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M E T H O D S I N M O L E C U L A R M E D I C I N ETM CCoolloorreeccttaall CCaanncceerr MMeetthhooddss aanndd PPrroottooccoollss EEddiitteedd bbyy SStteevveenn MM.. PPoowweellll,, MMDD HHuummaannaa PPrreessss Microdissection of Histologic Sections 1 1 Microdissection of Histologic Sections Manual and Laser Capture Microdissection Techniques Christopher A. Moskaluk 1. Introduction The molecular analysis of human cancer is complicated by the difficulty in obtaining pure populations of tumor cells to study. One traditional method of obtaining a pure representation has been establishing cancer cell lines from primary tumors. However, this technique is time consuming and of low yield. Artifacts of cell culture include the selection of genetic alterations not present in primary tumors (1,2) and the alteration of gene expression as compared to primary tumors (3). When molecular techniques move from experimental to diagnostic settings, the need for robust, reproducible and “real time” testing will probably therefore require the direct analysis of tissue samples. Problems with the study of primary tissue samples include the heterogeneity of cell types and the range in the ratio of neoplastic cells relative to benign cells (“tumor cellularity”). All tissues, even malignant tumors, are composed of a mixture of cell types. No tumors are free of supporting stromal cells (fibro- blasts, endothelial cells) and many tumors are invested with inflammatory cells and other residual benign tissue elements. Tumor cellularity and the degree of tumor necrosis not only varies between different neoplasms but can vary greatly between different areas in a single tumor mass. Molecular analyses of cancer in tissue samples may be hindered by insufficient number of viable target cells and a significant degree of contamination by nontarget cells. While it may be true that tests for specific genetic alterations may eventually make some histologic assessment superfluous (4), proposed “gene expression profiling” studies (e.g., microarray assays) will require molecular analysis on pure representations of cancer cells (5). Hence, histologic analysis of tumors will remain an From:Methods in Molecular Medicine, vol. 50: Colorectal Cancer: Methods and Protocols Edited by: S. M. Powell © Humana Press Inc., Totowa, NJ 1 2 Moskaluk important part of tissue procurement for molecular analysis and experimental correlation with molecular assays (6). To address these issues, various microdissection methodologies have been developed to obtain enriched and/or pure representations of target cells from histologic tissue sections. The methodologies can be separated into two basic strategies: selection of specific tissue elements for analysis, or the destruction of unwanted tissue elements. In the category of positive selection, the least complex methodology involves the manual dissection of tissue elements under direct microscopic visualization using scalpel blades, fine-gage needles, or drawn glass pipets (7). The precision with which manual microdissection can be performed depends greatly on the architectural arrangement of the target tissue and the skill of the dissector. An extension of this method is the attach- ment of steel or glass needles to micromanipulator devices that allow for more fine control, enabling the dissection of individual cells (8,9). The latter technique is quite laborious, which is a limitation to the procurement of large num- bers of cells. Recent advances have brought the power of laser technology to microdissection, which allow both precise and rapid procurement of tissue elements. There are two prevalent laser-based techniques: laser capture microdissection (LCM) and laser microbeam microdissection with laser pressure catapulting (LMM-LPC). In LCM a transparent ethylene vinyl acetate thermoplastic film covers the tissue section, which is melted over areas of interest by an infrared laser thus embedding the target tissue (10,11). When the film is removed from the histologic section the selected tissue remains on the film while unselected tissue remains in the tissue section (seeFigs. 