Proteomic and metabolomic features in patients with HCC responding to lenvatinib and anti-PD1 therapy.

Combination therapy (lenvatinib/programmed death-1 inhibitor) is effective for treating unresectable hepatocellular carcinoma (uHCC). We reveal that responders have better overall and progression-free survival, as well as high tumor mutation burden and special somatic variants. We analyze the proteome and metabolome of 82 plasma samples from patients with hepatocellular carcinoma (HCC; n = 51) and normal controls (n = 15), revealing that individual differences outweigh treatment differences. Responders exhibit enhanced activity in the alternative/lectin complement pathway and higher levels of lysophosphatidylcholines (LysoPCs), predicting a favorable prognosis. Non-responders are enriched for immunoglobulins, predicting worse outcomes. Compared to normal controls, HCC plasma proteins show acute inflammatory response and platelet activation, while LysoPCs decrease. Combination therapy increases LysoPCs/phosphocholines in responders. Logistic regression/random forest models using metabolomic features achieve good performance in the prediction of responders. Proteomic analysis of cancer tissues unveils molecular features that are associated with side effects in responders receiving combination therapy. In conclusion, our analysis identifies plasma features associated with uHCC responders to combination therapy.

Insight into the mechanism of gallstone disease by proteomic and metaproteomic characterization of human bile.

Introduction: Cholesterol gallstone disease is a prevalent condition that has a significant economic impact. However, the role of the bile microbiome in its development and the host's responses to it remain poorly understood. Methods: In this study, we conducted a comprehensive analysis of microbial and human bile proteins in 40 individuals with either gallstone disease or gallbladder polyps. We employed a combined proteomic and metaproteomic approach, as well as meta-taxonomic analysis, functional pathway enrichment, and Western blot analyses. Results: Our metaproteomic analysis, utilizing the lowest common ancestor algorithm, identified 158 microbial taxa in the bile samples. We discovered microbial taxa that may contribute to gallstone formation, including ß-glucuronidase-producing bacteria such as Streptococcus, Staphylococcus, and Clostridium, as well as those involved in biofilm formation like Helicobacter, Cyanobacteria, Pseudomonas, Escherichia coli, and Clostridium. Furthermore, we identified 2,749 human proteins and 87 microbial proteins with a protein false discovery rate (FDR) of 1% and at least 2 distinct peptides. Among these proteins, we found microbial proteins crucial to biofilm formation, such as QDR3, ompA, ndk, pstS, nanA, pfIB, and dnaK. Notably, QDR3 showed a gradual upregulation from chronic to acute cholesterol gallstone disease when compared to polyp samples. Additionally, we discovered other microbial proteins that enhance bacterial virulence and gallstone formation by counteracting host oxidative stress, including sodB, katG, rbr, htrA, and ahpC. We also identified microbial proteins like lepA, rtxA, pckA, tuf, and tpiA that are linked to bacterial virulence and potential gallstone formation, with lepA being upregulated in gallstone bile compared to polyp bile. Furthermore, our analysis of the host proteome in gallstone bile revealed enhanced inflammatory molecular profiles, including innate immune molecules against microbial infections. Gallstone bile exhibited overrepresented pathways related to blood coagulation, folate metabolism, and the IL-17 pathway. However, we observed suppressed metabolic activities, particularly catabolic metabolism and transport activities, in gallstone bile compared to polyp bile. Notably, acute cholelithiasis bile demonstrated significantly impaired metabolic activities compared to chronic cholelithiasis bile. Conclusion: Our study provides a comprehensive metaproteomic analysis of bile samples related to gallstone disease, offering new insights into the microbiome-host interaction and gallstone formation mechanism.

Effect of far-infrared radiation on inhibition of colonies on packaging during storage of sterilised surgical instruments.

