Correlation between secretion pattern and metastatic potential from drug-treated 3LL tumor cell line

In the present study, we described the effects of three different drugs, A23187 calcium ionophore (A23187), indomethacin (IND) and phorbol myristate acetate (PMA), on the secretion pattern and the metastatic potential in the low metastatic 3LL tumor cell line. The results evidence that a low molecular weight protein fraction is always secreted in an higher amount in drug treated cells than in untreated cells independently of the drug used. In addition to their effects on secretion pattern, the three drugs assayed enhance noticeably the metastatic rate in vivo of the 3LL cells. The data confirm our earlier observations showing that our high performance gel permeation chromatography (HPGPC) method is suitable for discriminating even between cells with a different degree (low and high) of metastatic potential by its chromatographic secretion pattern and that alterations in the secretion pattern from treated cells are related with changes in the degree of metastatic potential of these cells.     

Precipitation of dilute chromatographic samples (ng/ml) containing interfering substances for SDS-PAGE

SDS-PAGE of chromatographic fractions requires prior removal of salts, detergents, denaturants, or organic solvents which may perturb the electrophoretic separation. Likewise, to successfully visualize minute amounts of protein present in chromatographic fractions, they must often be concentrated before analysis by SDS-PAGE. In this study, we used a dye precipitation procedure for simultaneous removal of interfering substances and concentration of dilute samples (ng/ml) before analysis by SDS-PAGE. Nanogram amounts of protein (143 ng) were effectively precipitated with a pyrogallol red-molybdate reagent from commonly used chromatographic buffers containing various interfering solutes or solvents. Proteins were successfully precipitated from solution in the presence of organic solvents (acetonitrile, methanol, 2-propanol), chaotropic agents (6 M urea, 6 M guanidine-HCl), a protein stabilizer (40% sucrose), metal chelators (30 mM EDTA and 30 mM EGTA), or high salt (1.0 M NaCl). Detergents, at concentrations up to twice their critical micelle concentrations, from the nonionic class (Triton X-100, Tween 20) or from the zwitterionic class (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) did not inhibit protein precipitation. Some interference was observed when proteins were precipitated in the presence of ammonium sulfate (0. 5-2.0 M). Proteins did not precipitate in the presence of ionic detergents (SDS and cetyltrimethylammonium bromide). The sensitivity of the combined pyrogallol red-molybdate precipitation/SDS-PAGE procedure is approximately 7 ng. Two other methods of precipitating proteins (trichloroacetic acid and phenol-ether) both exhibited varying degrees of effectiveness, ranging from 714 to 7 ng/ml, in the precipitation of individual proteins. In summary, the pyrogallol red-molybdate protein precipitation procedure facilitates the SDS-PAGE analysis of dilute protein samples (ng/ml) from chromatographic fractions of various compositions. The method is useful for rapid pilot-scale protein fractionation and facilitates the ongoing propensity of researchers to work with minuscule amounts of protein.     

Liquid biopsy by NGS: Differential presence of exons (DPE) is related to metastatic potential in a colon-cancer model in the rat

Differential presence of exons (DPE) is a method of interpretation of exome sequencing, which has been proposed to design a predictive algorithm with clinical value in patients with colorectal cancer (CRC). The goal of the present study was to examine the reproducibility in a rat model of metastatic colon cancer. DHD/K12-TRb cells were injected in syngenic immunocompetent BD-IX rats. Cells were from two stocks with low and normal metastatic potential, and injected into two separate groups of rats. Five to ten weeks after injection, blood samples were taken prior euthanasia and whole exome sequencing performed. Through DPE analysis, we identified a set of exons whose differential presence in plasma allowed us to compare both groups of tumor-bearing animals. A verification test was performed to confirm that the algorithm was able to classify extracted samples into their corresponding groups of origin. The highest mean probability was 0.8954. In conclusion, the DPE analysis in tumor-bearing animals was able to discriminate between different disease status, which fully supports previous results in CRC patients.     

