Abstract
Using Affymetrix HG-U133 Plus 2.0 array and laser capture microdissection techniques, we determined whether different zones of the same pancreatic tumor exhibited differential expression of genes. Human L3.6pl pancreatic cancer cells were implanted into the pancreas of nude mice. Three weeks later when tumors were 7 to 9 mm in diameter, gene expression patterns in tumor cells within the central and peripheral zones were compared, and 1,222 genes showed statistically significant differences. Bioinformatic functional analysis revealed that 346 up-regulated genes in the peripheral zone were related to cytoskeleton organization and biogenesis, cell cycle, cell adhesion, cell motility, DNA replication, localization, integrin-mediated signaling pathway, development, morphogenesis, and IκB kinase/nuclear factor-κB cascade; 876 up-regulated genes in the central zone were related to regulation of cell proliferation, regulation of transcription, transmembrane receptor protein tyrosine kinase signaling pathways, response to stress, small GTPase-mediated signal transduction, hexose metabolism, cell death, response to external stimulus, carbohydrate metabolism, and response to wounding. The reliability of the microarray results were confirmed by in situ hybridization analysis of the expression of two genes. Collectively, the data showed zonal heterogeneity for gene expression profiles in tumors and suggest that characterization of zonal gene expression profiles is essential if microarray analyses of genetic profiles are to produce reproducible data, predict disease prognosis, and allow design of specific therapeutics. [Cancer Res 2007;67(16):7597–604]
Introduction
The genetic instability of neoplastic cells and the consequent biological heterogeneity of individual tumors has been well documented (1). Tumor growth, prognosis, and metastasis are dependent on multiple interactions of tumor cells with homeostatic factors in the microenvironment (1–4). A better understanding of the cross-talk between cancer cells and the organ microenvironment involving multiple genes requires in the first instance identification of gene expression profiles within the tumor. Among current methods for establishing these profiles, microarray analysis is the most powerful, and several such studies have been undertaken to predict disease outcome for individual patients (5–12) and to identify cancer patients who should receive chemotherapy (13).
Pretherapeutic gene expression profiling has been undertaken to identify breast cancer patients who should receive chemotherapy (13), to predict response to preoperative chemoradiotherapy in rectal carcinoma patients (14), and to predict response to docetaxel in breast cancer patients (15). A series of studies suggested that pretherapeutic gene expression profiles might predict the response to the treatment in various other tumors (10–16). For example, a specific gene expression pattern was correlated with recurrence in Dukes' B colon carcinomas (16). None of these studies, however, accounted for the biological heterogeneity of gene expression within a tumor. Tumor cells depend on multiple and redundant pathways for growth, survival, and adaptation to the host microenvironment (3, 17–19). Genetic instability of cancer cells frequently yields extensive intratumoral heterogeneity in the pattern of structural chromosome aberrations in highly malignant neoplasms (20–22). Three-dimensional tumor growth and uneven fractional dividing of tumor cells within a tumor mass cause biologically different zones, whether central or peripheral, within a single neoplasm. The microenvironment of the central zone differs significantly from that of the peripheral zone, and zonal heterogeneity for several molecules in different tumor systems has been reported (23–30). Among these, basic fibroblast growth factor, matrix metalloproteinase (MMP)-2, and MMP-9 are highly expressed at the invasive edge of tumors (23–27), whereas the cell-to-cell cohesion molecule, E-cadherin, was down-regulated at the periphery of the tumors (24–28). In fact, the ratio of expression of MMP-2 and MMP-9 to E-cadherin (MMP/E-cadherin ratio) at the periphery of the tumors correlated with metastatic potential and recurrent disease (23–27).
To provide reproducible data among different tumor specimens, clinical or experimental, it may be necessary to standardize the area zone of each specimen to be studied. The purpose of this study was therefore to compare gene expression profiles of pancreatic cancer cells growing in the peripheral zone with expression in cells in the central zone of the same tumor and to identify any zonal heterogeneity in a genome-wide gene expression analysis. We compared gene expression profiles generated from microarray analysis of cancer cells in the peripheral and central zones of the same tumor by laser capture microdissection (LCM) and determined whether the resulting gene ontogeny of pancreatic cancer cells in the two zones correlated with presumptive functions of cells at the periphery and the center of tumors.