The sterilisation of surgical instruments is a major factor in infection control in the operating room (OR). All items used in the OR must be sterile for patient safety. Therefore, the present study evaluated the effect of far-infrared radiation (FIR) on the inhibition of colonies on packaging surface during the long-term storage of sterilised surgical instruments. From September 2021 to July 2022, 68.2% of 85 packages without FIR treatment showed microbial growth after incubation at 35 °C for 30 days and at room temperature for 5 days. A total of 34 bacterial species were identified, with the number of colonies increasing over time. In total, 130 colony-forming units were observed. The main microorganisms detected were Staphylococcus spp. (35%) and Bacillus spp. (21%) , Kocuria marina and Lactobacillus spp. (14%), and mould (5%). No colonies were found in 72 packages treated with FIR in the OR. Even after sterilisation, microbial growth can occur due to movement of the packages by staff, sweeping of floors, lack of high-efficiency particulate air filtration, high humidity, and inadequate hand hygiene. Thus, safe and simple far-infrared devices that allow continuous disinfection for storage spaces, as well as temperature and humidity control, help to reduce microorganisms in the OR.

The Intratumoral Bacterial Metataxonomic Signature of Hepatocellular Carcinoma.

Microbiota is implicated in hepatocellular carcinoma (HCC). The spectrum of intratumoral microbiota associated with HCC progression remains elusive. Fluorescence in situ hybridization revealed that microbial DNAs were distributed in the cytosol of liver hepatocytes and erythrocytes. Viable anaerobic or aerobic bacteria were recovered in HCC tissues by fresh tissue culture. We performed a comprehensive DNA sequencing of bacterial 16S rRNA genes in 156 samples from 28 normal liver, 64 peritumoral, and 64 HCC tissues, and the DNA sequencing yielded 4.2 million high-quality reads. Both alpha and beta diversity in peritumor and HCC microbiota were increased compared to normal controls. The most predominant phyla in HCC were Patescibacteria, Proteobacteria, Bacteroidota, Firmicutes, and Actinobacteriota. phyla of Proteobacteria, Firmicutes, and Actinobacteriota, and classes of Bacilli and Actinobacteria, were consistently enriched in peritumor and HCC tissues, while Gammaproteobacteria was especially abundant in HCC tissues compared to normal controls. Streptococcaceae and Lactococcus were the marker taxa of HCC cirrhosis. The Staphylococcus branch and Caulobacter branch were selectively enriched in HBV-negative HCCs. The abundance of Proteobacteria, Gammaproteobacteria, Firmicutes, Actinobacteriota, and Saccharimonadia were associated with the clinicopathological features of HCC patients. The inferred functions of different taxa were changed between the microbiota of normal liver and peritumor/HCC. Random forest machine learning achieved great discriminative performance in HCC prediction (area under the curve [AUC] = 1.00 in the training cohort, AUC = 0.950 for top five class signature, and AUC = 0.943 for the top 50 operational taxonomy units [OTUs] in the validation cohort). Our analysis highlights the complexity and diversity of the liver and HCC microbiota and established a specific intratumoral microbial signature for the potential prediction of HCC. IMPORTANCE Gut microbiome is an important regulator of hepatic inflammation, detoxification, and immunity, and contributes to the carcinogenesis of liver cancer. Intratumoral bacteria are supposed to be closer to the tumor cells, forming a microenvironment that may be relevant to the pathological process of hepatocellular carcinoma (HCC). However, the presence of viable intratumoral bacteria remains unclear. It is worth exploring whether the metataxonomic characteristics of intratumoral bacteria can be used as a potential marker for HCC prediction. Here, we present the first evidence of the existence of viable intratumoral bacteria in HCC using the tissue culture method. We revealed that microbial DNAs were distributed in the cytosol of liver hepatocytes and erythrocytes. We analyzed the diversity, structure, and abundance of normal liver and HCC microbiota. We built a machine learning model for HCC prediction using intratumoral bacterial features. We show that specific taxa represent potential targets for both therapeutic and diagnostic interventions.

Enhancement of E-cadherin expression and processing and driving of cancer cell metastasis by ARID1A deficiency.

