least one cyanoacrylate layer. In some embodiments, the non-silylated fluoroalkyl layer comprises fluoroacrylate or a non-silylated perfluoroalkyl compound. In some embodiments, the perfluoroalkyltrichlorosilane is FOTS ((trifluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane). In some embodiments, the tissue slice comprises at least one region lacking a cyanoacrylate layer, the region being surrounded by a region having the cyanoacrylate layer. In some embodiments, the region is substantially elliptical, polygonal, or free-form. In some embodiments, an aqueous solution of a detergent is applied to the tissue slice that is capable of lysing cells within the region. In some embodiments, the area is less than about 1 mm in size. In some embodiments, the area is less than about 100 microns in size.

[0010] Incorporation by reference

[0011] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The novel features of the present invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and accompanying drawings which illustrate illustrative embodiments in which the principles of the invention are utilized, in which:

[0013] Figure 1 Shown are photographs of hematoxylin / eosin staining of FFPE hepatocellular carcinoma sections (top) without any further treatment (left) and with a hydrophobic mask as described herein (right); and (bottom) shows stained microtome liver sections (left) and microtome liver sections patterned with a hydrophobic mask and stained after patterning (right).

[0014] Figure 2A 、 2B , 2C, 2D, 2E, and 2F show bright field microscopy images (10x magnification in 2A, 2B, 2C, and 2D, 4x magnification in 2E) showing regions of interest of different sizes (~25 μm, ~100 μm, ~250 μm, 500 μm, and 1 mm) and shapes (square or circle) isolated from FFPE human liver microtome sections by applying the hydrophobic masking technique described herein.

[0015] Figure 3 Schematic diagram showing how a reagent transfer array device (B) is used to transfer a solution to a region of interest on a tissue section (A) via surface tension contact (C).

[0016] Figure 4 Shown is a schematic diagram illustrating how the hydrophobic mask approach enables the isolation and marking of regions of interest from tissue sections without specialized equipment such as microfluidics or lasers.

[0017] Figure 5 Shown is a diagram illustrating how the hydrophobic masking method can delineate customized regions of interest in tissue sections.

[0018] Figure 6 Exemplary hypothetical data are shown illustrating how application of the hydrophobic masking approach of the present disclosure in combination with microscopy analysis and spatially aware labeling allows gene expression and / or sequence data to be associated with specific cell types in tissue sections.

[0019] Figure 7Depicts two stained z-slices of FFPE breast cancer tissue sections (Biomax, huCAT299) containing areas of cancer and normal tissue. The left panel shows a section stained with hematoxylin and eosin (H&E); the right panel shows another section of the same tumor sample stained for the cancer marker HER2 using immunohistochemistry (IHC).

[0020] Figure 8 Describes the application Figure 7 The chemical masking method described herein is depicted for each serial section of the same FFPE breast cancer sample. The image on the left depicts various mask patterns all applied to the same image, while the image on the right depicts the section after chemical masking indicating the selected tissue area.

[0021] Figure 9 Describes the Figure 8 Results of qPCR analysis of samples extracted as shown and described in Example 8.

[0022] Figure 10A and 10B A flow chart illustrating the hydrophobic masking method described in Example 5 is depicted. DETAILED DESCRIPTION

[0023] Clinical analyses based on the detection of rare or numerous cell types within tissues are challenging. For example, precancerous tissue may contain only a small number of abnormal cells, diluting the signal from surrounding normal cells and hindering early detection of cancer. Conversely, advanced tumors may harbor a large number of abnormal cells but may contain a variety of cells with distinct genotypes that respond differently to chemotherapy. In these cases, the ability to selectively isolate and analyze morphologically abnormal cells would improve diagnosis and understanding of disease pathophysiology.

[0024] However, existing methods for isolating and analyzing small numbers of selected cells in tissue samples are laborious, expensive, and unsuitable for true high-throughput multiplex analysis of tissues (e.g., laser capture microdissection, which requires extensive manual manipulation and expensive machinery).

[0025] Therefore, there is a need for a simple, inexpensive, high-throughput method for multiplex separation and analysis of regions within tissue samples.

[0026] definition

[0027] The term "tissue" refers to the aggregation of cells and optional intercellular substances (such as ECM). Typically, the cells in a tissue are not free-floating, but adhere to each other to form a multicellular structure. Tissue types include, but are not limited to, muscle, nerve, epidermis, and connective tissue. Tissues as described herein can be derived from a variety of organisms, including but not limited to mammals, such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cattle, cats, dogs, primates (i.e., humans or non-human primates); plants, such as Arabidopsis thaliana, corn, sorghum, oats, wheat, rice, rapeseed, or soybeans; algae, such as Chlamydomonas reinhardtii; nematodes, such as Caenorhabditis elegans; insects, such as Drosophila melanogaster, mosquitoes, fruit flies, honeybees, or spiders; fish, for example, zebrafish; reptiles; or amphibians, such as frogs or clawed frogs.

[0028] The term "contact angle" refers to the shape of a liquid droplet resting on a solid surface. Assuming a liquid is placed on a solid surface in air, the term "contact angle" represents the angle between a tangent line to the liquid and a tangent line to the solid surface at the point of contact between the liquid, the solid surface, and the air.

[0029] The term "hydrophobic" refers to a surface or coating that is difficult to wet with water. A surface is considered hydrophobic if it has a receding water contact angle of at least 60°, very hydrophobic if it has a contact angle of at least 110°, and extremely hydrophobic if it has a receding water contact angle of at least 120°.

[0030] The term "superhydrophobic" refers to a surface or coating that is very difficult to wet with water. A superhydrophobic surface or coating typically has a contact angle exceeding 140°, often exceeding 150°.

[0031] The term "hydrophilic" is used to refer to a surface that is wetted by aqueous fluids. A surface is wetted by a fluid (hydrophilic) when the fluid tends to spread spontaneously across the surface rather than forming discrete droplets. A surface is considered hydrophilic if its contact angle is less than about 50 degrees.

[0032] "Inkjet delivery" is a non-contact method that can process 1-100 picoliter (pl) droplets into two- and three-dimensional structures. The method involves dissolving or dispersing the material of interest in a liquid to form an ink. The most popular method for inkjet delivery is the drop-on-demand (DOD) method, in which droplets are ejected from a micron-sized nozzle by one of the following means: (a) heating the liquid to above boiling temperature (thermal DOD) to generate vapor bubbles, which stimulate the release of droplets from the nozzle, or (b) applying a voltage to a piezoelectric transducer, which causes the material to vibrate and release droplets from the nozzle (piezoelectric DOD).

[0033] Overview

[0034] Provided herein is a method for separating cellular components from at least one region of interest in a planar tissue slice on a solid substrate. The method is in situ and does not require the pre-preparation of a fixed nucleotide array to define the region of tissue. Instead, the method includes preparing a hydrophobic mask in situ on the tissue slice to isolate the region of interest, and then optionally using a surface tension array to add and / or remove extraction and / or analysis reagents from the specific region of interest surrounded by the hydrophobic mask. The use of a surface tension array is particularly useful, for example, when there are a large number (e.g., greater than 10, greater than 100, greater than 1000) of regions separated by the hydrophobic mask. In some embodiments, the method involves (a) applying a hydrophobic mask to the planar tissue slice, the hydrophobic mask surrounding the at least one region of interest in the planar tissue slice on a solid substrate; (b) dispensing a solution comprising one or more extractants onto a reagent transfer device comprising at least one reagent transfer array element located on the surface of a second solid substrate; and (c) contacting at least one reagent transfer array element located on the surface of the second solid substrate with the planar tissue slice so that the at least one reagent transfer array element spatially corresponds to the at least one region of interest in the planar tissue slice. In some embodiments, the contacting allows the solution comprising the one or more extractants to transfer to the at least one region of interest in the planar tissue section, thereby solubilizing cellular components in the at least one region of interest in the solution comprising the one or more extractants.

[0035] The solution comprising one or more extractants may include one or more tags or labels suitable for labeling nucleic acids or proteins. Such tags or labels may include barcodes or be spatially addressable so that nucleic acid sequencing data or mass spectrometry data derived from the extracted nucleic acids or proteins can be subsequently associated with the region of interest from which they were isolated. Therefore, the methods herein also provide a scheme for spatially labeling nucleic acids or proteins from tissue sections.

[0036] Tissue sections

[0037] The tissue sections used in the method can be prepared by any standard method commonly used for immunohistochemical preparations and can be fixed or unfixed (e.g., freshly excised or prepared by a non-fixed tissue preparation method, such as frozen). Fixation of cells or tissues can involve the use of cross-linking agents, such as formaldehyde, glutaraldehyde, etc.

