EP1153300A2 - Procedes et dispositifs d'isolation et d'analyse de la teneur proteique des cellules - Google Patents

Procedes et dispositifs d'isolation et d'analyse de la teneur proteique des cellules

Info

Publication number
EP1153300A2
EP1153300A2 EP00911845A EP00911845A EP1153300A2 EP 1153300 A2 EP1153300 A2 EP 1153300A2 EP 00911845 A EP00911845 A EP 00911845A EP 00911845 A EP00911845 A EP 00911845A EP 1153300 A2 EP1153300 A2 EP 1153300A2
Authority
EP
European Patent Office
Prior art keywords
protein
cells
population
interest
proteins
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00911845A
Other languages
German (de)
English (en)
Inventor
Lance A. Liotta
Nicole Simone
Michael Emmert-Buck
Emmanuel F. Petricoin, Iii
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Department of Health and Human Services
US Government
Original Assignee
US Department of Health and Human Services
US Government
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by US Department of Health and Human Services, US Government filed Critical US Department of Health and Human Services
Publication of EP1153300A2 publication Critical patent/EP1153300A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G01N2001/2833—Collecting samples on a sticky, tacky, adhesive surface
    • G01N2001/284—Collecting samples on a sticky, tacky, adhesive surface using local activation of adhesive, i.e. Laser Capture Microdissection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2550/00—Electrophoretic profiling, e.g. for proteome analysis
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes

