Proteomic analysis of primary colon cancer-associated fibroblasts using the SELDI-ProteinChip platform

Proteomic analysis of primary colon cancer-associated fibroblasts using the SELDI-ProteinChip platform 1 first-author 2 1 1 1 1 3 1 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:Suzhan.zhang@hotmail.com †E-mail:Suzhan.zhang@hotmail.com Suzhan.zhang@hotmail.com Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education, Key Laboratory of Molecular Biology in Medical Sciences, Zhejiang Province, China), the Second Affiliated Hospital School of Medicine, Zhejiang University, Hangzhou 310009, China Cancer Institute (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education, Key Laboratory of Molecular Biology in Medical Sciences, Zhejiang Province, China), the Second Affiliated Hospital School of Medicine, Zhejiang University, Hangzhou 310009, China Department of Oncology, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Department of Oncology, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Department of Gastroenterology, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Department of Gastroenterology, the Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310009, China Objective: 2 Cancer-associated fibroblasts (CAFs) are one of the hallmarks of the cancer microenvironment. Recent evidence has indicated that CAFs are more competent in enhancing cancer cell growth and migration than normal fibroblasts. However, the unique protein expression of CAFs has not been fully elucidated. This study aims to investigate the characterizations of colon CAFs by comparing the differential protein expression between CAFs and normal fibroblasts. Methods: 2 Primary fibroblasts were isolated from surgical specimen of human colon cancer and matched normal colonic tissue. Purity of the cell population was verified through immunostain analysis. Total cell lysates and conditioned media from each group of cells were extracted, and protein expression analysis was conducted using the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) ProteinChip platform. Results: 2 Most primary cells showed typical fibroblast-like features after two weeks. Increased proportion of α-smooth muscle actin-positive myofibroblasts was detected within the CAFs in four of the six pairs of primary cells. Fibroblast activation protein was weakly expressed in most cells without differences. Using SELDI-TOF-MS ProteinChip platform, four protein peaks mass over charge ratio ( m / z ) 1142, 3011, 4035, and 4945 were detected in the total cell lysates, and two protein peaks m / z 1368 and 1389 were detected in the conditioned media. The potential candidate proteins found in the Swiss-Prot database include morphogenetic neuropeptides, FMRFamide-related peptides, insulin-like growth factor II, thymosin β-4-like protein 3, and tight junction-associated protein 1. Conclusions: 2 Using the SELDI-ProteinChip platform, differential protein expressions were identified in colon CAFs compared with normal colonic stromal fibroblasts. The complex proteomic alternations in colon CAFs may play important roles related to the colon cancer microenvironment. Colon cancer Cancer microenvironment Cancer-associated fibroblasts Proteomics Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) Introduction 1 Cancer invasion depends on many cellular and molecular changes, not only in epithelial cells, but also in surrounding stromal cells. The interaction between cancer cells and their stromal microenvironment is crucial for cancer development and progression. Changes in the cancer microenvironment during cancer progression involve angiogenesis, lymphogenesis, macrophage infiltration, release of cytokines/growth factors and proteolytic enzymes, and alterations in the extracellular matrix (Almholt and Johnsen, 2003 2003 ). The putative cancer stroma consists of different cellular elements, such as inflammatory cells, endothelial cells, and fibroblasts. One of the hallmarks of these elements is cancer-associated fibroblasts (CAFs). CAFs can be propagated in vitro for extended periods without changing their phenotypes; hence, a number of studies have been conducted to investigate their phenotype characteristics, tumor-enhancing functions, and distinctive gene expression profiles (Allinen et al., 2004 2004 ; Orimo et al., 2005 2005 ; Bauer e t al ., 2010 2010 ). However, gene and protein expression levels cannot be easily correlated because proteins could exist in different functional states through post-translational modification. Therefore, gaining an overall view of CAFs at the protein level is important. However, to date, few studies have investigated the protein expression that can reflect differences between CAFs and their counterpart, normal fibroblasts (NFs). Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is a proteomic technique particularly powerful for analyzing complex biologic samples. Using chromatographic surfaces to retain proteins and peptides on ProteinChip arrays based on their physicochemical properties, the platform performs direct analysis via TOF-MS. Peak patterns are formed via MS, which represent protein expression profiles. The location and intensity of every peak in the pattern reflect the mass over charge ratio ( m / z ) and abundance of the corresponding protein (Chen et al., 2004 2004 ; Kumar et al., 2008 2008 ). This high-throughput technique allows the screening of low molecular weight proteins compared with other traditional methods, such as 2D gel electrophoresis. Combined with bioinformatic approaches, SELDI-TOF-MS is valuable in establishing protein expression profiles and in discovering new biomarkers with high sensitivity and specificity (Chen et al., 2004 2004 ; Melle et al., 2006 2006 ; Xu et al., 2006 2006 ; Enkelmann et al., 2011 2011 ). The present study focuses on CAFs in colon cancer by establishing primary fibroblast cultures from surgical specimens of patients with colon cancers and deals with the common phenotypical features of CAFs in vitro. The protein profiles of both total cell lysate and conditioned media extracted from CAFs and NFs were compared via SELDI-TOF-MS to better understand the specific features of CAFs at the protein level. Materials and methods 1 Materials 2 Iscove’s modified Dulbecco medium (IMDM) and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA). Collagenase Type I and hyaluronidase were purchased from Sigma (St. Louis, MO, USA). The cell strainer was purchased from BD Biosciences (San Diego, CA). The antibodies used, including anti-Pan cytokeratin (ab78478), anti-desmin (ab15200), anti-α-smooth muscle actin (α-SMA, ab15734), and anti-fibroblast activation protein (FAP) (ab28244), were all from Abcam (HK, China); Dako EnVision system and anti-vimentin (clone V9) were from Dako (Glostrup, Denmark). The protease inhibitor cocktail was purchased from Thermo (Rockford, IL). Patients and tissue specimens 2 Fresh surgical specimens were obtained with informed consent from patients who underwent surgical resection for colon cancer. The study was approved by the Research Ethics Board at Zhejiang University. The cancer-associated regions selected were minimally necrotic regions of the tumor mass. The normal colonic mucosa selected from the same specimen was at least 5 cm distal to the outer margin of the cancer mass. The specimens were determined through cross-examination at the time of the surgical excision and confirmed via subsequent histological analysis. The tumor characteristics of the patients are listed in Table 1 1 . The World Health Organization (WHO) standard grading system containing four categories was used: well differentiated, moderately differentiated, poorly differentiated, and undifferentiated (Aaltonen et al., 2000 2000 ). Patient No. Sex Age (year) Tumour site TNM Stromal cell culture 1 Female 48 Sigmoid T3N2M0 CAF-1, NF-1 2 Female 69 Left colon T3N0M0 CAF-2, NF-2 3 Male 61 Sigmoid T3N0M0 CAF-3, NF-3 4 Female 88 Right colon T3N0M0 CAF-4, NF-4 5 Female 59 Sigmoid T3N0M1 CAF-5, NF-5 6 Male 53 Left colon T3N1M0 CAF-6, NF-6 Isolation of human colon fibroblasts and cell culture 2 To obtain the CAFs, the fresh specimens were immediately minced into 1-mm 3 sizes and extensively washed in 100 mmol/L ice-cold phosphate buffered saline (PBS) with 5% antibiotic-antimycotic solution (50 000 U/ml penicillin, 125 μg/ml amphotericin B, and 50 000 μg/ml streptomycin). Subsequently, the solution was placed in IMDM, 1% FBS, 1 mg/ml collagenase Type I, and 1 mg/ml hyaluronidase, and then incubated for 2 h at 37 °C. The solution was vigorously shaken every 10 min to encourage dissociation. Mucosa strips were dissected from the submucosa to isolate the normal colonic fibroblasts; the colonic smooth muscle and serosa layers were discarded. Using the technique previously described by Brenmoehl et al. ( 2009 2009 ), the mucosa strips were denuded of epithelial cells through sequential treatments with 2 mmol/L ethylenediaminetetraacetic acid (EDTA) followed by enzyme digestion. The dissociated cells were filtered through a 40-μm cell strainer, centrifuged, and resuspended in IMDM, 10% FBS, and 1% antibiotic-antimycotic solution. The cells were then plated into 10-cm petri dishes. Cell viability was detected via