Method, composition, and device for isolation and expression analysis of region of interest from tissue

Abstract

To provide a method for isolating cellular components from at least one region of interest in a planar tissue section.SOLUTION: A method is in-situ, and does not require prearrangement of immobilized nucleotide arrays for defining regions from a tissue. The method may include: preparing in situ a hydrophobic mask on a tissue section to isolate regions of interest, and subsequently, using a surface tension array as necessary to add and / or remove extraction and / or analysis reagents from particular regions of interest circumscribed by the hydrophobic mask.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority based on U.S. Provisional Application No. 62 / 637,998, filed on Mar. 2, 2018, with the title "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 Jun. 28, 2018, with the title "METHODS, COMPOSITIONS, AND DEVICES FOR ISOLATION AND EXPRESSION ANALYSIS OF REGIONS OF INTEREST FROM A TISSUE SECTION", the entireties of which are incorporated herein by reference.

Background Art

[0002] Background of the Invention Conventional clinical analysis of gene and protein expression from tissue samples requires extraction of nucleic acids and proteins from sections (e.g., tissue biopsies). Since there is no selection mechanism, the analysis based on these sections represents a composite of the expression profiles and DNA / RNA sequence data of various cell types (e.g., epithelial, connective, and immune) within the tissue.

Summary of the Invention

Means for Solving the Problems

[0003] Gist of the Invention In some aspects, the present disclosure provides a method for isolating biomolecules from at least one target region in a flat tissue section on a solid substrate for sequencing or analysis, the method comprising: (a) delineating a boundary with a polymer from a defined target region in the flat tissue section on the solid substrate to form a non-physical fluid barrier, wherein the target region is less than 1 mm in diameter; and (b) selectively lysing cells within the at least one defined target region in situ. In some aspects, the present disclosure provides a method for isolating biomolecules from at least one target region in a flat tissue section on a solid substrate for sequencing or analysis, the method comprising applying a chemical mask that delineates the boundary of the at least one target region in the flat tissue section on the solid substrate, wherein the chemical mask forms a barrier suitable for selective lysis of cells within the at least one target region and the chemical mask is not a physical fluid boundary. In some aspects, the present disclosure provides a method for isolating biomolecules from at least one target region in a flat tissue section on a solid substrate for sequencing or analysis, the method comprising dispensing a solvent suitable for lysing cells within the target region in the flat tissue section on the solid substrate to form droplets containing released cellular components, wherein the droplets are isolated from fluid communication with tissue outside the target region without a physical barrier. In some aspects, the present disclosure provides a method for isolating and detecting biomolecules from a plurality of target regions in a tissue section, the method comprising applying a hydrophobic mask to the tissue section such that at least 1000 target regions in the flat tissue section are isolated from fluid communication with each other without a physical barrier, and detecting in situ a plurality of proteins or nucleic acids released from cells within the at least 1000 target regions while retaining spatial position information of the proteins or nucleic acids. In some embodiments, the method can isolate target regions that are less than about 100 microns in diameter or equal to about 100 microns in diameter. In some embodiments, the tissue section is an FFPE tissue section.In some embodiments, the method includes detecting a plurality of proteins or nucleic acids released in situ from the cells, and the detecting step includes sequencing the nucleic acids released in situ from the cells. In some embodiments, the sequencing includes tagging 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 linked to a surface. In some embodiments, the tagging includes tagging RNA released in situ and includes (a) providing an oligonucleotide containing a unique nucleic acid sequence to the at least 1000 regions of interest; and (b) performing first-strand and second-strand cDNA synthesis on the at least 1000 regions of interest, wherein the regions of interest are isolated from each other. In some embodiments of the method, cross-contamination of the unique nucleic acid sequences among the cDNA molecules synthesized from the regions of interest is reduced. In some embodiments, the first-strand cDNA synthesis is performed in situ. In some embodiments, the first-strand and second-strand cDNA synthesis are performed in situ. In some embodiments, sequencing the nucleic acids released in situ from the cells includes sequencing both mRNA and genomic DNA. In some embodiments, sequencing the nucleic acids released in situ from the cells includes sequencing mRNA. In some embodiments, sequencing the mRNA includes a pre-amplification step performed on the cDNA generated from the mRNA. In some embodiments, the pre-amplification step is LM-PCR, PCR with random hexamer primers, PCR with poly-A specific primers, or any combination thereof. In some embodiments, sequencing the nucleic acids released in situ from the cells includes sequencing genomic DNA. In some embodiments, sequencing the genomic DNA includes a whole genome amplification (WGA) step performed on the genomic DNA.In some embodiments, the WGA process is degenerate oligonucleotide primer (DOP) PCR, multiple displacement amplification (MDA), multiple annealing and loop-based amplification cycles (MALBAC), PicoPlex, isothermal amplification with self-priming primers, or combinations thereof.

[0004] In some aspects, the present disclosure provides a tissue section comprising at least 1000 aqueous droplets that are isolated from fluid communication with each other without a physical barrier in the section. In some embodiments, the tissue section comprises a hydrophobic mask that isolates the 1000 aqueous droplets from fluid communication with each other. In some embodiments, the at least 1000 aqueous droplets comprise at least 1000 unique oligonucleotides. In some embodiments, the hydrophobic mask comprises at least a layer of cyanoacrylate polymer. In some embodiments, the hydrophobic mask comprises at least a non-silylated fluoroalkyl layer. In some embodiments, the non-silylated fluoroalkyl layer comprises a fluoroacrylate or a non-silylated perfluoroalkane compound. In some embodiments, the tissue section comprises at least a perfluoroalkylsilane layer. In some embodiments, the perfluoroalkylsilane layer comprises perfluoroalkyltrichlorosilane or perfluoroalkyltris(dimethylamino)silane. In some embodiments, the perfluoroalkyltrichlorosilane is FOTS (tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane). In some embodiments, the perfluoroalkyltris(dimethylamino)silane is PF10TAS (perfluorodecyltris(dimethylamino)silane). In some embodiments the oligonucleotide is not linked to the surface.

[0005] In some aspects, the present disclosure provides a method for isolating cellular components from at least one target region in a flat tissue section on a solid substrate, the method comprising: (a) applying a chemical mask to the flat tissue section that delineates the boundary of the at least one target region in the flat tissue section on the solid substrate; and (b) dispensing a solution containing one or more extraction agents onto the target region, thereby lysing the cellular components within the at least one target region by selectively lysing the cells delineated by the boundary within the at least one target region. In some embodiments, the step of dispensing the solution onto the target region comprises: (a) dispensing the solution containing one or more extraction agents onto a drug delivery device comprising at least one drug delivery array element positioned on the surface of a second solid substrate; (b) contacting the at least one drug delivery array element positioned on the surface of the second solid substrate with the flat tissue section, wherein the at least one drug delivery array element spatially corresponds to the at least one target region in the flat tissue section, and wherein contacting enables the solution containing one or more extraction agents to be transferred to the at least one target region in the flat tissue section, thereby lysing the cellular components within the at least one target region in the solution containing one or more extraction agents. In some embodiments, the method further comprises isolating at least one lysed cellular component from the at least one target region in the flat tissue section on the solid substrate. In some embodiments, the method further comprises sequencing nucleic acids among the cellular components lysed from the at least one target region. In some embodiments, the method further comprises performing tandem mass spectrometry on proteins among the cellular components lysed from the at least one target region. In some embodiments, the solution in (b) further comprises a soluble tag, wherein the soluble tag corresponds to the at least one target region.In some embodiments, the soluble tag is an oligonucleotide. In some embodiments, the oligonucleotide includes a sample index sequence corresponding to the region of interest and, optionally, a unique molecular identifier sequence. In some embodiments, the oligonucleotide includes 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 tag. In some embodiments, the one or more extraction agents include one or more surfactants, proteases, tonicity regulators, chaotropes, nucleases, buffers, protease inhibitors, phosphatase inhibitors, or nuclease inhibitors. In some embodiments, the chemical mask has a contact angle of 60 to 155 degrees. In some embodiments, the chemical mask is applied via a hydrophobic mask solution containing a fluoroalkane or a fluoroacrylic polymer. In some embodiments, the chemical mask solution is applied via a solution containing acrylate or cyanoacrylate. In some embodiments, the chemical mask solution further includes a solvent. In some embodiments, the solvent is a pyropylene 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 at least one region of interest has an area of approximately 7.8×10. 5It is less than one square micron. In some embodiments, the at least one drug transfer array element positioned on the surface of the second solid substrate includes at least one hydrophilic region delineated by a hydrophobic region. In some embodiments, the at least one drug transfer array element positioned on the surface of the second solid substrate can deliver 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 delineating the boundary of the at least one hydrophilic region has a contact angle of 60 to 155 degrees. In some embodiments, the method includes lysing cellular components from more than one target region in the flat tissue section. In some embodiments, the method includes lysing cellular components from at least 10, at least 100, at least 1,000, or at least 10,000 target regions in the flat tissue section. In some embodiments, the hydrophobic mask delineating the boundary of at least one target region in the flat tissue section includes a grid pattern. In some embodiments, the hydrophobic mask delineating the boundary of at least one target region in the flat tissue section includes an ellipse. In some embodiments, the hydrophobic mask delineating the boundary of at least one target region in the flat tissue section on the solid substrate is applied to the flat tissue section using a piezoelectric inkjet delivery device. In some embodiments, the flat tissue section is about 2 to about 50 μm thick. In some embodiments, the flat tissue section is about 1 to about 15 μm thick. In some embodiments, the flat tissue section is a formalin-fixed paraffin-embedded (FFPE) tissue section. The flat tissue section is an unfixed tissue section.

[0006] In some aspects, the present disclosure is a system for isolating cellular components from at least one target region in a flat tissue section on a first solid support, the system comprising: (a) a flat tissue section on a first solid support comprising at least one target region outlined by the hydrophobic mask; (b) a second solid support comprising at least one drug transfer array element positioned on the surface of the solid substrate for alignment with at least one target region in the flat tissue section, the at least one drug transfer array element comprising a solution containing one or more extraction drugs; and (c) an electrodynamic stage coupled to the solid support comprising the at least one drug transfer array element, capable of moving the solid support, and allowing the at least one drug transfer array element positioned on the surface of the solid substrate to contact the at least one target region in the flat tissue section outlined by the hydrophobic mask. In some aspects, the system further comprises: (i) a computer system configured to control the position of the electrodynamic stage, such that the at least one drug transfer array element positioned on the surface of the second solid substrate contacts at least one target region in the tissue section. In some embodiments, the hydrophobic mask solution is applied by: (i) a piezoelectric inkjet delivery device capable of delivering the hydrophobic mask solution to the tissue section on the solid support, and (ii) a computer system configured to control the piezoelectric inkjet delivery device to outline the boundary of at least one target region in the flat tissue section with the hydrophobic mask solution to generate the hydrophobic mask. In some embodiments, the at least one drug transfer array element positioned on the surface of the second solid substrate comprises at least one hydrophilic region outlined by a hydrophobic region. In some embodiments, the hydrophobic mask solution comprises at least one of C7F15CH2OCOC(CH3)=CH2, FC-722, PerFluoroCoat, or FluoroPel. In some embodiments, the hydrophobic mask solution comprises at least one of acrylate or cyanoacrylate.In some embodiments, the at least one drug transfer array element positioned on the surface of the second solid substrate can deliver 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 contact angle of the hydrophobic region delineating the boundary of the at least one hydrophilic region is between 60 and 155 degrees. In some embodiments, the system for applying the hydrophobic mask solution includes a UV light source capable of polymerizing the hydrophobic mask solution after application to the flat tissue section. In some embodiments, the system for applying the hydrophobic mask solution includes an apparatus for chemical vapor deposition of perfluoroalkyltrichlorosilane or perfluoroalkylsilane.