[0038] Fixed tissue can be further embedded in paraffin or a hydrogel (e.g., a polyacrylamide carrier matrix). A popular tissue preparation method is formalin-fixed, paraffin-embedded (FFPE), which involves fixing the tissue block in a formalin solution, dehydrating the tissue with increasing concentrations of alcohol, clearing the tissue with xylene, and then infiltrating the tissue with wax (usually paraffin, a mixture of linear or normal alkanes with carbon chain lengths ranging from 20 to 40 with various melting points) and embedding the tissue in the wax block. FFPE tissue can then be prepared into tissue sections using a microtome.

[0039] In some embodiments, the thickness of the tissue sections prepared by FFPE is from about 1 micron to about 50 microns. In some embodiments, the thickness of the tissue sections prepared by FFPE is at least about 1 micron. In some embodiments, the thickness of the tissue sections prepared by FFPE is at most about 50 microns. In some embodiments, the thickness of the tissue sections prepared by FFPE is from about 1 micron to about 3 microns, from about 1 micron to about 5 microns, from about 1 micron to about 10 microns, from about 1 micron to about 15 microns, from about 1 micron to about 20 microns, from about 1 micron to about 30 microns, from about 1 micron to about 40 microns, from about 1 micron to about 50 microns, from about 3 microns to about 5 microns, from about 3 microns to about 10 microns, from about 3 microns to about 15 microns, from about 3 microns to about 20 microns, from about 3 microns to about 30 microns, from about 1 micron to about 40 microns, from about 3 microns to about 50 microns, from about 5 microns to about 10 microns, from about 3 microns to about 15 microns, from about 5 microns to about 2 In some embodiments, the thickness of tissue sections prepared by FFPE is about 1 micron, about 3 microns, about 5 microns, about 10 microns, about 15 microns, about 20 microns, about 30 microns, about 40 microns, about 10 microns to about 50 microns, about 15 microns to about 20 microns, about 15 microns to about 30 microns, about 15 microns to about 40 microns, about 15 microns to about 50 microns, about 20 microns to about 30 microns, about 20 microns to about 40 microns, about 20 microns to about 50 microns, about 30 microns to about 40 microns, or about 40 microns to about 50 microns.

[0040] Embedded tissue sections can be deparaffinized prior to further processing (such as with the hydrophobic chemistry and sample extraction techniques described further herein). Deparaffinization can be performed, for example, by incubating the sections in a xylene bath, then incubating in a xylene / ethanol bath, then incubating in baths of gradually decreasing ethanol concentrations (from 100% to 50%), followed by drying or rinsing in water.

[0041] In some embodiments, non-fixed or fixed tissue can be prepared into slices by frozen sectioning. For non-fixed tissue, the tissue is first immersed in a frozen tissue matrix (e.g., OCT or Cryomatrix), frozen in an isopentane and / or 2-methylbutane bath in contact with liquid nitrogen, and then sliced ​​on a cryostat. For fixed tissue, before embedding / freezing into the tissue matrix, the tissue can be cryoprotected in sucrose, which involves incubation in a sucrose bath (up to 30%) with a gradually increasing concentration. The fixed tissue of cryoprotection can then be immersed in a frozen tissue matrix (e.g., OCT or Cryomatrix) and can be frozen in a) an isopentane bath in contact with liquid nitrogen, or b) frozen using a slower freezing method (e.g., powdered dry ice or dry ice methanol / ethanol slurry) instead. The frozen fixed tissue that is cryoprotected can then be sliced ​​on a cryostat.

[0042] In some embodiments, the thickness of tissue sections prepared by cryotomy can be from about 5 microns to about 50 microns. In some embodiments, the thickness of tissue sections prepared by cryotomy can be at least about 5 microns. In some embodiments, the thickness of tissue sections prepared by cryotomy can be at most about 50 microns. In some embodiments, the thickness of tissue sections prepared by cryosectioning can be about 5 microns to about 7 microns, about 5 microns to about 9 microns, about 5 microns to about 11 microns, about 5 microns to about 13 microns, about 5 microns to about 15 microns, about 5 microns to about 20 microns, about 5 microns to about 30 microns, about 5 microns to about 40 microns, about 5 microns to about 50 microns, about 7 microns to about 9 microns, about 7 microns to about 11 microns, about 7 microns to about 13 microns, about 7 microns to about 15 microns, about 7 microns to about 20 microns, about 5 microns to about 30 microns, about 5 microns to about 40 microns, about 50 microns, about 9 microns to about 11 microns, about 9 microns to about 13 microns, about 9 microns to about 15 microns, about 9 microns to about 20 microns, about 9 microns to about 30 microns, about 9 microns to about micrometers to about 40 micrometers, about 9 micrometers to about 50 micrometers, about 11 micrometers to about 13 micrometers, about 11 micrometers to about 15 micrometers, about 11 micrometers to about 20 micrometers, about 11 micrometers to about 30 micrometers, about 11 micrometers to about 40 micrometers, about 11 micrometers to about 50 micrometers, about 13 micrometers to about 15 micrometers, about 13 micrometers to about 20 micrometers, about 13 micrometers to about 30 micrometers, about 13 micrometers to about 40 micrometers, about 13 micrometers to about 50 micrometers, about 15 micrometers to about 20 micrometers, about 15 micrometers to about 30 micrometers, about 15 micrometers to about 40 micrometers, about 15 micrometers to about 50 micrometers, about 20 micrometers to about 30 micrometers, about 20 micrometers to about 40 micrometers, about 20 micrometers to about 50 micrometers, about 30 micrometers to about 40 micrometers, about 30 micrometers to about 50 micrometers, or about 40 micrometers to about 50 micrometers. In some embodiments, the thickness of tissue sections prepared by cryosectioning can be about 5 microns, about 7 microns, about 9 microns, about 11 microns, about 13 microns, about 15 microns, about 20 microns, about 30 microns, about 40 microns, or about 50 microns.

[0043] Chemical mask

[0044] A. Composition / Application

[0045] In some embodiments, the methods described herein include depositing a chemical mask to surround and isolate a region of interest in a tissue section (e.g., fresh frozen, fixed frozen, and / or dewaxed FFPE tissue section). The chemical mask allows for selective water extraction / hydration of cellular components from the region of interest without contamination by components of surrounding areas (because regions of the sample other than the region of interest repel the solution required to disrupt / extract / hydrate the cells). Thus, the chemical mask solution is hydrophobic (e.g., having a receding water contact angle of at least 60 degrees).

[0046] In some embodiments, the contact angle of the chemical mask can be from about 60 degrees to about 150 degrees. In some embodiments, the contact angle of the chemical mask can be at least about 60 degrees. In some embodiments, the contact angle of the chemical mask can be at most about 150 degrees. In some embodiments, the contact angle of the chemical mask can be from about 60 degrees to about 100 degrees, from about 60 degrees to about 110 degrees, from about 60 degrees to about 140 degrees, from about 60 degrees to about 150 degrees, from about 100 degrees to about 110 degrees, from about 100 degrees to about 140 degrees, from about 100 degrees to about 150 degrees, from about 110 degrees to about 140 degrees, from about 110 degrees to about 150 degrees, or from about 140 degrees to about 150 degrees. In some embodiments, the contact angle of the chemical mask can be about 60 degrees, about 100 degrees, about 110 degrees, about 140 degrees, or about 150 degrees.

[0047] A variety of surface coating polymers and copolymers have been described that can be used to produce surface receding water contact angles of at least 60 degrees and are therefore suitable for use in producing the chemical masks described herein.

[0048] An important class of such reagents are fluorocarbons, particularly fluorocarbon monomers containing at least one terminal trifluoromethyl group. In some embodiments, the fluorocarbon monomer containing at least one terminal trifluoromethyl group contains from about 3 to about 20 carbon atoms. In some embodiments, such fluorocarbons are substantially non-branched fluoroalkyl or perfluoroalkyl ethylenically unsaturated monomers. In some embodiments, the ethylenically unsaturated monomer is an acrylate, such as methacrylate. In some embodiments, the ethylenically unsaturated monomer is a cyanoacrylate. In some embodiments, the fluorocarbon monomer containing at least one terminal trifluoromethyl group is a fluorinated or perfluorinated acrylate, silicone, epoxy resin, polyurethane or oxime or any combination thereof. Such reagents include fluoroacrylates (or solutions thereof) or perfluoroalkylsilanes. Fluoroacrylates or solutions thereof include, but are not limited to, fluoroalkyl methacrylate monomers C7F 15CH2OCOC(CH3)=CH2, FC-722 (available from 3M), PerFluoroCoat (Cytonix), and FluoroPel (Cytonix, including but not limited to FluoroPel 800 and 800M products). The solution can be used at full strength, but can be diluted with a solvent (e.g., a fluorosolvent, alcohol, ethanol) to form a low concentration of coating polymer or fluoroalkane. The polymer solution used to prepare the coating of the present invention preferably has a coating polymer content of about 0.01% to about 50% by weight. Perfluoroalkylsilanes include perfluoroalkyltrichlorosilanes and perfluorotris(dimethylamino)silane (e.g., PF10TAS). Perfluoroalkyltrichlorosilanes are compounds comprising trichlorosilyl groups (e.g., C1-C 20 Perfluoroalkyl groups such as perfluoromethyl, perfluoroethyl, perfluoropropyl, perfluorobutyl, perfluoro-tert-butyl, perfluoropentyl, perfluorohexyl, perfluorooctyl, perfluorononyl, and perfluorodecyl are organic molecules. Examples of perfluoroalkyltrichlorosilanes include, but are not limited to, FOTS (trichloro(1H,1H,2H,2H-perfluorooctyl)silane), trichloro((1H,1H,2H,2H-perfluorodecyltrichlorosilane) and 1H,1H,2H,2H-perfluorododecyltrichlorosilane). Perfluorotris(dimethylamino)silane is an organic molecule comprising a tris(dimethylamino)silyl group (e.g., C1-C ... 20 Perfluoroalkyl groups such as perfluoromethyl, perfluoroethyl, perfluoropropyl, perfluorobutyl, perfluoro-tert-butyl, perfluoropentyl, perfluorohexyl, perfluorooctyl, perfluorononyl, perfluorodecyl) are organic molecules. Perfluoroalkylsilanes, perfluorotris(dimethylamino)silanes or perfluoroalkyltrichlorosilanes can be applied directly (e.g., dipping, spraying) or indirectly (e.g., chemical vapor deposition, CVD).

