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[Cancer Research 60, 1526-1530, March 15, 2000]
© 2000 American Association for Cancer Research

Advances in Brief

Layered Expression Scanning: Rapid Molecular Profiling of Tumor Samples

Chad R. Englert, Galina V. Baibakov and Michael R. Emmert-Buck1

Pathogenetics Unit, Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20892 [C. R. E., G. V. B., M. R. E-B.], and Howard Hughes Medical Institute-National Institutes of Health Research Scholars Program, Bethesda, Maryland 20892 [C. R. E.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results and Discussion
 REFERENCES
 
Layered expression scanning is a new approach to comprehensive molecular analysis of tumor samples that uses a layered array of capture membranes coupled to antibodies or DNA sequences to perform multiplex protein or mRNA analysis. Cell or tissue samples are transferred through a series of individual capture layers, each linked to a separate antibody or DNA sequence. As the biomolecules traverse the membrane set, each targeted protein or mRNA is specifically captured by the layer containing its antibody or cDNA sequence. The two-dimensional relationship of the cell populations is maintained during the transfer process, thereby producing a molecular profile of each cell type present. Reduction-to-practice of the technique is demonstrated by analysis of prostate-specific antigen (PSA) protein, gelatinase A protein, and POV1 (PB39) cDNA. As layered expression scanning technology progresses, we envision a laboratory method that will have multiple applications for high-throughput molecular profiling of normal and tumor samples.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results and Discussion
 REFERENCES
 
The success of the Human Genome Project and related gene discovery initiatives are dramatically increasing the information available regarding the number, genomic location, and sequences of human genes (1, 2, 3, 4, 5, 6, 7) . Accompanying the expanding base of genetic knowledge are several new technologies geared toward high-throughput molecular analysis, allowing a global view of the expression of gene products in biological systems (8, 9, 10, 11, 12) . Used together the expanding genetic database and developing analysis technologies hold tremendous potential to increase the understanding of normal cellular physiology and the alterations that underlie cancer progression in patients and related model systems (13, 14, 15, 16, 17, 18) . However, tumor samples remain uniquely difficult to analyze because of their complex cellular heterogeneity. To overcome this problem, several methods have recently been developed. For example, new tissue microdissection techniques facilitate the procurement of microscopic cell populations from histological sections, thus permitting investigators to recover and study specific normal or diseased cell types (19, 20, 21, 22, 23, 24) . Alternatively, tissue arrays permit individual molecules to be studied simultaneously in hundreds of separate tissue samples using standard immunohistochemistry or in situ hybridization methodology (25) . Layered expression scanning is a new technique that combines cell and/or tissue samples with a high-throughput array approach to provide a simple and rapid method for comprehensive molecular analysis.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results and Discussion
 REFERENCES
 
Tissue Samples.
Tissues were obtained from patients on Institutional Review Board-approved protocols of the NCI. All of the clinical and patient identifiers were removed from the samples prior to the study.

Capture Membranes.
Two types of capture layers were used to demonstrate feasibility and reduction to practice of the technique. Nitrocellulose membranes were used for the capture of PSA2 and MMP-2. Agarose gel layers were used in the experiments that demonstrated the capture of POV1 (PB39) cDNA and the transfer of an intact tissue section through multiple layers. In applications using nitrocellulose capture membranes, the resolution of the transfer process could be increased by coating each membrane with 1% agarose prior to creating the membrane stack.

Transfer Process.
Transfer of tissue specimens through the capture membranes was performed similar to a standard Southern blot with the following modifications: (a) the samples were transferred through the capture membranes overnight by capillary movement using a 1x Tris-glycine transfer buffer for proteins and a 6x SSC buffer for DNA; (b) the standard nitrocellulose membrane was replaced with a stack of capture layers (either agarose gel layers or nitrocellulose membranes), each linked to a separate capture antibody or DNA sequence; and (c) the standard agarose gel was replaced with a gel that was specially prepared and contained the samples to be analyzed. If the samples were solubilized cellular lysates, purified protein, or cDNA, then the gel was created as follows: a 2-mm thick, 2% agarose gel layer was made, and a series of 4-mm diameter holes were "punched" in the gel to create sample "wells." The biological samples were then added to 1% liquid agarose, placed into the wells, and allowed to solidify. The agarose gel was then placed into a standard Southern blot apparatus and the samples transferred by capillary action out of the gel and through the capture layers. If the sample to be transferred was an intact histological tissue section, then a 2-mm thick, 2% agarose gel was made, and the tissue section was placed directly on top of the gel. The section was then covered by a thin layer of 1% liquid agarose that was allowed to polymerize, creating a two-layered gel with the intact tissue section located inside. The gel/tissue was then placed into the blot apparatus and transferred similar to a standard Southern blot.