[0007] In some aspects, the present disclosure provides a kit for isolating cellular components from at least one target region in a flat tissue section, the kit comprising any one of the elements described in any one of the aspects of any of the methods, systems, or tissue sections described herein.

[0008] In some aspects, the present disclosure provides a tissue section comprising, from bottom to top, at least one cyanoacrylate layer in contact with the tissue section, followed by at least one perfluoroalkylsilane layer. In some aspects, the perfluoroalkylsilane layer comprises perfluoroalkyltrichlorosilane or perfluoroalkyltris(dimethylamino)silane. In some embodiments, the perfluoroalkyltrichlorosilane is FOTS. In some embodiments, the tissue section comprises at least one non-silylated fluoroalkyl layer under the at least one cyanoacrylate layer. In some embodiments, the non-silylated fluoroalkyl layer comprises a fluoroacrylate or a non-silylated perfluoroalkane compound. In some embodiments, the perfluoroalkyltrichlorosilane is FOTS ((tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane). In some embodiments, the tissue section comprises at least one region lacking the cyanoacrylate layer, delineated by a boundary line by the region having the cyanoacrylate layer. In some embodiments, the region is substantially elliptical, polygonal, or free-form. In some embodiments, the application of an aqueous solution of a surfactant to the tissue section can lyse the cells within the region. In some embodiments, the region is less than about 1 mm in size. In some embodiments, the region is less than about 100 microns in size. In certain embodiments, for example, the following are provided: (Item 1) A method for isolating biomolecules from at least one target region in a flat tissue section on a solid substrate for sequencing or analysis, the method comprising: (a) forming a non-physical fluid barrier by polymerically delineating a defined target region from the flat tissue section on the solid substrate, wherein the target region is less than 1 mm in diameter; and (b) selectively lysing the cells within the at least one defined target region in situ. A method comprising the above. (Item 2) A method for isolating biomolecules from at least one target region in a flat tissue section on a solid substrate for sequencing or analysis, said method comprising: Applying a chemical mask to the flat tissue section that delineates the boundary of the at least one target region in the flat tissue section on the solid substrate; wherein the chemical mask forms a barrier suitable for selective destruction of cells within the at least one target region, and the chemical mask is not a physical fluid boundary. (Item 3) A method for isolating biomolecules from at least one target region in a flat tissue section on a solid substrate for sequencing or analysis, said method comprising: Dispensing a solvent suitable for destroying cells within the target region in the flat tissue section on the solid substrate to form droplets containing released cellular components; wherein the droplets are isolated from the tissue outside the target region without a physical barrier from fluid communication. (Item 4) A method for isolating and detecting biomolecules from a plurality of target regions in a tissue section, said method comprising: Applying a hydrophobic mask to the tissue section such that at least 1000 target regions in the flat tissue section are isolated from fluid communication with each other without a physical barrier, and Detecting a plurality of proteins or nucleic acids released in situ from cells within the at least 1000 target regions, wherein the spatial position information of the proteins or nucleic acids is retained. A method comprising the above steps. (Item 5) The method according to any one of Items 1 to 4, wherein the method can isolate a target region having a diameter smaller than about 100 microns or equal to about 100 microns. (Item 6) The method according to any one of Items 1 to 5, wherein the tissue section is an FFPE tissue section. (Item 7) A method according to any one of items 1 to 6, comprising the step of detecting a plurality of proteins or nucleic acids released in situ from cells within the region of interest, wherein the step of detecting comprises sequencing the nucleic acids released in situ from the cells. (Item 8) The method according to item 6, wherein the sequencing comprises tagging the nucleic acids released in situ with a barcode that identifies the location of the region of interest. (Item 9) The method according to item 8, wherein the barcode is not linked to the surface. (Item 10) The tagging comprises tagging RNA released in situ, (a) attaching an oligonucleotide containing a unique nucleic acid sequence to the at least 1000 regions of interest; and (b) performing first-strand and second-strand cDNA synthesis on the at least 1000 regions of interest, wherein the regions of interest are isolated from each other, The method according to item 8, comprising. (Item 11) The method according to item 10, wherein cross-contamination of the unique nucleic acid sequence between cDNA molecules synthesized from the regions of interest is reduced. (Item 12) The method according to item 10, wherein the first-strand cDNA synthesis is performed in situ. (Item 13) The method according to item 10, wherein the first-strand and second-strand cDNA syntheses are performed in situ. (Item 14) The method according to item 6, wherein sequencing the nucleic acids released in situ from the cells comprises sequencing both mRNA and genomic DNA. (Item 15) The method according to item 14, wherein sequencing the nucleic acids released in situ from the cells comprises sequencing mRNA. (Item 16) The method according to item 15, wherein sequencing the mRNA includes a pre-amplification step performed on cDNA generated from the mRNA. (Item 17) The method according to item 16, wherein the pre-amplification step is LM-PCR, PCR with random hexamer primers, PCR with polyA-specific primers, or any combination thereof. (Item 18) The method according to item 14, wherein sequencing the nucleic acid released in situ from the cell includes sequencing genomic DNA. (Item 19) The method according to item 18, wherein sequencing the genomic DNA includes a whole genome amplification (WGA) step performed on the genomic DNA. (Item 20) The method according to item 19, wherein the WGA step is degenerate oligonucleotide primer (DOP) PCR, multiple displacement amplification (MDA), multiple annealing and loop-based amplification cycles (MALBAC), PicoPlex, isothermal amplification with self-priming primers, or a combination thereof. (Item 21) A tissue section comprising at least 1000 aqueous droplets that are isolated from fluid communication with each other without a physical barrier in the section. (Item 22) The tissue section according to item 21, comprising a hydrophobic mask that isolates the 1000 aqueous droplets from fluid communication with each other. (Item 23) The tissue section according to item 21 or 22, wherein the at least 1000 aqueous droplets contain at least 1000 unique oligonucleotides. (Item 24) The tissue section according to any one of items 21 to 23, wherein the hydrophobic mask includes at least a layer of cyanoacrylate polymer. (Item 25) The tissue section according to any one of items 21 to 24, wherein the hydrophobic mask includes at least a non-silylated fluoroalkyl layer. (Item 26) The non-silylated fluoroalkyl layer-containing tissue section according to item 24, wherein the non-silylated fluoroalkyl layer contains a fluoroacrylate or a non-silylated perfluoroalkane compound. (Item 27) The tissue section according to any one of items 21 to 26, including at least a perfluoroalkylsilane layer. (Item 28) The tissue section according to item 27, wherein the perfluoroalkylsilane layer contains perfluoroalkyltrichlorosilane or perfluoroalkyltris(dimethylamino)silane. (Item 29) The tissue section according to any one of items 23 to 28, wherein the oligonucleotide is not linked to the surface. (Item 30) A method for isolating cell components from at least one target region in a flat tissue section on a solid substrate, the method comprising: (a) Applying a chemical mask that outlines the boundary of the at least one target region in the flat tissue section on the solid substrate to the flat tissue section; (b) Dispensing a solution containing one or more extraction agents onto the target region, thereby lysing the cell components within the at least one target region by selectively lysing the cells outlined by the boundary within the at least one target region. A method comprising the above. (Item 31) The step of dispensing the solution onto the target region comprises: (a) Dispensing the solution containing one or more extraction agents onto a drug transfer device including at least one drug transfer array element located on the surface of a second solid substrate; (b) Position the at least one drug transfer array element located on the surface of the second solid substrate in contact with the flat tissue section, wherein the at least one drug transfer array element spatially corresponds to the at least one target region in the flat tissue section, and wherein contacting enables a solution containing the one or more extracted drugs to be moved to the at least one target region in the flat tissue section, thereby dissolving cell components in the at least one target region in the solution containing the one or more extracted drugs. The method according to item 30, including this. (Item 32) A step of isolating at least one dissolved cell component from the at least one target region in the flat tissue section on the solid substrate. The method according to item 30, further including this. (Item 33) A step of sequencing nucleic acids among the cell components dissolved from the at least one target region. The method according to any one of items 30 to 32, further including this. (Item 34) A step of performing tandem mass spectrometry on proteins among the cell components dissolved from the at least one target region. The method according to any one of items 30 to 33, further including this. (Item 35) The solution in (b) further includes a soluble tag, wherein the soluble tag corresponds to the at least one target region. The method according to any one of items 30 to 34. (Item 36) The method according to item 35, wherein the soluble tag is an oligonucleotide. (Item 37) The method according to item 36, wherein the oligonucleotide includes a sample index sequence corresponding to the target region and, optionally, a unique molecular identifier sequence. (Item 38) The method according to item 36, wherein the oligonucleotide contains a charge tag corresponding to the target region. (Item 39) The method according to item 36 or 37, wherein the oligonucleotide is double-stranded. (Item 40) The method according to any one of items 36 to 38, wherein the oligonucleotide is conjugated to beads. (Item 41) The method according to item 35, wherein the soluble tag is a tandem mass tag. (Item 42) The method according to any one of items 30 to 41, wherein the one or more extraction agents include one or more surfactants, proteases, tonicity regulators, chaotropes, nucleases, buffers, protease inhibitors, phosphatase inhibitors, or nuclease inhibitors. (Item 43) The method according to any one of items 30 to 42, wherein the chemical mask has a contact angle of 60 to 155 degrees. (Item 44) The method according to any one of items 30 to 43, wherein the chemical mask is applied via a hydrophobic mask solution containing a fluoropolymer or a fluoroacrylate polymer. (Item 45) The method according to item 44, wherein the chemical mask solution is applied via a solution containing acrylate or cyanoacrylate. (Item 46) The method according to item 45, wherein the chemical mask solution further contains a solvent. (Item 47) The method according to item 46, wherein the solvent is a pyropylene glycol derivative, a fluorocarbon, or an alcohol. (Item 48) The method according to item 46, wherein the solvent is perfluorooctane, perfluoro-2-methylpentane, perfluoro-1,3-dimethylcyclohexane, perfluorodecalin, or 1,3-difluoropropane. (Item 49) The at least one target region is circular, and the diameter of the target region 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, according to any one of items 30 to 48. (Item 50) The at least one target region has an area of less than about 7.8×10 5 square microns, according to any one of items 30 to 44. (Item 51) The at least one drug transfer array element located on the surface of the second solid substrate includes at least one hydrophilic region delineated by a hydrophobic region, according to any one of items 31 to 50. (Item 52) The at least one drug transfer array element located on the surface of the second solid substrate can deliver 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, according to item 51. (Item 53) The hydrophobic region delineating the boundary of the at least one hydrophilic region has a contact angle of 60 to 155 degrees, according to item 51. (Item 54) The method according to any one of items 30 to 53, which includes lysing cell components from more than one target region in the flat tissue section. (Item 55) The method according to any one of items 30 to 53, comprising lysing cellular components from at least 10, at least 100, at least 1000, or at least 10,000 target regions in the flat tissue section. (Item 56) The method according to any one of items 30 to 55, wherein the hydrophobic mask delineating the boundary of at least one target region in the flat tissue section comprises a grid pattern. (Item 57) The method according to any one of items 30 to 55, wherein the hydrophobic mask delineating the boundary of at least one target region in the flat tissue section comprises a circle. (Item 58) The method according to any one of items 30 to 57, wherein the hydrophobic mask delineating the boundary of at least one target region in the flat tissue section on the solid substrate is applied to the flat tissue section using a piezoelectric inkjet delivery device. (Item 59) The method according to any one of items 30 to 58, wherein the flat tissue section is about 2 to about 50 μm thick. (Item 60) The method according to any one of items 30 to 58, wherein the flat tissue section is about 1 to about 15 μm thick. (Item 61) The method according to any one of items 30 to 58, wherein the flat tissue section is a formalin-fixed paraffin-embedded (FFPE) tissue section. (Item 62) The method according to any one of items 30 to 58, wherein the flat tissue section is an unfixed tissue section. (Item 63) A system for isolating cellular components from at least one target region in a flat tissue section on a first solid support, the system comprising: (a) A flat tissue section on a first solid support comprising at least one target region delineated by the hydrophobic mask; (b) A second solid support including at least one agent transfer array element positioned on the surface of the solid substrate for alignment with at least one target region in the flat tissue section, wherein the at least one agent transfer array element comprises a solution containing one or more extraction agents; and (c) An electromechanical stage coupled to the solid support including the at least one agent transfer array element, capable of moving the solid support, and allowing the at least one agent transfer array element positioned on the surface of the solid substrate to contact the at least one target region in the flat tissue section delineated by a hydrophobic mask. A system comprising. (Item 64) (i) A computer system configured to control the position of the electromechanical stage, wherein the at least one agent transfer array element positioned on the surface of the second solid substrate contacts at least one target region in the tissue section. The system according to item 63, further comprising. (Item 65) The hydrophobic mask solution is (i) A piezoelectric inkjet delivery device capable of delivering the hydrophobic mask solution to the tissue section on the solid support, and (ii) Applied by a computer system configured to control the piezoelectric inkjet delivery device to delineate the boundary of at least one target region in the flat tissue section with the hydrophobic mask solution to generate a hydrophobic mask. The system according to item 63 or 64. (Item 66) The system according to items 63 - 65, wherein the at least one agent transfer array element positioned on the surface of the second solid substrate includes at least one hydrophilic region delineated by a hydrophobic region. (Item 67) The hydrophobic mask solution-containing system according to any one of items 58 to 66, wherein the hydrophobic mask solution contains at least one of C7F15CH2OCOC(CH3)=CH2, FC-722, PerFluoroCoat, or FluoroPel. (Item 68) The system according to any one of items 58 to 67, wherein the hydrophobic mask solution contains at least one of acrylate or cyanoacrylate. (Item 69) The system according to any one of items 58 to 68, wherein the at least one drug transfer array element located on the surface of the second solid substrate can deliver 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. (Item 70) The system according to any one of items 58 to 68, wherein the contact angle of the hydrophobic region delineating the boundary line of the at least one hydrophilic region is 60 to 155 degrees. (Item 71) The system for applying a hydrophobic mask solution according to any one of items 65 to 70, wherein the system includes a UV light source capable of polymerizing the hydrophobic mask solution after application to the flat tissue section. (Item 72) The system for applying a hydrophobic mask solution according to any one of items 65 to 71, wherein the system includes a device for chemical vapor deposition of perfluoroalkylsilane. (Item 73) A kit for isolating cell components from at least one target region in a flat tissue section, the kit including any one of the elements according to any one of items 63 to 72. (Item 74) A tissue section comprising, from bottom to top, at least one cyanoacrylate layer in contact with the tissue section, followed by at least one perfluoroalkylsilane layer. (Item 75) The tissue section according to item 74, comprising at least one non-silylated fluoroalkyl layer under the at least one cyanoacrylate layer. (Item 76) The tissue section according to item 75, wherein the non-silylated fluoroalkyl layer comprises a fluoroacrylate or a non-silylated perfluoroalkane compound. (Item 77) The tissue section according to any one of items 74 to 76, wherein the perfluoroalkylsilane layer comprises perfluoroalkyltrichlorosilane or perfluoroalkyltris(dimethylamino)silane. (Item 78) The tissue section according to any one of items 74 to 77, comprising at least one region lacking the cyanoacrylate layer, delineated by a boundary line formed by the region having the cyanoacrylate layer. (Item 79) The tissue section according to item 78, wherein the region is substantially elliptical, polygonal, or in a free form. (Item 80) The tissue section according to any one of items 78 to 79, wherein the application of an aqueous solution of a surfactant to the tissue section can lyse the cells within the region. (Item 81) The tissue section according to any one of items 78 to 80, wherein the region has a size of less than about 1 mm. (Item 82) The tissue section according to item 81, wherein the region has a size of less than about 100 microns. (Item 83) The tissue section according to any one of items 74 to 82, wherein the tissue section is a formalin-fixed paraffin-embedded (FFPE) tissue section. (Item 84) The tissue section according to item 83, wherein the tissue section is a deparaffinized FFPE tissue section.