A Non-Biological Method for Screening Active Components against Influenza Virus from Traditional Chinese Medicine by Coupling a LC Column with Oseltamivir Molecularly Imprinted Polymers

To develop a non-biological method for screening active components against influenza virus from traditional Chinese medicine (TCM) extraction, a liquid chromatography (LC) column prepared with oseltamivir molecularly imprinted polymer (OSMIP) was employed with LC-mass spectrometry (LC-MS). From chloroform extracts of compound TCM liquid preparation, we observed an affinitive component m/z 249, which was identified to be matrine following analysis of phytochemical literatures, OSMIP-LC column on-line of control compounds and MS/MS off-line. The results showed that matrine had similar bioactivities with OS against avian influenza virus H9N2 in vitro for both alleviating cytopathic effect and hemagglutination inhibition and that the stereostructures of these two compounds are similar while their two-dimensional structures were different. In addition, our results suggested that the bioactivities of those affinitive compounds were correlated with their chromatographic behaviors, in which less difference of the chromatographic behaviors might have more similar bioactivities. This indicates that matrine is a potential candidate drug to prevent or cure influenza for human or animal. In conclusion, the present study showed that molecularly imprinted polymers can be used as a non-biological method for screening active components against influenza virus from TCM.     

Exome Sequencing of Cell-Free DNA from Metastatic Cancer Patients Identifies Clinically Actionable Mutations Distinct from Primary Disease

The identification of the molecular drivers of cancer by sequencing is the backbone of precision medicine and the basis of personalized therapy; however, biopsies of primary tumors provide only a snapshot of the evolution of the disease and may miss potential therapeutic targets, especially in the metastatic setting. A liquid biopsy, in the form of cell-free DNA (cfDNA) sequencing, has the potential to capture the inter- and intra-tumoral heterogeneity present in metastatic disease, and, through serial blood draws, track the evolution of the tumor genome.In order to determine the clinical utility of cfDNA sequencing we performed whole-exome sequencing on cfDNA and tumor DNA from two patients with metastatic disease; only minor modifications to our sequencing and analysis pipelines were required for sequencing and mutation calling of cfDNA. The first patient had metastatic sarcoma and 47 of 48 mutations present in the primary tumor were also found in the cell-free DNA. The second patient had metastatic breast cancer and sequencing identified an ESR1 mutation in the cfDNA and metastatic site, but not in the primary tumor. This likely explains tumor progression on Anastrozole. Significant heterogeneity between the primary and metastatic tumors, with cfDNA reflecting the metastases, suggested separation from the primary lesion early in tumor evolution. This is best illustrated by an activating PIK3CA mutation (H1047R) which was clonal in the primary tumor, but completely absent from either the metastasis or cfDNA. Here we show that cfDNA sequencing supplies clinically actionable information with minimal risks compared to metastatic biopsies. This study demonstrates the utility of whole-exome sequencing of cell-free DNA from patients with metastatic disease. cfDNA sequencing identified an ESR1 mutation, potentially explaining a patient’s resistance to aromatase inhibition, and gave insight into how metastatic lesions differ from the primary tumor.     

Involvement of highly sulfated chondroitin sulfate in the metastasis of the Lewis lung carcinoma cells

The altered expression of cell surface chondroitin sulfate (CS) and dermatan sulfate (DS) in cancer cells has been demonstrated to play a key role in malignant transformation and tumor metastasis. However, the functional highly sulfated structures in CS/DS chains and their involvement in the process have not been well documented. In the present study, a structural analysis of CS/DS from two mouse Lewis lung carcinoma (3LL)-derived cell lines with different metastatic potentials revealed a higher proportion of Delta(4,5)HexUA-GalNAc(4,6-O-disulfate) generated from E-units (GlcUA-GalNAc(4, 6-O-disulfate)) in highly metastatic LM66-H11 cells than in low metastatic P29 cells, although much less CS/DS is expressed by LM66-H11 than P29 cells. This key finding prompted us to study the role of CS-E-like structures in experimental lung metastasis. The metastasis of LM66-H11 cells to lungs was effectively inhibited by enzymatic removal of tumor cell surface CS or by preadministration of CS-E rich in E-units in a dose-dependent manner. In addition, immunocytochemical analysis showed that LM66-H11 rather than P29 cells expressed more strongly the CS-E epitope, which was specifically recognized by the phage display antibody GD3G7. More importantly, this antibody and a CS-E decasaccharide fraction, the minimal structure recognized by GD3G7, strongly inhibited the metastasis of LM66-H11 cells probably by modifying the proliferative and invading behavior of the metastatic tumor cells. These results suggest that the E-unit-containing epitopes are involved in the metastatic process and a potential target for the diagnosis and treatment of malignant tumors.     