The ARID1A gene, which encodes a subunit of the SWI/SNF chromatin remodeling complex, has been found to be frequently mutated in many human cancer types. However, the function and mechanism of ARID1A in cancer metastasis are still unclear. Here, we show that knockdown of ARID1A increases the ability of breast cancer cells to proliferate, migrate, invade, and metastasize in vivo. The ARID1A-related SWI/SNF complex binds to the second exon of CDH1 and negatively modulates the expression of E-cadherin/CDH1 by recruiting the transcriptional repressor ZEB2 to the CDH1 promoter and excluding the presence of RNA polymerase II. The silencing of CDH1 attenuated the migration, invasion, and metastasis of breast cancer cells in which ARID1A was silenced. ARID1A depletion increased the intracellular enzymatic processing of E-cadherin and the production of C-terminal fragment 2 (CTF2) of E-cadherin, which stabilized ß-catenin by competing for binding to the phosphorylation and degradation complex of ß-catenin. The matrix metalloproteinase inhibitor GM6001 inhibited the production of CTF2. In zebrafish and nude mice, ARID1A silencing or CTF2 overexpression activated ß-catenin signaling and promoted migration/invasion and metastasis of cancer cells in vivo. The inhibitors GM6001, BB94, and ICG-001 suppressed the migration and invasion of cancer cells with ARID1A-deficiency. Our findings provide novel insights into the mechanism of ARID1A metastasis and offer a scientific basis for targeted therapy of ARID1A-deficient cancer cells.

Increased Collagen Type V α2 (COL5A2) in Colorectal Cancer is Associated with Poor Prognosis and Tumor Progression.

PURPOSE: Colorectal cancer (CRC) is the third most common cancer in males and the second in females worldwide with very poor prognosis. Extracellular matrix proteins like collagens play important roles in cancer progression. Collagen type V α2 (COL5A2) is increased in several cancers but its role in cancer remains unclear. METHODS: COL5A2 expression was evaluated by interrogation of public Oncomine gene microarray datasets and immunohistochemistry (IHC) analyses of two tissue microarrays containing 180 paired CRC cases. Survival analysis was performed using Kaplan-Meier survival curve and Cox proportional hazards regression methods. COL5A2 was ectopically expressed in CRC cells, and the cell proliferation was measured using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) method. RESULTS: COL5A2 gene was significantly upregulated in the most types of CRC comparing with the normal counterparts. The mRNA expression of COL5A2 was associated with cancer stages, gender, recurrence, microsatellite instability and KRAS status of CRC. COL5A2 protein increased in the cancer epithelial cells comparing with the normal counterpart and associated with age and T stage of CRC, whereas stromal expression of COL5A2 has no significant change between cancerous and normal tissues. COL5A2 gene and protein (epithelial expression) are independent risk factors and predict poor prognosis of CRC. Ectopic expression of COL5A2 drives colon cancer cell growth and upregulates WNT/ß-catenin and PI3K/mTOR signaling via binding DDR1. CONCLUSION: COL5A2 is a potential prognostic marker of CRC.

Development of amphiphile 4-armed PEO-based Ti4+ complex for highly selective enrichment of phosphopeptides.

Protein phosphorylation is a reversible and important post-translational modification. Identification of phosphopeptides without enrichment is difficult for the low-abundance of phosphopeptides in real complex biological samples. Therefore, the effective and selective concentration of phosphopeptides prior to proteomic identification by mass spectrometer is necessary. In this study, we synthesized a novel titanium-based immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides. To improve material hydrophilia to the maximum extent, titanium ions were immobilized on the 4-armed Poly(ethylene oxide)(4µ-PEO-Ti4+), a totally soluble polymer with large molecular weight (20000 g/mol). The 4µ-PEO-Ti4+ was used to enrich phosphopeptides from tryptic digests of standard proteins and real complex biological samples, followed by MALDI-TOF MS analysis. In enrichment of phosphopeptides from 4 pmol ß-casein, the 4µ-PEO-Ti4+ performed the best property with starting material of 99-132 µg, loading buffer of 50% ACN/5% TFA (v/v), elution buffer of 10% NH3·H2O (v/v) and elution time of 30 min. The 4µ-PEO-Ti4+ has a superior detection sensitivity as low as 2 fmol for phosphopeptides. The high selectivity of 4µ-PEO-Ti4+ allows a deep enrichment of phosphopeptides of ß-casein from a mixture with BSA of 1000-fold abundant. The 4µ-PEO-Ti4+ shows great stability and endurability and can be recycled up to at least 5 times. In addition, 4µ-PEO-Ti4+ could detect 10 and 15 phosphopeptides from non-fat milk and nonenzymatic human saliva, respectively. In total, 4µ-PEO-Ti4+ is a novel excellent material which shows great sensitive and selective enrichment of low-abundance phosphopeptides in real complex biological samples.