[0009] Citation indication All publications, patents, and patent applications mentioned in this specification are hereby 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

[0010] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained from the following detailed description which illustrates exemplary embodiments in which the principles of the invention are utilized and from the appended drawings.

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DETAILED DESCRIPTION OF THE INVENTION

[0021] DETAILED DESCRIPTION OF THE INVENTION Clinical analyses based on the detection of rare and / or diverse cell types within a tissue are difficult. For example, pre-cancerous tissue may contain only a small percentage of abnormal cells, and the surrounding normal cells may cause signal dilution, sometimes interfering with the early-stage detection of cancer. In contrast, late-stage tumors may have a high population of abnormal cells, but can contain a large diversity of cells with different genotypes that respond differently to chemotherapy strategies. In these cases, the ability to selectively isolate and analyze morphologically abnormal cells is a boon for improved diagnosis and understanding of disease pathophysiology.

[0022] However, existing methods for the isolation and analysis of small populations of selected cells within a tissue sample are cumbersome, expensive, and not suitable for true high-throughput multiplex analysis of tissues (e.g., laser capture microdissection, which requires a large amount of manual operation and expensive equipment).

[0023] Therefore, there is a need for simple, inexpensive, high-throughput methods for the multiplex isolation and analysis of regions within a tissue sample.

[0024] Definitions

[0025] The term "tissue" refers to an aggregate of cells and, optionally, intracellular substances (e.g., ECM). Typically, the cells in a tissue are not free-floating but are attached to each other to form a multicellular structure. Tissue types include, but are not limited to, muscle, nerve, epidermis, and connective tissue. The tissue as described herein can be derived from various organisms, including mammals such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cows, cats, dogs, primates (i.e., humans or non-human primates); plants such as Arabidopsis thaliana, corn, sorghum, oats, wheat, rice, canola, or soybean; algae such as Chlamydomonas reinhardtii; nematodes such as Caenorhabditis elegans; insects such as Drosophila melanogaster, mosquitoes, flies, bees, or spiders; fish such as zebrafish; reptiles; or amphibians such as frogs or Xenopus laevis, but are not limited thereto.

[0026] The term "contact angle" refers to the shape of a liquid droplet resting on a solid surface. Assuming that a liquid is present on a solid plane in air, the term "contact angle" indicates the angle between the tangent of the liquid and the tangent of the solid plane at the point of contact of the liquid, the solid plane, and air.

[0027] The term "hydrophobic" refers to a surface or coating that is difficult to wet with water. A surface is considered hydrophobic if it exhibits a receding water contact angle of at least 60°, very hydrophobic if it exhibits a contact angle of at least 110°, and extremely hydrophobic if it exhibits a receding water contact angle of at least 120°.

[0028] The term "superhydrophobic" refers to a surface or coating that is extremely difficult to wet with water. Superhydrophobic surfaces or coatings typically have contact angles greater than 140° and often greater than 150°.

[0029] The term "hydrophilic" is used to refer to a surface that can be wetted by an aqueous fluid. A surface is wetted by a fluid if the fluid has a tendency to spread spontaneously over the surface, as opposed to forming discrete droplets (hydrophilic). A surface is considered hydrophilic if it exhibits a contact angle of less than about 50 degrees.

[0030] "Inkjet delivery" is a non-contact approach that enables the handling of liquid droplets in the range of 1 to 100 picoliters (pl) onto two-dimensional and three-dimensional structures. This approach requires dissolving or dispersing the substance of interest in a liquid to form the ink. The most prevalent method used for inkjet delivery is the drop-on-demand (DOD) type method, where the drops are ejected from nozzles on the micrometer scale and are created either by (a) heating the liquid to a temperature above its boiling point (thermal DOD) to generate vapor bubbles that stimulate the release of the droplets from the nozzles, or (b) applying a voltage to a piezoelectric transducer that results in the vibration of the material and the release of the droplets from the nozzles (piezoelectric DOD).

[0031] Overview

[0032] Methods are provided herein for isolating cellular components from at least one region of interest in a flat tissue section on a solid substrate. The methods are in situ and do not require pre-preparation of an immobilized nucleotide array to define the region from the tissue. Rather, the methods in situ prepare a hydrophobic mask on the tissue section to isolate the region of interest, and subsequently optionally use a surface tension array to add and / or remove extraction and / or analysis reagents to a particular region of interest delineated by the hydrophobic mask. The use of a surface tension array is particularly useful, for example, when there are a large number of regions (e.g., more than 10, more than 100, more than 1000) isolated by the hydrophobic mask. In some embodiments, the methods include: (a) applying a hydrophobic mask to a flat tissue section to delineate the boundaries of at least one region of interest in the flat tissue section on the solid substrate; (b) dispensing a solution containing one or more extraction reagents to a reagent transfer device including at least one reagent transfer array element positioned on the surface of a second solid substrate; and (c) contacting at least one reagent transfer array element positioned on the surface of the second solid substrate with the flat tissue section, such that the at least one reagent transfer array element spatially corresponds to at least one region of interest in the flat tissue section. In some embodiments, the contacting step enables the solution containing the one or more extraction reagents to be transferred to at least one region of interest in the flat tissue section, thereby dissolving the cellular components in the at least one region of interest in the solution containing the one or more extraction reagents.