1and2). DNA, protein and RNA can all be subsequently isolated from the tissue attached to the film. In LMM-LPC, a pulsed ultraviolet nitrogen laser is used as a fine “optical scalpel” to cut out target tissue of interest (12,13). The laser beam cuts Fig. 1. (opposite page) Schematic diagram of laser capture microdissection. (A)The upper figure shows a side view of a histologic section and the microfuge tube cap which bears the thermoplastic ethylene vinyl acetate capture film (CapSure, Arc- turus Engineering Inc.). The middle figure shows the CapSure cap in contact with the tissue and a burst of the infrared laser (not drawn to scale) traveling through the cap, film, and target tissue. The laser energy is absorbed by the thermoplastic film that melts and embeds the target tissue. The target tissue is not harmed in this process. The lower figure shows the result of a successful laser capture microdissection. The target tissue remains embedded in the thermoplastic film, and is lifted away from nontarget tissue in the histologic section. (B)The tissue-bearing cap is placed on a microfuge tube that contains a lysis buffer. After inversion of the tube and incuba- tion, the desired biomolecules (DNA, RNA and/or protein) are released from the captured tissue into the solution. Microdissection of Histologic Sections 3 4 Moskaluk Fig. 2. Example laser capture microdissection of colon cancer. (A) Low power magnification of a histologic section of a human colon adenocarcinoma. Area 1 is an area of adenoma adjacent to the invasive carcinoma. Area 2 is an area of a typical moderately differentiated tubular adenocarcinoma in the region of the submucosa. Area 3 shows a more deeply invasive area of the carcinoma (in the serosa) with mucinous differ- entiation. Original magnification ×7.(B)In the left column, portions of a nondissected histologic section (same as in A) which is immediately adjacent to a histologic section used in laser capture microdissection are shown. The corresponding areas of the dissected Microdissection of Histologic Sections 5 the tissue by “ablative photodecomposition” without heat generation or lateral damage to adjacent material (14). The freed tissue is then catapulted from the surface of the histologic section into the cap of a microfuge tube by the force of a pulse of a high photon density laser microbeam. Both LCM and LMM-LPC have the precision to collect single cells, and the capacity to quickly collect thousands of targeted cells. Their drawback is the cost of the laser apparatuses, which range from $70,000 to $130,000. The second strategy, removal or destruction of unwanted tissue, uses many of the same methodologies for positive selection. With manual techniques, it is sometimes easier to remove unwanted tissue from foci of targeted tissue, rather than to precisely dissect out the target tissue (15). Laser photodecomposition can be used to destroy contaminating nontarget material (16). DNA can also be destroyed by exposure to conventional ultraviolet light sources. The technique known as selective ultraviolet radiation fractionation (SURF) uses this prin- ciple (17,18). Target tissue is covered with protective ink (either manually or with the aid of a micromanipulator), and then the histologic section is exposed to UV light. The integrity of the DNA in the target tissue is preserved and can be subsequently analyzed by polymerase chain reaction (PCR) assays. SURF has the advantages of being a rapid and relatively inexpensive technology, but has some of the limitations of other manual methods in terms of precision. It has also not been widely applied to analysis of RNA or protein content. Presented here are two methods for microdissection that have yielded enriched populations of tumor cells used successfully in analysis of tumor-specific genetic alterations and gene expression. The first is a manual method which can be applied with a minimum of specialized equipment or expense. The second is laser capture microdissection, which requires the use of specialized equipment but offers increased precision. Manual microdissection is performed on hydrated tissue, and LCM is performed on dehydrated tissue. Hence, the latter method also offers greater protection to RNA and protein samples, which are more prone to degradation than DNA. 2. Materials 2.1. Histology 1. Series of containers suitable for slide baths. 2. Histology slide holders. 3. Xylene. section are shown in the middle column. The tissue obtained from these areas by LCM is shown in the right column. The microdissected areas correspond to areas 1 (adenoma), 2 (tubular carcinoma) and 3 (mucinous carcinoma) shown in (A). Micro- dissection resulted in capture of neoplastic epithelium. Original magnification ×40. 