[0049] In some embodiments, a fluorocarbon is applied as a polymer (selectively to areas desired to be excluded, or non-specifically to the entire tissue area) directly to the tissue section to create a mask (e.g., a fluorinated carbon monomer such as a fluoroacrylate is added to the tissue section along with a photoinitiator). In some embodiments, the fluorocarbon is applied to the tissue section after a non-fluorinated acrylate or cyanoacrylate is initially applied to the tissue section along with a photoinitiator, such as by inkjet printing. In some embodiments, more than one fluorocarbon is applied to the tissue section as a copolymer (e.g., a fluorocarbon such as a fluoroacrylate is first applied to the tissue section, then a non-fluorinated acrylate or cyanoacrylate and a photoinitiator are applied thereto, then the same fluorocarbon or a different fluorocarbon such as a perfluoroalkylsilane is applied thereto).

[0050] In some embodiments, a fluorocarbon is applied as a polymer to a layer of hydrophobic material on top of a tissue section to create a mask (e.g., after an initial application, such as by spraying or dipping a hydrophobic polymer such as cyanoacrylate onto the tissue section). In some embodiments, the fluorocarbon is applied to the hydrophobic material layer in the same or a different pattern than the hydrophobic material layer, such that the fluorocarbon and the hydrophobic material form a copolymer mask where they meet.

[0051] In some embodiments, a layer of hydrophobic material is applied as a polymer to a chemical mask (e.g., after the mask material is initially applied to the tissue section as a polymer or copolymer). In some embodiments, the hydrophobic material is a fluorocarbon. In some embodiments, the hydrophobic material comprises a perfluoroalkylsilane, such as perfluoroalkyltrichlorosilane or perfluoroalkyltris(dimethylamino)silane (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane. In some embodiments, the hydrophobic material comprises a fluorocarbon. In some embodiments, the hydrophobic material comprises a perfluoroalkyltrichlorosilane, such as trichloro(1H,1H,2H,2H-perfluorooctyl)silane. In some embodiments, the hydrophobic material comprises a perfluoroalkyltris(dimethylamino)silane, such as PF10TAS.

[0052] Other classes of agents that can be used to create chemical masks include photocurable (UV curable) acrylates and cyanoacrylates—with or without photocatalysts.

[0053] The monomers comprising the chemical mask can be applied to the tissue section by a variety of methods, including but not limited to inkjet printing (e.g., DOD or piezoelectric inkjet printing methods) using the monomers suspended in a suitable solvent (e.g., an alcohol such as ethanol, propylene glycol, propylene glycol monomethyl ether acetate, methyl ethyl ketone, or a suitable fluorocarbon having a boiling point above room temperature such as perfluorooctane, perfluoro-2-methylpentane, perfluoro-1,3-dimethylcyclohexane, perfluorodecalin, 1,3-difluoropropane, etc.).

[0054] The chemical mask may include one or more monomer or polymer layers. One or more monomer or polymer layers may include the same or different monomers or polymers. One or more monomer or polymer layers may form a copolymer. One or more monomer or polymer layers may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 polymer layers or more. In some embodiments, the chemical mask includes a cyanoacrylate layer, followed by a perfluoroalkylsilane (e.g., perfluoroalkyltrichlorosilane or perfluoroalkyltris(dimethylamino)silane) layer. In some embodiments, the chemical mask includes multilayer cyanoacrylate, followed by a perfluoroalkylsilane (e.g., perfluoroalkyltrichlorosilane or perfluoroalkyltris(dimethylamino)silane). In some embodiments, the chemical mask includes a fluoroacrylate or non-silylated fluoroalkane layer, followed by a cyanoacrylate layer, followed by a perfluoroalkylsilane (e.g., perfluoroalkyltrichlorosilane or perfluoroalkyltris(dimethylamino)silane) layer. In some embodiments, the chemical mask comprises multiple layers of fluoroacrylate or non-silylated fluoroalkane, followed by cyanoacrylate, and then perfluoroalkylsilane (eg, perfluoroalkyltrichlorosilane or perfluoroalkyltris(dimethylamino)silane).

[0055] B. Enclosure of the region of interest (shape, size, number, separation, array, pre-selected ROI, correlation with previous images)

[0056] Chemical mask can be used to surround at least one region of interest on the tissue section for subsequent separation, and it can be a range of shapes and sizes. The region of interest can be various shapes, such as square, circular, oval, triangle, trapezoid, pentagon, hexagon or regular polyhedron. The region of interest can be about 176 square microns to about 780,000 square microns. The region of interest can be at least about 176 square microns. The region of interest can be at most about 780,000 square microns. The area of ​​interest can be about 176 square microns to about 12,000 square microns, about 176 square microns to about 49,000 square microns, about 176 square microns to about 190,000 square microns, about 176 square microns to about 780,000 square microns, about 12,000 square microns to about 49,000 square microns, about 12,000 square microns to about 190,000 square microns, about 12,000 square microns to about 780,000 square microns, about 49,000 square microns to about 190,000 square microns, about 49,000 square microns to about 780,000 square microns, or about 190,000 square microns to about 780,000 square microns. The area of ​​interest can be about 176 square microns, about 12,000 square microns, about 49,000 square microns, about 190,000 square microns, or about 780,000 square microns. The area of ​​interest can be about 490 square microns to about 780,000 square microns. The area of ​​interest can be at least about 490 square microns. The area of ​​interest can be at most about 780,000 square microns. The area of ​​interest can be about 490 square microns to about 12,000 square microns, about 490 square microns to about 49,000 square microns, about 490 square microns to about 190,000 square microns, about 490 square microns to about 780,000 square microns, about 12,000 square microns to about 49,000 square microns, about 12,000 square microns to about 190,000 square microns, about 12,000 square microns to about 780,000 square microns, about 49,000 square microns to about 190,000 square microns, about 49,000 square microns to about 780,000 square microns or about 190,000 square microns to about 780,000 square microns. The area of ​​interest can be about 490 square microns, about 12,000 square microns, about 49,000 square microns, about 190,000 square microns, or about 780,000 square microns.

[0057] The diameter of the circular area of ​​interest can be from about 15 microns to about 10,000 square microns. The diameter of the circular area of ​​interest can be at least about 15 microns. The diameter of the circular area of ​​interest can be at least about 25 microns. The diameter of the circular area of ​​interest can be at most about 1,000 microns. The diameter of the circular area of ​​interest can be from about 15 microns to about 50 microns, from about 15 microns to about 125 microns in diameter, from about 15 microns to about 250 microns in diameter, from about 15 microns to about 500 microns in diameter, from about 15 microns to about 750 microns in diameter, from about 15 square microns to about 1,000 microns in diameter, from about 15 microns to about 125 microns in diameter, from about 50 microns to about 250 microns in diameter, from about 50 microns to about 500 microns in diameter, from about 50 microns to about 750 microns in diameter, from about 50 microns to about 1,0 00 microns in diameter, about 125 microns to about 250 microns in diameter, about 125 microns to about 500 microns in diameter, about 125 microns to about 750 microns in diameter, about 125 microns to about 1,000 microns in diameter, about 250 microns to about 500 microns in diameter, about 250 microns to about 750 microns in diameter, about 250 microns to about 1,000 microns in diameter, about 500 microns to about 750 microns in diameter, about 500 microns to about 10,000 microns in diameter, or about 750 microns to about 1,000 microns in diameter. The circular area of ​​interest can have a diameter of about 15 microns, a diameter of about 25 microns, a diameter of about 50 microns, a diameter of about 125 microns, a diameter of about 250 microns, a diameter of about 500 microns, a diameter of about 750 microns, a diameter of about 1,000 microns, a diameter of about 2,000 microns, a diameter of about 3,000 microns, a diameter of about 4,000 microns, a diameter of about 5,000 microns, a diameter of about 6,000 microns, a diameter of about 7,000 microns, a diameter of about 8,000 microns, a diameter of about 9,000 microns, or a diameter of about 10,000 microns. The circular area of ​​interest can have a diameter of less than or equal to about 15 microns, a diameter of less than or equal to about 25 microns, a diameter of less than or equal to about 50 microns, a diameter of less than or equal to about 125 microns, a diameter of less than or equal to about 250 microns, a diameter of less than or equal to about 500 microns, a diameter of less than or equal to about 750 microns, a diameter of less than or equal to about 1,000 microns, a diameter of less than or equal to about 2,000 microns, a diameter of less than or equal to 3,000 microns, a diameter of less than or equal to 4,000 microns, a diameter of less than or equal to 5,000 microns, a diameter of less than or equal to 6,000 microns, a diameter of less than or equal to about 7,000 microns, a diameter of less than or equal to about 8,000 microns, a diameter of less than or equal to about 9,000 microns, or a diameter of less than or equal to about 10,000 microns.