[0033] The solution containing the one or more extraction reagents can include one or more tags or labels suitable for labeling nucleic acids or proteins. Such tags ma Alternatively, the label may include a barcode or other spatially addressable entity, such that nucleic acid sequencing data or mass spectrometry data obtained from the extracted nucleic acid or protein can then be associated with the region of interest from which the nucleic acid or protein can be isolated. Thus, the methods herein also provide a scheme for spatially tagging nucleic acids or proteins from tissue sections.

[0034] tissue section

[0035] The tissue sections utilized in the methods described herein can be prepared by any standard method commonly used for immunohistochemistry preparations and can be fixed or unfixed, either being acceptable (e.g., they may be freshly excised or prepared by non-fixed tissue preparation methods such as freezing). Fixation of cells or tissues may require the use of a cross-linking agent (e.g., formaldehyde, glutaraldehyde, etc.).

[0036] Fixed tissues can further be embedded in paraffin wax or a hydrogel (e.g., a polyacrylamide support matrix). A prevalent method of tissue preparation is FFPE (formalin-fixed paraffin-embedded), which requires fixing tissue pieces in a formalin solution, dehydrating the tissue in gradually increasing concentrations of alcohol, clearing the tissue with xylene, infiltrating the tissue with wax (typically a mixture of paraffin, straight-chain or n-alkanes having a carbon chain length between 20 and 40 that can be obtained with various different melting points), and then embedding the tissue in a wax block. The FFPE tissue can then be prepared into tissue sections using a microtome.

[0037] In some embodiments, the thickness of the tissue section prepared by FFPE is from about 1 micron to about 50 microns. In some embodiments, the thickness of the tissue section prepared by FFPE is at least about 1 micron. In some embodiments, the thickness of the tissue section prepared by FFPE is at most about 50 microns. In some embodiments, the thickness of the tissue section 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 3 microns to about 40 microns, from about 3 microns to about 50 microns, from about 5 microns to about 10 microns, from about 5 microns to about 15 microns, from about 5 microns to about 20 microns, from about 5 microns to about 30 microns, from about 5 microns to about 40 microns, from about 5 microns to about 50 microns, from about 10 microns to about 15 microns, from about 10 microns to about 20 microns, from about 10 microns to about 30 microns, from about 10 microns to about 40 microns, from about 10 microns to about 50 microns, from about 15 microns to about 20 microns, from about 15 microns to about 30 microns, from about 15 microns to about 40 microns, from about 15 microns to about 50 microns, from about 20 microns to about 30 microns, from about 20 microns to about 40 microns, from about 20 microns to about 50 microns, from about 30 microns to about 40 microns, from about 30 microns to about 50 microns, or from about 40 microns to about 50 microns. In some embodiments, the thickness of the tissue section 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, or about 50 microns.

[0038] Embedded tissue sections can be deparaffinized prior to further processing (e.g., treatment with hydrophobic chemicals and sample extraction techniques further described herein). Deparaffinization can be performed, for example, by incubating the sections in a xylene bath, followed by incubation in a xylene / ethanol bath, followed by incubation in baths of gradually decreasing ethanol concentration (from 100% to 50%), followed by drying or rinsing in water.

[0039] In some embodiments, unfixed or fixed tissue can be prepared into sections by cryosectioning. For unfixed tissue, the tissue is first immersed in a frozen tissue matrix (e.g., OCT or Cryomatrix) and frozen in an isopentane and / or 2-methylbutane bath in contact with liquid nitrogen. It is sectioned on a cryostat. For fixed tissue, the tissue can be cryoprotected in sucrose prior to embedding / freezing in a tissue matrix, which includes incubation in baths of gradually increasing concentration of sucrose (up to 30%). The cryoprotected, fixed tissue can then be immersed in a frozen tissue matrix (e.g., OCT or Cryomatrix) and frozen in a) an isopentane bath in contact with liquid nitrogen, or b) an alternative, slower freezing method (e.g., powdered dry ice or dry ice / methanol / ethanol slurry). The cryoprotected, frozen, fixed tissue can then be sectioned on a cryostat.

[0040] In some embodiments, the thickness of the tissue section prepared by cryosectioning can be from about 5 microns to about 50 microns. In some embodiments, the thickness of the tissue section prepared by cryosectioning can be at least about 5 microns. In some embodiments, the thickness of the tissue section prepared by cryosectioning can be at most about 50 microns. In some embodiments, the thickness of the tissue section prepared by cryosectioning can be from about 5 microns to about 7 microns, from about 5 microns to about 9 microns, from about 5 microns to about 11 microns, from about 5 microns to about 13 microns, from about 5 microns to about 15 microns, from about 5 microns to about 20 microns, from about 5 microns to about 30 microns, from about 5 microns to about 40 microns, from about 5 microns to about 50 microns, from about 7 microns to about 9 microns, from about 7 microns to about 11 microns, from about 7 microns to about 13 microns, from about 7 microns to about 15 microns, from about 7 microns to about 20 microns, from about 7 microns to about 30 microns, from about 7 microns to about 40 microns, from about 7 microns to about 50 microns, from about 9 microns to about 11 microns, from about 9 microns to about 13 microns, from about 9 microns to about 15 microns, from about 9 microns to about 20 microns, from about 9 microns to about 30 microns, from about 9 microns to about 40 microns, from about 9 microns to about 50 microns, from about 11 microns to about 13 microns, from about 11 microns to about 15 microns, from about 11 microns to about 20 microns, from about 11 microns to about 30 microns, from about 11 microns to about 40 microns, from about 11 microns to about 50 microns, from about 13 microns to about 15 microns, from about 13 microns to about 20 microns, from about 13 microns to about 30 microns, from about 13 microns to about 40 microns, from about 13 microns to about 50 microns, from about 15 microns to about 20 microns, from about 15 microns to about 30 microns, from about 15 microns to about 40 microns, from about 15 microns to about 50 microns, from about 20 microns to about 30 microns, from about 20 microns to about 40 microns, from about 20 microns to about 50 microns, from about 30 microns to about 40 microns, from about 30 microns to about 50 microns, or from about 40 microns to about 50 microns.In some embodiments, the thickness of the tissue section 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.

[0041] Chemical mask

[0042] A. Composition / Application

[0043] In some embodiments, the methods described herein include the deposition of a chemical mask to surround and isolate the region of interest in a tissue section (e.g., a fresh frozen, fixed and frozen, and / or deparaffinized FFPE tissue section). The chemical mask allows for the selective aqueous extraction / rehydration of cellular components from the region of interest without contamination by components of the surrounding region (because regions of the sample other than the region of interest repel the solutions necessary to disrupt / extract / rehydrate the cells). Thus, the chemical mask solution is hydrophobic (e.g., having a receding water contact angle of at least 60 degrees).

[0044] 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.

[0045] Can be used to generate a surface receding water contact angle of at least 60 degrees and thus can be suitable for use in generating chemical masks as described herein, and a wide variety of surface coating polymers and copolymers are described.

[0046] An important class of such agents are fluorocarbons, particularly fluorinated carbon monomers containing at least one terminal trifluoromethyl group. In some embodiments, the fluorinated carbon monomer containing at least one terminal trifluoromethyl group contains from about 3 to about 20 carbon atoms. In some embodiments, such fluorocarbons are substantially unbranched fluoroalkyl or perfluoroalkyl ethylenically unsaturated monomers. In some embodiments, the ethylenically unsaturated monomer is an acrylate (e.g., methacrylate). In some embodiments, the ethylenically unsaturated monomer is a cyanoacrylate. In some embodiments, the fluorinated carbon monomer containing at least one terminal trifluoromethyl group is a fluorinated or perfluorinated acrylate, silicone, epoxy, urethane, or oxime, or any combination thereof. Such agents include fluoroacrylate (or a solution thereof) or perfluoroalkylsilane. Examples of fluoroacrylate or a solution thereof include, but are not limited to, the fluoroalkyl methacrylate monomer C7F15CH2OCOC(CH3)=CH2, FC-722 (available from 3M), PerFluoroCoat (Cytonix), and FluoroPel (Cytonix, FluoroPel 800 and 800M products), among others. The solution can be used full strength, but may be diluted with a solvent (e.g., a fluorosolvent, alcohol, ethanol) to form a low concentration coating polymer or fluoroalkane. The polymer solution used to make the coatings of the present invention preferably has a coating polymer content of from about 0.01 wt% to about 50 wt%. Perfluoroalkylsilanes include perfluoroalkyltrichlorosilane and perfluorotris(dimethylamino)silane (e.g., PF10TAS).Perfluoroalkyltrichlorosilane is an organic molecule containing a trichlorosilyl group bonded to perfluorinated alkyl groups of various lengths and isomers (e.g., C such as perfluoromethyl, perfluoroethyl, perfluoropropyl, perfluorobutyl, perfluorotert-butyl, perfluoropentyl, perfluorohexyl, perfluorooctyl, perfluorononyl, perfluorodecyl). 1 -C 20 perfluoroalkyl group). Examples of perfluoroalkyltrichlorosilane include FOTS (trichloro(1H,1H,2H,2H-perfluorooctyl)silane),

Chemical formula

[0047] In some embodiments, the fluorocarbon is applied to the tissue section as a polymer, either directly (either selectively to the regions desired to be excluded or non-specifically to the entire tissue region) to generate the mask (e.g., a fluorinated carbon monomer such as fluoroacrylate is added to the tissue section together with a photoinitiator). In some embodiments, the fluorocarbon is applied to the tissue section after first applying a non-fluorinated acrylate or cyanoacrylate to the tissue section together with the photoinitiator, e.g., 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 fluoroacrylate is first applied to the tissue section, followed by applying a non-fluorinated acrylate or cyanoacrylate to it together with a photoinitiator, and then applying the same fluorocarbon or a different fluorocarbon such as perfluoroalkylsilane to it).

[0048] In some embodiments, the fluorocarbon is applied as a polymer on top of a layer of hydrophobic material on the tissue section to generate the mask (a hydrophobic polymer such as cyanoacrylate is applied to the tissue section, e.g., by spraying or dipping, e.g., after an initial application). In some embodiments, the fluorocarbon is applied to the layer of hydrophobic material in the same or a different pattern as the layer of hydrophobic material, such that the fluorocarbon and the hydrophobic material form a copolymer mask that they fill.