Materials and Methods
Human pancreatic cancer cell line. The human pancreatic cancer cells from line L3.6pl (31) were maintained in Eagle's minimal essential medium supplemented with 10% fetal bovine serum (FBS), sodium pyruvate, nonessential amino acids, l-glutamine, a 2-fold vitamin solution (Life Technologies, Inc.), and a penicillin-streptomycin mixture (Flow Laboratories). Adherent monolayer cultures were maintained on plastic and incubated at 37°C in a mixture of 5% CO2 and 95% air. The cultures were free of Mycoplasma and the following pathogenic murine viruses: reovirus type 3, pneumonia virus, K virus, Theiler's encephalitis virus, Sendai virus, minute virus, mouse adenovirus, mouse hepatitis virus, lymphocytic choriomeningitis virus, ectromelia virus, and lactate dehydrogenase virus (assayed by M.A. Bioproducts). The cultures were maintained for no longer than 12 weeks after recovery from frozen stocks.
Animals. Male athymic nude mice (NCI-nu) were obtained from the Animal Production Area of the National Cancer Institute Frederick Cancer Research and Development Center (Frederick, MD). The mice were maintained under specific pathogen-free conditions in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the U.S. Department of Agriculture, Department of Health and Human Services, and NIH. The mice were used in accordance with institutional guidelines when they were 8 to 12 weeks old.
Orthotopic pancreatic cancer model. To produce pancreatic tumors, L3.6pl cells were harvested from subconfluent cultures by a brief exposure to 0.25% trypsin and 0.02% EDTA. Trypsinization was stopped with medium containing 10% FBS, and then the cells were washed once in serum-free medium and resuspended in HBSS. Only single-cell suspensions of >90% viability (trypan blue exclusion) were used for injection into the pancreas.
The mice were anesthetized by i.m. injection of Nembutal (0.5 mg/kg). A small left abdominal flank incision was made, the spleen was exteriorized, and the pancreas was identified in a region just beneath the spleen. A 30-gauge needle was inserted into the pancreas and tumor cells (1 × 106/50 μL of HBSS) were injected under the capsule. The abdominal wound was closed in one layer with wound clips (Autoclip, Clay Adams). The animals tolerated the surgical procedure well, and no procedure-related deaths occurred.
After 3 weeks, the pancreatic tumors reached 7 to 9 mm in diameter. At this time, specimens were harvested, immediately embedded in ornithine carbamyl transferase compound (Miles), rapidly frozen in liquid nitrogen, and stored at −80°C.
LCM and RNA preparation. The frozen tissues from three different pancreatic tumors growing in three different mice were cut into 8-μm sections and stained with H&E for histologic examination. LCM was done by using Pixcel II LM200 (Arcturus). A total of 180 frozen tissue sections were prepared from three independent pancreatic tumor samples (60 sections each), and ∼20,000 tumor cells were captured from either the central or peripheral zones of each sample. The sample for the peripheral zone was taken from the edge, and the sample for the central zone was taken at the center, at least 3.5 mm from the tumor edge. Total RNA was extracted using an RNeasy mini kit (Qiagen) with on-column DNase I treatment done according to the manufacturer's protocol.
Affymetrix GeneChip hybridization. The Affymetrix human genome U133 Plus 2.0 GeneChip arrays were used for microarray hybridizations. This GeneChip carries 54,675 probe sets. For microarray hybridization, we followed the protocol described in the Affymetrix GeneChip eukaryotic one-cycle target preparation protocol (Affymetrix). In short, 200 ng of total RNA were used to prepare antisense RNA. A single-stranded cDNA was synthesized using a T7-oligo(dT) promoter primer followed by RNase H-facilitated second-strand cDNA synthesis. The cDNA was purified and served as a template for the subsequent in vitro transcription. The second in vitro transcription reaction was carried out in the presence of T7 RNA polymerase and a biotinylated nucleotide analogue/ribonucleotide mix for cRNA. The biotinylated cRNA targets were then cleaned up and fragmented. The fragmented cRNA was used for hybridization to the U133 Plus 2.0 chip at 42°C for 16 h. The chips were washed and stained using Affymetrix GeneChip Fluidic and scanned and visualized using a GeneArray scanner (Hewlett-Packard).