Definitions

  • the present invention relates to methods and devices for the analysis of cell samples where the samples are pure populations or subpopulations of desired types.
  • the present invention allows for direct comparison of protein content and protein characteristics between proteins isolated from tumor and normal cells from the same tissue sample.
  • the present invention describes devices and methods for extracting proteins from samples of microdissected cells, and applying various analytic processes to the extracted proteins, such as immunoassays, ID and 2D gel electrophoresis characterization, Western blotting, Matrix Assisted Laser Desorption lonization Time of Flight (MALDI/TOF), liquid chromatography quadrapole ion trap electrospray (LCQ-MS), and Surface Enhanced Laser Desorption Ionization Spectroscopy (SELDI).
  • MALDI/TOF Matrix Assisted Laser Desorption lonization Time of Flight
  • LCQ-MS liquid chromatography quadrapole ion trap electrospray
  • SELDI Surface Enhanced Laser Desorption Ionization Spectroscopy
  • the protein content of a selected population of cells from a tissue sample is analyzed, by extracting the population of cells from the tissue sample using laser capture microdissection (LCM).
  • the population of cells that is extracted can be, for example, cells of a particular cellular substructure (such as cells from epithelium on the lumen of an organ, or pockets of cells that have undergone malignant transformation against a background of a larger population of more normal cells).
  • Proteins are isolated from the isolated population of cells, and characteristics of the proteins may be analyzed to provide information about the protein characteristics of the selected isolated population of cells.
  • differential expression of proteins in isolated malignant cells can be used to study changing patterns of protein expression during malignant transformation.
  • cells at different stages of biological transformation such as neoplastic progression from normal cells to metaplastic cells to invasive carcinoma
  • Similar methods can be used to analyze cells for therapeutic purposes (such as selecting drugs targeted against expression of particular proteins), or in drug response assays (to assess changes in protein expression as an indication of drug response).
  • Figure 1 is a relative size comparison of the laser beam diameter and the epithelial tissue sample being microdissected.
  • Laser Capture Microdissection (LCM) was performed to selectively transfer only the epithelial lining of the prostate gland to the polymer film. The 30 micron laser beam spot size is shown in relation to the thickness of the gland.
  • EP epithelial
  • LU lumen
  • stroma are as marked.
  • Figure 2 shows how cells of interest are selected using LCM and transferred onto a cap, where the proteins are solubilized directly into a tube.
  • Figures 3A and B are graphic representations of the number of molecules of PSA as compared to the number of laser shots used to harvest the cells.
  • Figure 3 A shows the results of a 500 shot sample of tumor cells serially diluted to verify sensitivity. RLU values were converted to PSA molecule numbers. Linear regression indicated an r value of 0.998.
  • Figure 4 is a table showing the number of PSA molecules, the coefficient of variation of the process, and the mean number of cells used in the immunoassays. These sensitivity and precision analyses were done on 10 separated microdissection replicates. The mean readout in RLUs is reported as well as the corresponding number of PSA molecules. The sensitivity, defined as two standard deviations above background, was one laser shot. Imprecision (%CV) was inversely correlated with number of shots per specimen.
  • Figure 5 is a table comparing the number of PSA molecules with the immunohistochemistry score. Independent immunohistochemistry scoring of coded specimens was done by a pathologist and scored as high (+ + +), medium (+ +), and low (+). PIN: prostate intraepithelial neoplasia, Tumor: invasive carcinoma, Normal: histologically normal prostate glands. The relative concentrations of PSA per cell among different progression stages varies greatly and this corresponds directly to the semiquantitative immunohistochemistry scoring.
  • Figures 6A, B, and C are 2-D or 1-D gel comparisons of the proteins in microdissected normal and tumor epithelium. Fifty thousand cells were procured by LCM, directly lysed in IEF buffer and run on a 3-10 NL Pharmacia IPG IEF strip for lOOkVhr. The second dimension runs were performed on 8-18% linear gradient SDS-PAGE gels and the gels were stained with silver.
  • Figures 6A and 6B show match tumor and normal fingerprints for each patient. A representative pi and molecular weight ruler for direct comparison and alignment is shown in panel A.
  • Figure 6C shows the alpha-tubulin immunoblot that was used to normalize for relative protein load.
  • Figure 7 is a 2-D gel comparison of microdissected normal and tumor epithelium compared to a Western blot using an anti-pan-cytokeratin type II antibody. The gels were run as described for Figure 6. The completed gel was transferred to PVDF membrane and western blotted with a 1: 1000 dilution of anti-pan type II cytokeratin.
  • Figure 8 is a 2-D gel comparison of microdissected normal, tumor epithelium, and stroma tissue indicating the differences by circling the altered proteins. The gels were prepared and run as described in connection with Figure 6.
  • Figure 9 is a 2-D gel comparison of microdissected normal and tumor epithelium as compared to whole tissue cryostat where circles indicate the locations of the "altered" proteins. The gels were prepared and run as described in Figure 6.
  • Figure 10 is a ID-gel anti-PSA Western blot of cell lysates from microdissected normal and tumor prostatectomy specimens. Lanes 1 & 3 are benign epithelium and lanes 2 & 4 are from malignant epithelium.
  • Figures 11A and B are anti-PSA Western blots, where lanes 1-4 are benign epithelium and lanes 5-8 are malignant. Lanes 1 & 5 are untreated, lanes 2 & 6 are with ACT added, and lanes 3 & 7 are incubation controls, while lanes 4 & 8 are ACT added with 120 minutes of incubation at 37° C.
  • Figures 12A and B are anti-PSA Western blots showing complexed and non-complexed species.
  • Figures 13A, B, and C show data which indicates SELDI protein profiles of LCM-derived cellular lysates are reproducible and sensitive.
  • Figure 13 A shows two separate microdissections of prostate tumor epithelium form the same tissue section from the same patient (1200) cells each) which were analyzed by SELDI protein fmgerprinting. The raw mass spectroscopic mass map is shown for each microdissection along with the Gel- View * display from the same data set.
  • Figure 13B shows two separate microdissections of prostate tumor epithelium from a tissue section from two different patients (1200 cells each) which were analyzed by SELDI protein fingerprinting. The raw mass spectroscopic mass map is shown for each microdissection along with the Gel- View * display from the same data set.
  • Figure 13C shows four separate microdissections of decreasing number of cells that were analyzed by SELDI protein fingerprinting.
  • the Gel- View * display is shown as a representation of the direct alignment of each of these four mass spectra to each other.
  • Figures 14A, B, and C show that SELDI protem profiles of LCM-derived cellular lysates are discriminatory between different tumor epithelial cell types from different patients and between tumor and normal epithelial cells from the same patient.
  • LCM derived tumor epithelial cells (1200 cells) from prostate, breast, and colon frozen tissue sections were separately acquired and analyzed via SELDI .
  • the Gel-View * representation is shown in Figure 14A and the spectrographic mass profile is shown in panel B.
  • Figure 14C shows the SELDI analysis of four separate patient-matched microdissections of 1200 cells of colon normal epithelium, colon tumor epithelium, colon tumor epithelium from the colon tumor that has metastasized to the liver, and normal liver cells next to the metastasis.
  • the Gel- View * display is shown as a representation of the direct alignment of each of these four mass spectra to each other.
  • Figure 15 is a SELDI fingerprint comparison between a variety of tumor types.
  • Figure 16 shows a device developed for solubilizing proteins in cells taken from a biological specimen by LCM.
  • Figures 17A and 17B are SELDI analyses of microdissected esophageal epithelium showing proteins disregulated in a disease-specific manner.
  • Figures 18A and 18B are SELDI analyses of 8 different esophageal cancer cases, where three separate microdissections of eight different patients' matched tumor and normal cells were subjected to SELDI analysis via the use of a hydrophobic interaction C18 binding surface. Each replicate was run in triplicate, giving a total of 72 data points for each protein peak analyzed. The analysis of the protein fingerprint in the low mass region is shown in Figure 17A, the higher mass region in Figure 17B.
  • a representative mass map from one case (case #1) is shown on the left side of each panel with the normal and tumor fingerprint shown (top and bottom, respectively) for each mass region.
  • a gel-like representation is displayed for that particular case as well as the fingerprint for two other cases.
  • Proteins 1, 2, 6, and 7 are labeled for orientation. All cases analyzed in the study set were then subjected to analysis as a ration of relative intensity of the selected proteins to one another and the statistical results sonw on the right side of each figure.
  • Figure 18 shows the SELDI analysis of prostate carcinogenesis.
  • Figure 18A shows a mass map that represents the profile from 1500 normal, pre-invasive neoplasia (PIN) and invasive carcinoma cells acquired by LCM from one case (case #2). Additionally, the corresponding patient-matched stromal cells (1500 cells) were also microdissected for analysis.
  • Figure 18B shows a gel-like image of the raw mass data shown in Panael A. All samples from this patient were run in triplicate, with the representation of one experiment shown. Two proteins, A and B, having molecular weights of 28,000 and 32,000 respectively, were found to be reproducibly differentially expressed in this patient and are indicated in both Figures 18A and 18B.
  • Figure 19 is a diagram of the multiplexed tissue array method for a high throughput target validation and drug tissue interaction assay, using microdissected human breast cells in various stages of malignancy as an example biological sample.
  • Figures 20A, 20B and 20C are representative results using colorimetric and chemiluminescent detection methods for prostate soluble antigen (PSA).
  • PSA prostate soluble antigen
  • the rows of the upper arrays are in all three cases (1) a protein standard, (2) prostate stroma, and (3) normal prostate tissue, while the lower arrays include (4) prostate intraepithelial neoplasia (PIN), (5) tumor tissue, and (6) invasive tumor tissue. The amount of total protein loaded on the gel is reduced across the rows.