[0058] In some embodiments, more than one region of interest is surrounded in a tissue sample so that the regions of interest form a grid. In some embodiments, the grid includes about 10 regions of interest to about 10,000 regions of interest. In some embodiments, the grid includes at least about 10 regions of interest. In some embodiments, the grid includes at most about 10,000 regions of interest. In some embodiments, the grid includes about 10 regions of interest to about 100 regions of interest, about 10 regions of interest to about 1,000 regions of interest, about 10 regions of interest to about 10,000 regions of interest, about 100 regions of interest to about 1,000 regions of interest, about 100 regions of interest to about 10,000 regions of interest, or about 1,000 regions of interest to about 10,000 regions of interest. In some embodiments, the grid includes about 10 regions of interest, about 100 regions of interest, about 1,000 regions of interest, or about 10,000 regions of interest.

[0059] In some embodiments, the size, shape, and / or pattern of the region of interest can be user-defined. Such user-defined patterns can be achieved by first staining / imaging the corresponding tissue section from a higher or lower vertical position in the tissue block using a suitable visualization technique (e.g., hematoxylin / eosin staining, immunofluorescence, immunohistochemistry), defining the region of interest on the tissue image in a computer system, and transferring the desired pattern to a fresh, unprocessed corresponding tissue section using inkjet printing and guidance from the computer system.

[0060] extraction

[0061] A.Solution

[0062] After one or more regions of interest are defined on the tissue section by applying a chemical mask, an extraction solution can be transferred to the one or more regions of interest to isolate nucleic acids and / or proteins from the regions of interest. The extraction solution can include one or more ionic or nonionic surfactants (e.g., β-octylglucoside, Triton-X-100, SDS, Tween-20, CHAPS), proteases (e.g., proteinase-K, trypsin, chymotrypsin, Lys-C, Asp-N, collagenase), tonicity regulators (e.g., glucose, glycerol, mannitol, potassium chloride, sodium chloride), chaotropic agents (e.g., urea, thiourea, guanidine chloride), nucleases (deoxyribonucleases, ribonucleases), buffers (e.g., Tris, Trizma, MOPS, sodium phosphate, sodium bicarbonate, Bicine, CAPS, CAPSO, Tricine, HEPES, MOPSO), protease inhibitors (e.g., AEBSF·HCl, aprotinin, Bestatin, E-64d, leupeptin, pepstatin, EDTA, PMSF), phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophospholipase, sodium orthophosphate), or nuclease inhibitors (e.g., EDTA, EGTA, DEPC, RNaisin). In the case of frozen, non-fixed tissue sections, standard compositions for isolating nucleic acids or proteins from cultured mammalian cells (e.g., solutions containing buffers, salts, and surfactants) can be used. For retrieval of proteins or DNA from FFPE tissue sections, exemplary protocols can be found, for example, in Pikor et al., J Vis Exp. 2011;(49):2763 or in Paulo et al., JOP. 2013 Jul;14(4):405-414.

[0063] i. Soluble tags

[0064] In some embodiments, the extraction solution may further comprise a soluble tag that spatially corresponds to the region of interest, such that downstream multiplex sequencing or mass spectrometry data can be assigned to the region of interest.

[0065] For multiplex sequencing (e.g., NGS sequencing) of nucleic acids (e.g., mRNA, cDNA, genomic DNA) derived from regions of interest, suitable soluble tags include synthetic oligonucleotides. Such oligonucleotides contain a positional sequence (e.g., a non-random sequence corresponding to the region of interest) and, optionally, a specific molecular index (UMI) that allows each individual reverse transcribed cDNA molecule to be counted in downstream sequencing. Random sequence generation can be used to generate UMI sequences. UMI sequences can then be rigorously filtered by mapping to the genomes of all common reference species and have a preset Tm interval, GC content, and a distance defined by the difference from other barcode sequences to ensure that the barcode sequences do not interfere with the capture of nucleic acids (e.g., RNA from tissue samples) and can be distinguished from each other without difficulty. Synthetic oligonucleotides may include positional and / or UMI sequences and are used to label only the 5' or 3' end of the reverse transcribed cDNA or both the 5' and 3' end.

[0066] Labeling nucleic acids (e.g., RNA) isolated from each region of interest using such synthetic oligonucleotides can be accomplished by a variety of methods. In some embodiments, the synthetic oligonucleotide comprising the position / UMI sequence is double-stranded (optionally hairpin or Y-shaped and / or optionally includes a 3' terminal T-overhang (e.g., as described in Wei et al., Genetics. 2016 Jan;202(1):37-44)) and ligated to either end of a cDNA generated by a first round of reverse transcriptase synthesis using an oligo-dT universal primer by A-tailing followed by ligation. In some embodiments, the synthetic oligonucleotide is single-stranded and additionally comprises an oligo-dT sequence such that the position / UMI sequence is incorporated into the cDNA during the first round of reverse transcription. Other exemplary primer designs and protocols for cDNA library tagging can be found, for example, in Hashimshony et al., Genome Biology (2016) 17:77, Hashimony et al., Cell Rep. 2012 Sep 27; 2(3): 666-73, and Head et al., Biotechniques. 2014; 56(2): 61.

[0067] For multiplex mass spectrometry analysis (e.g., LC-MS / MS) of proteins derived from one or more regions of interest, suitable soluble tags include tandem mass spectrometry tags available from Thermo Scientific (e.g., Duplex TMT, Simplex TMT, 10plex TMT, 11plex TMT) and those described in WO2016196994A1. These are amine-reactive tags that are suitable for binding to tryptic peptides generated from proteins in the region of interest with slightly different molecular weights, which allows easy differentiation of tryptic peptides derived from multiple different samples on MS / MS spectra (for further details on the construction and detection of TMT, see WO2016196994A1 and Zhang et al., Methods Mol Biol. 2017; 1550: 185-198).

[0068] B. Reagent Transfer Device

[0069] In some embodiments, transferring an extraction solution or other component to or from an area of ​​interest on a masked tissue section involves the use of one or more reagent transfer devices. The reagent transfer device comprises an array comprising a plurality of hydrophilic regions (reagent transfer array elements) spatially corresponding to the area of ​​interest on the masked tissue section. The hydrophilic regions surrounded by hydrophobic areas represent a "surface tension array," wherein an aqueous solution added to the hydrophilic regions is contained within the surrounding hydrophobic regions, and wherein the maximum solution volume of the reagent transfer array element can be controlled by varying the area / diameter of the hydrophilic regions (controlling the "width" of the solution that can be added to the feature) and the difference between the contact angles of the hydrophilic and hydrophobic regions (controlling the "height" of the solution that can be added to the feature). Figure 3 Demonstrates how a reagent transfer array B containing an extraction solution (black) can be used to transfer the solution to a region of interest on slide A by touching the liquid dome of B to the tissue section and allowing surface tension to draw the liquid meniscus to the tissue section (C).

[0070] The reagent transfer device can be produced using a variety of different hydrophobic / hydrophilic chemistries on a variety of substrates. One convenient method involves derivatizing the glass with a hydrophilic compound and then protecting the hydrophilic features with a positive photoresist combined with a fluorocarbon coating. In one embodiment, an array containing hydrophilic reagent-transferred array features is produced by derivatizing a quantitatively cleaned glass slide with a monofunctional (one attachment site for silanization) organosilane such as 3-aminopropyldimethylethoxysilane (APDMS), then protecting the reagent-transferred array elements / hydrophilic areas with a positive photoresist and treating with a perfluoroalkyltrichlorosilane such as (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane or another suitable anti-sticking coating (e.g., dimethyldichlorosilane DDMS, perfluorododecyltris(dimethylamino)silane PF10TAS, perfluorodecanoic acid PFDA, or variants thereof with perfluoroalkyl chains of varying lengths and isomerisms; see, e.g., Ashurst et al., IEEE Transactions on Device and Materials Relatability. 3(4): 173-178 (2003)). Further chemical details of the derivatization of slides with hydrophilic and hydrophobic regions can be found in, for example, Butler et al., J. Am. Chem. Soc. 2001, 123, 8887-8894 and US20150268233A1.