Preparation of samples for fluorescence in situ hybridization. Frozen sections (4 μm) were cut on silane-treated ProbeOn slides. The slides were fixed for 15 min with 4% paraformaldehyde in PBS and then rinsed four times for 1 min each time with 50 mmol/L Tris buffer (pH 7.6). The tumor samples were then hybridized as described below.
Fluorescence in situ hybridization. Fluorescence in situ hybridization (FISH) was done as described previously (32–34) with modification. The FISH analysis was carried out by using the microprobe manual staining system (Fisher Scientific; ref. 35). The samples were hybridized with the probes at 45°C for 45 min, washed for 2 min with 5× SSC three times at 45°C, and then washed with PBS for 1 min three times at room temperature. Nuclei were stained with Hoechst. Samples were then rinsed three times with PBS and covered with a glass coverslip (Fisher Scientific). Mounting medium consisted of 90% glycerol, 10% PBS, and 0.1 mol/L propyl gallate.
Oligonucleotide probes. Specific antisense DNA oligonucleotide probes were designed to consist of sequences complementary to the mRNA transcripts based on the UniGene database of the National Center for Biotechnology Information (NCBI). The oligonucleotide sequence was initially confirmed with the NCBI basic local alignment search tool. All oligonucleotide sequences showed 100% homology with the target gene sequences, and any homology with nonspecific mammalian gene sequences was minimal. All DNA oligonucleotide probes, fluorescence labeled (Alexa 594 or Alexa 488) on the 5′-prime end, were commercially synthesized by Invitrogen custom DNA oligonucleotides. The probes were reconstituted to a stock concentration of 1 μg/μL with TE buffer [10 mmol/L Tris base, 1 mmol/L EDTA (pH 7.5)]. The final dilution of probes in the reaction mixture was 1:1,000 for polydeoxythymidylic acid [poly(dT)] 24 and 1:400 for adhesion molecule with Ig-like domain 2 (AMIGO2) and retinoic acid receptor responder (tazarotene-induced) 1 (RARRES1). The sequences of specific probes for each gene were as follows: 5′-CTTGCACCTCCCGACTTCCTTTCC-3′ (AMIGO2) and 5′-GACAGACACGGGCTCGGAGCGGGC-3′ (RARRES1).
The preservation of mRNA in the samples was confirmed by the hybridization of the samples with a poly(dT)24 probe. The specificity of the hybridization signal was confirmed by the negative controls pretreated with RNase or hybridized with a sense probe instead of the antisense probe. Negative controls yielded no signal or a markedly decreased signal. Water treated with diethyl pyrocarbonate (Sigma) was used in the preparation of all solutions to preserve RNAs.
Image analysis to quantify intensity of FISH. For quantification of the FISH reaction intensity, the absorbance of AMIGO2 and RARRES1 from three of different orthotopic human pancreatic tumors were measured at ×200 magnification using the Optimas image analysis software (version 5.2; refs. 25, 31). For each section, we determined the absorbance in 0.5 × 0.5–mm zones located at the center and periphery of the tumors. We quantified the fluorescence intensity in six different fields in the peripheral region and the central region of each of the three tumors. The samples for quantification were not nuclei stained, so that the absorbance was attributable solely to the product of the FISH reaction. The intensity of staining was standardized to that of the integrated absorbance of poly(dT)24. To analyze these data, we used a linear mixed effects model with location as a fixed effect and mouse as a random effect to account for the repeated measurements. (A more general model with an additional random effect for location within the orthotopic tumor was tested by the Akaike Information Criterion and found to be equivalent to the simpler model.) Mixed effects models were fit by maximum likelihood using the nlme package in the R statistical programming environment; significance was assessed by an F test.
Statistical analysis. Microarray data were loaded into the DNA chip analyzer (dChip 2006)3
for quality control and analysis. Data were normalized to a sample of median brightness using the invariant set method and then quantified using the perfect match–only model-based expression indices (36). All arrays passed quality control checks based on percentage present calls (>30%) and outliers (<5%). To identify differentially expressed genes, the confidence interval estimates produced by dChip were used. Specifically, the criteria were that the lower bound of the 90% confidence interval on the fold change should be at least 1.2-fold, and the difference in expression should be at least 100.Gene ontology analysis. Up-regulated genes in the central and peripheral zones of each tumor were analyzed for the functional categories using the database for annotation, visualization, and integrated discovery (DAVID).4
We used the Functional Annotation Tool program and reported only GOTERM-BP (Biological Process) that had corrected P values of <0.05.Results
Microarray analyses. We did six microarray experiments using human U133 Plus 2.0 GeneChip microarrays that contained 54,675 probe sets. Samples of human pancreatic cancer cells growing in the pancreas of nude mice were microdissected to obtain tumor cells from the central and peripheral zones of the tumor. Because necrosis, especially in the central zone of a growing tumor, could affect gene expression, nonnecrotic tissues were carefully selected under the microscope and dissected by the LCM (Fig. 1).