  • Figure 20A shows the colorimetric results
  • Figure 20B is the positive
  • Figure 20C is the negative image of the fluorescence results.
  • Figures 21A, 21B, and 21C diagram the reproducibility of protein analysis for samples microdissected from a mixed sample of epithelial cells of the esophagus. All the following data was obtained from normal cells present in the sample.
  • Figure 21A shows the reproducibility of the annexin I protein data in normal cells over a variation of "shot" size within one slide and between multiple slide sets.
  • Figure 21B shows a histogram of the coefficient of variance for these data sets.
  • Figure 21C graphically shows reproducibility of the total protein obtained from the cells as shown by fluorescence detection.
  • Figure 22 shows the results of interaction between immobilized binding agents and biotin- labeled lysates from microdissected normal and tumor cells.
  • Laser Capture Microdissection is a recently developed technology that enables the user to obtain pure cell populations from stained heterogeneous tissue under direct, high power microscopic visualization. See Emmert-Buck et al., Laser Capture Microdissection, 274 Science 998 (1996); Bonner et al., Laser Capture Microdissection: Molecular Analysis of Tissue, 278 Science 1481 (1997).
  • the film melts and fuses with the underlying cells of choice.
  • the chosen cell(s) are tightly held within the focally expanded polymer, while the rest of the tissue is left behind.
  • the exact morphology of the procured cells is retained and held on the transfer film, ensuring preservation of the cells' intracellular components such as DNA, RNA, and proteins for future analysis.
  • the removed transfer film and cells are transferred onto a plastic cap (referred to as the LCM cap) for subsequent analysis.
  • the present methods utilize a new procedure for extracting proteins from very small sample sizes, on the order of about 1500 to about 5 cells, the number obtained in a typical LCM laser shot.
  • This procedure generally involves the extraction of proteins in one solubilizing step, using a very small volume of a unique buffer.
  • a device in which the solubilization step can be conveniently performed was developed.
  • the results of this new procedure are intact proteins, substantially free of cross-contamination from other nontumor or normal cell types.
  • the isolated proteins maintain activity, allowing analysis through any number of immunological and biochemical assays.
  • the buffers for the protein isolation step can include one or more of buffer components, salt, detergents, protease inhibitors, and phosphatase inhibitors.
  • one effective buffer for extracting proteins to be analyzed by immunohistochemistry includes the buffer Tris-HCl, NaCl, the detergents Nonidet ® P-40, EDTA, and sodium pyrophosphate, the protease inhibitors aprotinin and leupeptin, and the phosphatase inhibitors sodium deoxycholate, sodium orthovanadate, and 4-2-aminoethyl benzenesulfonylfluroride (AEBSF).
  • AEBSF 4-2-aminoethyl benzenesulfonylfluroride
  • Another salt which could be used is LiCl, while glycerol is a suitable emulsifying agent that can be added to the fraction buffer.
  • Additional protease inhibitors include soybean trypsin inhibitor and pepstatin.
  • Other suitable phosphatase inhibitors include phenylmethylsufonyl fluoride, sodium molybdate, sodium fluoride, and beta-glycerol phosphate.
  • Triton-X-100 Triton-X-100, a detergent (Sigma, St.
  • a preferred 1:100 concentration buffer is as follows:
  • the buffer is made and diluted 1: 100 in distilled water for use.
  • the buffer must be kept frozen at -20°C. It can only be used unfrozen for a few hours. In all cases, the buffer is used in very low volumes, from about 1 ⁇ l to about 15 ⁇ l, and is applied directly to the laser capture dissected cells while still on the LCM cap.
  • This device includes a small chamber 1 where the solubilization process can occur.
  • the chamber has an upper opening 2 structured such that it can directly accept the LCM cap with its adhered cells.
  • the LCM cap is placed in the chamber 1 with the surface having cells upon it facing the interior of the chamber. Once the LCM cap is attached to the device, the cells are therefore strategically positioned within the chamber and solubilization of the proteins will occur upon introduction of the solubilization buffer.
  • the chamber is supplied with at least one inlet port 3, which can be equipped with a syringe 4 as a means of introducing the small volume of the solubilizing buffer into the chamber 1.
  • the inlet port 3 is connected to the chamber by a narrow inlet canal 5, where liquids placed into the canal will move toward the solubilization chamber by capillary action.
  • the device can include more than one inlet chamber, if desired, each equipped with a means of introducing liquid, such as a syringe.
  • the device also includes an outlet port 6, connected to the chamber 1 with a narrow outlet canal 7, which can be supplied with a means of collecting and removing the solubilized proteins.
  • One embodiment of this device has the chamber 1, the inlet ports 3, inlet canals 5, outlet port 6, and outlet canal 7, all machined in a hard plastic block, such as lucite.
  • a soluble immunoassay where an antibody specific for a protein of interest is used.
  • the antibody can be labeled with a variety of markers, such as chemiluminence, fluorescence and radioactivite markers.
  • the assay used should be of high sensitivity, such as a microparticle enzyme immunoassay (MEIA).
  • MEIA microparticle enzyme immunoassay
  • the presently described methods provide a quantitative immunoassay, which can measure the actual number of the protein molecules of interest in vivo.
  • a second type of assay that can be used to analyze the extracted proteins is two- dimensional polyacrylamide gel electrophoresis (2D PAGE).
  • differential protein expression By running both proteins extracted from normal cells of the sample and proteins extracted from tumors cells of a sample, and comparing the blots, differential protein expression can be seen.
  • the location of proteins that are present in one cell type and absent in the other can be determined.
  • these altered proteins can be isolated from the gel where they are present, and mass spectroscopy MS-MS sequencing can be used to identify the protein, if the sequence exists in a database. In this way, the protein differences between normal and tumor cells can be more fully understood.
  • proteins of interest isolated from a 2-D gel may be used in binding studies, where the protein is functionally tested for an alteration in the ability to bind with a putative or known ligand.
  • this comparative analysis need not be between normal and tumor cells, but can be between isolated stages of a tumor, where the different stages exhibit sufficient morphological differences to allow separate isolation of populations using the LCM technique.
  • a further analysis that may be performed involves the use of the surface enhanced laser desorption ionization spectroscopy technique, or SELDI (Ciphergen Biosystems Inc., Palo Alto, CA). This process can separate proteins which would not be separately focused by 2-D gel analysis, in particular those proteins which are very basic, very small ( ⁇ 7000 daltons) or are expressed at low or moderate levels in the cells. The lower level of expression becomes critical in these experiments because of the extremely small sample size of cells used. SELDI also separates proteins more rapidly than gel analysis.
  • SELDI utilizes a "protein chip” that allows for desorption and detection of intact proteins at the femtomole levels from crude samples. Proteins of interest are directly applied to a defined small surface area of the protein chip formatted in 8 to 24 predetermined regions on an aluminum support. These surfaces are coated with defined chemical "bait" matrices comprised of standard chromatographic supports, such as hydrophobic, cationic, or anionic or biochemical bait molecules such as purified protein ligands, receptors, antibodies, or DNA oligonucleotides. See Strauss, New ways to probe the molecules of life, 282 Science 1406 (1998). In the case of LCM collected samples, the solubilized proteins are applied to the surface of the SELDI chip.
  • Binding of the proteins to the surface is dependent on the nature of the bait surface and the wash conditions employed.
  • the mixture of bound proteins is then characterized by laser desorption and ionization and subsequent time of flight mass analysis generated from a sensitive molecular weight detector. This data produces a protein fingerprint for the sample, with SELDI having a practical resolution and detection working range of 1000 to 300,000 daltons, depending on the energy absorbing molecule utilized and the bait surface/wash conditions employed.
  • the produced protein fingerprints have proven to be both disease-specific and organ- specific. That is to say, the protein fingerprint of a tumor from a particular tissue type remains characteristic of that tissue type, whether it is normal, tumor, or a metastasis. This allows a determination of the origin of an unknown metastasis to be made, through comparison to protein fingerprints of normal or tumor tissue of likely organ sources.
  • Tissue was obtained following an IRB approved protocol from both the Urologic Oncology Branch in the National Cancer Institute, Bethesda, MD and the Mayo Clinic in Rochester MN. After surgery, the tissue samples were snap frozen in liquid nitrogen. The tissue was then embedded in Optical Coherence Tomography (O.C.T.) compound (Tissue Tek, Miles, Elkhart, IN) and stored at -80°C. Cases were selected based on the histology present in the tissue sections so that normal glands, Prostate Intraepithelial Neoplasia (PIN), and adjacent carcinoma, could be compared within the same patient. Prostate tissue cases were selected to include ample stroma to serve as a negative control. Lung tissue was used as a second negative control.
  • O.C.T. Optical Coherence Tomography
  • the O.C.T embedded tissue blocks were cut into 8 ⁇ m sections with a cryostat. After cutting, the sections were immediately placed on dry ice and then stored at -80°C. Only one section was thawed and dissected at a time, to minimize degradation of proteins. After fixation in 70% ethanol for 10 s, the section was stained with hematoxylin and eoxin, and dehydrated in xylene.
  • Laser Capture Microdissection The PixCell system incorporates an Olympus IX-50 Microscope containing a microscope slide stage which is moved by a joystick. The operator uses the joystick to position the tissue under a fixed laser beam that can be focused from 5 to 60 ⁇ m in diameter.
  • the LCM transfer film is fixed to the undersurface of a vial cap (CapSure * TF- 100 transfer film carrier, 5mm dia. optical grade transparent plastic; matching vial is Brinkmann #22 36 430-8).
  • LCM cell procurement time was always less than 15 minutes.