Capture of PSA.
Small tissue samples (0.25 cubic inches) from five patients were lysed in 250 µl of tissue protein extraction buffer (Pierce, Rockford, IL). The tissue samples included normal lung, lung tumor, esophageal tumor, normal prostate, and breast cancer. A 1:10 dilution (0.25 µl) of purified PSA (Scripps, San Diego, CA) was used as a positive control. One µl of each tissue sample lysate was placed on the top layer of a nitrocellulose membrane set (1.75 square inches, 0.45 µm pore size, Schleicher and Schuell, Keene, NH) as illustrated in Fig. 1Citation . The membranes were coupled to individual antibodies as follows. Each antibody was diluted 1:100 from the original titer and linked to individual membranes by gentle shaking in 1x PBS for 1 h at 4°C, followed by three 10-min washes in 1x PBS. The membranes were subsequently treated with a commercial blocking agent (Pierce) for 1 h at room temperature, followed by three repeat washes with 1x PBS. The identity and order of the antibodies was as follows: (a) layer 1, PB39–644 (NCI, Bethesda, MD); (b) layer 2, actin (Sigma, St. Louis, MO); (c) layer 3, PB39–645 (NCI); (d) layer 4, tubulin (Sigma); (e) layer 5, PB39–646 (NCI); (f) layer 6, cytokeratin (Sigma); (g) layer 7, PB39–655 (NCI); (h) layer 8, MMP-2 (NCI); (i) layer 9, PB39–656 (NCI); and (j) layer 10, polyclonal anti-PSA (Scripps). After tissue sample transfer, capture layer 10 was individually analyzed by a standard immunoblot procedure using a monoclonal anti-PSA antibody (Scripps, 1:1000 titer), and the membrane was visualized using ECL in accordance with the recommendations of the manufacturer (Pierce).



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  		Fig. 1. Schematic diagram showing the principle of layered expression scanning. A is a schematic of a whole-mount section of prostate from a patient with cancer. Contained within the section are multiple different cell populations and subpopulations, each of biological interest. For example, normal prostate epithelium adjacent to a tumor focus may show a distinct gene expression profile compared with normal epithelium distant from the cancer. Lymphocytes associated with the tumor may show a distinct molecular profile unique to the local environment and/or alter the expression pattern of nearby tumor cells. B shows the conceptual basis of layered expression scanning. A tissue section, microdissected cells, or cell lysates are transferred through a series of capture membranes linked to capture (hybridization) molecules. The membranes are subsequently analyzed and a measurement of each target molecule is obtained from the corresponding layer.

 
Specificity of PSA Capture.
A 20-um cryostat section of prostate tissue was placed on the top layer of a membrane set (see Fig. 1Citation ) and transferred overnight by capillary movement through nine layers of nitrocellulose (0.5 square inches), each coupled to a separate antibody as described above. The following antibodies were used: (a) layer 1, PB39–644 (NCI); (b) layer 2, actin (Sigma); (c) layer 3, tubulin (Sigma); (d) layer 4, PB39–645 (NCI); (e) layer 5, polyclonal anti-PSA (Scripps); (f) layer 6, CaIR 1 (NCI); (g) layer 7, PB39–656 (NCI); (h) layer 8, cytokeratin (Sigma); and (i) layer 9, CD3 (United States Food and Drug Administration, Bethesda, MD). After transfer of the tissue specimen through the nine layers, each membrane was placed into 30 µl of SDS-containing sample buffer (Novex) to remove captured molecules. The recovered molecules from each individual layer were separated by electrophoresis on a 4–20% Tris-glycine acrylamide gel (Novex) for 1.5 h at 110 V. The proteins recovered from layer 1 were run in Lane 1, the proteins recovered from layer 2 were run in Lane 2, and so forth. After electrophoresis, the proteins were transferred from the gel to a 0.2 µm pore size polyvinylidene difluoride membrane (Novex) for 2 h at 40 V and analyzed by a standard immunoblotting procedure using a 1:1000 titer of monoclonal anti-PSA antibody (Scripps). Subsequently, a repeat of the experiment was performed except the tissue was transferred through 101 membranes with polyclonal anti-PSA (Scripps) placed on membrane 100. In this experiment, membranes 1–99 and 101 were treated with a blocking reagent (Pierce) but were not linked to antibodies.