Mice were killed when pancreatic tumors reached 7 to 9 mm in diameter. The specimens were processed for H&E staining (A). Under the microscope, viable cancer cells were collected from the peripheral zone (B) and central zone (C) of the tumor mass by LCM.
Mice were killed when pancreatic tumors reached 7 to 9 mm in diameter. The specimens were processed for H&E staining (A). Under the microscope, viable cancer cells were collected from the peripheral zone (B) and central zone (C) of the tumor mass by LCM.
A different level of expression between the central and peripheral zones was identified for 1,222 genes, of which 346 were up-regulated in the peripheral zone and 876 were up-regulated in the central zone (Supplementary Table S1). To identify statistically significant functional categories by gene ontology, the up-regulated genes were placed into DAVID (Table 1). Many categories with statistically significant differences were identified. The patterns up-regulated in the peripheral zone were enriched for cytoskeleton organization and biogenesis, cell cycle, cell adhesion, cell motility, DNA replication, localization, integrin-mediated signaling pathway, development, morphogenesis, and IκB kinase/nuclear factor-κB (NF-κB) cascade (Table 1). The patterns up-regulated in the central zone were enriched for regulation of cell proliferation, regulation of transcription, transmembrane receptor protein tyrosine kinase signaling pathway, response to stress, small GTPase-mediated signal transduction, hexose metabolism, cell death, response to external stimulus, carbohydrate metabolism, and response to wounding. Three hypoxia-related genes were identified in the patterns up-regulated in the central zone, but the differences were not statistically significant (P = 0.062; Table 1).
Gene ontology analysis of gene expression patterns in pancreatic tumor cells from central versus peripheral zone
Zone . | GO category term . | No. genes . | P . |
---|---|---|---|
Peripheral | Cytoskeleton organization and biogenesis | 20 | 2.7e-05 |
Cell cycle | 28 | 3.4e-05 | |
Cell adhesion | 22 | 0.0031 | |
Cell motility | 10 | 0.015 | |
DNA replication | 9 | 0.017 | |
Localization | 63 | 0.026 | |
Integrin-mediated signaling pathway | 5 | 0.029 | |
Development | 41 | 0.030 | |
Morphogenesis | 17 | 0.033 | |
IκB kinase/NF-κB cascade | 6 | 0.039 | |
Central | Regulation of cell proliferation | 28 | 6.8e-06 |
Regulation of transcription | 112 | 2.3e-04 | |
Transmembrane receptor PTK signaling pathway | 13 | 0.0025 | |
Response to stress | 57 | 0.0029 | |
Small GTPase-mediated signal transduction | 19 | 0.0093 | |
Hexose metabolism | 13 | 0.012 | |
Cell death | 32 | 0.013 | |
Response to external stimulus | 29 | 0.015 | |
Carbohydrate metabolism | 29 | 0.017 | |
Response to wounding | 23 | 0.023 | |
Response to hypoxia | 3 | 0.062 |
Zone . | GO category term . | No. genes . | P . |
---|---|---|---|
Peripheral | Cytoskeleton organization and biogenesis | 20 | 2.7e-05 |
Cell cycle | 28 | 3.4e-05 | |
Cell adhesion | 22 | 0.0031 | |
Cell motility | 10 | 0.015 | |
DNA replication | 9 | 0.017 | |
Localization | 63 | 0.026 | |
Integrin-mediated signaling pathway | 5 | 0.029 | |
Development | 41 | 0.030 | |
Morphogenesis | 17 | 0.033 | |
IκB kinase/NF-κB cascade | 6 | 0.039 | |
Central | Regulation of cell proliferation | 28 | 6.8e-06 |
Regulation of transcription | 112 | 2.3e-04 | |
Transmembrane receptor PTK signaling pathway | 13 | 0.0025 | |
Response to stress | 57 | 0.0029 | |
Small GTPase-mediated signal transduction | 19 | 0.0093 | |
Hexose metabolism | 13 | 0.012 | |
Cell death | 32 | 0.013 | |
Response to external stimulus | 29 | 0.015 | |
Carbohydrate metabolism | 29 | 0.017 | |
Response to wounding | 23 | 0.023 | |
Response to hypoxia | 3 | 0.062 |
NOTE: Gene ontology was based on the DAVID database.