  • a droplet of the extraction buffer was applied to the surface of the film containing the selected cells.
  • the cap with the droplet on its undersurface was inserted into the mouth of the matching vial containing 50 ⁇ l of the immunoassay dilution buffer.
  • the sealed vial sample receptacle was frozen at -20°C and stored for less than 48 hours prior to assay. The thawed sample was held at 4°C for no longer than 2.5 hours prior to introduction into the immunoassay module.
  • Figure 1 compares the size of a 30 ⁇ m laser shot with the size of an example prostate gland that was microdissected by movement of the joystick.
  • the yield of procured pure epithelial or carcinoma cells, and the precision of cellular procurement, were tested by visually counting the number of cells removed from the tissue and transferred to the film.
  • Table 1 compares the number of laser shots with the average total number of microdissected cells.
  • a standard laser spot diameter of 30 ⁇ m encompasses 5-7 cells. Individual tissue cells can vary in their packing density, their shape and their volume. Consequently, the imprecision of the cell yield is greater with fewer laser shots. Solubilization of proteins from LCM procured cells
  • the final buffer composition was a 1/1000 dilution of the following stock: 50mM Tris HC1, 1% NP-40, 0. 1% Na Deoxycholate , 150mM NaCl, 4mM EDTA, Aprotinin (lOmg/ml), Leupeptin (lOmg/ml), Na Pyrophosphate (lOmM) Na orthovanate (2mM), and AEBSF (lOOmM). Assay Principle
  • the Immulite Third Generation PSA assay (Diagnostic Products Corp., Los Angeles, CA) was adapted to measure PSA solubilized from tissue cells procured by LCM. Photon production is measured by a luminometer (output: relative light units (RLUs)). Negative tissues or zero controls produced a value of 80140,000 RLUs, compared to PSA positive samples that ranged from 600,000 to 10,000,000 RLUs.
  • PSA immunoassay Microparticle Enzyme Immunoassay (MEIA)
  • PSA was measured by an automated two site chemnuminescent assay, using the ultrasensitive PSA reagent kit on the Immulite immunoassay analyzer (Diagnostic Products Corp., Los Angeles, CA). RLUs reflect the photons detected by the photomultiplier tube, which is proportional to the concentration of PSA.
  • the sensitivity of the assay is .004 ng/mL of PSA, or approximately 4 x 10 6 molecules of PSA per assay.
  • Immunohistochemistry staining for PSA was conducted as follows. The frozen sections were desiccated and then fixed in acetone. Following a wash with 3% goat serum, primary antibody incubation was 1 hour at room temperature. The secondary antibody was labeled by Avidin/Biotin and the substrate system was Peroxidase/DAB. The secondary reaction was performed essentially as suggested by the commercial product insert. Calibration Curve
  • the calibration curve used for measurement of PSA in serum according to the Immulite package insert was applied to the measurement of cellular tissue proteins procured by LCM.
  • the calibration curve designed for serum was reproduced with the LCM extracted cellular proteins in the buffer solution.
  • Serum or buffer samples with a known concentration of PSA were mixed with equal volumes of 100 laser shots of PSA negative tissue cells. There was approximately 100% recovery in all cases, with no detectable elevation of the zero dose or diminution of the signal.
  • the number of laser shots of actual tissue cells was compared to the calculated number of PSA molecules for individual patient cases as shown in Figure 3. Linear regression analysis yielded an r value of greater than 0.95 over a dynamic range of 0.004 ng/mL to 1 ng/mL. Sensitivity
  • the detection limit of the assay for microdissected cellular proteins was 0.004 ng/ml PSA as defined as the concentration two standard deviations above the signal response of a sample free of PSA or a PSA negative tissue.
  • the sensitivity achieved with this criteria was one 30 ⁇ m laser shot (5-7 cells). Imprecision
  • the immunohistochemistry scoring values paralleled the quantitation and mirrored the heterogeneity in PSA production by normal and neoplastic cell populations.
  • the normal epithelium contained 6.3 x 10 6 molecules and scored three plus by immunohistochemistry.
  • the PIN cells contained 3.7 x 10 5 molecules and scored two plus
  • the tumor cells contained 1.99 x 10 ⁇ and stained one plus.
  • Application of this new technology provides quantitative confirmation of the heterogeneity in PSA expression that was previously detected only by qualitative staining.
  • the average numbers of PSA molecules harvested per cell ranged over several logs. An immunohistochemical staining difference could be discriminated only if there was greater than a ten-fold difference in PSA molecule number per cell.
  • the present technology provides one of the first direct estimates of the actual number of protein molecules per tissue cell in vivo for a single specific known protein of moderate to low abundance.
  • the number of total PSA molecules in normal prostate epithelium ranged from 10 4 to 10 6 .
  • PSA is an important serum analyte used to clinically monitor prostate cancer, but it is not a specific maker of prostate cancer.
  • Previous investigators have reported great heterogeneity in the intensity of PSA immunohistochemical staining among various neoplastic and non-neoplastic populations in the prostate. Populations of microdissected cells of a pure histologic class would be expected to contain some level of heterogeneity in PSA expression among the population members, as presently seen.
  • LCM Pancell 100, Arcturus Engineering, Mountain View, CA
  • AEBSF Boeringer Manheim
  • IEF lysing solution containing 7M Urea, 2M Thiourea, 4% CHAPS, 1 % MEGA-10, 1% Octyl-b-Glucopyranoside, 40 mM Tris, 50 mM DTT, and 2 mM tri-butyl phosphine (TBP) and 0.5% (v/v) Pharmalytes was applied directly to the microdissected cells adhered on the LCM cap, placed into an eppendorf tube and vortexed vigorously for one minute until all cells were completely lysed.
  • TBP tri-butyl phosphine
  • the IEF lysing solution was then re-applied to another cap containing cells from the same microdissected material and the procedure repeated until each 100 ⁇ l contained lysate from 50,000 cells (approximately 7000 LCM transfer pulses). 2D PAGE and Image Analysis
  • First-dimension isoelectric focusing was carried out on a Pharmacia Immobiline IPG Dry-strip system essentially as described by the manufacturer. Pre-cast immobilized pH gradient strips (18 cm, 3-10 non-linear) were employed for the first dimensional separation for a total focusing time of 120kVh. The strips were re-equilibrated with a solution containing SDS and Tris pH 6.9, reduced with TBP (2 mM), alkylated with iodoactemide (2.5% w/v), and directly applied to a 8-18% linear gradient SDS-PAGE gel for electrophoresis overnight at 40 volts constant voltage.
  • the gels were stained with silver and direct scanning and image analysis was performed using an Umax scanner with Adobe Photoshop software and a Tektronix IISDX photographic-quality printer. Scanned images were analyzed and compared using the MELANIE II software package (BioRad). Comparison of protein fingerprints were performed using images representing protein spots readily apparent by direct visualization. Only spots that were present/completely absent between normal-tumor cells were defined as altered. Each experiment was performed in duplicate and produced similar results (data not shown). Normalization of sample load was by anti-alpha -tubulin immunoblot analysis prior to the first-dimension run. Scoring of the blots included comparison of multiple exposure times. Analysis of alpha - tubulin
  • IEF lysate Ten ⁇ l of the IEF lysate was diluted 1:1 in 2X SDS sample buffer, boiled for 5 minutes, and applied to a 4-20% NO VEX Tris-glycine SDS gel and electrophoresed for 1 hour. Immunoblotting was performed for 1.5 hour using a BIO-RAD Semi-dry blotting apparatus with Immobilon PVDF membrane as the capture surface. Blots were blocked with IX TBS containing
  • Two 2D gels were run simultaneously, both containing identical amounts of lysates of microdissected tumor from case #1.
  • One gel was silver stained and the other was immunoblotted to PVDF membrane as outlined above.
  • 2D western blot analysis was performed as described earlier except antibody to type II cytokeratin or annexin I was used as probe.
  • Anti-pan type II cytokeratin antibody was purchased from SIGMA and used at a final dilution of 1 : 1000.
  • Anti-annexin I antibody was purchased from Transduction Labs and used at a final dilution of 1:5000.
  • a male BALB C mouse was sacrificed with immediate surgical excision of the liver.
  • One piece was embedded in OCT compound (Tissue Tek, Miles, Elkhart, IN) and immediately frozen on dry ice.
  • Two pieces were fixed in 70% ethanol containing proteinase inhibitor (Complete, Mini Boehringer Mannheim Corp., Indianapolis, IN) for 1 hour at room temperature.
  • One of these pieces was then embedded and blocked in paraffin wax and the other in polyester wax (Gallard-Schlesinger Industries, Inc., Carle Place, NY).
  • the polyester wax embedding the tissue was dehydrated in 90% ethanol at 4° C for 1 hour followed by 99% ethanol at 4° C for 1 hour, then by 100% ethanol at room temperature for 1 hour.
  • proteins were scored as "altered” only if there was a clear-cut "on-off ' ' difference between the comparison groups. No quantitative evaluation was made of the level of those proteins which were present in both sample groups and showed subtle differences in levels.
  • Immunoblot analysis of alpha-tubulin using a small aliquot of each sample was used to verify that equal amounts of total protein were analyzed from each dissection (Figure 6C).
  • microdissected normal epithelium and tumor samples from case #2 were subjected to 2D PAGE separation and immunoblot analysis using an antibody directed against a human pan-type II cytokeratin, and an antibody against annexin I.
  • Figure 7 shows the results confirming the identity of the protein up-regulated in tumor as cytokeratin 1.
  • the identity of the protein down-regulated in tumor as annexin I was similarly confirmed (results not shown).
  • protein profiling may more easily detect certain types of alterations than genomic or expression-based approaches, for example, a tumor suppresser gene mutation which results in protein truncation. Therefore, high-throughput protein studies will be an important component of future efforts to determine the molecular anatomy of normal and diseased cells.
  • Dysregulated proteins of high or moderate abundance will have sustained utility for basic research and clinical applications as they will be the easiest to detect, study, and monitor.
  • proteomic studies of microdissected normal and tumor cells will also benefit from increased sensitivity enabling a larger percentage of the cellular proteins to be analyzed.
  • protein labeling with (for example) 125-1 labeling or biotinylation dramatically increases the number of proteins visualized from microdissected cells (data not shown).