Chromatographic determination of arylsulfatases A and B in human gastric mucosa]

In continuation of a previous work, we have confirmed the occurrence of arylsulfatase A in 4 samples of human gastric mucosa analysed by the chromatographic procedure described by Stevens et all. By using the chromatographic method we have also evidentiated the occurrence of arylsulfatase B, which was not detected by using the method of Baum et all. The B form was lower than the A form in 3 samples while it was higher in another sample. In the latter sample of gastric mucosa it was also detected the unusual form Bm of arylsulfatase. It was concluded that both forms A and B of arylsulfatase are present in human gastric mucosa, in variable amounts and that the simple procedure developed by Baum et all., although suitable for the analysis of these enzymes in the urine, is not useful for the determination of arylsulfate B in the gastric mucosa.     

Exosome‐mediated extracellular release of polyadenylate‐binding protein 1 in human metastatic duodenal cancer cells

Exosomes are small vesicles secreted from cells that transport their embedded molecules through bidirectional exocytosis‐ and endocytosis‐like pathways. Expression patterns of exosomal molecules such as proteins and RNAs can be indicative of cell type since their signature is thought to be unique among cells. Using human primary (AZ‐521) and metastatic (AZ‐P7a) duodenal cancer cell lines, we conducted a comparative exosomal proteome analysis to identify proteins with metastatic marker potential. As determined by LC‐MS/MS and Western blot analyses, polyadenylate‐binding protein 1 (PABP1) was found to be predominantly abundant in AZ‐P7a exosomes. The amount of exosomal PABP1 in AZ‐P7a cells increased by treating the cells with inhibitors for the classical ER/Golgi secretory pathway (brefeldin A and monensin) and the ubiquitin‐proteasome pathway (MG‐132 and PYR‐41). Treatment of AZ‐P7a cells with the neutral sphingomyelinase inhibitor GW4869, which suppresses exosome release, not only reduced the amount of exosomal PABP1 but also produced PABP1‐immunoreactive products cleaved via a proteolysis‐like process. Taken together, these results suggest that AZ‐P7a cells do not tolerate intracellular PABP1 accumulation and are thus exported into the extracellular milieu by the exosome‐mediated pathway. In addition, PABP1 has a potential use as a biomarker for metastatic duodenal cancer.     

Kinetic analysis of enzyme reactions with slow-binding inhibition.

In this paper we present a general kinetic study of slow-binding inhibition processes, i.e. enzyme reactions that do not respond instantly to the presence of a competitive inhibitor. The analysis that we present is based on the equation that describes the formation of products with time in each case on the experimental progress curve. It is carried out under the condition of limiting enzyme concentration and allows the discrimination between the different cases of slow-binding inhibition. The mechanism in which the formation of complex enzyme-inhibitor is a single or two slow steps or follow a rapid equilibrium, has been considered. The corresponding explicit equations of each case have been obtained and checked by numerical integration. A kinetic data analysis to evaluate the corresponding kinetic parameters is suggested. We illustrate the method, numerically by computer simulation, of the reaction and present some numerical examples that demonstrate the applicability of our procedure.     

Overexpression of Hsp25 in K1735 murine melanoma cells enhances susceptibility to natural killer cytotoxicity

In the present study we used a murine melanoma model to investigate the effect of the 25-kDa heat shock protein (Hsp25) on natural killer (NK) cytotoxicity. The melanoma lines K1735-C123 (low metastatic potential) and K1735-M2 (high metastatic potential) were transfected with hsp25 and a control plasmid. Highly purified interleukin (IL)-2-stimulated DX-5+ NK cells showed enhanced lysis of Hsp25-overexpressing K1735-C123 targets in comparison with controls. In contrast, there was no difference in susceptibility to lysis by purified IL-2-stimulated DX-5+ NK cells between Hsp25-overexpressing and control-transfected K1735-M2 targets. Fluorescence-activated cell sorter analysis revealed that Hsp25 is displayed on the cell surface independently of Hsp25 overexpression and metastatic phenotype. Thus, surface localization of Hsp25 does not correlate with the target cell susceptibility to killing. To sum up, a cytoplasmic overexpression of Hsp25 is associated with an increased susceptibility to lysis by DX-5+ NK cells in the low-metastatic murine melanoma model investigated.     