Chromatin remodeling gene AT-rich interactive domain-containing protein 1A suppresses gastric cancer cell proliferation by targeting PIK3CA and PDK1.

The tumor suppressor gene AT-rich interactive domain-containing protein 1A (ARID1A) was frequently mutated in cancers. The modulation mechanism of ARID1A for PI3K/AKT signaling in gastric cancer (GC) remains elusive. Here, we found that depletion of endogenous ARID1A enhanced the in vitro proliferation, colony formation, cellular growth, nutrient uptake and in vivo xenograft tumor growth of GC cells. PI3K/AKT activation by ARID1A-silencing was profiled using a phospho-protein antibody array. The phosphorylation of PDK1, AKT, GSK3ß and 70S6K, and the protein and mRNA expressions of PI3K and PDK1, were upregulated by ARID1A-silencing. Chromatin immunoprecipitation and luciferase reporter assay revealed that ARID1A-involved SWI/SNF complex inhibited PIK3CA and PDK1 transcription by direct binding to their promoters. Serial deletion mutation analyses revealed that the ARID1A central region containing the HIC1-binding domain, but not the ARID DNA-binding domain and the C-terminal domain, was essential for the inhibition of GC cell growth, PI3K/AKT pathway phosphorylation and its transcriptional modulation activity of PIK3CA and PDK1. The proliferation, cellular growth and glucose consumption of ARID1A-deficient GC cells were efficiently prohibited by allosteric inhibitors mk2206 and LY294002, which targeting AKT and PI3K, respectively. Both inhibitors also downregulated the phosphorylation of PI3K/AKT pathway in ARID1A-deficient GC cells. Such cells were sensitized to the treatment of LY294002, and AT7867, another inhibitor of AKT and p70S6K. The administration of LY294002 alone inhibited the in vivo growth of ARID1A- deficient GC cells in mouse xenograft model. Our study provides a novel insight into the modulatory function and mechanism of ARID1A in PI3K/AKT signaling in GC.

Epithelial but not stromal expression of collagen alpha-1(III) is a diagnostic and prognostic indicator of colorectal carcinoma.

Colorectal cancer (CRC) is the third most common cancer in males and the second in females worldwide with very poor prognosis. Collagen alpha-1(III) (COL3A1) gene, encoding an extracellular matrix protein, is upregulated in human cancers. Here, we revealed that COL3A1 was increased in CRC by analysis of five Oncomine gene expression datasets (n = 496). Immunohistochemistry analysis of a tissue microarray (n = 90) demonstrated that cancer epithelial but not stromal COL3A1 was significantly upregulated comparing with the normal counterparts. High COL3A1 mRNA and/or protein expression was accompanied with high stage, T stage, Dukes stage, grade and older age, as well as smoking and recurrence status. Upregulated COL3A1 predicted poor overall (p = 0.003) and disease-free (p = 0.025) survival. Increased epithelial but not stromal COL3A1 protein predicted worse outcome (p = 0.03). Older patients (age>65) with high COL3A1 had worse survival than younger (age≤65) with high COL3A1. Plasma COL3A1 was increased in CRC patients (n = 86) by 5.4 fold comparing with healthy individuals, enteritis and polyps patients. Plasma COL3A1 had an area under curve (AUC) of 0.92 and the best sensitivity/specificity of 98.8%/69.1%. While plasma CEA had a poorer prediction power (AUC = 0.791, sensitivity/selectivity = 70.2%/73.0%). Older patients (age≥60) had higher plasma COL3A1 than younger patients. The epithelial COL3A1 protein had an AUC of 0.975 and the best sensitivity/specificity of 95.2%/91.1%. Silencing of COL3A1 suppressed CRC cell proliferation in in vitro MTT assay and in in vivo Zebra fish xenograft model by downregulation of PI3K/AKT and WNT signaling. COL3A1 was a novel diagnosis and prognosis marker of CRC.