  • scanning immunoblotting with class-specific antibodies allows for more sensitive detection of specific subsets of proteins, for example, all known proteins involved with cell cycle regulation.
  • the small amount of material analyzed is not ideal for obtaining highly sensitive protein fingerprints, and identifying proteins of interest.
  • a useful strategy is to produce parallel "diagnostic" and “sequencing” 2D gels from each case.
  • the diagnostic fingerprints are derived from microdissected cells and provide maximal sensitivity for detection of normal-tumor differences. Sequencing fingerprints then allow for determination of protein identity.
  • the sequencing 2D gels are generated from serial, whole tissue section cryostat recuts which contain abundant amounts of protein representing all cell types present in the tissue, including the dissected cell population(s). Alignment of the diagnostic and sequencing 2D gels permits determination of proteins of interest for subsequent mass spectrometry or N-terminal sequence analysis.
  • the present invention can be used to provide interpatient analyses of both normal and diseased cells to help reveal patient-unique protein profiles related to disease susceptibility or progression.
  • the esophageal specimens utilized in this study were frozen in liquid nitrogen shortly after surgical resection and histologic tissue sections were prepared on a cryostat. Frozen sections are often used for molecular analyses of tissue due to the relatively high quality DNA, RNA, and proteins which can be recovered. Tissue processed through standard formalin fixation and paraffin embedding is less advantageous due to the molecular cross-linking which occurs during fixation and the prolonged exposure to elevated temperatures during embedding.
  • anti-alpha tubulin antibodies used at 1:1000, and HRP-coupled rabbit anti-mouse secondary antibodies, used at 1:10,000 were purchased from Sigma (St. Louis, MO). Blots were washed using conditions described above and ECL substrate (Amersham, Piscataway, NJ) was added for chemiluminescent detection via autoradiography on Kodak Bio-Max film.
  • ECL substrate Amersham, Piscataway, NJ
  • anti-PSA antibodies were purchased from Scripps Laboratories, San Diego, CA).
  • PSA was not detected in normal cells, PC3, or a tumor cell line developed from the in vivo dissected tumor cells.
  • LnCaP expressed PSA, but there was an alteration in the migration, reflective of a change in the qualitative change in the protein.
  • Frozen tissue was obtained from radical prostatectomy specimens and embedded in OCT compound (Tissue-Tek, Miles, Elkhart, IN). Eight micron sections were made with a standard cryostat and stained with hemotoxylin and eosin using standard protocols. Benign and malignant histology was identified by a pathologist and LCM was performed to obtain cells from each population by directing the laser at those populations of cells. LCM was perfomred as previously described, except AEBSF (Boeringer Manheim) was added to the staining baths at a final concentration of 2 mM to inhibit proteases.
  • OCT compound Tissue-Tek, Miles, Elkhart, IN. Eight micron sections were made with a standard cryostat and stained with hemotoxylin and eosin using standard protocols. Benign and malignant histology was identified by a pathologist and LCM was performed to obtain cells from each population by directing the laser at those populations of cells. LCM was perfomred as previously described, except AEBSF
  • 2X SDS buffer was used to lyse cells directly from the EVA film (from the LCM cap) and the lysates were run on 20% tris-glycine nondenaturing gels and transferred to a nylon membrane using the Novex system.
  • a murine monoclonal antibody purchased from Scripps Laboratories was used as the primary antibody at a concentration of
  • the protein was transferred to a nylon membrane and western blot performed using previously described reagents. Twenty 8 micron cryostat sections containing both malignant and benign epithelium were suspended in EF buffer and analyzed by 2D PAGE and western blotting as just described. PSA/ACT binding studies.
  • PSA is a serine protease that is produced as an inactive zymogen and then activated by release of a signal peptide of 17 amino acids followed by liberation of a 7 amino acid propeptide.
  • the catalytically active form of PSA is highly glycosylated with a molecular mass of approximately 30 kd.
  • the analyte was allowed to concentrate by air drying followed by the application of 0.3 ml of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, 98%, Sigma, St. Louis, MO) as the energy absorbing molecule of choice for all experiments in this study.
  • the results of this experiment are displayed in two of several possible formats offered by the SELDI software analysis package.
  • the first representation is a standard chromatographic mass map with the respective molecular weight range displayed on the x-axis. Each "peak” represents a protein isoform with a different molecular weight.
  • the second representation is a Gel- View * display which takes the peak data from the mass-map chromatogram and presents the data as if one is looking at a standard ID PAGE gel "stained" for proteins, with the molecular weight ranges displayed at the same scale as that seen in the chromatogram.
  • the protein profiles of the lysates of separate microdissected regions of the same tumor are nearly identical. These results indicate that reproducible protein profiles are generated from LCM-derived cells. Similar experiments were repeated several times with identical results (data not shown).
  • prostatic tumor epithelium from two different patients was dissected (1200 cells per dissection) and analyzed as above. These results are shown by both massmapping and Gel- ViewTM display in Figure 13B, and show that the protein fingerprint generated from case 1 and case 2 is highly reproducible. Similar results were obtained with multiple samples from the two cases (data not shown). The minor differences in protein expression patterns observed between case 1 and case 2 were no more variable than that seen between microdissections from the same case (data not shown).
  • lysing buffer alone, or LCM with a blank glass slide were analyzed.
  • the Gel-View * display normalizes the relative intensities of each separate spectra and does not reflect what was seen in the mass map; that is, as the total number of cells decreases, so does the relative intensity of the peaks seen (data not shown).
  • Diagnosis and prognosis from the limiting amounts of cells generated by fine needle aspirants or sentinel node analysis could be achieved very rapidly if a tumor-specific or grade-specific profiles could be generated and used as a template.
  • SELDI protein fingerprinting of colon cancer with liver metastasis The ability to assess changes in protein expression occurring during tumor progression will aid in the elucidation of the fundamental mechanisms underlying carcinogenesis in patients.
  • SELDI analysis of LCM derived cells To investigate the potential of SELDI analysis of LCM derived cells to study this process, we analyzed the colonic normal epithelium, primary cancer, and hepatic metastasis from one patient. As a comparison, we analyzed the normal liver cells, which were microdissected from the same case. The results are shown in Figure 14C, and show that a normal epithelial cell and tumor epithelial cell-specific fingerprint can be identified.
  • the protein profile from the colon tumor that had metastasized resembles the colonic epithelium, regardless of tumor or normal cell state, and not the liver cell protein profile.
  • the cells from the metastasis have their own distinct protein fingerprint. This uniqueness may arise from the fact that the metastatic tumor has changed its expression profile as a result of its new environment, or it may reflect protein expression changes that enabled the cells to metastasize, or a combination of both.
  • a direct application of this type of analysis would be to identify protein profiles of primary tumors which predict that a tumor has likely metastasized indicating a need for more aggressive patient treatment and follow-up.
  • tissue arrays The ability to compare protein content of finite cell samples as described above can be applied to high throughput assays utilizing tissue arrays.
  • tissue array techniques that can be used with the protein extraction and analysis methods of the present invention are disclosed in PCT publication W09944063 and W09944062, whose disclosures are hereby incorporated by reference.
  • the present methods in a tissue array format are useful for screening potential therapeutic agents and analyzing their impact on very small subsets of cells.
  • the methods are used to analyze the impact of therapeutic agents on specific cellular pathways, such as signaling pathways. These results can, for example, indicate the efficacy or toxicity of a particular agent on the cell.
  • the tissue from multiple patients is exposed to the agent to be tested.
  • the exposure can be done in vivo, prior to collection of the sample, or in vitro, after collection of the sample and/or after laser microdissection.
  • In vivo exposure would involve administration of the agent to the subject, while in vitro administration could be adminsitration to cultured cells.
  • Some agents that could be tested include pharmacological agents, imaging agents, labeled proteins, such as ligands, or other agents known to have particular effects on cells, such as cytokines.
  • microdissection techniques are used to isolate the cells of interest from the sample, the cells are lysed to allow isolation of the proteins or other cellular components, such as nucleic acids or other subcellular structures, from the sample, and the lysate contents are transferred to a confined zone of a substrate.
  • the ly state contents, or cellular components are placed in identifiable positions on a substrate, where such positions are confined zones.
  • a confined zone is the coordinates of an array.
  • the array is constructed by applying microspots of the isolated proteins on any suitable matrix, such as nitrocellulose, nylon, or silica.
  • the microspots are arranged on the matrix in any manner that produces meaningful data.
  • the micropsots can be placed on the matrix using, for example, a micropipette, and examples of the substrate include a glass or plastic slide, a section of embedding medium, or a nitrocellulose matrix.
  • the microspots can be arranged in the y- dimension by patient number and in the x-dimension by a criterion for categorizing the various samples obtained, such as by stage of malignancy. Other alternative criteria include before and after treatment samples, various cells types, such as epithelial and stromal, or stages of development for embryonic samples.
  • microspots are then subjected to some type of analysis, for example, to determine if an amount of a particular protein is altered.
  • analysis can include probing with an antibody as the binding agent.