Differences in transferrin response and numbers of transferrin receptors in rat and human mammary carcinoma lines of different metastatic potentials

We previously found that transferrin (Tf) differentially stimulated the growth of highly metastatic variant lines of murine melanoma and that these highly metastatic cells also had greater numbers of Tf receptors on their cell surfaces. In the present study we found that highly metastatic rat mammary adenocarcinoma cell lines also responded differentially to Tf in proliferation assays, and cell monolayers bound Tf in relation to their metastatic potential (MTPaB10 > MTPaB5 > MTLn3 > MTLn2 > MTC > MTF7 > MTPa). The brain-colonizing lines PaB10 and PaB5 were the most responsive to Tf and had the highest numbers of Tf receptors. Different human breast cancer cell lines also responded differentially to Tf in proliferation assays and bound different amounts of Tf to their cell surface Tf receptors. Transferrin binding, but not growth response, correlated with metastatic and invasive properties of lines selected from the human MCF-7 series (MCF7/LCC2 > MCF7/LCC1 > MCF7). In examining the transferrin binding and growth response of lines from the human MDA series, the Tf binding and growth response was MDA231 > MDA435 > MDA468. The lines MDA435 and MDA231 were metastatic in nude mouse assays, whereas the line MDA468 was not. Scatchard analysis indicated the presence of a single class of receptor for Tf on the rat and human mammary cell lines. The results suggest that neoplastic cells displaying various metastatic properties may express differing numbers of Tf receptors and respond differently to growth factors such as Tf. © 1993 Wiley-Liss, Inc.     

Monitoring phosphatase reactions of multiple phosphorylated substrates by reversed-phase HPLC

In an approach to gain insight into the sequence-dependent dephosphorylation of multiple phosphotyrosyl-containing peptides by the phosphatases SHP-1 and PTP1B, we applied a chromatographic technique for the analysis of the dephosphorylation products. Mono-, bi- and triphosphorylated reference peptides corresponding to positions 1999-2014 in the activation loop of the receptor tyrosine kinase Ros were first analyzed by reversed-phase HPLC and MALDI-TOF/TOF mass spectrometry. Then, the respective products from enzymatic treatment were investigated by HPLC and compared to the standard peptides. The results obtained in this study emphasize the advantage of monitoring phosphatase reactions for mono- and biphosphorylated peptides using the described procedure rather than spectrophotometric and fluorimetric methods that do not allow for a clear identification of the products formed.     

Comparison of fluorescent labels for oligosaccharides and introduction of a new postlabeling purification method

Labeling of oligosaccharides with fluorescent dyes is the prerequisite for their sensitive analysis by high-performance liquid chromatography (HPLC). In this work, we present a fast new postlabeling cleanup procedure that requires no device other than the reaction vial itself. The procedure can be applied to essentially all labeling reagents. We also compare the performance of 15 different labels for N-glycan analysis in various analytical procedures. We took special care to prevent obscuring influences from incomplete derivatization and signal quenching by impurities. Procainamide emerged as more sensitive than anthranilic acid for normal-phase HPLC, but its chromatographic performance was not convincing. 2-Aminopyridine was the label with the lowest retention on reversed-phase and graphitic carbon columns and, thus, appears to be most suitable for glycan fractionation by multidimensional HPLC. Most glycan derivatives performed better than native sugars in matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and electrospray ionization-MS (ESI-MS), but the gain was small and hardly sufficient to compensate for sample loss during preparation.     