trypan blue dye exclusion assay. All the experiments were performed in cells between passages 5 and 10. Morphologic features and immunostaining of primary cultured fibroblasts 2 The morphologic features and sizes of the colon CAFs and NFs were assessed as cells passaged for five population doublings. For the immunostain experiments, the cells were cultured overnight on chamber slides, fixed with 4% paraformaldehyde, and then permeabilized with 0.25% Triton X-100 for the detection of vimentin, pan cytokeratin, desmin, α-SMA, and FAP. To quantify the percentage of α-SMA-positive cells, the positive cell numbers relative to the total cell numbers (>100 counted cells) were evaluated in 10 independent fields from three different wells of each fibroblast type. Paraffin sections prepared from human colon tissues were used as the control. Preparation of total cell lysates and conditioned media 2 For total cell lysate extraction, the primary cells of both CAF and NF groups were harvested after PBS washing and then resuspended in lysis buffer composed of 7 mol/L urea, 2 mol/L thiourea, 100 mol/L dithiothreitol, 4% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate, and protease inhibitor cocktail. After 60 min lysis on ice, the total cell lysates were obtained through centrifugation at 20 000× g for 15 min to remove insoluble debris. For secreted protein analysis, both CAF and NF groups were grown in standard culture media (IMDM with 10% FBS) until they reached a confluency state of approximately 80%, washed three times with PBS, and then incubated in conditioned media (serum-free IMDM) for 24 h. The conditioned media were collected, cooled on ice, centrifuged at 200× g for 10 min to remove floating cells and cellular debris, and then subjected to sterile filtration (pore size: 0.22 mm). The conditioned media were dialyzed, concentrated through lyophilization, and then reconstituted in a buffer. The protein concentration was measured using a BioRad DC protein assay kit (BioRad, Hercules, CA) and adjusted to 3.5 mg/ml (total cell lysate) and 1.5 mg/ml (conditioned media) for expression analysis. SELDI protein profiling 2 Up to 200 μl sodium acetate was added to each spot of the bioprocessor (Ciphergen Biosystems) that contains the ProteinChip arrays. The bioprocessor was sealed and agitated on a platform shaker for 5 min at 4 °C; the process was repeated. The chips were removed from the bioprocess, and diluted samples (5 μl) were applied to each spot of the protein arrays. Each sample was added to three different spots. The chips were plugged into a wet box at room temperature for an hour. The excess of the mixtures was discarded. The chip arrays were placed in the bioprocessor; the chips were then washed thrice with 200 μl sodium acetate and twice with deionized water. Finally, the chips were removed from the bioprocessor and air-dried. Prior to SELDI-TOF-MS analysis, 1 μl semi-saturated solution of the scintillation proximity assay in 50% cerium (IV) ammonium nitrate and 0.5% trifluoroacetic acid was applied twice onto each spot, and the chips were again air-dried. The chips were analyzed using a PBS-II plus mass spectrometer reader (Ciphergen Biosystems). Data were obtained by averaging 140 laser shots with an intensity of 170, a detector sensitivity of 5, a high mass of 100 000 Da, and an optimized range of 1 000 to 20 000 Da. Mass accuracy was calibrated using the all-in-one peptide molecular mass standard (Ciphergen Biosystems). Data analysis 2 Data of every spectrum were collected and analyzed using the ProteinChip Software version 3.0 (Ciphergen Biosystems) with integrated Biomarker Wizard software (Ciphergen Biosystems) as previously described (Ge et al., 2008 2008 ). The baselines were subtracted, and normalization was performed according to the software instructions. Biomarker Wizard was used to identify the corresponding peaks in the spectra. Significant differences in the peak intensity of particular proteins between each group were calculated through the software using the Mann-Whitney non-parametrical test and then further examined via paired Student’s t -test or paired Wilcoxon test if the data did not follow normal distribution. P < 0.05 was considered to indicate statistical significance. Based on the adjustment of the isoelectric point ranging from 4 to 14 and the specific mass provided by the ProteinChip software with a variation of 0.3% in the mass range, the possible protein candidates were searched using the protein database TagIdent provided by ExPASy molecular biology server. Results 1 Isolation of primary fibroblastic population from human