[0071] The size of the hydrophilic area / reagent transfer array element on the reagent transfer device corresponds to the size of the region of interest of the mask on the tissue sample. The reagent transfer array element can be various shapes, for example square, circular, oval, triangle, trapezoid, pentagon, hexagon or regular polyhedron. The reagent transfer array element can be approximately 490 square microns to approximately 780,000 square microns. The reagent transfer array element can be at least approximately 490 square microns. The reagent transfer array element can be approximately 780,000 square microns at the most. The reagent transfer array elements can be about 490 square microns to about 12,000 square microns, about 490 square microns to about 49,000 square microns, about 490 square microns to about 190,000 square microns, about 490 square microns to about 780,000 square microns, about 12,000 square microns to about 49,000 square microns, about 12,000 square microns to about 190,000 square microns, about 12,000 square microns to about 780,000 square microns, about 49,000 square microns to about 190,000 square microns, about 49,000 square microns to about 780,000 square microns, or about 190,000 square microns to about 780,000 square microns. The reagent transfer array element can be about 490 square microns, about 12,000 square microns, about 49,000 square microns, about 190,000 square microns, or about 780,000 square microns. The diameter of a circular reagent transfer array element can be about 25 square microns to about 1,000 square microns. The diameter of a circular reagent transfer array element can be at least about 25 square microns. The diameter of a circular reagent transfer array element can be at most about 1,000 square microns.The diameter of the circular reagent transfer array element can be from about 25 square microns to about 50 square microns, from about 25 square microns to about 125 square microns, from about 25 square microns to about 250 square microns, from about 25 square microns to about 500 square microns, from about 25 square microns to about 750 square microns, from about 25 square microns to about 1,000 square microns, from about 50 square microns to about 125 square microns, from about 50 square microns to about 250 square microns, from about 50 square microns to about 500 square microns, from about 50 square microns to about 750 square microns, from about 50 square microns to about 2 microns in diameter, about 125 to about 250 square microns in diameter, about 125 to about 500 square microns in diameter, about 125 to about 750 square microns in diameter, about 125 to about 1,000 square microns in diameter, about 250 to about 500 square microns in diameter, about 250 to about 750 square microns in diameter, about 250 to about 1,000 square microns in diameter, about 500 to about 750 square microns in diameter, about 500 to about 1,000 square microns in diameter, or about 750 to about 1,000 square microns in diameter. Circular reagent transfer array elements can have a diameter of about 25 square microns, about 50 square microns in diameter, about 125 square microns in diameter, about 250 square microns in diameter, about 500 square microns in diameter, about 750 square microns in diameter, or about 1,000 square microns in diameter.

[0072] In some embodiments, more than one reagent transfer array element is printed on the reagent transfer device so that the reagent transfer array element forms a grid. In some embodiments, the grid comprises approximately 10 reagent transfer array elements to approximately 10,000 reagent transfer array elements. In some embodiments, the grid comprises at least approximately 10 reagent transfer array elements. In some embodiments, the grid comprises approximately 10,000 reagent transfer array elements at the most. In some embodiments, the grid comprises approximately 10 reagent transfer array elements to approximately 100 reagent transfer array elements, approximately 10 reagent transfer array elements to approximately 1,000 reagent transfer array elements, approximately 10 reagent transfer array elements to approximately 10,000 reagent transfer array elements, approximately 100 reagent transfer array elements to approximately 1,000 reagent transfer array elements, approximately 100 reagent transfer array elements to approximately 10,000 reagent transfer array elements, approximately 100 reagent transfer array elements to approximately 10,000 reagent transfer array elements or approximately 1,000 reagent transfer array elements to approximately 10,000 reagent transfer array elements. In some embodiments, the grid includes about 10 reagent transfer array elements, about 100 reagent transfer array elements, about 1,000 reagent transfer array elements, or about 10,000 reagent transfer array elements.

[0073] The reagent transfer array element can be designed to adapt to a wide range of volumes. In some embodiments, the maximum volume of the reagent transfer array element is about 2 picoliters to about 10,000 picoliters. In some embodiments, the maximum volume of the reagent transfer array element is at least about 2 picoliters. In some embodiments, the maximum volume of the reagent transfer array element is at most about 10,000 picoliters. In some embodiments, the maximum volume of a reagent transfer array element is about 2 picoliters to about 10 picoliters, about 2 picoliters to about 50 picoliters, about 2 picoliters to about 100 picoliters, about 2 picoliters to about 250 picoliters, about 2 picoliters to about 500 picoliters, about 2 picoliters to about 1,000 picoliters, about 2 picoliters to about 10,000 picoliters, about 10 picoliters to about 50 picoliters, about 10 picoliters to about 100 picoliters, about 10 picoliters to about 250 picoliters, about 10 picoliters to about 500 picoliters, about 10 picoliters to about 1,000 picoliters, about 10 picoliters to about 10,000 picoliters, about 50 picoliters to about 100 picoliters, about 50 picoliters to about 1 About 250 picoliters, about 50 picoliters to about 500 picoliters, about 50 picoliters to about 1,000 picoliters, about 50 picoliters to about 10,000 picoliters, about 100 picoliters to about 250 picoliters, about 100 picoliters to about 500 picoliters, about 100 picoliters to about 1,000 picoliters, about 100 picoliters to about 10,000 picoliters, about 250 picoliters to about 500 picoliters, about 250 picoliters to about 1,000 picoliters, about 250 picoliters to about 10,000 picoliters, about 500 picoliters to about 1,000 picoliters, about 500 picoliters to about 10,000 picoliters, or about 1,000 picoliters to about 10,000 picoliters. In some embodiments, the maximum volume of a reagent transfer array element is about 2 picoliters, about 10 picoliters, about 50 picoliters, about 100 picoliters, about 250 picoliters, about 500 picoliters, about 1,000 picoliters, or about 10,000 picoliters.

[0074] C. Detection Technology

[0075] The biomolecules extracted from at least one region of interest are then detected to analyze the biomolecule expression profile of the cells within the region of interest, or to analyze the genomic DNA composition of the cells within the region of interest (e.g., in the case of a tissue sample containing cancer cells, where the genomic DNA of the cells within the tissue section is heterogeneous due to mutations, deletions, and / or translocations). Thus, the methods, devices, and compositions encompassed herein can be used to analyze the genetic and epigenetic characteristics of cells within a region of interest.

[0076] In some methods, the expression level of a biomolecule (e.g., mRNA or cDNA derived from mRNA by reverse transcription, or genomic DNA) within at least one region of interest is determined by sequencing. Sequencing methods can include: next-generation sequencing, high-throughput sequencing, pyrosequencing, classical Sanger sequencing, ligation sequencing, synthesis sequencing, hybridization sequencing, RNA-Seq (Illumina), digital PCR, digital gene expression (Helicos), next-generation sequencing, single molecule sequencing by synthesis (SMSS) (Helicos), Ion Torrent sequencer (Life Technologies / Thermo-Fisher), massively parallel sequencing, clonal single molecule arrays (Solexa), shotgun sequencing, Maxim-Gilbert sequencing, and primer walking.

[0077] In some methods, the expression level of a biomolecule (e.g., mRNA or cDNA derived from mRNA by reverse transcription) within at least one region of interest is determined by so-called "real-time amplification" methods, also known as quantitative PCR (qPCR) or Taqman (see, e.g., U.S. Pat. Nos. 5,210,015 to Gelfand, 5,538,848 to Livak et al., and 5,863,736 to Haaland, and Heid, CA et al., Genome Research, 6:986-994 (1996); Gibson, U. et al., Genome Research 6:995-1001 (1996); Holland, PM et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, (1991); and Livak, KJ et al., PCR Methods and Applications 357-362 (1995)). The basis of the method that this monitoring amplification product forms is to use the fluorescent oligonucleotide probe of double labeling to measure the accumulation of PCR product continuously.The probe used in such mensuration is normally short (about 20-25 bases) polynucleotide with two different fluorescent dye labels.The 5 ' end of probe is usually attached on reporter dye, and 3 ' end is attached to quencher dye.Probe is designed to have at least substantial sequence complementarity with the site on target mRNA or derivative nucleic acid.Also upstream and downstream PCR primers that are combined with the flanking region of locus are added to reaction mixture.When probe is complete, energy transfer occurs between two fluorophores, and quencher quenches the emission from reporter.In the extension phase of PCR, probe is cut by 5 ' nuclease activity of nucleic acid polymerase (such as Taq polymerase), thus discharges reporter from polynucleotide-quencher, thus causes the increase of reporter emission intensity, it can be measured by suitable detector.Then, the value recorded can be used for continuous calculation normalization reporter emission intensity increase, and finally quantifies the amount of amplified mRNA.

