Bioorthogonal Chemical Labeling Probes Targeting Sialic Acid Isomers for N-Glycan MALDI Imaging Mass Spectrometry of Tissues, Cells, and Biofluids.

Sialic acid isomers attached in either α2,3 or α2,6 linkage to glycan termini confer distinct chemical, biological, and pathological properties, but they cannot be distinguished by mass differences in traditional mass spectrometry experiments. Multiple derivatization strategies have been developed to stabilize and facilitate the analysis of sialic acid isomers and their glycoconjugate carriers by high-performance liquid chromatography, capillary electrophoresis, and mass spectrometry workflows. Herein, a set of novel derivatization schemes are described that result in the introduction of bioorthogonal click chemistry alkyne or azide groups into α2,3- and α2,8-linked sialic acids. These chemical modifications were validated and structurally characterized using model isomeric sialic acid conjugates and model protein carriers. Use of an alkyne-amine, propargylamine, as the second amidation reagent effectively introduces an alkyne functional group into α2,3-linked sialic acid glycoproteins. In tissues, serum, and cultured cells, this allows for the detection and visualization of N-linked glycan sialic acid isomers by imaging mass spectrometry approaches. Formalin-fixed paraffin-embedded prostate cancer tissues and pancreatic cancer cell lines were used to characterize the numbers and distribution of alkyne-modified α2,3-linked sialic acid N-glycans. An azide-amine compound with a poly(ethylene glycol) linker was evaluated for use in histochemical staining. Formalin-fixed pancreatic cancer tissues were amidated with the azide amine, reacted with biotin-alkyne and copper catalyst, and sialic acid isomers detected by streptavidin-peroxidase staining. The direct chemical introduction of bioorthogonal click chemistry reagents into sialic acid-containing glycans and glycoproteins provides a new glycomic tool set to expand approaches for their detection, labeling, visualization, and enrichment.

Heterogeneity of glycan biomarker clusters as an indicator of recurrence in pancreatic cancer.

Introduction: Outcomes following tumor resection vary dramatically among patients with pancreatic ductal adenocarcinoma (PDAC). A challenge in defining predictive biomarkers is to discern within the complex tumor tissue the specific subpopulations and relationships that drive recurrence. Multiplexed immunofluorescence is valuable for such studies when supplied with markers of relevant subpopulations and analysis methods to sort out the intra-tumor relationships that are informative of tumor behavior. We hypothesized that the glycan biomarkers CA19-9 and STRA, which detect separate subpopulations of cancer cells, define intra-tumoral features associated with recurrence. Methods: We probed this question using automated signal thresholding and spatial cluster analysis applied to the immunofluorescence images of the STRA and CA19-9 glycan biomarkers in whole-block sections of PDAC tumors collected from curative resections. Results: The tumors (N = 22) displayed extreme diversity between them in the amounts of the glycans and in the levels of spatial clustering, but neither the amounts nor the clusters of the individual and combined glycans associated with recurrence. The combined glycans, however, marked divergent types of spatial clusters, alternatively only STRA, only CA19-9, or both. The co-occurrence of more than one cluster type within a tumor associated significantly with disease recurrence, in contrast to the independent occurrence of each type of cluster. In addition, intra-tumoral regions with heterogeneity in biomarker clusters spatially aligned with pathology-confirmed cancer cells, whereas regions with homogeneous biomarker clusters aligned with various non-cancer cells. Conclusion: Thus, the STRA and CA19-9 glycans are markers of distinct and co-occurring subpopulations of cancer cells that in combination are associated with recurrence. Furthermore, automated signal thresholding and spatial clustering provides a tool for quantifying intra-tumoral subpopulations that are informative of outcome.

Heterogeneity of Glycan Biomarker Clusters as an Indicator of Recurrence in Pancreatic Cancer.