Stroma derived COL6A3 is a potential prognosis marker of colorectal carcinoma revealed by quantitative proteomics.

Colorectal cancer (CRC) represents the third most common cancer in males and second in females worldwide. Here, we performed a quantitative 8-plex iTRAQ proteomics analysis of the secreted proteins from five colonic fibroblast cultures and three colon cancer epithelial cell lines. We identified 1114 proteins at 0% FDR, including 587 potential secreted proteins. We further recognized 116 fibroblast-enriched proteins which were significantly associated with cell movement, angiogenesis, proliferation and wound healing, and 44 epithelial cell-enriched proteins. By interrogation of Oncomine database, we found that 20 and 8 fibroblast-enriched proteins were up- and downregulated in CRC, respectively. Western blots confirmed the fibroblast-specific secretion of filamin C, COL6A3, COL4A1 and spondin-2. Upregulated mRNA and stroma expression of COL6A3 in CRC, which were revealed by Oncomine analyses and tissue-microarray-immunohistochemistry, indicated poor prognosis. COL6A3 expression was significantly associated with Dukes stage, T stage, stage, recurrence and smoking status. Circulating plasma COL6A3 in CRC patients was upregulated significantly comparing with healthy peoples. Receiver operating characteristic curve analysis revealed that COL6A3 has better predictive performance for CRC with an area under the curve of 0.885 and the best sensitivity/specificity of 92.9%/81.3%. Thus we demonstrated that COL6A3 was a potential diagnosis and prognosis marker of CRC.

Upregulation of spondin-2 predicts poor survival of colorectal carcinoma patients.

Colorectal cancer (CRC) is the third and second most common cancer in males and females worldwide, respectively. Spondin-2 is a conserved secreted extracellular matrix protein and a candidate cancer biomarker. Here we found that Spondin-2 mRNA was upregulated in CRC tissues using quantitative RT-PCR and data-mining of public Oncomine microarray datasets. Spondin-2 protein was increased in CRC tissues, as revealed by immunohistochemistry analyses of two tissue microarrays containing 180 cases. Spondin-2 gene expression was significantly associated with CRC stage, T stage, M stage and Dukes stage, while its protein was associated with age and M stage. Kaplan-Meier analysis revealed that the upregulated Spondin-2 mRNA and protein predicted poor prognosis of CRC patients. Univariate and multivariate Cox regression analyses indicated that grade, recurrence, N stage and high Spondin-2 were independent predictors of overall survival of CRC patients. ELISA revealed that plasma Spondin-2 was upregulated in CRC and dropped after surgery. Receiver operating characteristic curve analysis demonstrated that plasma Spondin-2 has superior predictive performance for CRC with an area under the curve of 0.959 and the best sensitivity/specificity of 100%/90%. Furthermore, ectopic expression of Spondin-2 enhanced colon cancer cell proliferation. Spondin-2 could be an independent diagnostic and prognostic biomarker of colon cancer.

Filamin C, a dysregulated protein in cancer revealed by label-free quantitative proteomic analyses of human gastric cancer cells.

Gastric cancer (GC) is the fourth and fifth most common cancer in men and women, respectively. We identified 2,750 proteins at false discovery rates of 1.3% (protein) and 0.03% (spectrum) by comparing the proteomic profiles of three GC and a normal gastric cell lines. Nine proteins were significantly dysregulated in all three GC cell lines, including filamin C, a muscle-specific filamin and a large actin-cross-linking protein. Downregulation of filamin C in GC cell lines and tissues were verified using quantitative PCR and immunohistochemistry. Data-mining using public microarray datasets shown that filamin C was significantly reduced in many human primary and metastasis cancers. Transient expression or silencing of filamin C affected the proliferation and colony formation of cancer cells. Silencing of endogenous filamin C enhanced cancer cell migration and invasion, whereas ectopic expression of filamin C had opposing effects. Silencing of filamin C increased the expression of matrix metallopeptidase 2 and improved the metastasis of prostate cancer in a zebrafish model. High filamin C associated with better prognosis of prostate cancer, leukemia and breast cancer patients. These findings establish a functional role of filamin C in human cancers and these data will be valuable for further study of its mechanisms.