[0078] In some embodiments, for qPCR or Taqman assays, an RT-PCR step is first performed to generate cDNA from cellular RNA. Such RT-PCR amplification can be general (e.g., amplification using partially / completely degenerate oligonucleotide primers) or targeted (e.g., amplification using oligonucleotide primers directed to a specific gene to be analyzed in a subsequent step).

[0079] In some embodiments, qPCR or Taqman is used immediately after a reverse transcriptase reaction on isolated cellular mRNA; this format is used to quantify the levels of individual mRNAs during qPCR.

[0080] For qPCR or Taqman, the level of a particular gene can be expressed relative to one or more internal control genes measured from the same sample using the same detection methodology. Internal control genes can include so-called "housekeeping" genes (e.g., ACTB, B2M, UBC, GAPD, and HPRT1).

[0081] In some embodiments, for qPCR or Taqman detection or RNA sequencing, a "pre-amplification" step is first performed on cDNA transcribed from cellular RNA. This helps to increase the signal under conditions where the natural level of the RNA / cDNA to be detected is very low. Suitable pre-amplification methods include, but are not limited to, LM-PCR, PCR using random oligonucleotide primers (e.g., random hexamer PCR), PCR using polyadenylation-specific primers, and any combination thereof. Pre-amplification can be conventional amplification in the same manner as the reverse transcription reaction described above, or targeted amplification.

[0082] mRNA levels can also be measured without amplification by hybridization to a probe, for example using branched nucleic acid probes such as those from Panomics. Reagent System.

[0083] Alternatively or additionally, the expression level of the gene from at least one region of interest can be determined at the protein level, which means measuring the level of the protein encoded by the above-mentioned gene. Several methods and devices for measuring protein levels are well known, including immunoassays, for example, described in U.S. Patents 6,143,576, 6,113,855, 6,019,944, 5,985,579, 5,947,124, 5,939,272, 5,922,615, 5,885,527, 5,851,776, 5,824,799, 5,679,526, 5,525,524 and 5,480,792. These assays include various sandwich, competitive or non-competitive assay formats to generate a signal relevant to the presence or content of a protein analyte of interest. Any suitable immunoassay can be utilized, for example, lateral flow, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), competitive binding assay, etc. Various forms of antibody arrays employing antibodies have been proposed. Such arrays typically include different antibodies specific for the different proteins intended to be detected. For example, typically at least one hundred different antibodies are used to detect one hundred different protein targets, each antibody being specific for one target. Other ligands specific for a particular protein target can also be used, such as the synthetic antibodies disclosed in WO / 2008 / 048970. Other compounds with desired binding specificity can be selected from random libraries of peptides or small molecules. U.S. Patent No. 5,922,615 describes a device for detecting multiple target antigens in an array using multiple discrete regions of antibodies fixed on a membrane. U.S. Patent Nos. 5,458,852, 6,019,944, and 6,143,576. Microtiter plates or automation can be used to facilitate the detection of a large number of different proteins. Protein levels can also be determined by mass spectrometry (e.g., tandem LC / MS / MS). In some embodiments, genomic DNA isolated from the region of interest is analyzed (e.g., by any of the sequencing techniques described above, or by qPCR). Such analysis can be used to detect genomic variation, including but not limited to nucleotide sequence variation in and around genes, the presence of SNPs, insertions, deletions, and / or copy number variation. In some variations of this embodiment, the isolated DNA is first amplified by whole genome amplification (WGA) technology to improve the ability to extract DNA sequences from limited samples.

[0084] In some embodiments, the WGA technique is a two-stage technique known as degenerate oligonucleotide-primed (DOP) PCR. In the first stage, DOP-PCR uses primers containing: a) a randomized 6-nucleotide sequence at its 3' end, and b) a fixed adapter sequence (optionally containing a barcode containing a sample index and / or a specific molecular tag), with an optimal T for PCR amplification. mRandom primers are hybridized to the sample at a lower annealing temperature, and then chain extension is performed using a polymerase at a higher temperature. In the second stage, products from the first stage are amplified using PCR annealing at a higher temperature than in the first stage using primers containing a 3' end complementary to the 5' fixed adapter sequence of the first stage primer (and optionally including a barcode at its 5' end containing a sample index and / or specific molecular tag).

[0085] In some embodiments, WGA technology is a one-stage isothermal technique referred to as multiple displacement amplification (MDA).MDA can use primers similar to the first stage of DOP-PCR, for example, primers comprising a) 6 random nucleotide sequences at their 3' ends, and b) fixed adapter sequences (optionally comprising a barcode, the barcode comprising a sample index and / or a specific molecular tag). However, instead of traditional PCR DNA polymerases (such as Taq), MDA uses strand displacement polymerases (such as DNA polymerase). Use primers and DNA polymerases can amplify isolated DNA without the need for cycling under isothermal conditions.

[0086] In some embodiments, the WGA technique is a quasi-linear amplification technique called multiple annealing loop amplification technique (MALBAC). In the first stage, MALBAC uses specially designed primers that have a common 27-nucleotide sequence at the 5' end (e.g., GTG AGT GAT GGT TGA GGT AGT GTG GAG) and 8 random nucleotides at the 3' end; these primers are used to produce half-amplicons by first annealing at a low temperature (e.g., 15-20°C) and then extending at a higher temperature (e.g., 70-75°C) using a chain displacement polymerase (e.g., Bst DNA polymerase). After the primers are melted from the template (e.g., at 95°C or higher), cycles of low temperature (e.g., 15-20°C), high temperature (e.g., 70-75°C), denaturation temperature (e.g., 95°C or higher) are repeated (e.g., more than 10 cycles), and hairpin formation (e.g., 58°C) is used to further amplify the half-amplicons into complete amplicons. Because the complete amplicons contain the same 5'27 nucleotide sequence (e.g., GTG AGT GAT GGT TGA GGT AGT GTG GAG), once formed, the hairpin formation step removes them from further participation in subsequent amplification cycles. This ensures more linear amplification because copies of the original DNA are not made.

[0087] In some embodiments, the WGA technology is Pico-Plex TM(Takara Bio) and / or derivatives described in U.S. Patent No. 8,206,913. These techniques and derivatives attempt to correct some problems associated with completely random primer amplification of the entire genome (e.g., insufficient representation of certain regions). In some embodiments, in the first step of the method, self-inert (e.g., non-self-primer) degenerate primers are used, and the first step can be an isothermal amplification step or an initial annealing step of ~15-20°C, followed by an extension step at ~75°C (e.g., using a thermophilic DNA polymerase). In some embodiments, these primers include a) a 5' fixed region, b) a 3' variable region, and are designed so that the primers do not cross-hybridize or self-hybridize. In some embodiments, the variable region also includes a partially degenerate sequence (e.g., 10nt long, which can be all Y, R, K, or M, wherein Y = random C or T; R = random A or G; M = random A or C; K = random G or T) and a completely degenerate sequence (e.g., 2nt long, Ns, random A / C / T / G). In some embodiments, the constant region and the variable region of the self-inert degenerate primers consist essentially of only two types of non-complementary nucleotides selected from adenine and guanine; adenine and cytosine; guanine and thymidine. In some embodiments, the self-inert degenerate primers used in the isothermal amplification step are according to any one of SEQ ID NOs 1-8 in Table 1 below.

[0088] Table 1: Exemplary primer sequences

[0089]

[0090] In some embodiments, the DNA amplified above is subjected to an additional amplification step using a thermostable DNA polymerase. An additional amplification step can be performed using a primer consisting of the 5' fixed region of the primer used only in the isothermal amplification step. In some embodiments, these primers are any one of SEQ ID NO 8-12 in Table 1. The additional amplification step may comprise cycles of >10 normal PCR cycles (e.g., denaturation, annealing, extension) using a thermostable DNA polymerase. In some embodiments, similar to the method for above-mentioned MALBAC, cycling conditions have added an additional lower temperature step to promote the formation of hairpin DNA after the extension step.

[0091] Example

[0092] Example 1A.—Protocol for using a hydrophobic mask in tissue sections

[0093] A grid pattern of coating is applied to the tissue sample using a computer-assisted inkjet system, creating multiple regularly spaced, non-coated areas. The coating comprises a non-fluorinated acrylate or cyanoacrylate mixed with a fluoroalkyl acrylate monomer described herein in an alcohol or ketone solvent plus a photoinitiator. The section is then briefly exposed to ultraviolet light to cure the coating.

[0094] Example 1B. - Alternatives to using a hydrophobic mask in tissue sections

[0095] A grid pattern of a first coating is applied to a tissue sample using a computer-assisted inkjet system, thereby generating a plurality of regularly spaced, uncoated areas. The first coating comprises a non-fluorinated acrylate or cyanoacrylate plus a photoinitiator. The first coating is cured by briefly exposing it to ultraviolet light. Next, also using a computer-assisted inkjet system, a second coating comprising a fluoroalkyl acrylate monomer as described herein and a photoinitiator is added to the first coating. The second coating is then cured by briefly exposing it to ultraviolet light.