Outcomes following tumor resection vary dramatically among patients with pancreatic cancer. A challenge in defining predictive biomarkers is to discern within the complex tumor tissue the specific subpopulations and relationships that drive recurrence. Multiplexed immunofluorescence is valuable for such studies when supplied with markers of relevant subpopulations and analysis methods to sort out the intra-tumor relationships that are informative of tumor behavior. We hypothesized that the glycan biomarkers CA19-9 and STRA, which detect separate subpopulations of cancer cells, define intra-tumoral features associated with recurrence. We probed this question using automated signal thresholding and spatial cluster analysis applied to the immunofluorescence images of the STRA and CA19-9 glycan biomarkers in whole-block tumor sections. The tumors (N = 22) displayed extreme diversity between them in the amounts of the glycans and in the levels of spatial clustering, but neither the amounts nor the clusters of the individual and combined glycans associated with recurrence. The combined glycans, however, marked divergent types of spatial clusters, alternatively only STRA, only CA19-9, or both. The co-occurrence of more than one cluster type within a tumor associated significantly with disease recurrence, in contrast to the independent occurrence of each type of cluster. In addition, intra-tumoral regions with heterogeneity in biomarker clusters spatially aligned with pathology-confirmed cancer cells, whereas regions with homogeneous biomarker clusters aligned with various non-cancer cells. Thus, the STRA and CA19-9 glycans are markers of distinct and co-occurring subpopulations of cancer cells that in combination are associated with recurrence. Furthermore, automated signal thresholding and spatial clustering provides a tool for quantifying intra-tumoral subpopulations that are informative of outcome.

Effects of straw return and straw biochar on soil properties and crop growth: A review.

Straw return is an effective method for disposing agricultural residues. It not only utilizes agricultural waste but also improves soil. In the current review, different crop straw and its characteristics were highlighted, and patterns of straw return were explored (including straw return, straw biochar return, and their combined with fertilizer return), as well as their environmental impacts were outlined. In addition, the effects of straw return and straw biochar amendment on soil properties [e.g., pH, soil organic carbon (SOC), soil nitrogen (N)/phosphorus (P)/potassium (K), soil enzyme activities, and soil microbes] were discussed. Information collected from this review proposed that straw return and straw biochar return or in combination with fertilizer is an applicable way for improving soil fertility and enhancing crop production. Straw return is beneficial to soil physicochemical properties and soil microbial features. The rice straw has positive impacts on crop growth. However, there are different climate types, soil types and crops in China, meaning that the future research need long-term experiment to assess the complex interactions among straw, soil, and plant eco-systems. Accordingly, this review aims to provide available information on the application of straw return in terms of different patterns of its to justify and to expand their effective promotion.

Effect of combined wet alkaline mechanical pretreatment on enzymatic hydrolysis of corn stover and its mechanism.

BACKGROUND: To further optimize the mechanochemical pretreatment process, a combined wet alkaline mechanical pretreatment of corn stover was proposed with a short time and less chemical consumption at room temperature. RESULTS: The combined alkaline mechanical pretreatment significantly enhanced enzymatic hydrolysis resulting a highest glucose yield (YG) of 91.9% with 3% NaOH and ball milling (BM) for 10 min. At this optimal condition, 44.4% lignin was removed and major portion of cellulose was retained (86.6%). The prehydrolysate contained by-products such as monosaccharides, oligosaccharides, acetic acid, and lignin but no furfural and 5-HMF. The alkaline concentration showed a significant impact on glucose yield, while the BM time was less important. Quantitative correlation analysis showed that YG (%) = 0.68 × BM time (min) + 19.27 × NaOH concentration (%) + 13.71 (R2 = 0.85), YG = 6.35 × glucan content - 231.84 (R2 = 0.84), and YG = - 14.22 × lignin content + 282.70 (R2 = 0.87). CONCLUSION: The combined wet alkaline mechanical pretreatment at room temperature had a boosting effect on the yield of enzymatic hydrolysis with short treatment time and less chemical consumption. The impact of the physical and chemical properties of corn stover pretreated with different BM times and/or different NaOH concentrations on the subsequent enzymatic hydrolysis was investigated, which would be beneficial to illustrate the effective mechanism of the mechanochemical pretreatment method.

