(12) United States Patent (10) Patent No.: US 8,304,196 B2 Caprioli (45) Date of Patent: *Nov
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USOO83 041.96B2 (12) United States Patent (10) Patent No.: US 8,304,196 B2 Caprioli (45) Date of Patent: *Nov. 6, 2012 (54) INSITUANALYSIS OF TISSUES 6,809,315 B2 10/2004 Ellson et al. .................. 250/288 7,534.338 B2 5/2009 Hafeman et al. ... 205/288 O O 2003.0049701 A1* 3, 2003 Muraca .......... 435/723 (75) Inventor: Richard Caprioli, Brentwood, TN (US) 2003/0186287 A1 10, 2003 Lin et al. 435/6 2004.0007673 A1* 1 2004 Coon et al. .. 250,424 (73) Assignee: Vanderbilt University, Nashville, TN 2007/0082356 A1 4/2007 Strom et al. ...................... 435/6 (US) FOREIGN PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this WO WOO1,26460 4/2001 patent is extended or adjusted under 35 WO WOO3,O34024 4/2003 U.S.C. 154(b) by 0 days. OTHER PUBLICATIONS This patent is Subject to a terminal dis- Schwartz et al. (J. Mass Spectrometry 2003 vol. 38, p. 699-708).* claimer. Pauletti et al. (J. Clin. Oncology 2000 vol. 18, 3651-3664).* Office Action issued in U.S. Appl. No. 1 1/355.912, mailed Apr. 3, (21) Appl. No.: 12/942,840 2008. Office Action issued in U.S. Appl. No. 1 1/355.912, mailed Dec. 8, 1-1. 2009. (22) Filed: Nov. 9, 2010 Office Action issued in U.S. Appl. No. 1 1/355.912, mailed May 22, O O 2009. (65) Prior Publication Data Yanagisawa et al., “Proteomic patterns of tumour Subsets in non US 2011 FO190145 A1 Aug. 4, 2011 Small-cell lung cancer.” The Lancet, 362:433-439, 2003. Related U.S. Application Data * c1tedcited bby examiner (63) Continuation of application No. 1 1/355,912, filed on Primary Examiner — Jacob Cheu Feb. 16, 2006, now Pat. No. 7,829,291. (74) Attorney, Agent, or Firm — Parker Highlander PLLC (60) sal application No. 60/653,665, filed on Feb. (57) ABSTRACT s The present invention provides for the simultaneous assess (51) Int. Cl. ment of a plurality of tissue regions or microregions, the GOIN33/53 (2006.01) benefit being homogeneity of the sampling, both in terms of (52) U.S. Cl. ......................................... 435/7.1436/518 tissue content and timing. Discrete regions of a tissue sample, (58) Field of Classification Search s None such as those demarcated by microwells formed within the See application file for complete search history tissue itself or tissue plugs removed from the tissue in a spatially referenced fashion, can be treated with one or more (56) References Cited physical or chemical treatments to liberate target molecules of interest. Subsequent analysis of said target molecules by, U.S. PATENT DOCUMENTS e.g., mass spectroscopy, permits identification of a variety of biological parameters, including those associated with dis 4,932,412 A 6/1990 Goldenberg .................. 600,431 5,384,260 A * 1/1995 Osborne et al. .. ... 436/64 ease or therapy. 6,677,503 B1 1/2004 Bidney et al. ... 800,279 6,707,038 B2 3/2004 Ellson et al. .................. 250/288 8 Claims, 20 Drawing Sheets U.S. Patent Nov. 6, 2012 Sheet 1 of 20 US 8,304,196 B2 U.S. Patent Nov. 6, 2012 Sheet 2 of 20 US 8,304,196 B2 : U.S. Patent Nov. 6, 2012 Sheet 3 of 20 US 8,304,196 B2 s U.S. Patent Nov. 6, 2012 Sheet 4 of 20 US 8,304,196 B2 r Y U.S. Patent Nov. 6, 2012 Sheet 5 of 20 US 8,304,196 B2 U.S. Patent Nov. 6, 2012 Sheet 6 of 20 US 8,304,196 B2 ? U.S. Patent Nov. 6, 2012 Sheet 7 of 20 US 8,304,196 B2 U.S. Patent Nov. 6, 2012 Sheet 8 of 20 US 8,304,196 B2  U.S. Patent Nov. 6, 2012 Sheet 9 of 20 US 8,304,196 B2 U.S. Patent Nov. 6, 2012 Sheet 10 of 20 US 8,304,196 B2 U.S. Patent Nov. 6, 2012 Sheet 11 of 20 US 8,304,196 B2 U.S. Patent Nov. 6, 2012 Sheet 12 of 20 US 8,304,196 B2 i 9886/a GZO/92 o SN CC'972 LOCGdz isS. k- N sc 60.18 v B s (f) 98008 68'06/ke CD : CD 68'0A 2n s (5s GA629 eas s 71,097 CO "689 CO A72 2 d '86 ke 98.1 99.80 gig 190 a 3 sk Se N vv Li-6 E 67'998 9792. SSA cr'84 verg ---,-,-,-,-,-,-,-,-, -, -8 3 s. s is is a s is is se o' U.S. Patent Nov. 6, 2012 Sheet 13 of 20 US 8,304,196 B2 b2(+1), FM(+1)- 28 296.8 LHFFMPGFAPLTSR 100 251,0399 