[0096] Example 2. – Use hydrophobic chemicals to isolate regions of interest in tissue samples

[0097] FFPE hepatocellular carcinoma tissue sections were stained with hematoxylin / eosin using fluoroalkyl acrylate monomers, with or without deparaffinization of the region of interest and with or without hydrophobic masking. Figure 1 Deparaffinization of FFPE tissue sections followed by treatment with the hydrophobic chemical of Example 1 effectively water-isolated the regions of interest of the mask, thereby preventing them from staining with hematoxylin / eosin.

[0098] Example 3. - Isolation of regions of different sizes in FFPE mouse liver sections

[0099] FFPE mouse liver sections were deparaffinized and masked to isolate regions of varying size (100, 250, 500, 1000 μm in diameter) and shape (square and circle) and stained with hematoxylin / eosin to demonstrate the accessibility of the hydrophilic region of interest (and inaccessibility of the surrounding hydrophobic mask) and the precision / accuracy of the inkjet application of the mask. Figure 2A It was shown that inkjet printing using a chemical mask can clearly define regions with diameters of either -160 or -550 microns. Figure 2B It was shown that inkjet printing using a chemical mask can clearly define regions with diameters of ∼300 or ∼1000 microns. Figure 2C Prove that both circles and polygons can be isolated from slices. Figure 2E It was shown that an area with a diameter of 2 mm could be clearly defined on the slice. Figure 2DIt is shown that areas of different diameters (100, 250, 500, 1 mm) and shapes (square and circle) can be printed in a clearly defined grid on a single tissue section.

[0100] Example 4. - A hydrophobic mask prevents nucleic acid extraction from the masked area

[0101] FFPE tissue sections were masked according to the method of Example 1A or Example 1B to generate regions of interest of varying sizes.

[0102] Dewax the slides by baking them in an oven at 55-60°C for 1 hour, immerse them twice in xylene for 3 minutes, and then twice in 100% ethanol for 3 minutes. Slides can optionally be stored in ethanol for up to one month.

[0103] For further analysis, the slides were air-dried to remove the ethanol and the tissue was lysed. An adhesive chamber was attached to the slide surrounding the tissue area, 600 microliters of Qiagen Tissue Lysis Buffer ATL with Proteinase K was added, the chamber was sealed with adhesive film, and the slide was incubated on a heating block at 56°C for 1 hour. The liquid (~450 microliters) was collected from the slide cavity of the microcentrifuge tube, and the microcentrifuge tube was then heated at 90°C for 1 hour. The microcentrifuge tube was then centrifuged to precipitate insoluble material, and the supernatant was transferred to a new tube and treated with 45 microliters of RNase A (100 mg / ml) and then incubated at room temperature for 2 minutes. An equal volume of Qiagen Buffer AL (~500 microliters) was added to the supernatant and the sample was mixed by vortexing. After vortexing, an equal amount of 100% ethanol (~500 microliters) was added to the supernatant, and the entire solution was transferred to a Qiagen minimal elution column. The column was washed with buffers AW1 and AW2 according to the manufacturer's instructions, and nucleic acids were eluted in Qiagen buffer ATE. Nucleic acids in each sample were then quantified on a Qubit spectrophotometer. Table 2 shows the relationship between region of interest size and extracted DNA. Data comparing DNA yield to uncovered surface area indicate that DNA yield is proportional to the area covered, indicating that DNA cannot be extracted from areas of the tissue section covered by the hydrophobic coating.

[0104] Table 2 Relationship between the uncoated surface area of ​​hydrophobically coated slides and DNA yield

[0105]

[0106] Example 5.-Optimized hydrophobic coating process:

[0107] A) Processing of small features (diameter < 1mm)

[0108] If the tissue sample is an FFPE sample, the tissue sample is first dewaxed by standard procedures (e.g., baking in an oven at approximately 60 degrees for 1 hour, followed by incubation in xylene for 3 minutes, incubation in xylene for 3 minutes, incubation in 100% ethanol for 3 minutes, incubation in 100% ethanol for 3 minutes, and air drying for 5 minutes). An initial hydrophobic coating is applied to the tissue surface by spraying or immersion in an ethanolic solution of a fluoroacrylate or micron-sized fluorine particles; examples include, but are not limited to, Fluoro-pel 800 or 800M. The slide is then air-dried (this initial treatment can improve the clarity of small features through the subsequent cyanoacrylate printing step).

[0109] A cyanoacrylate solution containing a photoinitiator is inkjet printed on a tissue sample to form a mask over areas that are desired to be excluded or to isolate desired features. During the printing process, the cyanoacrylate is exposed to ultraviolet light (e.g., 395 nm @ ~400 mJ / cm 2 ) for at least one second to be fixed. The slide is then removed from the printing apparatus and immediately subjected to extended UV curing (e.g., at 400 mJ / cm 2 395 nm for about 5 min).

[0110] After UV curing, the entire slide is subjected to vacuum-assisted vapor deposition of a perfluoroalkyltrichlorosilane (e.g., FOTS, trichloro(1H,1H,2H,2H-perfluorooctyl)silane). This technique has been used to generate circular regions of interest on FFPE human liver tissue sections with diameters as small as 25 microns.

[0111] B) Processing of large features (about 1mm or larger)

[0112] If the tissue sample is an FFPE sample, the tissue sample is first dewaxed by standard processes (e.g., baking in an oven at about 60 degrees for 1 hour, then incubating in xylene for 3 minutes, incubating in xylene for 3 minutes, incubating in 100% ethanol for 3 minutes, incubating in 100% ethanol for 3 minutes, and air drying for 5 minutes). Larger features can forgo the initial fluoroalkyl coating. A cyanoacrylate solution containing a photoinitiator is inkjet printed on the tissue sample in areas where it is desired to exclude or to isolate the desired features. During the printing process, the cyanoacrylate is dewaxed after deposition by exposure to ultraviolet light (e.g., 395nm @ ~400mJ / cm 2 ) for at least one second to be fixed. The slide is then removed from the printing apparatus and immediately subjected to extended UV curing (e.g., at 400 mJ / cm 2 395 nm for about 5 min).

[0113] After UV curing, the entire slide was subjected to vacuum-assisted vapor deposition of a perfluoroalkylsilane (eg, trichloro(1H,1H,2H,2H-perfluorooctyl)silane).

[0114] Example 6.—Cleavage and separation of hydrophobic separation regions

[0115] A surface tension array is prepared as described in US20150268233 A1 or Butler et al., J.Am.Chem.Soc.2001,123,8887-8894, wherein the hydrophilic characteristics correspond to the non-exclusion areas of the mask applied to the above tissue sample. The derivatization process includes coating a quantitatively cleaned glass slide with a monofunctional (one silanized attachment site) organosilane such as 3-aminopropyldimethylethoxysilane (APDMS), then protecting the non-exclusion areas / hydrophilic areas with a positive photoresist and treating with an anti-adhesion coating such as perfluoroalkylsilane (e.g., tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane.

[0116] The surface tension array is then tested. The surface tension array is then tested for hydrophilicity. A suitable lysis buffer solution containing a detergent is applied to the hydrophilic characteristics of the surface tension array. The surface tension array containing the hydrophilic region with the lysis buffer solution is contacted with a tissue sample prepared as described in Example 1A, 1B, or 5. A suitable period of contact is allowed to proceed with cell lysis. The surface tension array (now comprising the lysis buffer solution containing the cracked cellular components) is removed from contact with the tissue section and the individual points on the surface tension array are asked whether they have the desired characteristic (e.g., protein or nucleic acid abundance, sequence, or both).

[0117] Example 7.— Contact angle measurements of coated surfaces.

[0118] FFPE tissue sections or glass slides were subjected to different stages of the coating process shown in Example 5B, using FOTS ((tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane) for different lengths of vapor phase coating. Contact angle measurements were made directly using a telescopic goniometer (see, e.g., Bracco and Holst. Surface Science Techniques. 2013. ISBN 978-3-642-34243-1. pp. 3-34). The data showed that the addition of cyanoacrylate on top of the tissue sections enhanced the FOTS coating, and the combination of the two increased the contact angle of the combined FOTS / cyanoacrylate tissue surface to the very hydrophobic (contact angle greater than 110) range.