Systematic comparison for effects of different scale mechanical-NaOH coupling treatments on lignocellulosic components, micromorphology and cellulose crystal structure of wheat straw.

In order to compare the effect of different mechanical-chemical coupling treatment on wheat straw and provide guidance for the subsequent preparation of cellulose nanomaterials, this paper systematically explored the impact of different scale mechanical fragmentation coupling various NaOH concentration treatment on the lignocellulosic components, micromorphology and cellulose crystal structure of wheat straw. The results showed that the relationship between hemicellulose and lignin removal with NaOH concentration can be expressed as exponential function Y = ai(1-exp(-biX)), and micro-nano-scale ball-milling coupling NaOH treatment can facilitate the removal of hemicellulose and lignin. Micromorphology analysis found that wet ball milling coupling NaOH one-step treatment can disintegrate cellulose fiber into crosslinked network structure of cellulose microfibrils. XRD results indicated that wet ball milling with NaOH solution was contributed to retaining cellulose crystal structure and conducive to cellulose crystalline transformation. In conclusion, wet ball milling coupling NaOH simultaneous treatment can be a promising pretreatment for cellulose nanomaterials preparation.

Detection of Chemotherapy-resistant Pancreatic Cancer Using a Glycan Biomarker, sTRA.

PURPOSE: A subset of pancreatic ductal adenocarcinomas (PDACs) is highly resistant to systemic chemotherapy, but no markers are available in clinical settings to identify this subset. We hypothesized that a glycan biomarker for PDACs called sialylated tumor-related antigen (sTRA) could be used for this purpose. EXPERIMENTAL DESIGN: We tested for differences between PDACs classified by glycan expression in multiple systems: sets of cell lines, organoids, and isogenic cell lines; primary tumors; and blood plasma from human subjects. RESULTS: The sTRA-expressing models tended to have stem-like gene expression and the capacity for mesenchymal differentiation, in contrast to the nonexpressing models. The sTRA cell lines also had significantly increased resistance to seven different chemotherapeutics commonly used against pancreatic cancer. Patients with primary tumors that were positive for a gene expression classifier for sTRA received no statistically significant benefit from adjuvant chemotherapy, in contrast to those negative for the signature. In another cohort, based on direct measurements of sTRA in tissue microarrays, the patients who were high in sTRA again had no statistically significant benefit from adjuvant chemotherapy. Furthermore, a blood plasma test for the sTRA glycan identified the PDACs that showed rapid relapse following neoadjuvant chemotherapy. CONCLUSIONS: This research demonstrates that a glycan biomarker could have value to detect chemotherapy-resistant PDAC in clinical settings. This capability could aid in the development of stratified treatment plans and facilitate biomarker-guided trials targeting resistant PDAC.

Quantitative and qualitative characterization of dual scale mechanical enhancement on cellulosic and crystalline-structural variation of NaOH treated wheat straw.

In order to explore the effects of different mechanical fragmentation on cellulose separation and cellulose polymorphic transformation of wheat straw during alkali treatment, one coarse milled (CM) and two ball milled wheat straw samples (BM30 and BM120) were treated with different NaOH concentrations (1%-10%), and the lignocellulosic compositions and crystalline-structural various were quantitative and qualitative characterized. The quantitative equations between cellulose content and NaOH concentration of different mechanical treated samples were YCM = 69.8-35.1exp(-0.64X)), YBM30 = 71.3-35.1exp(-0.86X)) and YBM120 = 73.5-35.1exp(-1.82X)). The enhancement effect of cellulose separation with the increasing mechanical fragmentation intensity is mainly due to the increasing hemicellulose solubilization. X-ray diffraction results reveals that the NaOH concentration required for cellulose crystalline transformation of CM, BM30 and BM120 is 10%, 8% and 2%, respectively. In conclusion, mechanical fragmentation contributes to cellulose separation and cellulose crystalline transformation under lower NaOH concentration.

Impacts of carbonization temperature on the Pb(II) adsorption by wheat straw-derived biochar and related mechanism.