296.8 y5(+1), MPGFAP(+1)-28 y9(+1), FMPGFAPLT(+1)- 18 1104.309 1580.1340 70,0855 20 KE2 1452.0557 60 10 d13a, 577.1033 50 398,0202 oil. Availabilitial. WAAA E. E. 69.0 397.2 725.4 1053.6 1381.8 1710.0 40 3.0781 85,0494 361.0090 5730519 945.2168 9.0709 644.0821 28.2119 1104.3091 1580.1340 395.0302.0238 21 545.041 76.0421 862 31.4859 1308.4983 1505A.' O 3.01796, 854B553 392 e 69.0 397.2 725.4 1053.6 1381.8 1710.0 FIG. 11B U.S. Patent Nov. 6, 2012 Sheet 14 of 20 US 8,304,196 B2 100 L. 90 0.0677 YTVAAIFR 542.9 80 1(+1) 86.0976 70 10070, 60 542.9 90 50 V7 an 40 80 175,1194 by b2(+1) b3b3(1) - 18(+1) 70 20 A +1) 60 10 838 b6(+1) 897.2039 1010.6800 O : E. EEAA s s 50 72.083 69.0 277.6 486.2 694.8 903.4 1112.0 40 120,0935 30 B4O675 59,1082 29,0943 78.2990 20 15.0947 77.2423 10 B i3.1384,720,2926T 619.7313 B97.2039 1010.680 O O P. t y dolski a E.R.AEAlish kill, 69.0 277.6 4862 6948 903.4 1112.0 FIG. 11C U.S. Patent Nov. 6, 2012 Sheet 16 of 20 US 8,304,196 B2 HRDTGILDSGR 1586.5 110.1055 100. b11 + H2O(+1) 1586.5 90 40 112.1.194 y1 (+1),DS(+1) - 28 y9(+1) 8O 86.1396 ye1) y9 - 17(+1) y10(+1)11(1) 1299. 432 70 7O 1126 situal SA4. M91) 295.353 60 69 338 6O7 876 1145 1414 50 1183.9572 40 112.1194 908.54O2 30 175.1152 1204 20 129.1276.277.1223432.2048 931,6248 1299.5432 10 4620 6412934 8104.5" 165.8959 549.2264 1291.4321 69 338 607 876 1145 1414 F.G. 12B U.S. Patent Nov. 6, 2012 Sheet 17 of 20 US 8,304,196 B2 FG 13 U.S. Patent US 8,304,196 B2 U.S. Patent Nov. 6, 2012 Sheet 19 of 20 US 8,304,196 B2 U.S. Patent Nov. 6, 2012 Sheet 20 of 20 US 8,304,196 B2 S. US 8,304,196 B2 1. 2 INSITU ANALYSIS OF TISSUES comprising (a) providing an intact tissue comprising a first spatially discrete microregion; (b) Subjecting the microregion This application is a Continuation Application of U.S. to one or more physical or chemical treatments; and (c) ana patent application Ser. No. 1 1/355,912 filed Feb. 16, 2006, lyzing a protein sample from the microregion, thereby pro now U.S. Pat. No. 7,829,291 which claims benefit of priority to U.S. Provisional Application Ser. No. 60/653,665, filed viding analysis of protein content in the tissue. The intact Feb. 17, 2005, the entire contents of both applications are tissue may further comprise at least a second spatially dis hereby incorporated by reference in their entirety. crete microregion, and the method further comprises Subject ing the second spatially discrete microregion to one or more BACKGROUND OF THE INVENTION physical or chemical treatments, and analyzing discrete a 10 protein sample from the second spatially discrete microre 1. Field of the Invention gion, and the protein content of the first and second spatially The present invention relates generally to the fields of discrete microregions may be compared. Protein encom molecular biology. More particularly, it concerns measure passes both peptides and polypeptides. One or more of the ment of biological molecules in situ in a tissue sample. Spe steps may be automated. cifically, the invention provides for creation of a series of 15 microwells in a single tissue or tissue region, thereby permit The provision of a microregion may involve different ting analysis of proteins, nucleic acids, lipids, carbohydrates, aspects. In one embodiment, the microregion may comprise a drugs and other biomolecules in a homogeneous set of reac well in a larger tissue section. The well may be a true depres tions. sion or hole in the tissue, or a channel structure created in the 2. Description of Related Art tissue. Multiple wells, depressions or holes can be created in With the completion of the Human Genome Project, a spatially distinct manner. Alternatively, the intact tissue may emphasis is shifting to examining the protein complement of be extracted from a larger tissue section, i.e., a tissue plug. the human organism. This has given rise to the Science of The plug may then be transferred to a support for further proteomics, the study of all the proteins produced by cell type treatment and analysis, and may be placed adjacent to other and organism.