Identification of colonic fibroblast secretomes reveals secretory factors regulating colon cancer cell proliferation.

Stromal microenvironment influences tumor cell proliferation and migration. Fibroblasts represent the most abundant stromal constituents. Here, we established two pairs of normal fibroblast (NF) and cancer-associated fibroblast (CAF) cultures from colorectal adenocarcinoma tissues and the normal counterparts. The NFs and CAFs were stained positive for typical fibroblast markers and inhibited colon cancer (CC) cell proliferation in in vitro cocultures and in xenograft mouse models. The fibroblast conditioned media were analyzed using LC-MS and 227 proteins were identified at a false discovery rate of 1.3%, including 131 putative secretory and 20 plasma membrane proteins. These proteins were enriched for functional categories of extracellular matrix, adhesion, cell motion, inflammatory response, redox homeostasis and peptidase inhibitor. Secreted protein acidic and rich in cysteine, transgelin, follistatin-related protein 1 (FSTL1) and decorin was abundant in the fibroblast secretome as confirmed by Western blot. Silencing of FSTL1 and transgelin in colonic fibroblast cell line CCD-18Co induced an accelerated proliferation of CC cells in cocultures. Exogenous FSTL1 attenuates CC cell proliferation in a negative fashion. FSTL1 was upregulated in CC patient plasma and cancerous tissues but had no implication in prognosis. Our results provided novel insights into the molecular signatures and modulatory role of CC associated fibroblasts. BIOLOGICAL SIGNIFICANCE: In this study, a label-free LC-MS was performed to analyze the secretomes of two paired primary fibroblasts, which were isolated from fresh surgical specimen of colorectal adenocarcinoma and adjacent normal colonic tissues and exhibited negative modulatory activity for colon cancer cell growth in in vitro cocultures and in vivo xenograph mouse models. Follistatin-related protein 1 was further revealed to be one of the stroma-derived factors of potential suppression role for colon cancer cell proliferation. Our results provide novel insights into the molecular signatures and the modulatory role of colon cancer associated fibroblasts, and establish a valuable resource for the development of therapeutic agents or novel clinic biomarker.

Reduced expression of the chromatin remodeling gene ARID1A enhances gastric cancer cell migration and invasion via downregulation of E-cadherin transcription.

The chromatin remodeling gene AT-rich interactive domain-containing protein 1A (ARID1A) encodes the protein BAF250a, a subunit of human SWI/SNF-related complexes. Recent studies have identified ARID1A as a tumor suppressor. Here, we show that ARID1A expression is reduced in gastric cancer (GC) tissues, which are significantly associated with local lymph node metastasis, tumor infiltration and poor patient prognosis. ARID1A silencing enforces the migration and invasion of GC cells, whereas ectopic expression of ARID1A inhibits migration. The adhesive protein E-cadherin is remarkably downregulated in response to ARID1A silencing, but it is upregulated by ARID1A overexpression. E-cadherin overexpression significantly inhibits GC cell migration and invasion, whereas CDH1 (coded E-cadherin) silencing promotes migration. Restored expression of CDH1 in ARID1A-silenced cell lines restores the inhibition of cell migration. Luciferase reporter assays and chromatin immunoprecipitation indicate that the ARID1A-associated SWI/SNF complex binds to the CDH1 promoter and modulates CDH1 transcription. ARID1A knockdown induces evident morphological changes of GC cells with increased expression of mesenchymal markers, indicating an epithelial-mesenchymal transition. ARID1A silencing does not alter the level of ß-catenin but induces a subcellular redistribution of ß-catenin from the plasma membrane to the cytoplasm and nucleus. Immunohistochemical studies demonstrate that reduced expression of E-cadherin is associated with local lymph node metastasis, tumor infiltration and poor clinical prognosis. ARID1A and E-cadherin expression show a strong correlation in 75.4% of the analyzed GC tissues. They are synergistically downregulated in 23.5% of analyzed GC tissues. In conclusion, ARID1A targets E-cadherin during the modulation of GC cell migration and invasion.