[0119] Table 3: Contact angle measurements of tissue or glass with various coatings

[0120]

[0121]

[0122] Example 8. - qPCR of RNA isolated from hydrophobic masked FFPE tissue sections

[0123] The optimized coating process of Example 5 was performed on each serial section from the same FFPE breast cancer tissue sample (Biomax, huCAT299) to select a sample that included non-cancerous tissue, transitional tissue (e.g., some non-cancerous tissue and some cancerous tissue), and cancerous tissue (see Figure 7 and Figure 8 ,in Figure 8 The left side of the figure shows different mask patterns applied to various tissue layers / sections, and the right side shows various tissue regions of interest isolated after masking. After coating, the regions of interest were lysed by applying lysis buffer containing detergent and proteinase K to the slide (lysis buffer was also applied to the coated area as a control). Lysis buffer was applied directly to the area and incubated at 56°C for 1 hour. Figure 8 Total RNA was extracted from the samples indicated. Total RNA was purified using the NucleoSpin total RNA FFPE XS kit (Takara Bio) according to the manufacturer's protocol, and the amount of RNA in the samples was quantified by Qubit fluorescence assay. A series of qPCR analyses were performed on RNA extracted from the samples to detect genes known to be enriched in breast cancer (see Figure 9 , which shows the qPCR Ct values ​​for each known tumor-enriched gene in the sample. This analysis showed that, as expected, in tumor samples, levels of genes detected by qPCR were more characteristic of cancerous tissue than healthy tissue, demonstrating that the mask effectively isolates tissue regions for selective lysis, while the hydrophobic coating process does not interfere with downstream amplification of nucleic acids released from defined regions of interest.

[0124] The RNA samples isolated by the masking process described above were also compared with the RNA samples isolated by the masking process described above. Figure 9 Conventional manual spatial separation was performed using the qPCR method (labeled "tube" in Figure B). qPCR Ct numbers were calculated relative to the housekeeping gene GAPDH and the data were plotted ( Figure 9 ). Gene expression levels by both methods were found to be highly correlated, indicating that the hydrophobic masking process is equivalent to the manual separation workflow.

[0125] Additionally, for exemplary masked tissue regions, the amount of nucleic acid released by application of lysis buffer to masked or unmasked areas was compared ( Figure 9 (C) This analysis shows that nucleic acids isolated from the covered area are significantly reduced, indicating that the hydrophobic mask effectively excludes unwanted tissue from lysis during the process.

[0126] In addition, a panel of 177 qPCR analyses was performed on RNA isolated from masked tumor samples, aiming to detect genes selectively expressed in breast cancer. 177 uniquely expressed qPCR products were detected in RNA released from cancerous samples by the masking method, indicating that a large portion of the expressed gene set was isolated from cancer cells isolated by the masking method.

[0127] Although preferred embodiments of the present invention have been shown and described herein, it will be readily understood by those skilled in the art that such embodiments are provided by way of example only. Those skilled in the art will now appreciate that many modifications, variations, and substitutions can occur without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the present invention. The following claims are intended to define the scope of the present invention and therefore cover methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for isolating cellular components from at least one region of interest in a planar tissue section on a solid substrate, comprising: (a) contacting the planar tissue section with a fluorocarbon; (b) applying a chemical mask comprising a non-fluorinated acrylate or cyanoacrylate and a photoinitiator to the planar tissue section by inkjet printing, wherein the chemical mask surrounds the at least one region of interest in the planar tissue section on a solid substrate by inkjet printing and isolates the region of interest from regions outside thereof by hydrophobicity; as well as (c) selectively lysing cells enclosed within the at least one region of interest by dispensing a solution comprising one or more extractants onto the region of interest to solubilize cellular components within the at least one region of interest, The inkjet printing is selected from the group consisting of piezoelectric inkjet printing, drop-on-demand inkjet printing and combinations thereof.

2. The method of claim 1 , wherein dispensing the solution onto the area of ​​interest comprises: (a) dispensing the solution comprising one or more extractants onto a reagent transfer device comprising at least one reagent transfer array element disposed on a surface of a second solid substrate; (b) contacting at least one reagent transfer array element located on the surface of the second solid substrate with the planar tissue slice such that the at least one reagent transfer array element spatially corresponds to the at least one region of interest in the planar tissue slice, wherein the contacting causes the solution comprising the one or more extractants to be transferred to the at least one region of interest in the planar tissue slice, thereby solubilizing cellular components in the at least one region of interest in the solution comprising the one or more extractants.

3. The method according to claim 1, further comprising: At least one lysed cellular component is isolated from the at least one region of interest in the planar tissue section on the solid substrate.

4. The method according to any one of claims 1 to 3, further comprising: Nucleic acids in the lysed cellular fraction from the at least one region of interest are sequenced.

5. The method according to any one of claims 1 to 3, further comprising: Tandem mass spectrometry analysis is performed on proteins in the cellular fraction solubilized from the at least one region of interest. 6 . The method according to any one of claims 1 to 3 , wherein the solution in (b) further comprises a soluble tag, wherein the soluble tag corresponds to the at least one region of interest. The method of claim 6 , wherein the soluble tag is an oligonucleotide.

8. The method of claim 7, wherein the oligonucleotide comprises a sample index sequence corresponding to the region of interest and optionally a specific molecular tag sequence.

9. The method of claim 7, wherein the oligonucleotide comprises a charge tag corresponding to the region of interest.

10. The method of claim 7 or 8, wherein the oligonucleotide is double-stranded.

11. The method of any one of claims 7-9, wherein the oligonucleotide is conjugated to beads.

12. The method of claim 1 , comprising dispensing a solution comprising one or more extractants onto the area of ​​interest to form droplets containing released cellular components, wherein the droplets are isolated from fluid communication with tissue outside the area of ​​interest in the absence of a physical barrier.

13. The method of any one of claims 1-3, 7-9, and 12, wherein the one or more extractants comprise one or more surfactants, proteases, tonicity adjusters, chaotropic agents, nucleases, buffers, protease inhibitors, phosphatase inhibitors, or nuclease inhibitors.

14. The method of any one of claims 1-3, 7-9, and 12, wherein the chemical mask has a contact angle of 60-155 degrees.

15. The method of any one of claims 1-3, 7-9, and 12, further comprising applying a chemical mask comprising a fluoropolymer or a fluoroacrylic polymer.

16. The method of claim 1, wherein the chemical mask is cyanoacrylate. The method of claim 16 , wherein the chemical mask further comprises a solvent.

18. The method of claim 17, wherein the solvent is a propylene glycol derivative, a fluorocarbon, or an alcohol.

19. The method of claim 17, wherein the solvent is perfluorooctane, perfluoro-2-methylpentane, perfluoro-1,3-dimethylcyclohexane, perfluorodecalin, or 1,3-difluoropropane.

20. The method of any one of claims 1-3, 7-9, 12, and 16-19, wherein the at least one region of interest is circular and the diameter of the region of interest is 1 mm or less, 500 microns or less, 250 microns or less, 125 microns or less, 100 microns or less, 80 microns or less, 50 microns or less, 25 microns or less, or 15 microns or less.

21. The method of any one of claims 1-3, 7-9, and 12, wherein the area of ​​the at least one region of interest is less than about 7.8×10 5 square micrometers.

22. The method of any one of claims 2-3, 7-9, 12, and 16-19, wherein the at least one reagent transfer array element located on the surface of the second solid substrate comprises at least one hydrophilic region defined by a hydrophobic region.

23. The method of claim 22, wherein the at least one reagent transfer array element located on the surface of the second solid substrate is capable of delivering a volume of solution of 10,000 picoliters or less, 1,000 picoliters or less, 500 picoliters or less, 250 picoliters or less, 100 picoliters or less, 50 picoliters or less, 10 picoliters or less, or 2 picoliters or less.

24. The method of claim 22, wherein the hydrophobic region surrounding the at least one hydrophilic region has a contact angle of 60-155 degrees.

25. The method of any one of claims 1-3, 7-9, 12, 16-19, and 23-24, comprising simultaneously lysing cellular components from more than one region of interest in the planar tissue section.

26. The method of any one of claims 1-3, 7-9, 12, 16-19, and 23-24, comprising lysing cellular components from at least 10, at least 100, at least 1000, or at least 10,000 regions of interest in the planar tissue section.

27. The method of any one of claims 1-3, 7-9, 12, 16-19, and 23-24, wherein the chemical mask comprises a grid pattern.

28. The method of any one of claims 1-3, 7-9, 12, 16-19, and 23-24, wherein the chemical mask comprises a circular shape.

29. The method of any one of claims 1-3, 7-9, 12, 16-19, and 23-24, wherein the chemical mask is applied to the planar tissue section using a piezoelectric inkjet delivery device.

30. The method of any one of claims 1-3, 7-9, 12, 16-19, and 23-24, wherein the planar tissue section has a thickness of about 2 to about 50 μιη.

31. The method of any one of claims 1-3, 7-9, 12, 16-19, and 23-24, wherein the planar tissue section has a thickness of about 1 to about 15 μιη.

32. The method of any one of claims 1-3, 7-9, 12, 16-19, and 23-24, wherein the planar tissue section is a formalin-fixed, paraffin-embedded (FFPE) tissue section.

33. The method of any one of claims 1-3, 7-9, 12, 16-19, and 23-24, wherein the planar tissue section is a non-fixed tissue section.

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