[0049] In some embodiments, the layer of hydrophobic material is applied as a polymer to the chemical mask (e.g., after first applying the mask material as a polymer or copolymer to the tissue section). In some embodiments, the hydrophobic material is a fluorocarbon. In some embodiments, the hydrophobic material includes a perfluoroalkylsilane such as perfluoroalkyltrichlorosilane or perfluoroalkyltris(dimethylamino)silane ((tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane). In some embodiments, the hydrophobic material includes a fluorocarbon. In some embodiments, the hydrophobic material includes a perfluoroalkyltrichlorosilane such as trichloro(1H,1H,2H,2H-perfluorooctyl)silane. In some embodiments, the hydrophobic material includes a perfluoroalkyltris(dimethylamino)silane such as PF10TAS.

[0050] Other classes of agents that can be applied to generate the chemical mask include acrylates and cyanoacrylates that are photocurable with or without a photocatalyst (curable with UV).

[0051] The monomer containing the chemical mask can be applied to the tissue section by various approaches (not limited to, for example, inkjet printing (e.g., DOD or piezoelectric inkjet printing methods) using a monomer suspended in a suitable fluorocarbon having a boiling point higher than room temperature such as a standard solvent (e.g., an alcohol such as ethanol, propylene glycol, propylene glycol monomethyl ether acetate, methyl ethyl ketone, or perfluorooctane, perfluoro-2-methylpentane, perfluoro-1,3-dimethylcyclohexane, perfluorodecalin, 1,3-difluoropropane, etc.)).

[0052] The chemical mask may include one or more monomer or polymer layers. The one or more monomer or polymer layers may include the same or different monomers or polymers. The one or more monomer or polymer layers may form a copolymer. The one or more monomer or polymer layers may be one, two, three, four, five, six, seven, eight, nine, or ten polymer layers, or more. In some embodiments, the chemical mask includes a layer of cyanoacrylate, followed by a layer of perfluoroalkylsilane (e.g., perfluoroalkyltrichlorosilane or perfluoroalkyltris(dimethylamino)silane). In some embodiments, the chemical mask includes a layer of cyanoacrylate, followed by multiple layers of perfluoroalkylsilane (e.g., perfluoroalkyltrichlorosilane or perfluoroalkyltris(dimethylamino)silane). In some embodiments, the chemical mask includes a layer of fluoroacrylate or non-silylated fluoroalkane, followed by a layer of cyanoacrylate, followed by a layer of perfluoroalkylsilane (e.g., perfluoroalkyltrichlorosilane or perfluoroalkyltris(dimethylamino)silane). In some embodiments, the chemical mask includes fluoroacrylate or non-silylated fluoroalkane, followed by cyanoacrylate, followed by multiple layers of perfluoroalkylsilane (e.g., perfluoroalkyltrichlorosilane or perfluoroalkyltris(dimethylamino)silane).

[0053] B. Format of the targeted region outlined (shape, size, number, separation, array, pre-selected ROI, association with previous images)

[0054] The above chemical mask can be used to delineate the boundary lines of at least one target region, which can be of a range of shapes and sizes, on a tissue section for subsequent isolation. The above target region can be of various shapes, such as rectangular, circular, oval, triangular, trapezoidal, pentagonal, hexagonal, or n - sided. The above target region can be from about 176 square microns to about 780,000 square microns. The above target region can be at least about 176 square microns. The above target region can be up to about 780,000 square microns. The above target region can be from about 176 square microns to about 12,000 square microns, from about 176 square microns to about 49,000 square microns, from about 176 square microns to about 190,000 square microns, from about 176 square microns to about 780,000 square microns, from about 12,000 square microns to about 49,000 square microns, from about 12,000 square microns to about 190,000 square microns, from about 12,000 square microns to about 780,000 square microns, from about 49,000 square microns to about 190,000 square microns, from about 49,000 square microns to about 780,000 square microns, or from about 190,000 square microns to about 780,000 square microns. The above target region 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 above target region can be from about 490 square microns to about 780,000 square microns. The above target region can be at least about 490 square microns. The above target region can be up to about 780,000 square microns.The area of the above-mentioned target region can be approximately 490 square microns to approximately 12,000 square microns, approximately 490 square microns to approximately 49,000 square microns, approximately 490 square microns to approximately 190,000 square microns, approximately 490 square microns to approximately 780,000 square microns, approximately 12,000 square microns to approximately 49,000 square microns, approximately 12,000 square microns to approximately 190,000 square microns, approximately 12,000 square microns to approximately 780,000 square microns, approximately 49,000 square microns to approximately 190,000 square microns, approximately 49,000 square microns to approximately 780,000 square microns, or approximately 190,000 square microns to approximately 780,000 square microns. The area of the above-mentioned target region can be approximately 490 square microns, approximately 12,000 square microns, approximately 49,000 square microns, approximately 190,000 square microns, or approximately 780,000 square microns.

[0055] The circular target area can be from about 15 microns in diameter to about 10,000 square microns. The circular target area can be at least about 15 microns in diameter. The circular target area can be at least about 25 microns in diameter. The circular target area can be up to about 1,000 microns in diameter. The circular target area can be from about 15 microns in diameter to about 50 microns, from about 15 microns in diameter to about 125 microns, from about 15 microns in diameter to about 250 microns, from about 15 microns in diameter to about 500 microns, from about 15 microns in diameter to about 750 microns, from about 15 square microns in diameter to about 1,000 microns, from about 15 microns in diameter to about 125 microns, from about 50 microns in diameter to about 250 microns, from about 50 microns in diameter to about 500 microns, from about 50 microns in diameter to about 750 microns, from about 50 microns in diameter to about 1,000 microns, from about 125 microns in diameter to about 250 microns, from about 125 microns in diameter to about 500 microns, from about 125 microns in diameter to about 750 microns, from about 125 microns in diameter to about 1,000 microns, from about 250 microns in diameter to about 500 microns, from about 250 microns in diameter to about 750 microns, from about 250 microns in diameter to about 1,000 microns, from about 500 microns in diameter to about 750 microns, from about 500 microns in diameter to about 10,000 microns, or from about 750 microns in diameter to about 1,000 microns. The circular target area can be about 15 microns, about 25 microns, about 50 microns, about 125 microns, about 250 microns, about 500 microns, about 750 microns, about 1,000 microns, about 2,000 microns, about 3,000 microns, about 4,000 microns, about 5,000 microns, about 6,000 microns, about 7,000 microns, about 8,000 microns, about 9,000 microns, or about 10,000 microns in diameter.The circular target region may have a diameter of less than about 15 microns or equal thereto, less than about 25 microns or equal thereto, less than about 50 microns or equal thereto, less than about 125 microns or equal thereto, less than about 250 microns or equal thereto, less than about 500 microns or equal thereto, less than about 750 microns or equal thereto, less than about 1,000 microns or equal thereto, less than about 2,000 microns or equal thereto, less than about 3,000 microns or equal thereto, less than about 4,000 microns or equal thereto, less than about 5,000 microns or equal thereto, less than about 6,000 microns or equal thereto, less than about 7,000 microns or equal thereto, less than about 8,000 microns or equal thereto, or less than about 9,000 microns or equal thereto, or less than about 10,000 microns or equal thereto.

[0056] In some embodiments, more than one target region has a boundary line drawn in the tissue sample such that the target regions form a grid. In some embodiments, the grid includes from about 10 to about 10,000 target regions. In some embodiments, the grid includes at least about 10 target regions. In some embodiments, the grid includes up to about 10,000 target regions. In some embodiments, the grid includes from about 10 to about 100 target regions, from about 10 to about 1,000 target regions, from about 10 to about 10,000 target regions, from about 100 to about 1,000 target regions, from about 100 to about 10,000 target regions, or from about 1,000 to about 10,000 target regions. In some embodiments, the grid includes about 10 target regions, about 100 target regions, about 1,000 target regions, or about 10,000 target regions.

[0057] In some embodiments, the size, shape, and / or pattern of the above-described target region can be user-defined. Such a user-defined pattern can be achieved by first staining / imaging corresponding tissue sections from the higher or lower vertical position in the tissue block using appropriate visualization techniques (e.g., hematoxylin / eosin staining, immunofluorescence, immunohistochemistry), defining the target region on the tissue image in a computer system, and transferring the desired pattern to the corresponding fresh, unprocessed tissue section using inkjet printing and guidance from the computer system.

[0058] Extraction

[0059] A. Solution

[0060] After defining one or more target regions on a tissue section through the application of the chemical mask described above, an extraction solution can be transferred to the one or more target regions to isolate nucleic acids and / or proteins from the target regions. The extraction solution can include one or more ionic or non-ionic surfactants (e.g., β-octyl glucoside, Triton-X-100, SDS, Tween-20, CHAPS), proteases (e.g., proteinase-K, trypsin, chymotrypsin, Lys-C, Asp-N, collagenase), tonicity regulators (e.g., dextrose, glycerol, mannitol, potassium chloride, sodium chloride), chaotropes (e.g., urea, thiourea, guanidinium hydrochloride), nucleases (DNAse, RNAse), buffers (e.g., Tris, Trizma, MOPS, sodium phosphate, bicarbonate, Bicine, CAPS, CAPSO, tricine, HEPES, MOPSO), protease inhibitors (e.g., AEBSF·HCl, Aprotenin, bestatin, E-64d, leupeptin, pepstatin, EDTA, PMSF), phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, beta-Glycerophosphase, sodium orthophosphate), or nuclease inhibitors (e.g., EDTA, EGTA, DEPC, RNaisin). In the case of frozen, unfixed tissue sections, standard compositions for the isolation of nucleic acids or proteins from cultured mammalian cells can be used (e.g., solutions containing buffers, salts, and surfactants). To search for 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 Paulo et al. JOP. 2013 can be found in Jul; 14(4): 405-414.

[0061] i. Soluble tags In some embodiments, the extraction solution may further include 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.

[0062] For multiplex sequencing (e.g., NGS sequencing) of nucleic acids (e.g., mRNA, cDNA, genomic DNA) derived from the region of interest, suitable soluble tags include synthetic oligonucleotides. Such oligonucleotides include a positional sequence (e.g., a non-random sequence corresponding to the region of interest) and, optionally, a unique molecular identifier (UMI) that enables counting of each individual reverse-transcribed cDNA molecule in downstream sequencing. The UMI sequence can be generated using random sequence generation. The UMI sequences are mapped to the genomes of all common reference species with a preset Tm interval, GC content, and defined distance difference to other barcode sequences to ensure that the barcode sequences do not interfere with the capture of nucleic acids, e.g., RNA, from the tissue sample and are distinguishable from each other without difficulty, followed by stringent filtering. The synthetic oligonucleotide may include a positional sequence and / or a UMI sequence for tagging of only the 5' end or 3' end label, or both the 5' end and 3' end labels of the reverse-transcribed cDNA.

