Unknown,Transcriptomics,Genomics,Proteomics

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Glyco gene profiling of two Hela cell lines to help identify NKp30L expressed on tumoral cells

ABSTRACT: NKp30 is one of the first NK-specific triggering receptor involved in tumour cell lysis to be identified. It is part of the so-called Natural Cytotoxicity Receptors (NCRs) also including NKp44, NKp46 and NKp80 (Bottino, 2005). Human NKp30 is a 190 amino-acids transmembrane protein with an extracellular V-type Ig-like domain containing two putative N-glycosylation sites (Pende, 1999). Three alternative splices can yield three different intracellular domains. An additional alternative splice can induce the deletion of 25 AA in the extracellular domain leading to the formation of a predicted C2-type Ig-like domain instead of a V-type Ig-like domain (Hollyoake, 2005; Neville, 1999). Dr. Romagne lab's aim is to identify NKp30L expressed on tumoral cells. Preliminary Results: Two in vitro systems allow us to detect the NKp30L on the surface of target cells. The first reporter system is a reporter cell line expressing a NKp30 chimeric protein. Upon engagement of this chimeric protein with the NKp30L, cell activation specific for this interaction is measured. The second reporter systems uses the NKp30Fc recombinant protein as a detection tool or eventually as a blocking reagent. It is made with the sequence encoding the V-type Ig-like domain of NKp30. Work to qualify and define cell lines for the expression of NKp30L was carried out in order to identify in our two reporter systems the best cells lines to use. We finally identified the Hela EV2 and Hela PF cell lines as a positive and negative for NKp30L respectively whereas other NK receptor ligands expression is identical in these two cells lines. The NKp30L is sensitive to trypsin digestion and to PNGase F digestion in the NKp30Fc FACS staining assay. These results indicate that at least one NKp30L is probably a N-Glycan carried by a trypsin-sensitive protein at the cell surface. We performed a glycan gene expression profiling of these two Hela cell lines as this will be of major importance to help characterizing the differences in glycans between these two variants of Hela cells and the nature of the NKp30L. RNA preparations of Hela EV2 and Hela cells from EV23, EV2B, EV2III(positive) and PF4, PFB, PFIII (negative) cells were sent to the Microarray Core (E). The RNA was amplified, labeled, and hybridized to the GLYCOv3 microarrays.

ORGANISM(S): Homo sapiens

SUBMITTER: Steven Head

PROVIDER: E-GEOD-29930 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:NKp30 is one of the first NK-specific triggering receptor involved in tumour cell lysis to be identified. It is part of the so-called Natural Cytotoxicity Receptors (NCRs) also including NKp44, NKp46 and NKp80 (Bottino, 2005). Human NKp30 is a 190 amino-acids transmembrane protein with an extracellular V-type Ig-like domain containing two putative N-glycosylation sites (Pende, 1999). Three alternative splices can yield three different intracellular domains. An additional alternative splice can induce the deletion of 25 AA in the extracellular domain leading to the formation of a predicted C2-type Ig-like domain instead of a V-type Ig-like domain (Hollyoake, 2005; Neville, 1999). Dr. Romagne lab's aim is to identify NKp30L expressed on tumoral cells. Preliminary Results: Two in vitro systems allow us to detect the NKp30L on the surface of target cells. The first reporter system is a reporter cell line expressing a NKp30 chimeric protein. Upon engagement of this chimeric protein with the NKp30L, cell activation specific for this interaction is measured. The second reporter systems uses the NKp30Fc recombinant protein as a detection tool or eventually as a blocking reagent. It is made with the sequence encoding the V-type Ig-like domain of NKp30. Work to qualify and define cell lines for the expression of NKp30L was carried out in order to identify in our two reporter systems the best cells lines to use. We finally identified the Hela EV2 and Hela PF cell lines as a positive and negative for NKp30L respectively whereas other NK receptor ligands expression is identical in these two cells lines. The NKp30L is sensitive to trypsin digestion and to PNGase F digestion in the NKp30Fc FACS staining assay. These results indicate that at least one NKp30L is probably a N-Glycan carried by a trypsin-sensitive protein at the cell surface. We performed a glycan gene expression profiling of these two Hela cell lines as this will be of major importance to help characterizing the differences in glycans between these two variants of Hela cells and the nature of the NKp30L.

Project description:This data set was downloaded from MetaboLights (http://www.ebi.ac.uk/metabolights/) accession number MTBLS227 Abstract:"Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between a2,3- and a2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation."

Project description:Various cancers such as colorectal cancer (CRC) are associated with alterations in protein glycosylation. CRC cell lines are frequently used to study these (glyco)biological changes and their mechanisms. However, differences between CRC cell lines with regard to their glycosylation have hitherto been largely neglected. Here, we comprehensively characterized the N-glycan profiles of 25 different CRC cell lines, derived from primary tumors and metastatic sites, in order to investigate their potential as glycobiological tumor model systems and to reveal glycans associated with cell line phenotypes. We applied an optimized, high-throughput membrane-based enzymatic glycan release for small sample amounts. Released glycans were derivatized to stabilize and differentiate between a2,3- and a2,6-linked N-acetylneuraminic acids, followed by N-glycosylation analysis by MALDI-TOF(/TOF)-MS. Our results showed pronounced differences between the N-glycosylation patterns of CRC cell lines. CRC cell line profiles differed from tissue-derived N-glycan profiles with regard to their high-mannose N-glycan content but showed a large overlap for complex type N-glycans, supporting their use as a glycobiological cancer model system. Importantly, we could show that the high-mannose N-glycans did not only occur as intracellular precursors but were also present at the cell surface. The obtained CRC cell line N-glycan features were not clearly correlated with mRNA expression levels of glycosyltransferases, demonstrating the usefulness of performing the structural analysis of glycans. Finally, correlation of CRC cell line glycosylation features with cancer cell markers and phenotypes revealed an association between highly fucosylated glycans and CDX1 and/or villin mRNA expression that both correlate with cell differentiation. Together, our findings provide new insights into CRC-associated glycan changes and setting the basis for more in-depth experiments on glycan function and regulation.