Capture of MMP-2.
The experiment showing capture and analysis of MMP-2 was performed similar to the capture of PSA described above ("Specificity of PSA Capture") except a polyclonal antibody against MMP-2 (NCI) was used in place of anti-PSA, and purified MMP-2 was used in place of prostate tissue. After sample transfer, the membranes were placed in 30 µl of SDS-containing buffer to remove captured molecules, and each sample was subsequently analyzed by gelatin zymography as described previously (19) .

Capture of POV1 (PB39) cDNA.
33P-labeled PCR products (200 bp) were amplified from plasmids containing cDNAs of the POV1 (PB39, NCI) and ß-actin (Clontech, Palo Alto, CA) genes, respectively. The PCR products were excised from an agarose gel, and 5% of each product was placed in a 4-mm diameter spot on top of a set of 10 thin (<50-µm) 2% agarose gel layers. During the preparation of layer 5, the POV1 cDNA-containing plasmid was added to the agarose prior to gel polymerization at a final concentration of 30 ng/µl The POV1 and actin PCR products were transferred through the 10 agarose layers by capillary fluid movement overnight at room temperature using 6x SSC. After transfer, the layers were separated and visualized by X-OMAT radiography.

Transfer of an Intact Tissue Section.
Ten-µm-thick whole-mount cryostat sections of human prostate from radical prostatectomy specimens were placed on top of either a 10-layer (Fig. 4Citation , top) or a 100-layer (Fig. 4Citation , bottom) agarose gel layer set as illustrated in Fig. 1Citation . The intact tissue section was transferred through the layers by capillary fluid movement overnight at room temperature to a 1.75-square-inch, 0.45-µm pore size nitrocellulose membrane (Schleicher and Schuell). In this experiment, the layers consisted of <50-µm-thick, 0.5% agarose gels without antibodies or DNAs attached. After transfer of the tissue sections, the nitrocellulose membranes were probed with an antibody against cytokeratin (Sigma, 1:1000 dilution) to selectively identify epithelial elements and were visualized by ECL according to the recommendations of the manufacturer (Pierce).



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  		Fig. 4. Transfer of an intact tissue section. Whole-mount prostate tissue sections were transferred through either 10 (top) or 100 (bottom) agarose gel layers to a nitrocellulose membrane and were subsequently probed with a monoclonal antibody against cytokeratin. Similarities can be seen in the corresponding H&E-stained sections.

 

   Results and Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
REFERENCES
 
Fig. 1Citation shows the principle of layered expression scanning in schematic form. A biological sample (Fig. 1Citation , left, tissue section; middle, dissected cell populations; right, lysates from cells) is placed adjacent to a set of capture layers, each containing an individual hybridization molecule (antibody or DNA sequence). The specimen(s) is transferred through the membrane set, and—importantly—the overall two-dimensional architecture within the sample(s) is maintained. As the proteins and nucleic acids are transferred, each target molecule specifically hybridizes to the membrane containing its antibody or cDNA sequence. After hybridization, each of the membranes is analyzed, which provides a measurement of the level of expression of each targeted molecule in all of the cell types present in the sample.