6 Moskaluk 4. 100% Ethanol. 5. 95% Ethanol. 6. 70% Ethanol. 7. Deionized water. 8. Harris hematoxylin (Sigma-Aldrich Co., St. Louis, MO). 9. Eosin Y solution, alcoholic (Sigma-Aldrich Co.). 10. Bluing solution (Richard-Allen medical, Richland, MI). 11. loTE buffer: 3 mM Tris-HCl (pH 7.5), 0.2 mM EDTA. Store at 4°C. 12. loTE/glycerol solution (100:2.5, v/v). Store at 4°C. 2.2. Manual Microdissection 1. Standard binocular light microscope with 4×, 10×, and 20× objectives and 10× oculars. 2. 30-gauge hypodermic needles. 3. 1 cc TB syringes. 4. #11 dissecting scalpel blades and scalpel handle. 2.3. Laser Capture Microdissection 1. Pixcell™ Laser Capture Microdissection System (Arcturus Engineering Inc., Mountain View, CA). 2. CapSure™ ethylene vinyl acetate film carriers (Arcturus Engineering Inc.). 3. 0.5 mL Eppendorf™ microfuge tubes. 2.4. DNA Isolation 1. 5% suspension (w/v) of Chelex 100 resin (19)(BioRad, Hercules, CA) in loTE buffer. Store at 4°C. 2. 10X TK buffer: 0.5 M Tris-HCl (pH 8.9), 20 mM EDTA, 10 mM NaCl, 5% Tween-20, 2 mg/mL proteinase K. Store at –20°C. 2.5. RNA Isolation (see Note 1) 1. Denaturing solution: 4 Mguanidine isothiocyanate, 0.02 Msodium citrate, 0.5% sarcosyl. Store at room temperature. 2. 2 M sodium acetate (pH 4.0). Store at room temperature. 3. Chloroform:isoamyl alcohol (24:1). Store at room temperature. 4. Isopropanol. Store at room temperature. 5. Phenol equilibrated to pH 5.3–5.7 with 0.1 M succinic acid. Store at 4°C. 6. β-mercaptoethanol. Store at 4°C. 7. 2 mg/mL glycogen. Store at –20°C. 2.6. Protein Isolation 1. SDS sample buffer: 75 mMTris-HCl (pH 8.3), 2% sodium dodecyl sulfate, 10% glycerol, 0.001% bromophenol blue, 100 mM dithiothreitol. 2. IEF sample buffer: 9 M urea, 4% NP40, 2% β-mercaptoethanol. Microdissection of Histologic Sections 7 3. Methods 3.1. Preparation of Histologic Sections Seven micron-thick sections are cut from formalin-fixed paraffin embedded tissue (FFPE) or frozen tissue using standard histologic techniques and placed on clean standard glass slides (seeNote 2). 3.2. Staining of FFPE Histologic Sections for Manual Microdissection (DNA Isolation) (see Note 3) 1. Deparaffinization: place the sections in a xylene bath for 5 min. Repeat in a second xylene bath. 2. Removal of xylene and hydration: 100% ethanol bath for 2 min, 70% ethanol bath for 2 min, deionized water bath for 2 min. 3. Place in hematoxylin stain for 30 s (seeNote 4). 4. Rinse in deionized water, repeat rinse. 5. Place in bluing solution for 15 s. 6. Dehydration: 70% ethanol bath for 30 s, 95% Ethanol bath for 30 s. 7. Place in eosin stain for 30 s. 8. Rinse in deionized water, repeat rinse. 9. Place in loTE 2.5% glycerol bath for 2 min (seeNote 5). 10. Allow slides to air dry (seeNote 6). 3.3. Staining of Frozen Sections for Manual Microdissection (DNA Isolation) 1. Fixation: 100% ethanol bath for 2 min. 2. Hydration: 70% ethanol bath for 30 s, deionized water bath for 30 s. 3. Continue from step 3 in Subheading 3.2. 3.4. Staining of FFPE Histologic Sections for LCM (DNA Isolation) 1. Perform steps 1–7 in Subheading 3.2. 2. After staining in eosin, rinse in a 95% ethanol bath, then repeat rinse in a second 95% ethanol bath. 3. 100% Ethanol bath for 1 min (use a clean ethanol bath, not the one used after xylene deparaffinization). 4. Xylene bath for 5 min (use a clean xylene bath, not the one used to deparaffinize sections). 5. Allow slides to air dry. 3.5. Staining of Frozen Histologic Sections for LCM (DNA Isolation) 1. Fixation: 100% ethanol bath for 2 min. 2. Hydration: 70% ethanol bath for 30 s, deionized water bath for 30 s. 3. Steps 3–7 in Subheading 3.2., followed by steps 2–5 in Subheading 3.4. 8 Moskaluk 3.6. Staining of Frozen Histologic Sections for LCM (RNA and Protein Isolation) (see Note 7) 1. Ethanol-fixed frozen sections are dipped 15 times in RNase-free water using gloved hands or a slide holder. 2. 15 dips in hematoxylin stain. 3. The slide is dipped a few times in a deionized water bath to remove the majority of the stain, and is then dipped a few times in a fresh deionized water bath until the slide is clear of stain. 4. 15 dips in bluing reagent. 5. 15 dips in 70% ethanol. 6. 15 dips in 95% ethanol. 7. 15 dips in eosin stain. 8. 15 dips in 95% ethanol, then repeat in a fresh 95% ethanol bath. 9. 15 dips in 100% ethanol. 10. 5 min in xylene bath. 11. Air dry for at least 2 min or until the xylene is completely evaporated. 