  • Alternative analyses include probing with other binding agents such as nucleic acids, labeled or unlabeled DNA or RNA, or aptameric or phage display screening.
  • a consistent alteration in the cellular content of a protein is then correlated to exposure of the tissue or cells to the agent of interest.
  • the matrix can be a general capture matrix, such that all isolated proteins from the sample are present, or embedded with a specific binding agent that would result in the maintenance of only proteins of interest.
  • the microdissected proteins in a soluble form can be labeled, for example with a radioactive or fluorescent tag, and capture by the binding agent is detected by the presence of the label.
  • the protein spots themselves can act as a binding agent or attractant for an interaction, such as detection of a labeled target or ligand within the protein spot after treatment with a labeled test solution. Because of the method of isolation of the proteins, the test is highly sensitive, as each spot may be the protein extracted from a pure population of cells from an individual tissue, or a combination of particular populations of interest.
  • tissue arrays can include a body tissue set, which allows comparison of the results obtained from the experimental tissue set to samples from other tissues of the patient's body.
  • FIG. 19 A schematic drawing illustrating a sample tissue array for examining protein content of breast tissue of multiple patients is illustrated in Figure 19.
  • the tissue from multiple patients is used to examine the effect of a particular therapeutic agent administered to a subject on protein content of breast tissue in various stages of malignancy, from normal tissue to stromal invasion.
  • These tissues are microdissected from a tissue specimen using LCM. Because of the sensitivity of the test, detailed questions about effects on the level of cellular biochemical pathways in each stage subset can be obtained.
  • tissue array technology to the methods of the present invention is the use of the protein content assay to prescreen various tissue types for the target of diagnostic or therapeutic moieties.
  • a therapeutic or diagnostic agent is being evaluated for its appropriateness in a patient's treatment. If the target of this agent is known, the presence or absence of this target in various cell types of the patient can be tested.
  • the breast tissue of Figure 19 as an example, if it was questionable whether a target enzyme for a chemotherapeutic agent was present in a particular patient's cancerous cells, the samples are generated and placed in an array as illustrated. Then an antibody or other means of identification of the target is used to determine if the target agent is present. This allows diagnostic, imaging, or therapeutic agents to be specifically selected for patients or disease types based on the presence of the target molecule in the diseased tissue.
  • tissue may, for example, be preserved by fixation in 70% ethanol and embedded in a paraffin block.
  • the paraffin block is cut in 5 to 10 um sections and mounted on microscope slides.
  • the tissue on these slides is deparaffinized by placing the slide in room temperature xylene for 5 minutes, followed by xylene heated to 70°C for 5 minutes.
  • the slide is next placed in acetone for 10 seconds, Diff Quick Solution II (Dade Behringer) for 5 seconds, and acetone for 10 seconds.
  • the stained slide is allowed to rinse in xylene for a rninimum of 30 seconds to dehydrate.
  • Cells of interest are dissected from the slide by LCM using the PixCell II (Arcturus Engineering). Using the 30 ⁇ m spot size, 3000 laser pulses are fired to collect cells from a subcellular structure of interest, and the cells are embedded in a thermoplastic polymer film supported on a plastic cap.
  • An extraction buffer consisting of one part SDS sample buffer (Novex) with 4% BME and one part T-PER (Pierce) is heated to boiling.
  • a 40 ⁇ l aliquot of the hot extraction buffer is added to a standard 500 ⁇ l microcentrifuge tube. The cap containing the tissue embedded on the thermoplastic film is inserted into the tube and inverted to allow the extraction buffer to cover the tissue.
  • the inverted tube is vortexed for 20-30 seconds to aid in the disruption of the cells and release of the cellular components.
  • the tube and cap assembly is placed in a 70°C incubator for overnight incubation. After the incubation, the tube is once more vortexed in an inverted position, after which it is turned upright and centrifuged for 10 seconds. Dilutions are made from the resulting lysate using T-PER. A minimum of 10 ⁇ l of lysate or dilution is placed in the bottom of a well of a 96-well round- bottom microtiter plate.
  • This plate is positioned on the stage of Genetic Micro Systems 417 Arrayer. Oncyte Slides (Schleicher & Schuell) covered in pure nitrocellulose are placed in the appropriate positions on the arrayer. After the desired number of slides and replicates has been programmed into the accompanying software, the instrument is activated. A metal micro tube lowers into the appropriate well and lifts 1 ⁇ l of the lysate, trapping it inside the bore of the tube. After positioning the tube over the nitrocellulose slide, a pin pierces the trapped lysate, spotting a minute aliquot onto the nitrocellulose surface measuring 500 ⁇ m in diameter.
  • the Western-Star Chemuuminescent Detection System (TROPIX, Inc., Bedford, MA) is an immunoblot detection system designed for membrane-bound proteins.
  • the protocol outlined for nitrocellulose membranes is followed for the Oncyte slides. After the nitrocellulose slide is prepared on the arrayer and dried, it is submerged in I-Block blocking buffer (provided in the kit) prepared as directed, and agitated on an orbital mixer for at least 1 hour.
  • the primary antibody of choice is diluted of the recommended concentration using I-Block buffer.
  • the slides are drained after the blocking step and the diluted primary antibody is poured over the slides and for incubation, with agitation, overnight at 4°C.
  • the slides are washed with agitation in a IX PBS/0.1% TWEEN washing buffer, one time for 15 minutes and twice for 5 minutes.
  • the secondary antibody which is conjugated to alkaline phosphatase is diluted in I-Block blocking buffer and poured over the slide. It is allowed to incubate for 4-5 hours at room temperature on an orbital mixer. At the end of this incubation, the slide is washed with PBS washing as described above.
  • the 10X Assay Buffer (provided in the kit) is diluted with deionized water and the slide is incubated twice for two minutes submerged in the IX Assay Buffer. The slides are blotted dry on a piece of tissue paper.
  • Nitro-Block-II (provided in the kit) is prepared using the CDP-St ⁇ r Ready-To-Use substrate solution. This substrate solution is pooled on the slide and allowed to sit undisturbed for 5 minutes. At the end of this incubation, the slide is blotted and placed in a plastic development folder, after which it is exposed to x-ray film. Representative results using this system for analysis of the content of prostate soluble antigen in prostate tissue is shown in Figures 20B and 20C. System Two
  • the second illustrative example uses the DAKO Autostainer Universal Staining System (DAKO Corporation, Carpinteria, CA).
  • This automated slide processing system uses a primary antibody followed by the addition of a patented horse-radish peroxidase labeled polymer (DAKO EnVisionTM System). This is an example of a colorimetric-detectable labeled antibody.
  • the prepared Oncyte slide is blocked in I-Block (TROPIX, Inc.) for a minimum of 1 hour.
  • the DAKO Autostainer is programmed with the incubation times and volumes of the reagents to be dispensed on the slides.
  • the nitrocellulose slide is washed with a Wash Buffer wash (prepackaged by
  • the primary antibody diluted as specified for the antibody being used, is applied in the predetermined amount of 600 ⁇ l, and allowed to incubate for 30 minutes. At the end of the incubation, the slide is washed with 600 ⁇ l of the Wash Buffer. This step is followed by the addition of the HRP labeled polymer for 10 minutes, followed by washing with the Wash Buffer. The substrate is added to the slide and allowed to sit for 5 minutes, followed by a final wash, and is then allowed to dry for optimal visualization. The positive results are visible staining of the spotted proteins on the slide. Representative results using this system for analysis of the content of prostate soluble antigen (PSA) in prostate tissue are shown in Figure 20A.
  • PSA prostate soluble antigen
  • the tissue array method for analyzing protein content of small sample sizes is very reproducible, even when applied to very small numbers of cells.
  • Experiments to examine the reproducibility of the technique were done using five separate slides of normal esophageal tissue, and detection for annexin I was performed.
  • the size of the samples varied in the number of microdissection "shots," from a high of 500 shots to a low of 16 shots.
  • Samples of varying cell numbers were collected, the proteins were isolated as described above, and the isolated proteins were placed on a matrix of nitrocellulose paper.
  • the microspotted proteins were probed using the antibody systems described above. The results of these tests are diagrammed in Figures 21A, 21B, and 21C.
  • Figure 21A indicates that the results from each data set (average of triplicate results for each slide) remains consistent within the set. As is expected, the amount of variance increases with the smaller number of "shots" included in the sample. However, as indicated graphically in Figure 21B, the results for multiple samples containing as few as 125 shots varies only by 18%. These results indicate that this method of analyzing protein content of small tissue samples is reliable. The results shown in Figure 21C also support this conclusion and indicate that the relative value between the various series remains generally consistent between samples of varying numbers of shots. In summary, the results indicate that decreasing the number of "shots" in a sample affects only the quantitative, not qualitative, character of the results.
  • Example results of method of present invention where the binding agent has been immobilized, and the cell lysates placed in contact with the immobilized agent are shown in Figure 22.
  • lysates from microdissected normal and tumor esophageal cells were labeled with biotin.
  • An array of binding agents, including anti-annexin I, anti-alpha tubulin, anti- phospho ERK, and mouse IgGl, and normal rabbit serum were placed on nitrocellulose paper.
  • Biotin labeled lysates were placed in contact with the binding agent arrays, and the interaction was detected by avidin-peroxidase.
  • the tumor lysates contained a reduced concentration of annexin I, and an increased concentration of phospho ERK when compared to the normal cell lysates.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