Fig. 1ACitation shows a schematic representation of a whole-mount prostate tissue section from a patient with cancer who was treated at the NCI. Contained within the section are multiple cell populations of biological interest, including normal epithelium, premalignant lesions, high- and low-grade tumor foci, stromal components, and important tumor-host interactions such as lymphocytes associated with cancer cells. Additionally, each of these cell populations contains many distinct subpopulations. For example, in a given region of the prostate that contains cancer, there are several hundred individual tumor foci invading through the stroma. Presumably, these tumor glands share molecular alterations of the transformed phenotype. However, it is also likely that important molecular heterogeneity exists, either inherent to the tumor cells themselves (such as a tumor focus that has progressed to the metastatic phenotype) or secondary to their location within the prostate gland and to the influence of neighboring cell types or structures. Thus, a critical need in furthering our understanding of the molecular profiles of normal and tumor cells in tissues is a methodology that permits simultaneous molecular profiling of all of the cell types that are present. For investigators, this will facilitate a deeper understanding of tumorigenesis at a molecular level. For clinicians, this will permit molecular assessment of the entire tumor with the potential to identify aggressive subpopulations that are not evident based on cellular phenotype observed under the microscope (26) . Layered expression scanning is a simple technique that was designed to permit concurrent molecular profiling of multiple cell populations. To demonstrate the feasibility and initial reduction to practice of the technique, six experiments of a prototype system were performed and analyzed.

Capture of PSA.
To demonstrate molecular capture, cell samples from five separate patients were procured from tissue specimens and solubilized in standard protein extraction buffer. The samples included normal lung, lung cancer, esophageal cancer, normal prostate, and breast cancer. Each of the cell lysates was placed within a discrete 4-mm-diameter spot on the top layer of a capture membrane set (see Fig. 1BCitation , cell lysate example). Additionally, purified PSA was used as a positive control sample. In this experiment, the capture membranes consisted of 10 nitrocellulose layers, each coupled to a separate antibody. Polyclonal anti-PSA was linked to layer 10. The six tissue samples were transferred through the capture membranes by capillary action, and each membrane was subsequently analyzed. Fig. 2ACitation shows capture layer 10 after probing with a monoclonal antibody against PSA and visualization by ECL. Samples 1 (purified PSA) and 5 (normal prostate tissue) show a positive signal, which indicates that PSA has been successfully captured. Samples 2 (normal lung), 3 (lung tumor), 4 (esophageal tumor), and 6 (breast cancer) do not contain PSA and are appropriately negative.



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  		Fig. 2. Selective and Specific Capture of Proteins. In A, cell lysates from five tissue samples were transferred through 10 antibody-linked capture layers and analyzed for PSA. Sample 1, purified PSA control; sample 2, lung tumor; sample 3, normal lung; sample 4, esophageal tumor; sample 5, normal prostate; sample 6, breast tumor. Positive staining for PSA was observed only in samples 1 and 5. B, specific capture of PSA from a prostate tissue section. Lane 5 (representing the layer linked to anti-PSA antibody) shows a single, distinct PSA band at Mr 30,000 (30 kDa). The remaining capture membranes are negative for PSA. C, specific capture of PSA after transfer through 101 layers. Layer 100 was linked to anti-PSA antibody. D, specific capture of active MMP-2 enzyme. The analysis was performed by gelatin zymography. Lane 5 (representing the layer linked to anti-MMP-2 antibody) shows a single, distinct MMP-2 band at Mr 72,000.

 
Specificity of PSA Capture.
To show the specificity of the capture process, a single sample of prostate tissue was solubilized and transferred through a set of capture layers as described in the section "Capture of PSA," except that polyclonal anti-PSA was placed on membrane 5. After the transfer of the prostate tissue through the layers, each membrane was placed in denaturing buffer to remove captured molecules. The proteins recovered from every membrane were subsequently separated by gel electrophoresis (the proteins recovered from layer 1 were run in Lane 1, the proteins recovered from layer 2 were run in Lane 2, and so forth) and analyzed by immunoblot using a monoclonal anti-PSA antibody. Fig. 2BCitation shows the results from each of the capture layers. Lane 5 (representing layer 5, linked to anti-PSA) shows a single, distinct PSA band at Mr 30,000 (30 kDa). The remaining capture membranes are negative for PSA. This experiment demonstrates that PSA was captured only on the membrane containing its antibody. Moreover, the single band on the immunoblot indicates that the ECL signal derived from the capture membrane in the previous experiment (see section above) was specific for PSA.

To illustrate the potential of the method for high-throughput analysis, a repeat of the experiment was performed, except the tissue was transferred through 101 capture layers with anti-PSA placed on layer 100. Successful and specific capture of PSA is shown in Fig. 2CCitation . There does not seem to be a limit to the number of capture membranes that can be used; thus, it is possible that, when fully developed, layered expression scanning will allow the simultaneous measurement of hundreds of molecular species.