Abbreviations: GO, gene ontology; PTK, protein tyrosine kinase.
These data suggest that gene expression of cells growing in the peripheral zone of the tumor were optimized for cell proliferation and invasion, whereas the gene expression of the cells in the central zone of the same tumor were related to stress, a unique cascade of signaling genes whose expression helps the cell to adapt to or undergo programmed cell death. The top 20 overexpressed genes in each category are shown in Table 2.
Top 20 up-regulated genes in peripheral zone and central zone of human pancreatic cancer growing in the pancreas of nude mice
Zone . | Probe set . | Gene . | Fold difference . |
---|---|---|---|
Peripheral | 216405_at | Lectin, galactoside-binding, soluble, 1 (galectin 1) | 8.83 |
201852_x_at | Collagen, type III, α1 (Ehlers-Danlos syndrome type IV) autosomal dominant | 8.04 | |
214416_at | Transcribed locus, strongly similar to NP_033799.1 amylase 2, pancreatic [Mus musculus] | 6.55 | |
215076_s_at | Collagen, type III, α1 (Ehlers-Danlos syndrome type IV, autosomal dominant) | 6.45 | |
217683_at | ESTs moderately similar to B chain B, crystal structure of deoxy-human hemoglobin β6 | 6.26 | |
222108_at | Adhesion molecule with Ig-like domain 2 | 4.84 | |
216442_x_at | Fibronectin 1 | 4.39 | |
204619_s_at | Chondroitin sulfate proteoglycan 2 (versican) | 4.34 | |
209312_x_at | MHC, class II, DRβ1 | 4.29 | |
212233_at | Microtubule-associated protein 1B | 4.28 | |
211719_x_at | Fibronectin 1 | 4.26 | |
226084_at | Microtubule-associated protein 1B | 4.1 | |
239176_at | Nebulin-related anchoring protein | 4.1 | |
210926_at | Similar to FKSG30 | 4.07 | |
210495_x_at | Fibronectin 1 | 3.8 | |
224833_at | v-ets erythroblastosis virus E26 oncogene homolog (avian) | 3.78 | |
226535_at | FLJ23083 fis, cl. LNG06541, highly similar to IR2005735 Homo sapiens mRNA full-length insert cDNA clone EUROIMAGE 2005735 | 3.7 | |
239430_at | Insulin growth factor-like family member 1 | 3.63 | |
233149_at | cDNA clone IMAGE:4750272, containing Frame-shift errors | 3.57 | |
1552411_at | Defensin, β106 | 3.55 | |
Central | 212977_at | Chemokine orphan receptor 1 | 13.7 |
209395_at | Chitinase 3-like 1 (cartilage glycoprotein-39) | 9.75 | |
209396_s_at | Chitinase 3-like 1 (cartilage glycoprotein-39) | 8.14 | |
206392_s_at | Retinoic acid receptor responder (tazarotene-induced) 1 | 7.86 | |
212143_s_at | Insulin-like growth factor binding protein 3 | 6.84 | |
202917_s_at | S100 calcium binding protein A8 (calgranulin A) | 6.08 | |
223278_at | Gap junction protein, β2, 26-kDa (connexin 26) | 5.98 | |
206697_s_at | Haptoglobin | 5.83 | |
218960_at | Transmembrane protease, serine 4 | 5.75 | |
217109_at | Mucin 4, tracheobronchial | 5.74 | |
205476_at | Chemokine (C-C motif) ligand 20 | 5.61 | |
209821_at | Chromosome 9 open reading frame 26 (NF-HEV) | 5.53 | |
221872_at | Retinoic acid receptor responder (tazarotene-induced) 1 | 5.34 | |
206391_at | Retinoic acid receptor responder (tazarotene-induced) 1 | 5.26 | |
203921_at | Carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2 | 4.64 | |
202948_at | Interleukin-1 receptor, type 1 | 4.6 | |
213432_at | Mucin 5, subtype B, tracheobronchial | 4.28 | |
204637_at | Glycoprotein hormones, α polypeptide | 4.23 | |
222830_at | Transcription factor CP2-like 2 | 4.13 | |
213693_s_at | Mucin 1, transmembrane | 4.06 | |
242868_at | Hypoxia-inducible factor 2α | 2.06 | |
202986_at | Aryl-hydrocarbon receptor nuclear translocator 2 | 1.82 | |