La présente invention concerne des dispositifs et des procédés d'analyse protéique de cellules ayant subi une microdissection par capture au laser, permettant l'analyse protéomique de cellules provenant de populations variées. Des exemples particuliers de cette invention se rapportent à l'analyse de cellules normales par rapport aux cellules malignes, ou à la comparaison de l'expression protéique différentielle dans des cellules évoluant de l'état normal à l'état malin. La teneur protéique des cellules microdisséquées peut être analysée avec des techniques comme le dosage immunologique, la caractérisation par électrophorèse unidimensionnelle et bidimensionnelle en gélose, le 'Western blotting', l'électronébulisation à piégeage d'ions quadrupôles par chromatographie liquide (LCQ-MS), la désorption-ionisation par impact laser assistée par matrice/ temps de vol (MALDI/TV), la spectroscopie à ionisation/désorption avec exaltation de surface (SELDI). Par ailleurs, en plus de permettre la comparaison directe entre la teneur qualitative et quantitative protéique des cellules tumorales et normales provenant d'un même prélèvement tissulaire, ces procédés permettent également la recherche des caractéristiques protéiques des cellules tumorales, comme par exemple la capacité de liaison et la séquence d'acide aminé, et l'expression différentiée des protéines dans des populations de cellules particulières en réponse à un traitement par des médicaments. Grâce aux empreintes protéiques, ces procédés constituent un moyen efficace et rapide d'identification du tissu responsable de métastases.
EP00911845A 1999-02-16 2000-02-16 Procedes et dispositifs d'isolation et d'analyse de la teneur proteique des cellules Withdrawn EP1153300A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US12028899P 1999-02-16 1999-02-16
US120288P 1999-02-16
PCT/US2000/004023 WO2000049410A2 (fr) 1999-02-16 2000-02-16 Procedes et dispositifs d'isolation et d'analyse de la teneur proteique des cellules