colon tissue 2 Fibroblasts were extracted from six human colon carcinoma tissues obtained from surgical resection. The tumor masses were dissociated, and various cell types were separated to obtain populations of CAFs. The matched NFs were isolated from the normal colonic mucosa of the same six patients. The cultures reached confluency after two weeks and were stored in −80 °C at cell passages 2 to 3. The cultured primary cells showed typical fibroblast-like features, with spindle-like shapes and elongated projections (Fig. 1 1 ). Representative microscopic fields show the cancer-associated fibroblasts CAF-3 (b), CAF-5 (d), and CAF-6 (f), and their counterpart normal fibroblasts NF-3 (a), NF-5 (c), and NF-6 (e) Characteristics of CAFs and NFs 2 In cell passage 5, the purities of the various fibroblast populations were verified through immunostaining. The population expressed the fibroblastic marker vimentin (Fig. 2a 2a ) but lacked the epithelial cell marker cytokeratin (Fig. 2b 2b ) and the smooth muscle cell marker desmin (Fig. 2c 2c ). A small fraction in both groups of cells expressed the myofibroblast marker α-SMA (Fig. 2e 2e ). An enhanced expression of α-SMA in CAFs in four out of the six pairs was observed. In patient 2, the expression of α-SMA was more enhanced in NFs compared with CAFs, whereas the expression was equal in patient 5 (Table 2 2 ). FAP, another myofibroblast marker, was weakly expressed in most primary fibroblasts. No obvious difference was observed in the expression of FAP between CAFs and NFs (Fig. 2d 2d ). All the primary fibroblastic cells strongly expressed fibroblastic marker vimentin (a) but were negative for epithelial cell marker cytokeratin (b) and smooth muscle cell marker desmin (c). Both CAFs and NFs weakly expressed FAP (d), but CAFs showed enhanced expression of α-SMA (e) compared with NFs. Paraffin sections prepared from human colon tissues were used as the control 2 Patient No. 2 α-SMA-positive cells in cultures (%) CAFs NFs 1 25.90 14.70 2 4.06 21.20 3 7.53 5.16 4 15.30 10.70 5 4.63 4.87 6 19.00 6.40 Protein profiling of total cell lysates and conditioned media 2 After cluster analysis through Biomarker Wizard (Ciphergen Inc., Fremont, CA; ProteinChip software versions 3.0), 398 peaks of the total cell lysates and 547 peaks of the conditioned media were detected for discriminating CAFs and NFs. The peaks were between m / z 1 000 and 30 000. Peaks with m / z < 1 000 were mainly ion noise from the matrix; thus, they were excluded. The peaks were further examined using paired Student’s t -test or paired Wilcoxon test if the data did not follow normal distribution. Four protein peaks m / z 1 142, 3 011, 4 035, and 4 945 in the total cell lysates and two protein peaks m / z 1 368 and 1 389 in the conditioned media distinguished CAFs from NFs ( P < 0.05, Fig. 3 3 ). Table 3 3 presents the descriptive statistics of the six peaks. The spectra in peak view and gel view of the protein peaks are shown in Fig. 4 4 . (a) In conditioned media, protein peaks m / z 1 368 and 1 389 were decreased in CAFs compared with NFs, with P < 0.05; (b) In the total cell lysates, protein peaks were sorted by m / z , with P < 0.05, among which protein peaks m / z 3 011, 4 035, and 4 945 were increased in CAFs and protein peak m / z 1 142 was decreased in CAFs compared with NFs The protein peaks corresponding to m / z 1 142, 1 368, and 1 389 were weakly expressed in the CAFs, but highly expressed in the NFs. The protein peaks corresponding to m / z 3 011, 4 035, and 4 945 were highly expressed in the CAFs, but weekly expressed in the NFs Values are expressed as mean±SD 2 Solution 2 Peak m / z 2 Relative peak intensity * * 2 P value NFs CAFs 4 Total cell lysates 1142 15.1±3.76 11.2±2.70 0.040 3011 5.68±0.92 9.74±3.56 0.020 4035 3.72±0.64 4.71±1.46 0.026 4945 5.97±2.48 9.24±1.94 0.028 5 2 Conditioned media 1368 8.27±2.70 4.99±1.27 0.046 1389 3.08±1.68 1.03±0.91 0.016 Database search for potential protein candidates 2 The Swiss-Prot database was searched using the TagIdent tool, along with the pI and mass information, to obtain matching proteins. Six pieces of matching information were obtained from the database. These potential candidate proteins include morphogenetic neuropeptide, FMRFamide-related peptides, insulin-like growth factor II, thymosin β-4-like protein 3, and tight junction-associated protein 1. Therefore, protein peaks with m/z 1 142, 1 368, 4 035, and 4 945 have greater potential to identify colon cancer-associated stroma in future research (Table 4 4 ). Searching was conducted using TagIdent tool with mass and the pI (4.0–14.0) information