202221_s_at | E1A binding protein p300 | 1.42 |
Zone . | Probe set . | Gene . | Fold difference . |
---|---|---|---|
Peripheral | 216405_at | Lectin, galactoside-binding, soluble, 1 (galectin 1) | 8.83 |
201852_x_at | Collagen, type III, α1 (Ehlers-Danlos syndrome type IV) autosomal dominant | 8.04 | |
214416_at | Transcribed locus, strongly similar to NP_033799.1 amylase 2, pancreatic [Mus musculus] | 6.55 | |
215076_s_at | Collagen, type III, α1 (Ehlers-Danlos syndrome type IV, autosomal dominant) | 6.45 | |
217683_at | ESTs moderately similar to B chain B, crystal structure of deoxy-human hemoglobin β6 | 6.26 | |
222108_at | Adhesion molecule with Ig-like domain 2 | 4.84 | |
216442_x_at | Fibronectin 1 | 4.39 | |
204619_s_at | Chondroitin sulfate proteoglycan 2 (versican) | 4.34 | |
209312_x_at | MHC, class II, DRβ1 | 4.29 | |
212233_at | Microtubule-associated protein 1B | 4.28 | |
211719_x_at | Fibronectin 1 | 4.26 | |
226084_at | Microtubule-associated protein 1B | 4.1 | |
239176_at | Nebulin-related anchoring protein | 4.1 | |
210926_at | Similar to FKSG30 | 4.07 | |
210495_x_at | Fibronectin 1 | 3.8 | |
224833_at | v-ets erythroblastosis virus E26 oncogene homolog (avian) | 3.78 | |
226535_at | FLJ23083 fis, cl. LNG06541, highly similar to IR2005735 Homo sapiens mRNA full-length insert cDNA clone EUROIMAGE 2005735 | 3.7 | |
239430_at | Insulin growth factor-like family member 1 | 3.63 | |
233149_at | cDNA clone IMAGE:4750272, containing Frame-shift errors | 3.57 | |
1552411_at | Defensin, β106 | 3.55 | |
Central | 212977_at | Chemokine orphan receptor 1 | 13.7 |
209395_at | Chitinase 3-like 1 (cartilage glycoprotein-39) | 9.75 | |
209396_s_at | Chitinase 3-like 1 (cartilage glycoprotein-39) | 8.14 | |
206392_s_at | Retinoic acid receptor responder (tazarotene-induced) 1 | 7.86 | |
212143_s_at | Insulin-like growth factor binding protein 3 | 6.84 | |
202917_s_at | S100 calcium binding protein A8 (calgranulin A) | 6.08 | |
223278_at | Gap junction protein, β2, 26-kDa (connexin 26) | 5.98 | |
206697_s_at | Haptoglobin | 5.83 | |
218960_at | Transmembrane protease, serine 4 | 5.75 | |
217109_at | Mucin 4, tracheobronchial | 5.74 | |
205476_at | Chemokine (C-C motif) ligand 20 | 5.61 | |
209821_at | Chromosome 9 open reading frame 26 (NF-HEV) | 5.53 | |
221872_at | Retinoic acid receptor responder (tazarotene-induced) 1 | 5.34 | |
206391_at | Retinoic acid receptor responder (tazarotene-induced) 1 | 5.26 | |
203921_at | Carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2 | 4.64 | |
202948_at | Interleukin-1 receptor, type 1 | 4.6 | |
213432_at | Mucin 5, subtype B, tracheobronchial | 4.28 | |
204637_at | Glycoprotein hormones, α polypeptide | 4.23 | |
222830_at | Transcription factor CP2-like 2 | 4.13 | |
213693_s_at | Mucin 1, transmembrane | 4.06 | |
242868_at | Hypoxia-inducible factor 2α | 2.06 | |
202986_at | Aryl-hydrocarbon receptor nuclear translocator 2 | 1.82 | |
202221_s_at | E1A binding protein p300 | 1.42 |