[0063] Tagging of nucleic acids (e.g., RNA) isolated from each region of interest using such synthetic oligonucleotides can be achieved by various methods. In some embodiments, the synthetic oligonucleotide containing the positional sequence / UMI sequence is double-stranded (optionally hairpin or Y-shaped, and / or optionally, e.g., Wei et al. As described in Genetics. 2016 Jan; 202(1): 37-44, it contains a 3'-terminal T overhang, and after A-tailing, an oligo dT universal primer is used by ligation to bind to either end of the cDNA generated from the first reverse transcriptase synthesis. In some embodiments, the synthetic oligonucleotide is single-stranded and further contains an oligo dT sequence, such that the positional sequence / UMI sequence is incorporated during the first reverse transcription into cDNA. 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-passim.

[0064] Regarding the multiplex mass spectrometry (e.g., LC-MS / MS) of proteins derived from the above one or more target regions, suitable soluble tag sequences include tandem mass 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 suitable for conjugation to tryptic peptides generated from proteins from the above target regions with slight differences in molecular weight, which enable tryptic peptides originating from multiple distinct samples to be easily distinguishable in the MS / MS spectra (for further details on TMT construction and detection, see WO2016196994A1 and Zhang et al. Methods Mol Biol. 2017; 1550:185-198).

[0065] B. Drug Delivery Device

[0066] In some embodiments, transferring the extraction solution or other components to or from the region of interest on the masked tissue section requires the use of one or more drug transfer devices. The drug transfer device includes an array (drug transfer array element) that includes a plurality of hydrophilic regions that spatially correspond to the region of interest on the masked tissue section. The hydrophilic regions facing the hydrophobicity represent a "surface tension array", where the aqueous solution added to the hydrophilic region is contained by the hydrophobic region it faces, and the maximum solution volume of the drug transfer array element is the area / diameter of the hydrophilic region (controlling the "width" of the solution that can be added to the feature) as well as the difference between the contact angle of the hydrophilic region and the hydrophobic region (controlling the "height" of the solution that can be added to the feature). FIG. 3 shows how a drug transfer array B filled with an extraction solution (black) can be used to transfer the solution to the region of interest on slide A by bringing the liquid dome of B into contact with the tissue section, allowing surface tension to draw a liquid meniscus on the tissue section (C).

[0067] Such drug delivery devices can be produced using a variety of different hydrophobic / hydrophilic chemicals on a variety of substrates. One convenient process involves derivatizing the glass with a hydrophilic compound, followed by protecting the hydrophilic features with a positive photoresist along with a coating using a fluorocarbon. In one embodiment, an array containing hydrophilic drug delivery array features is produced by derivatizing a quantitatively clean glass slide with a monofunctional (one binding site for silanization) organic silane (e.g., 3-aminopropyldimethylethoxysilane (APDMS), followed by protecting the drug delivery array elements / hydrophilic regions with a positive photoresist and treating with a perfluoroalkyltrichlorosilane (e.g., (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane) or another suitable wear-resistant coating (e.g., dimethyldichlorosilane DDMS, perfluorodecyltris(dimethylamino)silane PF10TAS, perfluorodecanoic acid PFDA. Or variants with perfluoroalkyl chains of various lengths and isomers); see, for example, Ashurst et al. IEEE Transactions on Device and Materials Relatability. 3(4):173-178 (2003)). Further chemical details of the derivatization of glass slides with hydrophilic and hydrophobic regions can be found, for example, in Butler et al. J. Am. Chem. Soc. 2001, 123, 8887-8894 and US20150268233 A1.

[0068] The dimensions of the hydrophilic region / drug transfer array elements on the drug transfer device correspond to the dimensions of the region of interest masked on the tissue sample. The drug transfer array elements can be of various shapes, such as rectangular, circular, oval, triangular, trapezoidal, pentagonal, hexagonal, or n-sided. The drug transfer array elements can be from about 490 square microns to about 780,000 square microns. The drug transfer array elements can be at least about 490 square microns. The drug transfer array elements can be at most about 780,000 square microns. The drug transfer array elements can be from about 490 square microns to about 12,000 square microns, from about 490 square microns to about 49,000 square microns, from about 490 square microns to about 190,000 square microns, from about 490 square microns to about 780,000 square microns, from about 12,000 square microns to about 49,000 square microns, from about 12,000 square microns to about 190,000 square microns, from about 12,000 square microns to about 780,000 square microns, from about 49,000 square microns to about 190,000 square microns, from about 49,000 square microns to about 780,000 square microns, or from about 190,000 square microns to about 780,000 square microns. The drug transfer array elements 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. Circular drug transfer array elements can be from a diameter of about 25 square microns to a diameter of about 1,000 square microns. Circular drug transfer array elements can be at least about 25 square microns in diameter. Circular drug transfer array elements can be at most about 1,000 square microns in diameter.The circular drug transfer array elements can be from approximately 25 square microns in diameter to approximately 50 square microns in diameter, from approximately 25 square microns in diameter to approximately 125 square microns in diameter, from approximately 25 square microns in diameter to approximately 250 square microns in diameter, from approximately 25 square microns in diameter to approximately 500 square microns in diameter, from approximately 25 square microns in diameter to approximately 750 square microns in diameter, from approximately 25 square microns in diameter to approximately 1,000 square microns in diameter, from approximately 50 square microns in diameter to approximately 125 square microns in diameter, from approximately 50 square microns in diameter to approximately 250 square microns in diameter, from approximately 50 square microns in diameter to approximately 500 square microns in diameter, from approximately 50 square microns in diameter to approximately 750 square microns in diameter, from approximately 50 square microns in diameter to approximately 1,000 square microns in diameter, from approximately 125 square microns in diameter to approximately 250 square microns in diameter, from approximately 125 square microns in diameter to approximately 500 square microns in diameter, from approximately 125 square microns in diameter to approximately 750 square microns in diameter, from approximately 125 square microns in diameter to approximately 1,000 square microns in diameter, from approximately 250 square microns in diameter to approximately 500 square microns in diameter, from approximately 250 square microns in diameter to approximately 750 square microns in diameter, from approximately 250 square microns in diameter to approximately 1,000 square microns in diameter, from approximately 500 square microns in diameter to approximately 750 square microns in diameter, from approximately 500 square microns in diameter to approximately 1,000 square microns in diameter, or from approximately 750 square microns in diameter to approximately 1,000 square microns in diameter. The circular drug transfer array elements can be approximately 25 square microns in diameter, approximately 50 square microns in diameter, approximately 125 square microns in diameter, approximately 250 square microns in diameter, approximately 500 square microns in diameter, approximately 750 square microns in diameter, or approximately 1,000 square microns in diameter.

[0069] In some embodiments, more than one drug transfer array element is printed on the drug transfer device, such that the drug transfer array elements form a grid. In some embodiments, the grid includes from about 10 drug transfer array elements to about 10,000 drug transfer array elements. In some embodiments, the grid includes at least about 10 drug transfer array elements. In some embodiments, the grid includes up to about 10,000 drug transfer array elements. In some embodiments, the grid includes from about 10 drug transfer array elements to about 100 drug transfer array elements, from about 10 drug transfer array elements to about 1,000 drug transfer array elements, from about 10 drug transfer array elements to about 10,000 drug transfer array elements, from about 100 drug transfer array elements to about 1,000 drug transfer array elements, from about 100 drug transfer array elements to about 10,000 drug transfer array elements, or from about 1,000 drug transfer array elements to about 10,000 drug transfer array elements. In some embodiments, the grid includes about 10 drug transfer array elements, about 100 drug transfer array elements, about 1,000 drug transfer array elements, or about 10,000 drug transfer array elements.

[0070] The above-described drug transfer array element can be designed to accommodate a wide range of volumes. In some embodiments, the maximum volume of the drug transfer array element is from about 2 picoliters to about 10,000 picoliters. In some embodiments, the maximum volume of the drug transfer array element is at least about 2 picoliters. In some embodiments, the maximum volume of the drug transfer array element is at most about 10,000 picoliters. In some embodiments, the maximum volume of the drug transfer array element is from about 2 picoliters to about 10 picoliters, from about 2 picoliters to about 50 picoliters, from about 2 picoliters to about 100 picoliters, from about 2 picoliters to about 250 picoliters, from about 2 picoliters to about 500 picoliters, from about 2 picoliters to about 1,000 picoliters, from about 2 picoliters to about 10,000 picoliters, from about 10 picoliters to about 50 picoliters, from about 10 picoliters to about 100 picoliters, from about 10 picoliters to about 250 picoliters, from about 10 picoliters to about 500 picoliters, from about 10 picoliters to about 1,000 picoliters, from about 10 picoliters to about 10,000 picoliters, from about 50 picoliters to about 100 picoliters, from about 50 picoliters to about 250 picoliters, from about 50 picoliters to about 500 picoliters, from about 50 picoliters to about 1,000 picoliters, from about 50 picoliters to about 10,000 picoliters, from about 100 picoliters to about 250 picoliters, from about 100 picoliters to about 500 picoliters, from about 100 picoliters to about 1,000 picoliters, from about 100 picoliters to about 10,000 picoliters, from about 250 picoliters to about 500 picoliters, from about 250 picoliters to about 1,000 picoliters, from about 250 picoliters to about 10,000 picoliters, from about 500 picoliters to about 1,000 picoliters, from about 500 picoliters to about 10,000 picoliters, or from about 1,000 picoliters to about 10,000 picoliters. In some embodiments, the maximum volume of the drug 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.

[0071] C. Detection technology

[0072] Next, the biomolecules extracted from the at least one target region can be detected to analyze the biomolecule expression profile of the cells in the target region or the genomic DNA composition of the cells in the target region (for example, in the case of a tissue sample containing cancerous cells, the genomic DNA of the cells in the tissue section may be heterogeneous due to mutations, deletions, and / or translocations). Therefore, the methods, devices, and compositions included herein can be used to analyze both the genetic and epigenetic characteristics of the cells in the target region.

[0073] In some methods, the expression level of biomolecules (e.g., mRNA, or cDNA obtained from mRNA by reverse transcription, or genomic DNA) in the at least one target region can be determined by sequencing. Examples of sequencing methods include: next-generation sequencing, high-throughput sequencing, pyrosequencing, classical Sanger sequencing, sequencing-by-ligation, sequencing by synthesis, sequencing-by-hybridization, RNA-Seq (Illumina), digital PCR, digital gene expression (Helicos), next-generation sequencing, single molecule sequencing by synthesis (SMSS) (Helicos), Ion Torrent Sequencing Machine (Life Technologies / Thermo-Fisher), massively-parallel sequencing, clonal single molecule Array (Solexa), shotgun sequencing, Maxam-Gilbert sequencing, and primer walking.