To determine the quantitative correlations between physicochemical characteristics and Pb(II) adsorption amounts of biochar fractions, we prepared wheat straw-derived biochar under various carbonization temperatures (300-900 °C). The different fractions of the wheat straw-derived biochar, water-soluble material (WM), acid-soluble material (AM), and organic material (OM), were acquired. The ash content, ultimate analysis, pH, ion strength (IS), cation exchange capacity (CEC), and acidic functional groups (AFG) were characterized. The Pb(II) adsorption amounts of different biochars and their fractions were determined. The results revealed that the proportions of biochar fractions (WM, AM, and OM) varied with various carbonization temperatures. The maximum Pb(II) adsorption amount of wheat straw-derived biochar (qTotal) was 157.95 ±â€¯0.13 mg/g obtained at 800 °C, and the quantitative correlations between Pb(II) adsorption amount (q) and carbonization temperature (T) can be elaborated by qTotal = 170.72-336.62exp(-0.0035T) (R2 = 0.97), qWM = 106.18-390.10exp(-0.0046T) (R2 = 0.98), qAM = 496.16-477.74exp(-0.0001T) (R2 = 0.79), and qOM = 1.80 + 34.69exp(-0.0038T) (R2 = 0.85). For rate of contribution (RC) for Pb(II) adsorption, when T < 400 °C, the order was AM (60.72 ±â€¯7.33%) > OM (23.41 ±â€¯7.33%) > WM (15.87 ±â€¯0.30%); however, when T ≥ 400 °C, the order was WM (52.31 ±â€¯0.85% - 67.65 ±â€¯2.99%) > AM (29.65 ±â€¯0.46% - 35.77 ±â€¯0.12%) > OM (2.30 ±â€¯0.47% - 12.02 ±â€¯2.43%). Moreover, qWM and qAM exhibited significant positive linear correlations with ash (qWM = 9.92Ash - 123.65, and qAM = 2.13Ash - 0.49), qTotal was predominantly affected by ash content (qTotal = 10.97 Ash - 95.49). The EDX, XRD, and FTIR analysis results further clarified that ion exchange and precipitation were the main adsorption mechanisms for Pb(II) adsorption by wheat straw-derived biochar.

Mechanochemical deconstruction of lignocellulosic cell wall polymers with ball-milling.

In this work, the deconstruction mechanism of corn stover cell wall polymers during ball milling was evaluated. The characterization showed that ball milling not only brought about the dissociation of the cross-linked cellulose-hemicellulose-lignin complex but also led to the depolymerization of the cell-wall polymers especially the carbohydrates. Micromorphology characterization revealed that mechanical treatment disrupted the orderly fibrillar matrices with a porous structure. The breakage of ß-1,4 glycosidic bonds in cellulose and the decomposition of arabinoxylans indicated the modification in polysaccharide chains. The degradation of lignin-carbohydrate complex (LCC) linkages and the cleavage of ß-O-4' linkages in lignin approved the partial degradation of lignin. In conclusion, mechanochemistry is an efficient force to make the polymers in plant fibers more digestible.

The sTRA Plasma Biomarker: Blinded Validation of Improved Accuracy Over CA19-9 in Pancreatic Cancer Diagnosis.

PURPOSE: The CA19-9 biomarker is elevated in a substantial group of patients with pancreatic ductal adenocarcinoma (PDAC), but not enough to be reliable for the detection or diagnosis of the disease. We hypothesized that a glycan called sTRA (sialylated tumor-related antigen) is a biomarker for PDAC that improves upon CA19-9. EXPERIMENTAL DESIGN: We examined sTRA and CA19-9 expression and secretion in panels of cell lines, patient-derived xenografts, and primary tumors. We developed candidate biomarkers from sTRA and CA19-9 in a training set of 147 plasma samples and used the panels to make case-control calls, based on predetermined thresholds, in a 50-sample validation set and a blinded, 147-sample test set. RESULTS: The sTRA glycan was produced and secreted by pancreatic tumors and models that did not produce and secrete CA19-9. Two biomarker panels improved upon CA19-9 in the training set, one optimized for specificity, which included CA19-9 and 2 versions of the sTRA assay, and another optimized for sensitivity, which included 2 sTRA assays. Both panels achieved statistical improvement (P < 0.001) over CA19-9 in the validation set, and the specificity-optimized panel achieved statistical improvement (P < 0.001) in the blinded set: 95% specificity and 54% sensitivity (75% accuracy), compared with 97%/30% (65% accuracy). Unblinding produced further improvements and revealed independent, complementary contributions from each marker. CONCLUSIONS: sTRA is a validated serological biomarker of PDAC that yields improved performance over CA19-9. The new panels may enable surveillance for PDAC among people with elevated risk, or improved differential diagnosis among patients with suspected pancreatic cancer.