Verification of expression of genes by FISH. FISH analysis was done for confirmation of data obtained from the microarray analysis. We did the FISH analysis on human pancreatic cancer L3.6pl cells growing in the pancreas of nude mice (Figs. 2 and 3). We examined AMIGO2 and RARRES1 because AMIGO2 was highly expressed in the peripheral zone and RARRES1 was highly expressed in the central zone according to microarray analysis (see Table 2). The staining intensity with AMIGO2 probes was strong in the peripheral zone of the tumor (Fig. 2B) but weak in the center of the tumor (Fig. 2D) and vice versa with RARRES1 probes (Fig. 3B and D). A poly(dT) probe was stained as a control for mRNA (Fig. 2C and E; Fig. 3C and E). We quantified the FISH data by measuring the fluorescence intensity in six different fields in both the peripheral regions and the central regions of three different orthotopic pancreatic tumors. The quantification of the FISH reaction intensity was summarized in Table 3. For the RARRES1 gene, the intensity was significantly lower in the peripheral zone by 51.8 units (F = 914; P < 0.0001). For the AMIGO2 gene, the intensity was significantly higher in the periphery by 51.5 units (F = 903; P < 0.0001). The results of FISH analysis were coincident to those of microarray analysis showing the heterogeneity of gene expression in the same tumor.
FISH analysis of AMIGO2. Pancreatic tumors 7 to 9 mm in diameter were analyzed. AMIGO2 probe was labeled for red fluorescence, and poly(dT) (control for mRNA) probe was labeled for green fluorescence. A, H&E staining of specimen showing the zones from which images of FISH were captured. Expression of AMIGO2 was high in the peripheral zone (B) and low in the central zone (D). Preservation of mRNA was confirmed by hybridization with poly(dT) probe (C and E).
FISH analysis of AMIGO2. Pancreatic tumors 7 to 9 mm in diameter were analyzed. AMIGO2 probe was labeled for red fluorescence, and poly(dT) (control for mRNA) probe was labeled for green fluorescence. A, H&E staining of specimen showing the zones from which images of FISH were captured. Expression of AMIGO2 was high in the peripheral zone (B) and low in the central zone (D). Preservation of mRNA was confirmed by hybridization with poly(dT) probe (C and E).
FISH analysis of RARRES1. Pancreatic tumors 7 to 9 mm in diameter were analyzed. RARRES1 probe was labeled for red fluorescence, and poly(dT) (control for mRNA) probe was labeled for green fluorescence. A, H&E staining of specimen showing the zones from which images of FISH were captured. Expression of RARRES1 was high in the central zone (B) and low in the peripheral zone (D). Preservation of mRNA was confirmed by hybridization with poly(dT) probe (C and E).
FISH analysis of RARRES1. Pancreatic tumors 7 to 9 mm in diameter were analyzed. RARRES1 probe was labeled for red fluorescence, and poly(dT) (control for mRNA) probe was labeled for green fluorescence. A, H&E staining of specimen showing the zones from which images of FISH were captured. Expression of RARRES1 was high in the central zone (B) and low in the peripheral zone (D). Preservation of mRNA was confirmed by hybridization with poly(dT) probe (C and E).
FISH analysis showing intratumoral heterogeneity in orthotopic pancreatic cancer
Intratumoral region . | No. tumors . | Gene expression median (range) . | . | |
---|---|---|---|---|
. | . | TIG-1 . | AMIGO2 . | |
Center | 3 | 73.6 (61.8–83.0)* | 18.6 (16.0–32.4)* | |
Periphery | 3 | 19.7 (15.1–31.3)* | 74.9 (51.2–85.3)* |
Intratumoral region . | No. tumors . | Gene expression median (range) . | . | |
---|---|---|---|---|
. | . | TIG-1 . | AMIGO2 . | |
Center | 3 | 73.6 (61.8–83.0)* | 18.6 (16.0–32.4)* | |
Periphery | 3 | 19.7 (15.1–31.3)* | 74.9 (51.2–85.3)* |
NOTE: Median (range) fluorescence intensities of each field. Intensity was measured in six different fields in both the peripheral and central regions of three different orthotopic tumors.
P < 0.0001, F test.