Apatinib inhibits migration and invasion as well as PD-L1 expression in osteosarcoma by targeting STAT3

The cure rate of osteosarcoma has not improved in the past 30 years. The new treatments and drugs is urgently needed, especially for metastatic osteosarcoma. Anti-angiogenesis therapy and immunotherapy has got promising anti-tumor effects in various tumors. It is hypothesised that combining checkpoint inhibitor immunotherapies with antiangiogenic treatment may have a synergistic effect and enhance the efficacy of both treatments. However, its underlying mechanism remain largely uninvestigated. To investigate the clinical significance of vascular endothelial growth factor receptor-2 (VEGFR2) and programmed death ligand-1 (PD-L1) in osteosarcoma, we analyzes their expression levels in 93 osteosarcoma specimens by immunohistochemistry. Meanwhile, we analyzes their correlation with the metastatic behavior and overall survival (OS). We also investigate the effects of Apatinib on migration and invasion of osteosarcoma cells and its underlying mechanism in vitro and in vivo. In our study, the positive rates of the VEGFR2 and PD-L1 expression are 64.5% (60/93) and 35.5% (33/93), respectively. A significant correlation is detected between VEGFR2 and PD-L1 expression (P = 0.009). Receiver-operating characteristic (ROC) curves analysis indicates the predictive value of the two markers in tumor metastasis, and both PD-L1 and VEGFR2 are negatively correlated with OS. Transwell assays reveals that VEGFR2 inhibition attenuates migration and invasion of osteosarcoma cells. Mechanistically, we demonstrate that Apatinib attenuates migration and invasion by suppressing epithelial-mesenchymal transition (EMT) and inactivating STAT3. Additionally, Apatinib reduces PD-L1 expression in osteosarcoma cells. Apatinib markedly weakens pulmonary metastatic potential of osteosarcoma in vivo. In conclusion, our study reveals a pro-metastatic functional mechanism for VEGFR2 in osteosarcoma. Furthermore, we demonstrate that Apatinib exerts anti-tumor effect not only through antiangiogenic effect, but also via suppressing immune escape, which may represent a potential therapeutic target for metastatic osteosarcoma.     

Processing and analysis of potentiometric data.

Potentiometric titration curves have traditionally been collected as the difference in absorbance at two wavelengths, and analyzed by plotting voltage vs. log (oxidized/reduced). The collection method, designed to monitor changes in local peak height, is effective for that purpose only when spectral backgrounds do not change slope as voltage changes, and the analysis method is valid only for a single isolated component (one whose midpoint potential is far from that of anything else in the mixture). Yet these methods are commonly used where such restrictions do not pertain, e.g. the study of cytochromes in mitonchondria. In this paper, we present more appropriate methods of collection and analysis, and suggest that, even with the best available methods, any conclusion should be confirmed in several ways. Experimental results are presented in accompanying papers.     

Tracking improves performance of biological collision avoidance models

Collision avoidance models derived from the study of insect brains do not perform universally well in practical collision scenarios, although the insects themselves may perform well in similar situations. In this article, we present a detailed simulation analysis of two well-known collision avoidance models and illustrate their limitations. In doing so, we present a novel continuous-time implementation of a neuronally based collision avoidance model. We then show that visual tracking can improve performance of these models by allowing an relative computation of the distance between the obstacle and the observer. We compare the results of simulations of the two models with and without tracking to show how tracking improves the ability of the model to detect an imminent collision. We present an implementation of one of these models processing imagery from a camera to show how it performs in real-world scenarios. These results suggest that insects may track looming objects with their gaze.     

Changes in the coding sequence of the H-2Dk gene of metastatic cells that might account for immunogenic abnormality of its encoded antigen.

In previous studies we have demonstrated that metastatic cells, derived from T-10 fibrosarcoma, express an immunogenically abnormal H-2Dk glycoprotein which is involved in manifesting their metastatic phenotype. In the present study we show that these cells contain a remarkably high level of H-2Dk specific mRNA. Moreover, by cloning cDNA of this gene and analyzing its nucleotide sequence, we found 4 single nucleotide changes. Two of them did not change the encoded amino acids, whereas the others resulted in two amino acid substitutions in the alpha-2 domain of the protein product that might account for its immunogenic abnormality.     