Carbonization and ball milling on the enhancement of Pb(II) adsorption by wheat straw: Competitive effects of ion exchange and precipitation.

Straw biomass is a promising adsorbent for the removal of heavy metals. To improve its Pb(II) adsorption capacity and elucidate competition of adsorption mechanisms (e.g., ion exchange and precipitation), the Pb(II) adsorption mechanisms for wheat straw (WS-CK), wheat straw-biochar (WS-BC), and ball-milled wheat straw-biochar (WS-BC + BM) samples were investigated in detail by EDX, XRD, and FTIR. The results implied that the Pb(II) adsorption capacities at an adsorbent dosage of 0.2 g/L onto WS-CK, WS-BC, and WS-BC + BM were 46.33, 119.55, and 134.68 mg/g, respectively. This indicates that carbonization and ball milling are efficient techniques for improving the adsorption capacity of Pb(II) onto wheat straw, as WS-BC and WS-BC + BM exhibited adsorption capacities comparable to other commonly used bioadsorbents. Carbonization contributed significantly to precipitation (e.g., PbCO3 and Pb3(CO3)2(OH)2). Furthermore, competition existed between ion exchange and precipitation during the Pb(II) adsorption process. With relative lower adsorbent dosages, carbonization and ball milling enhanced ion exchange capacity.

Regularity and mechanism of wheat straw properties change in ball milling process at cellular scale.

To investigate the change of structure and physicochemical properties of wheat straw in ball milling process at cellular scale, a series of wheat straws samples with different milling time were produced using an ultrafine vibration ball mill. A multitechnique approach was used to analyze the variation of wheat straw properties. The results showed that the characteristics of wheat straw powder displayed regular changes as a function of the milling time, i.e., the powder underwent the inversion of breakage to agglomerative regime during wheat straw ball milling process. The crystallinity index, bulk density and water retention capacity of wheat straw were exponential relation with ball milling time. Moreover, ball milling continually converted macromolecules of wheat straw cell wall into water-soluble substances resulting in the water extractives proportional to milling time.

Quantitative approaches for illustrating correlations among the mechanical fragmentation scales, crystallinity and enzymatic hydrolysis glucose yield of rice straw.

Mechanical fragmentation is an important pretreatment in the biomass biotransformation process. Mechanical fragmentation at the tissue scale significantly reduced the particle size of rice straw but did not significantly change its crystalline properties; the increase in the glucose yield was limited from 28.75% (95.55mg/g substrate) to 35.29% (115.28mg/g substrate). Mechanical fragmentation at the cellular scale destroyed the cell wall structure and reduced its crystalline properties. Thus, the glucose yield also showed a significant increase from 35.29% (115.28mg/g substrate) to 81.71% (287.07mg/g of substrate). The quantitative equations among the particle size, crystalline properties and glucose yield (mg/g substrate) are as follows: CrI=44.14×[1-exp(-0.03658×D50)] and CP=(8.403×logD50-24.1836)/(1-4.225/D50^0.5); GY=-5.636CrI+343.7 and GY=-14.62CP+512.1; and GY=97.218+247.5×exp(-0.03824×D50). The quantitative correlations among the mechanical fragmentation scales and crystalline properties can determine the effect and mechanism of mechanical fragmentation on biomass and can further promote the construction of a cost-competitive biotransformation process for biomass.