Data from a proteomic analysis of colonic fibroblasts secretomes.

The tumor cell proliferation, migration and invasion were influenced by the interaction between the cancer cells and their microenvironment. In current study, we established two pairs of the primary fibroblast cultures from colorectal adenocarcinoma tissues and the normal counterparts and identified 227 proteins in the colonic fibroblast secretomes; half of these proteins were novel. The mass spectrometry data and analyzed results presented here provide novel insights into the molecular characteristics and modulatory role of colon cancer associated fibroblasts. The data is related to "Identification of colonic fibroblast secretomes reveals secretory factors regulating colon cancer cell proliferation" by Chen et al. [1].

Serum GFAP autoantibody as an ELISA-detectable glioma marker.

Glioma is the most common primary brain tumor, yet the high cost of diagnostic imaging has made early detection of asymptomatic glioma a formidable challenge. Thus, the development of a convenient, sensitive, and cost-effective diagnostic strategy, such as enzyme-linked immunosorbent assay (ELISA) based on glioma-specific and World Health Organization (WHO) grade-specific autoantibody serum markers, is necessary. To this end, a comparative proteomic analysis based on two-dimensional western blotting was carried out with the sera of glioma patients and normal controls. Of the 11 novel glioma-expressed autoantibodies, the autoantibody against glial fibrillary acidic protein (GFAP) showed the highest differential expression. To investigate the potential clinical utility of the GFAP autoantibody as an early diagnostic marker for glioma, an ELISA-based assay was developed and validated with sera from glioma patients with WHO grades II (n = 19), III (n = 17), and IV (n = 24). The GFAP autoantibody level directly correlated with WHO grade and tumor volume. Sera from patients of non-glioma brain tumors, as well as non-brain tumors, showed much lower levels of GFAP autoantibody than those of the glioma patients, indicating that elevated GFAP autoantibody is specific to glioma patients. Analysis of the receiver operating characteristics curve suggested that the new ELISA has good distinguishing power and sensitivity for diagnosing glioma patients. This is the first ELISA assay developed for an autoantibody of a glioma antigen and may prove valuable for the clinical detection of glioma.

[Tumor proteomics: the past, present and future].

The application of proteomics techniques to tumor biological research promoted the emergence of Tumor Proteomics. Its major goal is to screen biomarkers for tumor detection, diagnosis, prognosis, as well as tumor treatment using proteomics technologies, such as two dimensional gel electrophoresis (2-DE), stable isotope labeling of amino acid in cell culture (SILAC) and matrix assisted laser desorption/ionization (MALDI). Three stages of tumor proteomics study, including expression proteomics, post-translation modification proteomics and activity-based proteomics, have shed lights on not only the possible tumor markers but also the underlying mechanisms. We reviewed the methods as well as the achievements of recent tumor proteomics studies on major tumor types. The challenges are to sift out tumor markers for clinical applications, and to reveal novel mechanisms of tumorigenesis that may help to fight this deadly disease.

[Human pot1 gene exon12 mutation screening in cultured human carcinoma cell strains (lines)].

OBJECTIVE: To screen the exon12 mutation of pot1 gene in cultured human carcinoma cell strains (lines). METHODS: The chromosomal DNA was extracted from 27 cultured carcinoma cell strains (lines). The exon 12 of pot1 gene was amplified by PCR, and the product was purified and screened. The screening results were compared with the data of GenBank and NCBI and the exon 12 mutations in cultured human carcinoma cell strains (lines) analyzed. RESULTS: The exon12 sequence of pot1 could be specifically amplified using the designed primers. Direct sequence analysis of the PCR products after purification showed that 4 of the 5 carcinoma cell lines of the female genital system such as Hela and HO8910-PM cells shared the same transition (G17722-->C) in exon12 of human pot1 gene resulting in a conversion of G1385-->C in the cDNA and amino acid change of Leu454-->Phe in the translated polypeptide. The rest of the 23 cell strains (lines) from different origins showed no such mutation. CONCLUSION: The exon12 (17,722 bp) is a mutant region specific for female genital system tumor.