for CM10 2 Protein peak m / z 3 Meaningful matching information in Swiss-Prot database 2 Comments Protein name M w pI 1 142 Morphogenetic neuropeptide chain: 1–11 1 142 8.75 An autocrine growth factor 1 368 FMRFamide-related peptides chain: 66–76 1 369 9.47 Morphine modulating peptides, wide-ranging physiologic effects 4 035 Insulin-like growth factor II chain: 93–126 4 030 8.42 Possesses growth-promoting activity 4 945 Thymosin β-4-like protein 3 chain: 2–44 4 932 5.03 Plays an important role in the organization of the cytoskeleton, inhibits actin polymerization Tight junction-associated protein 1 chain: 1–43 4 959 8.16 Cell junction, Golgi apparatus, tight junction Discussion 1 Primary stromal cell cultures isolated from colon cancer might in part represent the corresponding cancer microenvironment, thus providing a useful complement to the current cellular biochemistry and therapeutic research in colon cancer. Previous studies have investigated colon CAFs by establishing CAFs in primary fibroblast cultures from metastatic colon cancer in the liver (Nakagawa et al., 2004 2004 ; Mueller et al., 2007 2007 ). However, few studies have exploited the characteristics of CAFs in primary colon cancer. In the present study, an optimized method was used to isolate and culture the paired stromal fibroblasts derived from the human colon cancer and from the adjacent normal tissue in the same specimen. To avoid bias due to individual differences, all experiments were performed by comparing paired CAFs and corresponding NFs. High serum media suitable for fibroblast growth were used. With the growth advantage of fibroblasts, most tumor cells and colonic epithelial cells diminished around cell passages 2 and 3. To avoid cross-contamination of smooth muscle cells in the NF cultures, the colonic mucosa was dissected from the submucosa, and the smooth muscle layers were discarded. Individual differences among the different specimens due to age, stage, and status resulted in different cultivating characteristics. Generally, the primary CAFs have an advantage in proliferation rate. Morphologically, all established stromal cultures grew as a monolayer of cells with the same fibroblast-like appearance. The CAFs were slightly more slender than the NFs, but without significant differences (Fig. 1 1 ). The features of these cells were further characterized with the fibroblastic marker vimentin, epithelial cell marker cytokeratin, smooth muscle cell marker desmin, putative myofibroblast marker α-SMA, and FAP. Vimentin, a common mesenchymal cytoskeletal marker, was highly expressed in all primary cells (Fig. 2a 2a ), whereas cytokeratin was negative in most cells (Fig. 2b 2b ). This result indicates that vimentin is expressed in cells of mesenchymal origin. Very few cytokeratin-positive cells were detected, which may be due to epithelial-mesenchymal transition. Monoclonal desmin antibody was used as the smooth muscle cell marker. The negative expression of desmin in most primary cells indicated minimal contamination of the smooth muscle cells (Fig. 2c 2c ). Myofibroblasts, a prominent component of CAFs, distinguish CAFs from NFs. α-SMA, a protein marker of myogenic fibroblast differentiation, is a common marker of myofibroblasts (Bhowmick et al., 2004 2004 ; Orimo et al., 2005 2005 ). In the present study, monoclone α-SMA and FAP antibodies were used in the immunostain analysis to check for possible presence of myofibroblast cells (Fig. 2e 2e ). An increased proportion of α-SMA-positive cells was found in four of the six pairs; this result indicates that the isolated CAFs contain a high proportion of myofibroblasts (Table 2 2 ). This high expression of α-SMA in cancer stromal cells is in agreement with a previous study (Orimo et al., 2005 2005 ). However, a larger sample analysis is required for statistical analysis. FAP is a good myofibroblast marker in immunohistochemical analyses; it was mainly expressed in the invasive colon cancer stroma, but negative in the normal colonic mucosa (Henriksson et al., 2011 2011 ). In the present study, FAP was weakly expressed in most primary fibroblasts (Fig. 2d 2d ); the different expressions of FAP between CAFs and NFs were not observed. To further investigate the protein profile, SELDI-MS analyses of total cell lysates and conditioned media taken from CAFs and NFs were used. A range of peaks across CM10 surfaces increased ( m / z 3 011, 4 035, and 4 945) or decreased ( m / z 1 142, 1 368, and 1 389) in the CAFs compared with their counterpart NFs (Table 3 3 and Fig. 3 3 ). However, no direct relationship was found in the protein profiles of the total