Publications (1)

Publication Number Publication Date
EP1153300A2 true EP1153300A2 (fr) 2001-11-14

Family

ID=22389357

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00911845A Withdrawn EP1153300A2 (fr) 1999-02-16 2000-02-16 Procedes et dispositifs d'isolation et d'analyse de la teneur proteique des cellules

Country Status (5)

Country Link
EP (1) EP1153300A2 (fr)
JP (1) JP2002537561A (fr)
AU (1) AU772680B2 (fr)
CA (1) CA2359649A1 (fr)
WO (1) WO2000049410A2 (fr)

Families Citing this family (62)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003534533A (ja) * 2000-02-08 2003-11-18 ザ リージェンツ オブ ザ ユニバーシティ オブ ミシガン タンパク質のディファレンシャルディスプレイのマッピング
CN1426536A (zh) * 2000-03-20 2003-06-25 东弗吉尼亚医学院 前列腺癌标记
BR0111742A (pt) 2000-06-19 2004-02-03 Correlogic Systems Inc Método heurìstico de classificação
EP1386275A2 (fr) 2000-07-18 2004-02-04 Correlogic Systems, Inc. Procede de distinction d'etats biologiques sur la base de types caches de donnees biologiques
DE10043470B4 (de) 2000-09-04 2006-01-19 Mpb Meltec Patent- Und Beteiligungsgesellschaft Mbh Verfahren zur Identifizierung von zellspezifischen Proteinen
US20020132260A1 (en) * 2001-02-22 2002-09-19 Erlander Mark G. Quantitative immunohistochemistry (QIHC)
US7049397B2 (en) * 2001-04-30 2006-05-23 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 1211 daltons
US6599877B2 (en) 2001-04-30 2003-07-29 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 1020 daltons
US20020160434A1 (en) * 2001-04-30 2002-10-31 George Jackowski Biopolymer marker indicative of disease state having a molecular weight of 1777 daltons
US7294688B2 (en) 2001-04-30 2007-11-13 Nanogen Inc. Biopolymer marker indicative of disease state having a molecular weight of 1348 daltons
US6602855B2 (en) * 2001-04-30 2003-08-05 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 1449 daltons
US20040224423A1 (en) * 2001-04-30 2004-11-11 George Jackowski Biopolymer marker indicative of disease state having a molecular weight of 2056 daltons
US20020160532A1 (en) * 2001-04-30 2002-10-31 George Jackowski Biopolymer marker indicative of disease state having a molecular weight of 1998 daltons
US6627607B2 (en) 2001-04-30 2003-09-30 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 1845 daltons
US6998243B2 (en) 2001-04-30 2006-02-14 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 1793 daltons
US20020160533A1 (en) 2001-04-30 2002-10-31 George Jackowski Biopolymer marker indicative of disease state having a molecular of weight of 1525 daltons
US20040198950A1 (en) * 2001-04-30 2004-10-07 George Jackowski Biopolymer marker indicative of disease state having a molecular weight of 1518 daltons
US6890763B2 (en) * 2001-04-30 2005-05-10 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 1350 daltons
US20020160421A1 (en) * 2001-04-30 2002-10-31 George Jackowski Method for monitoring and validating stress induction of disease state
US6627608B2 (en) 2001-04-30 2003-09-30 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 1206 daltons
US6703366B2 (en) 2001-04-30 2004-03-09 George Jackowski Biopolymer marker indicative of disease state having a molecular weight of 1,896 daltons
US7008800B2 (en) * 2001-04-30 2006-03-07 Artemis Proteomics, Ltd. Biopolymer marker indicative of disease state having a molecular weight of 1077 daltons
US20020160423A1 (en) * 2001-04-30 2002-10-31 George Jackowski Biopolymer marker indicative of disease state having a molecular weight of 1536 daltons
US7314717B2 (en) * 2001-04-30 2008-01-01 Nanogen Inc. Biopolymer marker indicative of disease state having a molecular weight of 1562 daltons
US20030040602A1 (en) * 2001-04-30 2003-02-27 George Jackowski Biopolymer marker indicative of disease state having a molecular weight of 1406 daltons
US6593298B2 (en) * 2001-04-30 2003-07-15 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 1690 daltons
US6627606B2 (en) 2001-04-30 2003-09-30 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 1465 daltons
US6677303B2 (en) 2001-04-30 2004-01-13 Syn X Pharma Biopolymer marker indicative of disease state having a molecular weight of 1097 daltons
US6620787B2 (en) 2001-04-30 2003-09-16 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 2267 daltons
US20020160420A1 (en) * 2001-04-30 2002-10-31 George Jackowski Process for diagnosis of physiological conditions by characterization of proteomic materials
US20020160418A1 (en) * 2001-04-30 2002-10-31 George Jackowski Biopolymer marker indicative of disease state having a molecular weight of 1949 daltons
US6617308B2 (en) 2001-04-30 2003-09-09 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 1865 daltons
US7087435B2 (en) * 2001-04-30 2006-08-08 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 2753 daltons
US6756476B2 (en) 2001-04-30 2004-06-29 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having a molecular weight of 2021 daltons
US20020160417A1 (en) * 2001-04-30 2002-10-31 George Jackowski Biopolymer marker indicative of disease state having a molecular weight of 1424 daltons
US6693080B2 (en) 2001-04-30 2004-02-17 Syn X Pharma Biopolymer marker indicative of disease state having a molecular weight of 1521 daltons
US6620786B2 (en) * 2001-04-30 2003-09-16 Syn X Pharma, Inc. Biopolymer marker indicative of disease state having molecular weight of 2937 daltons
US7314762B2 (en) * 2001-11-21 2008-01-01 Nanogen, Inc. Apolipoprotein biopolymer markers indicative of insulin resistance
AU2002336845A1 (en) * 2001-11-23 2003-06-10 Syn.X Pharma, Inc. Globin biopolymer markers indicative of insulin resistance
US20070003974A1 (en) * 2001-11-23 2007-01-04 George Jackowski Cene-E biopolymer marker indicative of age matched control
US20090093000A1 (en) * 2001-11-23 2009-04-09 George Jackowski Glycoprotein and apolipoprotein biopolymer markers predictive of Alzheimers disease
US7125678B2 (en) 2001-11-23 2006-10-24 Nanogen, Inc. Protein biopolymer markers predictive of type II diabetes
US7097989B2 (en) * 2001-11-23 2006-08-29 Syn X Pharma, Inc. Complement C3 precursor biopolymer markers predictive of type II diabetes
US20030113808A1 (en) * 2001-12-13 2003-06-19 George Jackowski Apolipoprotein biopolymer markers predictive of alzheimers disease
WO2004011905A2 (fr) 2002-07-29 2004-02-05 Correlogic Systems, Inc. Assurance de la qualite/controle de la qualite pour processus d'ionisation par electronebulisation
SE0202346D0 (sv) * 2002-07-30 2002-07-30 Astrazeneca Ab Profiling process
AU2003256803B2 (en) 2003-01-24 2009-09-17 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target activated microtransfer
US7695752B2 (en) 2003-01-24 2010-04-13 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Target activated microtransfer
US8168568B1 (en) 2003-03-10 2012-05-01 The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Combinatorial therapy for protein signaling diseases
US20050124965A1 (en) * 2003-12-08 2005-06-09 Becton, Dickinson And Company Phosphatase inhibitor sample collection system
EP1709442A4 (fr) 2003-12-11 2010-01-20 Correlogic Systems Inc Procede de diagnostic d'etats biologiques mettant en oeuvre un modele adaptateur centralise, et un traitement a distance d'echantillons
US8962302B2 (en) 2005-11-07 2015-02-24 Canon Kabushiki Kaisha Biological tissue processing substrate for fixing proteins or protein degradation products in tissue for analysis
EP2071313A4 (fr) * 2006-09-28 2012-10-31 Shimadzu Corp Procédé de déparaffinage d'un échantillon noyé dans de la paraffine et procédé d'analyse d'un échantillon noyé dans de la paraffine
US8664358B2 (en) 2007-06-29 2014-03-04 Vermillion, Inc. Predictive markers for ovarian cancer
EP2863226A1 (fr) 2007-11-02 2015-04-22 Humanix Co., Ltd. Procédé de capture d'un fluide et d'analyse de composants de celui-ci et système pour capturer et analyser un fluide
US9502227B2 (en) 2007-11-02 2016-11-22 Humanix Co., Ltd. Capturing of cell fluid and analysis of its components under observation of cells and instruments for the cell fluid capturing and the analysis
EP2309262A1 (fr) * 2009-10-06 2011-04-13 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé pour la quantification de biomolécules
CN102621301B (zh) * 2011-01-28 2014-10-08 上海铭源数康生物芯片有限公司 一种基于硝酸纤维素膜的蛋白芯片封闭液的制备方法
WO2015199976A1 (fr) 2014-06-24 2015-12-30 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Microdissection activée par une cible
CN106644632B (zh) * 2016-11-17 2019-08-27 中国科学院苏州生物医学工程技术研究所 用于单层细胞平铺的人工基质
CN113740544A (zh) * 2021-07-29 2021-12-03 山东第一医科大学(山东省医学科学院) 鉴定人垂体组织泌乳激素蛋白质存在形式生物标志物方法
WO2023250212A1 (fr) * 2022-06-24 2023-12-28 The Johns Hopkins Univeristy Systèmes de microdissection d'expression

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4976957A (en) * 1974-03-12 1990-12-11 Samuel Bogoch Process for the production of recognins and their chemoreciprocals
JPS61275221A (ja) * 1985-05-30 1986-12-05 Mitsui Pharmaceut Inc タンパク質
US5843657A (en) * 1994-03-01 1998-12-01 The United States Of America As Represented By The Department Of Health And Human Services Isolation of cellular material under microscopic visualization

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0049410A3 *

Also Published As

Publication number Publication date
WO2000049410A3 (fr) 2001-03-08
AU3367100A (en) 2000-09-04
CA2359649A1 (fr) 2000-08-24
WO2000049410A9 (fr) 2001-08-30
WO2000049410A2 (fr) 2000-08-24
AU772680B2 (en) 2004-05-06
JP2002537561A (ja) 2002-11-05

Similar Documents

Publication Publication Date Title
AU772680B2 (en) LCM (Laser capture microdissection) for cellular protein analysis
US6969614B1 (en) Methods for the isolation and analysis of cellular protein content
Wulfkuhle et al. Proteomic approaches to the diagnosis, treatment, and monitoring of cancer
Somiari et al. Proteomics of breast carcinoma
Somiari et al. High‐throughput proteomic analysis of human infiltrating ductal carcinoma of the breast
Posadas et al. Proteomic analysis for the early detection and rational treatment of cancer—realistic hope?
JP5624079B2 (ja) マススペクトルを用いた化学的に架橋された細胞サンプルの分類方法
US20110105337A1 (en) Method of discovering and analyzing secreted biomarkers of disease from solid tissue
Sikaroodi et al. Tumor markers: the potential of “omics” approach
WO2009126969A2 (fr) Biomarqueurs pour maladie endométriale
WO2002077176A2 (fr) Proteines presentant une expression aberrante dans des tumeurs microdissequees capturees au laser
US20160054323A1 (en) SRM/MRM Assay for Subtyping Lung Histology
Antonucci et al. Two‐dimensional molecular profiling of mantle cell lymphoma
Craven et al. [4] Use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis
Jain [14] Application of laser capture microdissection to proteomics
US20120109530A1 (en) Method of classifying chemically crosslinked cellular samples using mass spectra
EP2630498A2 (fr) Biomarqueurs pronostiques chez des patientes atteintes d'un cancer ovarien
Gromov et al. A single lysis solution for the analysis of tissue samples by different proteomic technologies
Lee et al. Revealing urologic diseases by proteomic techniques
CN116148482A (zh) 用于乳腺癌患者鉴定的设备及其制备用途
Vimalachandran et al. Proteomic technologies and their application to pancreatic cancer
US20150126384A1 (en) Prognostic Biomarkers in Patients with Ovarian Cancer
US8951720B2 (en) Markers for transformed epithelium and potential targets for therapy of cancer of the gingivo buccal complex
CA2525740A1 (fr) Biomarqueurs permettant d'effectuer un diagnostic differentiel de la pancreatite et du cancer du pancreas
WO2010017331A1 (fr) Altérations de voie de signalisation et élévations de médicament cible dans le cancer colorectal métastasique métachrone primaire comparé à une maladie non métastasique

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20010914

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

17Q First examination report despatched

Effective date: 20030916

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20060503