Capture of MMP-2.
To demonstrate the ability of layered expression scanning to capture and analyze active enzymes a repeat of the "Specificity of PSA Capture" experiment was performed, except the anti-PSA antibody that was linked to capture layer 5 was replaced by an antibody against MMP-2. Purified MMP-2 protein was transferred through the capture layers, and each membrane was subsequently analyzed by gelatin zymography. Fig. 2DCitation shows successful capture of MMP-2 represented by a single band at Mr 72,000 (72 kDa).

Capture of POV1 (PB39) cDNA.
To show the ability of layered expression scanning to analyze nucleic acids, the following experiment was performed. Radiolabeled PCR products from transcripts from the POV1 (PB39) and actin genes were placed within discrete 4-mm diameter spots on the top layer of a capture membrane set. The PCR products were transferred through 10 capture layers overnight by capillary transfer. In this experiment, the capture layers consisted of ultrathin (<50-µm) 2% agarose gels. Capture layer five contained a plasmid containing the entire cDNA for the POV1 gene. Fig. 3Citation shows successful and selective capture of POV1 cDNA.



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  		Fig. 3. Capture of POV1 (PB39) cDNA. Radiolabeled PCR products from POV1 and ß-actin transcripts were transferred as discrete spots through 10 capture layers. Layer 5 was linked to a plasmid containing the entire POV1 cDNA. Successful and specific capture of POV1 is evident in layer 5. In contrast, the actin PCR product moved through the entire set of layers and was not captured. A nonblocked nitrocellulose membrane (top) was used to bind the noncaptured POV1 and actin PCR products after they traversed the membrane set.

 
Transfer of an Intact Tissue Section.
The experiments described above show the feasibility of layered expression scanning to analyze tissue samples after they have been appropriately procured and solubilized. We think that this approach holds great potential to facilitate high-throughput molecular studies of microdissected cell populations, cells grown in vitro, and bulk tissue specimens. Perhaps the best long-term potential of layered expression scanning, however, is for the analysis of intact tissue sections. This will permit investigators to simultaneously determine molecular profiles of all of the cell populations present in a histological section, without the need to mechanically separate or procure individual cell types. It would be ideal to perform these investigations at a single-cell level of resolution to permit individual cells to be analyzed. However, investigations performed at a "microscopic structure" level of resolution will also be of interest, particularly in the study of tumors. In prostate cancer, for example, all of the individual normal glands, premalignant foci, and high- and low-grade tumor glands could be simultaneously analyzed, as well as important subpopulations, such as tumor glands, that are invading through the prostate capsule.

The following two experiments were performed as a first step in assessing the feasibility of transferring histological sections through a large number of capture membranes while maintaining basic tissue architecture. Ten-µm-thick whole-mount sections of human prostate, representing cross-sections of the entire organ, were placed on top of a capture membrane set consisting of either 10 or 100 agarose gel layers (as illustrated in Fig. 1Citation ). The sections were transferred through the capture layers and onto a nitrocellulose membrane. The membrane was subsequently probed with an antibody against cytokeratin and visualized by ECL. Retention of the basic organization of the tissue section throughout the transfer process is demonstrated in Fig. 4Citation by comparing the transferred sections with a H&E-stained slide of an adjacent recut section. The overall architecture of the transferred sections is highly similar to the corresponding H&E-stained slide, and the location of individual glandular epithelial elements within the tissue sections can be determined. Thus, it appears feasible that layered expression scanning can be used for analyzing intact tissue sections, although the precise cellular resolution that can be obtained with captured molecules remains to be determined.

In summary, layered expression scanning is a newly developing analysis method that integrates procured cell populations and/or histopathological tissue sections with a high-throughput array approach. The technique may have utility for a wide range of studies investigating molecular profiles of defined cell types.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Pathogenetics Unit, Laboratory of Pathology, National Cancer Institute, Building 10, Room 2A33, 9000 Rockville Pike, Bethesda, MD 20892. E-mail: mbuck{at}helix.nih.gov Back

2 The abbreviations used are: PSA, prostate-specific antigen; MMP-2, matrix metalloproteinase-2; ECL, enhanced chemiluminescence; NCI, National Cancer Institute. Back

Received 9/24/99. Accepted 2/ 2/00.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results and Discussion
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