3.7. Manual Microdissection 1. Seat yourself squarely and comfortably in front of a standard light microscope (seeNote 8). 2. Place the glass slide containing the tissue under the 4× objective and focus. Use either the 4×, 10×, or 20×objectives for the dissection, depending on the tissue target and your preferences. 3. Place a 30-gauge needle on the end of a 1 cc TB syringe, or if doing a broader dissection, place a fine tip scalpel blade at the end of a scalpel handle. When using the needle, tap the end of the needle against a hard surface to bend it into a small hook (you will see the hook only under the microscope). 4. Rest your hand on the microscope stage and bring your instrument to bear on the tissue. Perform as clean a dissection as possible by gently scraping the target tissue into a small heap (seeNote 9). Keep a running estimate of the number of cells dissected. 5. Affix the dissected tissue to the end of your instrument, and place into a 1.5 mL microfuge tube. Disperse the tissue into the appropriate volume of buffer (see Subheading 3.9. for specific applications). If you are interrupted during the dissection, store tube at –20°C. 3.8. Laser Capture Microdissection (see Note 10) 1. Turn on the power to the laser control, the microscope and the video monitor components of the Pixcell LCM apparatus (Arcturus Engineering Inc.). 2. Place the slide to be dissected on the microscope stage over the 4× objective (tissue side up). 3. Adjust focus and light levels on the microscope so that the histologic image is seen clearly on the video monitor. Choose an appropriate microscope objective for the dissection and then refocus. Microdissection of Histologic Sections 9 4. Position the histologic section so that the tissue of interest is on the monitor. Keep the stage controls set in their central position and move the slide around on the stage while doing this. Once the slide is positioned, activate the vacuum mechanism to hold the slide firmly in place on the stage. 5. Set the amplitude and laser pulse width on the laser control to the manufacturer’s recommended settings initially (these values can be adjusted according to the requirements for the individual tissue section). 6. Place an ethylene vinyl acetate film-bearing microcentrifuge tube cap (CapSure, Arcturus Engineering Inc.) on the tissue section. 7. An aiming beam is projected onto the slide surface that allows pre-capture visualization. Lower the microscope light level until you can see the outline of the aiming beam on the video monitor. Position this target spot over the tissue area to be captured by moving the microscope stage (seeNote 11). 8. Fire the laser beam. This administers a laser pulse of the power and duration selected on the laser control, which briefly melts the thermoplastic film allowing it to permeate the target tissue. Continue moving the microscope stage, position- ing the aiming beam, and firing the laser until all the tissue of interest is captured (seeNote 12). 9. After dissection, lift the CapSure cap off of the tissue, move the slide so that a blank area of glass is in the viewing area. Place the CapSure cap down on the blank area and inspect the captured tissue. 10. Place the CapSure cap on a 0.5 mL Eppendorf microcentrifuge tube. Label the tube, not the cap, with an indelible marker. The tube may contain extraction buffer for the specific applications outlined below. 3.9. DNA Isolation from Manual Microdissection 1. Prior to microdissection, place 15 µL of 5% Chelex resin per 100 cells expected to be dissected. If you decide to harvest more cells than the target number during the dissection, then add additional buffer after the dissection. 2. After the dissection, add 10X TK buffer to make tube contents 1X. 3. Vortex tube for 5 s, then spin briefly in a microcentrifuge to settle the contents. 4. Incubate in a 56°C waterbath overnight. 5. Vortex and centrifuge tube as above. 6. Add 1/10 the volume of 10X TK that was added initially. 7. Vortex 5 s, incubate at 56°C overnight. 8. Place in dry heating block set at 100°C for 10 min. Alternatively, incubate the tubes in a boiling water bath for 10 min (seeNote 13). 9. Store at –20°C. 3.10. DNA Isolation from LCM 1. Place freshly diluted 1X TK buffer in a 0.5 mL Eppendorf microfuge tube at a ratio of 15 µL per 100 cells captured. Using the capping tool provided with the LCM apparatus, push the tissue-bearing CapSure cap to the prescribed distance into the tube on all sides. Invert the tube and shake.