[0074] In some methods, the expression level of a biomolecule (e.g., mRNA, or cDNA obtained from mRNA by reverse transcription) within at least one of the target regions described above is determined by a so-called "real time amplification" method, also known as quantitative PCR (qPCR) or Taqman (e.g., U.S. Patent Nos. 5,210,015 (Gelfand), 5,538,848 (Livak, et al.), and 5,863,736 (Haaland), as well as Heid, C.A., et al., Genome Research, 6:986-994 (1996); Gibson, U.E.M, et al., Genome Research 6:995-1001 (1996); Holland, P. M., et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, (1991); and Livak, K.J., (See, e.g., et al., PCR Methods and Applications 357-362 (1995)). The basis of this method for monitoring the formation of amplification products is to continuously measure the accumulation of PCR products using a dual-labeled fluorescent oligonucleotide probe. The probes used in such assays are typically short (about 20-25 bases) polynucleotides labeled with two different fluorescent dyes. The 5' end of the probe is typically conjugated to a reporter dye, and the 3' end is conjugated to a quenching dye. The probe is designed to have at least substantial sequence complementarity with a site on the target mRNA or nucleic acid derived therefrom. Upstream and downstream PCR primers that bind to adjacent regions of the locus are also added to the reaction mixture. When the probe is intact, energy transfer occurs between its two fluorophores, and the quencher quenches the emission from the reporter. During the elongation phase of PCR, the probe is cleaved by the 5' nuclease activity of a nucleic acid polymerase, such as Taq polymerase, thereby releasing the reporter from the polynucleotide-quencher and resulting in an increase in reporter emission intensity that can be measured by an appropriate detector. The recorded values can then be used to calculate the increase in reporter emission intensity normalized on a continuous basis, and ultimately, the amount of amplified mRNA can be quantified.

[0075] In some embodiments, for qPCR or Taqman detection, the RT-PCR step is first performed to generate cDNA from cellular RNA. Such amplification by RT-PCR can be either global (e.g., amplification with degenerate oligonucleotide primers, partially / fully) or targeted (e.g., amplification with oligonucleotide primers directed against a specific gene that will be analyzed in a later step).

[0076] In some embodiments, qPCR or Taqman is used immediately after a reverse transcriptase reaction is performed on isolated cellular mRNA; this diversity serves to quantify the levels of individual mRNAs during qPCR.

[0077] For qPCR or Taqman, the level of a particular gene can be expressed relative to one or more internal control genes measured using the same detection methodology from the same sample. Examples of internal control genes can include so-called "housekeeping" genes (e.g., ACTB, B2M, UBC, GAPD, and HPRT1).

[0078] 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 serves to increase the signal under conditions where the natural levels of the RNA / cDNA to be detected are very low. Suitable methods for pre-amplification include, but are not limited to, LM-PCR, PCR with random oligonucleotide primers (e.g., random hexamer PCR), PCR with poly-A specific primers, and any combination thereof. The pre-amplification can be either inclusive or can be targeted in the same manner as the reverse transcription reaction described above.

[0079] mRNA levels can also be measured without amplification by hybridization to a probe, for example, using branched nucleic acid probes (e.g., Panomics' QuantiGene® Reagent System).

[0080] Alternatively or additionally, the expression level of the gene from the at least one target region can be determined at the protein level, which means that the level of the protein encoded by the gene discussed above is measured. Several methods and devices are well known for determining the level of protein, including, for example, the immunoassays described in U.S. Patent Nos. 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 regarding the presence or amount of the protein analyte of interest. Any suitable immunoassay can be utilized (e.g., lateral flow, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), competitive binding assay, etc.). Many formats regarding antibody arrays have been described and the use of antibodies has been advocated. Such arrays typically include different antibodies having specificity for different proteins intended to be detected. For example, usually at least 100 different antibodies are used to detect 100 different protein targets, and each antibody is specific for one target. Other ligands having specificity for a particular protein target can also be used (e.g., synthetic antibodies disclosed in WO / 2008 / 048970). Other compounds having the desired binding specificity can be selected from peptide or small molecule random libraries. U.S. Patent No. 5,922,615 describes a device that utilizes a number of separate zones of antibodies immobilized on a membrane for detecting a number of target antigens in an array. U.S. Patent Nos. 5,458,852; 6,019,944; 6,143,576. Microtiter plates or automation can be used to facilitate the detection of a number of different proteins. The protein level can also be determined by mass spectrometry (e.g., tandem LC / MS / MS).In some embodiments, genomic DNA isolated from a target region is analyzed (e.g., by any of the sequencing techniques described above or by qPCR). Such analysis can serve to detect genomic variations including, but not limited to, variations, SNPs, insertions, deletions, and / or copy number variations in the nucleotide sequence in or around a gene. In some variations of this embodiment, the isolated DNA is first amplified by whole genome amplification (WGA) techniques to improve the ability to determine DNA sequences from limited samples.

[0081] In some embodiments, the WGA technique is a two-step technique known as degenerate-oligonucleotide-primed (DOP) PCR. In the first step, DOP-PCR uses a primer that includes a) a random 6-nucleotide sequence at the 3’ end, and b) a fixed adapter sequence having a T m optimal for PCR amplification (optionally including a barcode containing a sample index and / or unique molecular identifier); the random primer hybridizes to the sample at a low annealing temperature and is subsequently strand extended using polymerase at a high temperature. In the second step, a primer containing a 3’ end complementary to the 5’ fixed adapter sequence of the primers of the first step (and optionally including a barcode containing a sample index and / or unique molecular identifier at their 5’ ends) is used to amplify the product from the first step using PCR annealing at a higher temperature than the first step.

[0082] In some embodiments, the WGA technique is a one-step isothermal technique known as multiple displacement amplification (MDA). MDA can use primers similar to those of the first stage of DOP-PCR, for example, primers comprising a) a random 6-nucleotide sequence at their 3' end, and b) a fixed adapter sequence (optionally including a barcode containing a sample index and / or unique molecular identifier). However, instead of conventional PCR DNA polymerases such as Taq, MDA uses a strand-displacing polymerase such as φ29 DNA polymerase. The isolated DNA is amplified without cycling under isothermal conditions using the primers and φ29 DNA polymerase.

[0083] In some embodiments, the WGA technique is a quasi-linear amplification technique known as multiple annealing and loop-based amplification cycles (MALBAC). In the first stage, MALBAC has a common 27-nucleotide sequence at the 5' end (e.g., GTG Use a specially designed primer having (AGT GAT GGT TGA GGT AGT GTG GAG) and 8 random nucleotides at the 3' end; semi-amplicons are generated using these primers by first annealing at a low temperature (e.g., 15 - 20°C), followed by extension using a strand-displacing polymerase (e.g., Bst DNA polymerase) at a higher temperature (e.g., 70 - 75°C). After melting the primers from the template (e.g., at 95°C or higher), repeated cycles of low temperature (e.g., 15 - 20°C), high temperature (e.g., 70 - 75°C), denaturation temperature (e.g., 95°C or higher), and hairpin formation (e.g., 58°C) (e.g., 10+ cycles) are used to further amplify the semi-amplicons into full amplicons. Since the full amplicons contain the same 5' 27 nucleotide sequence (e.g., GTG AGT GAT GGT TGA GGT AGT GTG GAG), once they are created, the hairpin formation step is removed since it would otherwise involve them in subsequent cycles of amplification. This can ensure more linear amplification since copies of copies of the original DNA are not made.

[0084] In some embodiments, the WGA technique is Pico-Plex TM (Takara Derived from (Bio) and / or described in U.S. Patent No. 8,206,913. These techniques and derivatives attempt to correct some of the problems associated with whole-genome random primer amplification (e.g., underrepresentation of certain regions). In some embodiments, self-inactivating (e.g., non-self-priming) degenerate primers are used in the first step of this method, which can be an isothermal amplification step or an initial annealing step at about 15-20°C, followed by an extension step at about 75°C (e.g., with a mesophilic DNA polymerase). In some embodiments, these primers include a) a 5'-fixed region and b) a 3'-variable region, and are designed such that the primers do not cross-hybridize or self-hybridize. In some embodiments, the variable region further includes a partially degenerate sequence (e.g., a 10 nt length that can all be Y, R, K, or M, where Y = random C or T; R = random A or G; M = random A or C; K = random G or T) and a fully degenerate sequence (e.g., a 2 nt length, Ns, random A / C / T / G). In some embodiments, the constant region and the variable region of the self-inactivating degenerate primer consist essentially of only two types of non-complementary nucleotides selected from the group consisting of adenine and guanine; adenine and cytosine; guanine and thymidine. In some embodiments, the self-inactivating degenerate primer used in the isothermal amplification step follows any one of SEQ ID NOs: 1-8 in Table 1 below. [Table 1] In some embodiments, the further amplification step with the thermostable DNA polymerase is performed on the DNA amplified above. The further amplification step can be performed using primers consisting of a fixed region immediately 5' to the primers used in the isothermal amplification step. In some embodiments, these primers are any of SEQ ID NOs: 8-12 in Table 1. The further amplification step can include >10 cycles (e.g., denaturation, annealing, extension) of normal PCR cycling using a thermostable DNA polymerase. In some embodiments, the cycling conditions incorporate an additional low temperature step after the extension step to promote the formation of hairpin DNA similar to the MALBAC method described above.

Example

[0085] Example 1A. - Protocol for the Application of a Hydrophobic Mask to Tissue Sections A computer-assisted inkjet system is used to apply a grid pattern of coating to a tissue sample such that a plurality of regularly spaced uncoated regions are created. The coating contains a non-fluorinated acrylate or cyanoacrylate mixed with a fluoroalkyl acrylate monomer as described herein in an alcohol or ketone solvent + photoinitiator. The section is then briefly exposed to UV light to cure the coating.

[0086] Example 1B. - Alternative Protocol for the Application of a Hydrophobic Mask to Tissue Sections A computer-assisted inkjet system is used to apply a first coating grid pattern to a tissue sample such that a plurality of regularly spaced uncoated areas are created. The first coating includes a non-fluorinated acrylate or cyanoacrylate + photoinitiator. The first coating is exposed to UV light for a short time to cure it. Next, again using a computer-assisted inkjet system, a second coating including a fluoroalkyl acrylate monomer + photoinitiator as described herein is added over the first coating. The second coating is then cured by a short exposure to UV light.

[0087] Example 2. - Isolation of regions of interest in tissue samples using hydrophobic chemicals FFPE hepatocellular carcinoma tissue sections were subjected to hematoxylin / eosin staining with or without deparaffinization and with or without formation of a hydrophobic mask of the regions of interest using a fluoroalkyl acrylate monomer. Photographs of the resulting slides are shown in Figure 1. Deparaffinization of the FFPE tissue sections followed by treatment with the hydrophobic chemical of Example 1 caused effective aqueous isolation of the masked regions of interest and prevented staining with hematoxylin / eosin.