Zinc finger E-box binding homeobox-1 (Zeb1) drives anterograde lysosome trafficking and tumor cell invasion via upregulation of Na+/H+ Exchanger-1 (NHE1).

Tumor cell invasion through the extracellular matrix is facilitated by the secretion of lysosome-associated proteases. As a common mechanism for secretion, lysosomes must first traffic to the cell periphery (anterograde trafficking), consistent with invasive cells often containing lysosomes closer to the plasma membrane compared to non-invasive cells. Epithelial to mesenchymal transition (EMT) is a transcriptionally driven program that promotes an invasive phenotype, and Zeb1 is one transcription factor that activates the mesenchymal gene expression program. The role of lysosome trafficking in EMT-driven invasion has not been previously investigated. We found that cells with increased levels of Zeb1 displayed lysosomes located closer to the cell periphery and demonstrated increased protease secretion and invasion in 3-dimensional (3D) cultures compared to their epithelial counterparts. Additionally, preventing anterograde lysosome trafficking via pharmacological inhibition of Na+/H+ exchanger 1 (NHE1) or shRNA depletion of ADP-ribosylation like protein 8b (Arl8b) reversed the invasive phenotype of mesenchymal cells, thus supporting a role for lysosome positioning in EMT-mediated tumor cell invasion. Immunoblot revealed that expression of Na+/H+ exchanger 1 correlated with Zeb1 expression. Furthermore, we found that the transcription factor Zeb1 binds to the Na+/H+ exchanger 1 promoter, suggesting that Zeb1 directly controls Na+/H+ transcription. Collectively, these results provide insight into a novel mechanism regulating Na+/H+ exchanger 1 expression and support a role for anterograde lysosome trafficking in Zeb1-driven cancer progression. © 2016 Wiley Periodicals, Inc.

Chromosome instability drives phenotypic switching to metastasis.

Chromosome instability (CIN) is the most striking feature of human cancers. However, how CIN drives tumor progression to metastasis remains elusive. Here we studied the role of chromosome content changes in generating the phenotypic dynamics that are required for metastasis. We isolated epithelial and mesenchymal clones from human carcinoma cell lines and showed that the epithelial clones were able to generate mesenchymal variants, which had the potential to further produce epithelial revertants autonomously. The successive acquisition of invasive mesenchymal and then epithelial phenotypes recapitulated the steps in tumor progression to metastasis. Importantly, the generation of mesenchymal variants from clonal epithelial populations was associated with subtle changes in chromosome content, which altered the chromosome transcriptome and influenced the expression of genes encoding intercellular junction (IJ) proteins, whereas the loss of chromosome 10p, which harbors the ZEB1 gene, was frequently detected in epithelial variants generated from mesenchymal clones. Knocking down these IJ genes in epithelial cells induced a mesenchymal phenotype, whereas knocking down the ZEB1 gene in mesenchymal cells induced an epithelial phenotype, demonstrating a causal role of chromosome content changes in phenotypic determination. Thus, our studies suggest a paradigm of tumor metastasis: primary epithelial carcinoma cells that lose chromosomes harboring IJ genes acquire an invasive mesenchymal phenotype, and subsequent chromosome content changes such as loss of 10p in disseminated mesenchymal cells generate epithelial variants, which can be selected for to generate epithelial tumors during metastatic colonization.

Mechanical fragmentation of corncob at different plant scales: Impact and mechanism on microstructure features and enzymatic hydrolysis.

In this work, corncob samples at different scales, i.e., plant scale (>1mm), tissue scale (500-100µm) and cellular scale (50-30µm), were produced to investigate the impact and mechanisms of different mechanical fragmentations on microstructure features and enzymatic hydrolysis. The results showed that the microstructure features and enzymatic hydrolysis of corncob samples, either at a plant scale or tissue scale, did not change significantly. Conversely, corncob samples at a cellular scale exhibited some special properties, i.e., an increase in the special surface area with the inner mesopores and macropores exposed to the surface; breakage of crystalline cellulose and linkages in polysaccharides; and a higher proportion of polysaccharides on the surface, which significantly enhanced enzymatic digestibility resulting in a 98.3% conversion yield of cellulose to glucose which is the highest conversion ever reported. In conclusion, mechanical fragmentation at the cellular scale is an effective pretreatment for corncob.