Discussion
In this study, we clearly showed zonal heterogeneity for gene expression profiles of cells in the same tumor depending on the zones where they are growing (dividing). Hundreds of genes showed differential expression in the central and the peripheral zones of pancreatic tumor. Differences in gene expression profiles result from the processes of selection, adaptation, or both. Clonal populations expressing genes suited for specific microenvironments may survive (selection) or certain gene expression patterns may be induced by interactions between the tumor cells and a different microenvironment (adaptation; ref. 1). Characterization of gene expression profiles is very important for predicting the prognosis of the disease and whether the patient may respond to therapy.
To produce reproducible and reliable data, the collection of viable cells from a specific zone of a tumor and extraction of fresh mRNAs were the most important and critical steps of this study. Tumors of similar size were selected, anoxic time was minimized by immediate extraction of mRNAs, and cells were collected by laser microdissection from the corresponding zones of each tumor. Microscopic dissection allowed us to exclude necrotic areas.
Our results from the gene ontology analysis were biologically relevant in that the cells at the edge of the tumor expressed genes related to cell division, attachment, and cell migration because tumor cells at the periphery are dividing, attaching to the next tissue, and changing their shape in preparation for invading. In particular, overexpression of MMP-2 in the peripheral zone agrees with the previous data showing the biological function of MMP-2 in cells within the invasive edge of the tumors (23–27).
By contrast, the central zone had many genes involved in regulation of cell proliferation, response to stress, hexose metabolism, carbohydrate metabolism, and cell death (Table 1). We expected that genes related to response to hypoxia would appear on the top of the list for up-regulated genes in the central zone of the tumor; however, only three genes were included and the ratio was low (Table 2), falling below the cutoff for statistical significance in our study (P = 0.062; Table 1). Either surviving cells depend on other pathways (adaptation) or cells that are independent of this pathway survive (selection).
We confirmed the reliability of microarray data by FISH analysis for AMIGO2 and RARRES1. AMIGO2 is the novel cell adhesion molecule related to AMIGO, a leucine-rich repeat superfamily member expressed in fiber tracts of neuronal tissues (37). AMIGO2 is also associated with tumorigenesis and cell adhesion signaling in gastric cancer (38). Overexpression of AMIGO2 in pancreatic cancer is a novel finding in this report. Further study of it may lead to development of novel and effective therapeutic strategies against pancreatic cancer.
Decreased expression of RARRES1, a potential tumor suppressor gene for human prostate cancer, was associated with an increase in malignant characteristics of both prostate cancer cell lines and tissues (39). RARRES1 may be related to the ability of cell to react to retinoid stimulation (39). Strong association of loss of RARRES1 expression with RARRES1 promoter hypermethylation was observed in half of the cases tested in various cancer cell lines and primary tumor samples (40).
L3.6pl, the human pancreatic cancer cell line used in this study, has an aggressive phenotype (31). Whether endowing a highly metastatic phenotype and candidate genes producing an antiproliferation phenotype were expressed depended on the zonal microenvironment of the tumor. Further study to reveal whether those differences arise from adaptation or selection would be required to understand the mechanism of disease progression and allow development of treatment modalities.
Collectively, our present results predict that unless the zone of the tumor used to analyze gene expression is standardized, the results will be heterogeneous. Indeed, Moroni et al. (41) examined human colorectal cancers and reported intratumoral heterogeneity in EGFR gene amplification as correlated with protein level. Michiels et al. (42) reanalyzed data from the seven largest published studies that have attempted to predict prognosis of cancer patients based on DNA microarray analysis and concluded that five of the seven studies did not classify patients better than chance. To minimize intratumoral heterogeneity for gene expression among different cervical cancer patients, Bachtiary et al. (43) recommended analyzing multiple biopsies of each tumor.
In summary, the present results showing regional differences in gene expression between the peripheral zone and central zone of tumors are not surprising. Whereas earlier data derived from nondissected tumor samples differed among different studies (41–43), our results clearly indicate the absolute necessity of using LCM from specific locations of a neoplasm for collection of reproducible data.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
Acknowledgments
Grant support: Cancer Center Support Core grant CA16672 and National Cancer Institute, NIH Specialized Programs in Research Excellence in Prostate Cancer grant CA90270.
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.
We thank Walter Pagel for critical editorial review and Lola López for expert preparation of this manuscript.