[0088] Example 3. - Isolation of regions of various sizes in FFPE mouse liver sections

[0089] FFPE mouse liver sections were deparaffinized, masked, and regions of various sizes (100 microns, 250 microns, 500 microns, 1000 microns in diameter) and shapes (rectangular and circular) were isolated, stained with hematoxylin / eosin, and the hydrophilic accessibility (and hydrophobic mask inaccessibility) of the regions of interest, as well as the accuracy / precision of inkjet application of the masks, were shown. Figure 2A shows that regions of approximately 160 microns or approximately 550 microns in diameter can be clearly defined using inkjet printing of the chemical mask. Figure 2B shows that regions of approximately 300 or approximately 1000 microns in diameter can be clearly defined using inkjet printing of the chemical mask. Figure 2C shows that both circular and polygonal shapes can be isolated from the sections. Figure 2E shows that a region of 2 mm in diameter can be clearly defined on the section. Figure 2D shows that regions of various diameters (100 mm, 250 mm, 500 mm, 1 mm) and shapes (rectangular and circular) can be printed with a clearly defined grid on a single tissue section.

[0090] Example 4. - Hydrophobic mask formation blocks nucleic acid extraction from masked regions

[0091] FFPE tissue sections were masked according to the method of Example 1A or Example 1B to generate regions of interest having various sizes.

[0092] Slides were deparaffinized by baking in an oven at 55 - 60 °C for 1 hour, immersing twice in xylene for 3 minutes each, and immersing twice in 100% ethanol for 3 minutes each. The slides were stored in ethanol for up to 1 month if necessary.

[0093] For further analysis, the slides were air-dried to remove ethanol and the tissue was lysed. An adhesive chamber was attached to the slide delineating the boundary of the tissue area, 600 microliters of Qiagen tissue lysis buffer ATL containing proteinase K was added, the chamber was sealed with an adhesive film, and the slide was incubated on a heat block at 56 °C for 1 hour. The liquid (about 450 microliters) was collected from the slide chamber in a microcentrifuge tube, and then the microcentrifuge tube was heated at 90 °C for 1 hour. The microcentrifuge tube was then centrifuged to pellet the insoluble material, the supernatant was removed to a new tube, treated with 45 microliters of RNAse A (100 mg / ml), and subsequently incubated at room temperature for 2 minutes. An equal volume of Qiagen buffer AL (about 500 microliters) was added to the supernatant and the sample was mixed by vortexing. After vortexing, an equal volume of 100% ethanol (about 500 microliters) was added to the supernatant and the entire solution was transferred to a Qiagen min-elute column. The column was washed using buffer AW1 and AW2 according to the manufacturer's instructions, and the nucleic acids were eluted in Qiagen buffer ATE. The nucleic acids derived from each sample were then quantified using a Qubit spectrophotometer. The relationship between the size of the region of interest and the extracted DNA is shown in Table 2. Data on DNA yield versus uncoated surface area indicate that the DNA yield is proportional to the area covered, suggesting that DNA is not extracted from regions of the tissue section covered by the hydrophobic coating.

Table 2

[0094] Example 5. - Optimized hydrophobic coating procedure:

[0095] A) Procedure for small features (<1 mm diameter)

[0096] If the tissue sample is an FFPE sample, the tissue sample is first subjected to deparaffinization by standard procedures (e.g., baking in an oven at approximately 60 degrees for 1 hour, followed by incubation in xylene for 3 minutes, in xylene for 3 minutes, in 100% EtOH for 3 minutes, in 100% EtOH for 3 minutes, and air drying for 5 minutes). The tissue surface is coated with a first hydrophobic coating by spraying or dipping it into an alcohol solution containing fluoroacrylate together with micron-sized fluoroparticulates; examples include, but are not limited to, Fluoro-pel 800 or 800M. The slide is then air dried (this first treatment improves the definition of small features in the subsequent cyanoacrylate printing step).

[0097] A solution of cyanoacrylate containing a photoinitiator is inkjet printed onto the tissue sample to form a mask covering regions to be excluded or regions separating desired features. During printing, the cyanoacrylate is pinned via exposure to UV light (e.g., 395 nm @ approximately 400 mJ / cm2) for a minimum number of seconds after deposition. The slide is then removed from the printing apparatus and immediately subjected to long-term UV curing (e.g., 395 nm at approximately 400 mJ / cm2 for about 5 minutes).

[0098] After UV curing, the entire slide is subjected to vacuum-assisted vapor deposition of perfluoroalkyltrichlorosilane (e.g., FOTS, trichloro(1H,1H,2H,2H-perfluorooctyl)silane)). The application of this technique enabled the generation of circular regions of interest on FFPE human liver tissue sections with a small diameter of about 25 microns.

[0099] B) Procedure for large features (approximately 1 mm or larger)

[0100] When the tissue sample is an FFPE sample, the tissue sample is first subjected to deparaffinization by standard procedures (e.g., baking in an oven at approximately 60 degrees for 1 hour, followed by incubation in xylene for 3 minutes, in xylene for 3 minutes, in 100% EtOH for 3 minutes, in 100% EtOH for 3 minutes, and air drying for 5 minutes). A larger feature can proceed ahead of the first fluoroalkyl coating. A solution of cyanoacrylate containing a photoinitiator is inkjet printed onto the tissue sample in regions that isolate regions or features that are desired to be excluded. During printing, the cyanoacrylate is retained via exposure to UV light (e.g., 395 nm @ about 400 mJ / cm2) for a minimum number of seconds after deposition. The slide is then removed from the printing apparatus and immediately subjected to long-term UV curing (e.g., 395 nm at about 400 mJ / cm2 for about 5 minutes).

[0101] After UV curing, the entire slide is subjected to vacuum-assisted vapor deposition of a perfluoroalkylsilane (e.g., trichloro(1H,1H,2H,2H-perfluorooctyl)silane).

[0102] Example 6. - Dissolution and Isolation of Hydrophobically Separated Regions

[0103] A surface tension array is prepared in a state corresponding to the non-excluded regions of a mask in which hydrophilic features are imparted to the tissue sample, as in US20150268233 A1 or Butler et al. J. Am. Chem. Soc. 2001, 123, 8887-8894. This derived procedure involves coating a quantitatively clean glass slide with a monofunctional (one binding site for silanization) organic silane (e.g., 3-aminopropyldimethylethoxysilane (APDMS)), followed by protecting the non-excluded regions / hydrophilic regions with a positive photoresist and treating with an abrasion-resistant coating such as tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane.

[0104] An appropriate lysis buffer containing a surfactant is imparted to the hydrophilic features of the surface tension array. A surface tension array containing a hydrophilic region having the above lysis buffer is contacted with a tissue sample prepared as in Example 1A, 1B, or 5. The contact is provided for a suitable period to allow cell lysis. The surface tension array (which here contains the lysis buffer containing the lysed cell components) is removed from contact with the tissue section, and the individual spots on the surface tension array are interrogated for the desired properties (e.g., abundance, sequence, or both of proteins or nucleic acids).

[0105] Example 7. - Contact Angle Measurement on Coated Surfaces

[0106] FFPE tissue sections or glass slides were subjected to different stages of the coating procedure shown in Example 5B and to vapor phase coatings of various lengths with FOTS ((tridecafluoro-1,1,2,2-tetrahydrooctyl) trichlorosilane). Contact angle measurements were performed using direct measurements with a Telescope-Goniometer (see, for example, Bracco and Holst. Surface Science Techniques. 2013. ISBN 978-3-642-34243-1. pp.3-34). The data show that when cyanoacrylate is added on top of the tissue section, the coating with FOTS is enhanced, and the combination of the two increases the contact angle of the combined FOTS / cyanoacrylate tissue surface into a very hydrophobic range (contact angle greater than 110). [Table 3]

[0107] Example 8. - qPCR of RNA Isolated from FFPE Tissue Sections with Hydrophobic Mask Formed

[0108] Various consecutive sections from the same FFPE breast cancer tissue sample (Biomax, huCAT299) were subjected to the optimized coating procedure of Example 5 to select various different regions of interest including non-cancerous tissue, transitional tissue (e.g., tissue that is somewhat non-cancerous and somewhat cancerous), and cancerous tissue (see FIGS. 7 and 8. Here, the left side of FIG. 8 shows different mask patterns applied to various tissue layers / sections, and the right side shows the regions of interest of various tissues isolated after mask formation). After coating, the regions of interest were lysed by applying a surfactant-containing lysis buffer + proteinase K to the slides (the lysis buffer was also applied to the coated regions as a control). Total RNA was extracted from the samples shown in FIG. 8 by placing the lysis buffer directly on the regions and incubating at 56° C. for 1 hour. Total RNA was purified using the NucleoSpin totalRNA FFPE XS kit (Takara Bio) according to the manufacturer's protocol, and the amount of RNA from the samples was quantified by Qubit fluorescence assay. A panel of qPCR assays was performed on the RNA extracted from the samples to detect genes known to be enriched in breast cancer (see FIG. 9 showing the qPCR Ct values of genes enriched in individual known tumors from the above tissue). The analysis showed that in tumor samples, as expected, the levels of genes detected by qPCR were characteristic of cancerous tissue rather than healthy tissue, indicating that the mask was effective in isolating tissue regions for selective lysis and that the hydrophobic coating procedure did not interfere with downstream amplification of nucleic acids released from the defined regions of interest.

[0109] The RNA samples isolated by the above mask formation procedure were also compared to a conventional manual spatial isolation technique (denoted as "tube" in Panel B of Figure 9) performed on the same samples. qPCR Ct counts were calculated for the housekeeping gene GAPDH, and the data were graphed (Figure 9). The gene expression levels via both methods were found to be highly correlated, indicating that the above hydrophobic mask formation procedure is equivalent to the above manual isolation workflow.

[0110] Furthermore, for exemplary mask-formed tissue regions, the amount of nucleic acid released by the lysis buffer applied to the mask-formed or unmasked regions was compared (Figure 9 Panel C). The analysis shows that dramatically less nucleic acid was isolated from the covered regions. This indicates that the above hydrophobic mask is effective in excluding unwanted tissue from lysis in the above procedure.

[0111] Furthermore, a series of 177 qPCR assays designed to detect genes selectively expressed in breast cancer were performed on RNA isolated from the above mask-formed tumor samples. 177 unique expressed qPCR products were detected in the RNA released from the cancerous samples by the above mask formation procedure. This indicates that a broad portion of the expressed genome was isolated from the cancerous cells isolated by the above mask formation procedure.

[0112] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

[Claim 1] The invention as depicted in the drawings.

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