Various effects of hepatoma-derived growth factor on cell growth, migration and invasion of breast cancer and prostate cancer cells.

Hepatoma-derived growth factor (HDGF) has been implicated in the growth and metastasis of various types of human cancer, but the role of HDGF expression in prostate cancer or breast cancer has not been documented. To assess the role of HDGF in the proliferation, migration and invasion by prostate and breast cancer cells, HDGF expression in DU145 and MCF7 cells was knocked down using siRNA, and the effect of such knockdown was assessed by MTS and [3H]-thymidine incorporation Transwell assays. Moreover, we identified differentially expressed genes that might mediate the HDGF-induced cellular effects. Our results demonstrate that down-regulation of HDGF expression significantly reduces the proliferation of both DU145 and MCF7 cells. However, down-regulation of HDGF expression in DU145 inhibited cell migration and invasion, but in MCF7 cells it stimulated cell migration and invasion. This differential effect might result from the differential induction of PIK3R1 or SERPINE1 in the two cell lines upon HDGF-siRNA treatment. In conclusion, HDGF may participate in the pathogenesis of prostate and breast cancer by promoting cell growth and it may be a therapeutic target for these cancers.

Repeated hepatocyte growth factor neutralizing antibody treatment leads to HGF/SF unresponsiveness in human glioblastoma multiforme cells.

The purpose of this work is to seek putative markers for multi-targeted therapeutic treatment of human glioblastoma. We previously developed an anti-HGF neutralizing antibody cocktail Amix that inhibits human glioblastoma growth in mouse xenograft models. When these treated tumors were re-injected into nude mice and treatment with the neutralizing antibody cocktail plus heparin was repeated, the growth of the twice-treated tumors became HGF-independent, suggesting a possible switch in dominant signaling pathways. Microarray of the tumor cells revealed a number of genes elevated in the twice-treated tumor cells relative to untreated control tumors, including BAI1, CASP8, IL8, IGF1, TGFB1 and TNF. Our analyses provide a series of putative markers for additional evaluation in treating glioblastoma. Multi-targeted therapeutic approach might be a better solution for treating this disease.

Therapeutic potential of hepatocyte growth factor/scatter factor neutralizing antibodies: inhibition of tumor growth in both autocrine and paracrine hepatocyte growth factor/scatter factor:c-Met-driven models of leiomyosarcoma.

Hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, c-Met, have been implicated in the growth and progression of a variety of solid human tumors. Thus, inhibiting HGF/SF:c-Met signaling may provide a novel therapeutic approach for treating human tumors. We have generated and characterized fully human monoclonal antibodies that bind to and neutralize human HGF/SF. In this study, we tested the effects of the investigational, human anti-human HGF/SF monoclonal antibody, AMG 102, and a mixture of mouse anti-human HGF/SF monoclonal antibodies (Amix) on HGF/SF-mediated cell migration, proliferation, and invasion in vitro. Both agents had high HGF/SF-neutralizing activity in these cell-based assays. The HGF/SF:c-Met pathway has been implicated in the growth of sarcomas; thus, we also investigated the effect of AMG 102 on the growth of human leiomyosarcoma (SK-LMS-1) in HGF/SF transgenic C3H severe combined immunodeficient mice engineered to express high levels of human HGF/SF, as well as tumor growth of an autocrine variant of the SK-LMS-1 cell line (SK-LMS-1TO) in nude mice. The results indicate that interrupting autocrine and/or paracrine HGF/SF:c-Met signaling with AMG 102 has profound antitumor effects. These findings suggest that blocking HGF/SF:c-Met signaling may provide a potent intervention strategy to treat patients with HGF/SF:c-Met-dependent tumors.