cell lysates and conditioned media. The Swiss-Prot database was searched using the TagIdent tool with pI and mass information to obtain matching information for each protein peak (Table 4 4 ). The search results suggest that morphogenetic neuropeptide is the candidate for protein mass 1 142 Da, decreased in CAFs (Fig. 4 4 ). Morphogenetic neuropeptide is an autocrine growth factor found in neural cells, mammalian intestine, and hypothalamus; it is also involved in cell cycle, cell division, and mitosis. FMRFamide-related peptides are the candidates for protein mass 1 368 Da, decreased in CAFs (Fig. 4 4 ). They have wide-ranging physiologic effects, including the modulation of morphine-induced analgesia, elevation of arterial blood pressure, and increased somatostatin secretion from the pancreas. For protein mass 4 035 Da, increased in CAFs (Fig. 4 4 ), the candidate protein is insulin-like growth factor II (4 030 Da). Insulin-like growth factors possess growth-promoting activity. In vitro, they are potent mitogens for cultured cells. For protein mass 4 945 Da, increased in CAFs (Fig. 4 4 ), the database provided two candidate proteins. One is thymosin β-4-like protein (4 932 Da), which plays an important role in the organization of the cytoskeleton; the expression of thymosin β-4 promotes tumorigenicity and metastatic potential and induces drug resistance of malignant cells. The second candidate is tight junction-associated protein 1 (4 959 Da), which is related to cell junction, Golgi apparatus, and tight junction. Tight junction-associated protein modulates cell proliferation and is useful in the diagnosis of soft tissue sarcoma (Facchetti et al., 2007 2007 ; Jayagopal et al., 2011 2011 ). Other protein masses, including 1 389 and 3 011 Da, were searched across the database; however, no meaningful matching proteins were found, indicating that the functions of these putative uncharacterized proteins are unknown. CAFs are heterogeneous and possess complex mechanisms in facilitating tumor development, which express different sets of protein markers. α-SMA, FAP, S100A4, platelet derived growth factor receptor-β, and chondroitin sulfate proteoglycan are markers of CAFs, but none of them can exclusively reflect the characteristics of CAFs (Sugimoto et al., 2006 2006 ). The primarily cultured CAFs and NFs in the present study are a heterogeneous population, which to some extent, share similar features. The possible protein peaks detected in the present study include peptides or small proteins with low abundance, which can hardly be detected using traditional methods, but are essential for associated research. Further verification and function investigation are planned. Although the SELDI-ProteinChip platform is good for protein profiling research, it does not allow direct purification and functional identification, and still lacks resolution for the detection of higher molecular weight proteins. Most expected growth factors or matrix proteins have larger molecular weights beyond the limits of SELDI detection. Conclusions 1 A cell culture method was developed for the isolation and characterization of colon CAFs and their normal counterpart colonic fibroblasts. The current study is the first to reveal complex proteomic changes using the SELDI-ProteinChip platform in distinguishing colon CAFs from colonic NFs. These proteomic alternations may play important roles in the interaction between cancer cells and cancer stroma. The current study provided better understanding of the complex colon cancer microenvironment. Further studies on altered protein in colon cancer progression are currently in progress. References LA Aaltonen , SR Hamilton . World Health Organization and International Agency for Research on Cancer Pathology and Genetics of Tumours of the Digestive System , 2000 . : Lyon IARC Press , : Oxford Oxford University Press (Distributor) , . 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Preoperatively molecular staging with CM10 ProteinChip and SELDI-TOF-MS for colorectal cancer patients We are very grateful to Prof. Xun HU, Prof. Rong-zhen XU, Dr. Wei-ting GE, and Dr. Jia-wei ZHANG in Cancer Institute, School of Medicine, Zhejiang University for their assistance and suggestions. 10.1631/jzus.B1100266 Project supported by the National Natural Science Foundation of China (Nos. 81000892, 81071801, and 30801341) and the Research Fund for the Doctoral Program of Higher Education of China (No. 200803351107) 2011-08-17 2011-12-14 2011-11-30 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2012 2012 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 03 13