Synthesis of platelet-activating factor (PAF) in transformed cell lines of a different origin

Interest in the possible involvement of the platelet-activating factor (PAF) in tumor growth and invasiveness has been stimulated by the recognition that PAF influences various biological responses relevant to metastatic diffusion, such as angiogenesis, adhesiveness to endothelia and cellular motility. In the present study, we investigated the extent to which PAF is synthesized by a series of human and murine transformed cell lines of a different histotype. Synthesis of PAF was studied by combining the 14C-acetate incorporation into PAF with the quantitative analysis of PAF performed by a procedure based on gas chromatography-mass spectrometry with a negative ion chemical ionization. In the presence of the Ca2+ ionophore A23187, cultures of human melanoma (Hs294T), fibrosarcoma (HT1080) and colon carcinoma (LS180) cell lines synthesized conspicuous amounts of PAF, comparable to those produced by resident peritoneal macrophages. Substantial quantities of PAF were also synthesized by the murine melanoma (F10-M3 cells). PAF synthesis was rather limited in RSV-transformed Balb/c3T3 (B77-3T3) cells and in one of their high metastatic variants (B77-AA6 cells), although it was more abundant in the latter. We also investigated whether certain cytokines, such as TNFalpha and IFNgamma might induce PAF synthesis in our systems of cell lines which we found to express mRNAs encoding receptors for these cytokines. We observed that PAF synthesis was enhanced in human melanoma and colon carcinoma cell lines and in the murine B77-AA6 cells to levels comparable to those obtained with the Ca2+ ionophore. Synthesis of PAF was not inducible by TNFalpha in murine F10-M3 melanoma cells. IFNgamma also stimulated PAF synthesis in human and murine melanoma lines, and in human LS180 colon carcinoma line, but not in the B77-AA6 cells. PAF synthesis was also inducible by exogenous PAF in the human and murine melanoma lines, and in the human LS180 colon carcinoma line, all of which expressed cell surface PAF receptors. PAF synthesis was not inducible by exogenous PAF in the B77-AA6 cells, which do not express PAF receptors.     

Involvement of reactive nitrogen oxides for acquisition of metastatic properties of benign tumors in a model of inflammation-based tumor progression.

The cells of a weakly tumorigenic and non-metastatic murine fibrosarcoma (QR-32) are converted into highly malignant tumors (acquiring metastatic potential) once they have grown in vivo after being co-implanted with gelatin sponge which induces inflammation. In the present study, we examined whether nitric oxide (NO) is involved in the inflammation-based tumor progression by administrating a specific inhibitor to inducible nitric oxide synthase, aminoguanidine (AG). First, we co-implanted 1 x 10(5) QR-32 cells with gelatin sponge (10 x 5 x 3 mm piece) into a subcutaneous space in C57BL6 mice. Administration of AG in drinking water (1%) had started 2 days before the tumor implantation and continued until the termination of the experiment. The incidence of tumor formation and the tumor growth did not differ between AG-treated group and -untreated group. On day 28, we excised the arising tumors to establish culture cell lines for evaluation of their acquisition of metastatic phenotype in other normal mice. Metastasis incidence and the number of metastatic colonies were significantly reduced in the tumor cell lines obtained from AG-treated mice compared to those from non-treated mice (p < 0.05). Immunohistochemical analysis demonstrated that inducible nitric oxide synthase and nitrotyrosine in the inflamed lesion were reduced in the AG-administered mice. However, intensity of 8-hydroxy-2-deoxyguanosine was not different between the groups. These results showed that nitric oxide and its reactive nitrogen oxide species cooperatively play a pivotal role in the progression of benign tumor cells in inflamed lesions.     

Identification and quantification of host cell protein impurities in biotherapeutics using mass spectrometry

Residual host cell proteins (HCPs) in biotherapeutics can present potential safety risks to patients or compromise product stability. As such, their levels are typically monitored using a multicomponent HCP enzyme-linked immunosorbent assay (ELISA) to ensure adequate removal. However, it is not possible to guarantee ELISA coverage of every possible HCP impurity, and the specific HCPs remaining following purification are rarely identified. In the current study, we characterized the ability of an advanced two-dimensional liquid chromatography/mass spectrometry platform (2D-LC/MS(E)) to identify and quantify known low-level spiked protein impurities in a therapeutic peptide Fc fusion protein. The label-free quantification procedure based on the "top 3" intensity tryptic peptides per protein was applied and improved on for this application. Limits of detection for unknown HCPs were approximated from the spiked protein data along with estimates for the quantitative accuracy of the method. In all, we established that most protein impurities present at 13±4ppm can be identified with a quantitative error of less than 2-fold using the more sensitive of two tested method formats. To conclude the study, we characterized all detectable Escherichia coli proteins present in this Fc fusion protein drug substance and discuss future applications of the method.