Project description:Purpose: There is evidence that therapeutic cancer vaccines can lengthen survival for some cancer patients, but responses vary widely from one person to another. Methods to predict clinical outcomes will advance the field and provide new insights into critical determinants of in vivo efficacy. This study uses a high-throughput glycan microarray to assess correlations between a subject's overall survival after receiving PROSTVAC-VF and his baseline serum anti-glycan antibody levels. Results: Pre-vaccination antibody levels to blood group A trisaccharide (BG-Atri) were found to have a statistically significant correlation with survival. Long-term survival was approximately doubled in subjects with abundant anti-BG-Atri IgM relative to subjects with little or no pre-existing IgM for BG-Atri. This survival correlation was specific to vaccine treatment, as no correlation was observed in control patients immunized with wild-type poxviruses lacking the key tumor antigen, prostate specific antigen (PSA). Moreover, anti-BG-Atri IgM levels were not correlated with general measures of disease severity, such as PSA levels, Gleason score, or Halabi predicted survival. Conclusion: In addition to reporting a new potentially predictive biomarker for PROSTVAC-VF, this study highlights the potential of glycan microarray technology for personalized medicine. The overall study was a retrospective analysis of 141 subjects from phase II trials of PROSTVAC-VF, a poxvirus based cancer vaccine currently in phase III clinical trials for advanced prostate cancer. The subjects were divided into a training set (n=28) and a validation set (n=113). A glycan microarray was used to profile pre-vaccination anti-glycan antibody populations in sera as potential biomarkers for PROSTVAC-VF. For both the training set and validation set, the anti-glycan profiles were measured using four variations of serum dilution and isotype specific secondary antibody (IgM at 1:50, IgG at 1:50, total Ig at 1:50, and total Ig at 1:200). IgG levels for the training set measured at serum dilution of 1:50 are detailed here. The screen for predictive biomarkers identified anti-glycan antibodies that consistently stratified subjects into groups with different Kaplan Meier survival estimates. Due to the potential for overfitting, a permutation test was used to estimate the false discovery rate. Raw data on superSeries GSE42184 record.

Project description:Purpose: There is evidence that therapeutic cancer vaccines can lengthen survival for some cancer patients, but responses vary widely from one person to another. Methods to predict clinical outcomes will advance the field and provide new insights into critical determinants of in vivo efficacy. This study uses a high-throughput glycan microarray to assess correlations between a subject's overall survival after receiving PROSTVAC-VF and his baseline serum anti-glycan antibody levels. Results: Pre-vaccination antibody levels to blood group A trisaccharide (BG-Atri) were found to have a statistically significant correlation with survival. Long-term survival was approximately doubled in subjects with abundant anti-BG-Atri IgM relative to subjects with little or no pre-existing IgM for BG-Atri. This survival correlation was specific to vaccine treatment, as no correlation was observed in control patients immunized with wild-type poxviruses lacking the key tumor antigen, prostate specific antigen (PSA). Moreover, anti-BG-Atri IgM levels were not correlated with general measures of disease severity, such as PSA levels, Gleason score, or Halabi predicted survival. Conclusion: In addition to reporting a new potentially predictive biomarker for PROSTVAC-VF, this study highlights the potential of glycan microarray technology for personalized medicine. The overall study was a retrospective analysis of 141 subjects from phase II trials of PROSTVAC-VF, a poxvirus based cancer vaccine currently in phase III clinical trials for advanced prostate cancer. The subjects were divided into a training set (n=28) and a validation set (n=113). A glycan microarray was used to profile pre-vaccination anti-glycan antibody populations in sera as potential biomarkers for PROSTVAC-VF. For both the training set and validation set, the anti-glycan profiles were measured using four variations of serum dilution and isotype specific secondary antibody (IgM at 1:50, IgG at 1:50, total Ig at 1:50, and total Ig at 1:200). IgM levels for the training set measured at serum dilution of 1:50 are detailed here. The screen for predictive biomarkers identified anti-glycan antibodies that consistently stratified subjects into groups with different Kaplan Meier survival estimates. Due to the potential for overfitting, a permutation test was used to estimate the false discovery rate. Raw data on superSeries GSE42184 record.

Project description:Purpose: There is evidence that therapeutic cancer vaccines can lengthen survival for some cancer patients, but responses vary widely from one person to another. Methods to predict clinical outcomes will advance the field and provide new insights into critical determinants of in vivo efficacy. This study uses a high-throughput glycan microarray to assess correlations between a subject's overall survival after receiving PROSTVAC-VF and his baseline serum anti-glycan antibody levels. Results: Pre-vaccination antibody levels to blood group A trisaccharide (BG-Atri) were found to have a statistically significant correlation with survival. Long-term survival was approximately doubled in subjects with abundant anti-BG-Atri IgM relative to subjects with little or no pre-existing IgM for BG-Atri. This survival correlation was specific to vaccine treatment, as no correlation was observed in control patients immunized with wild-type poxviruses lacking the key tumor antigen, prostate specific antigen (PSA). Moreover, anti-BG-Atri IgM levels were not correlated with general measures of disease severity, such as PSA levels, Gleason score, or Halabi predicted survival. Conclusion: In addition to reporting a new potentially predictive biomarker for PROSTVAC-VF, this study highlights the potential of glycan microarray technology for personalized medicine. The overall study was a retrospective analysis of 141 subjects from phase II trials of PROSTVAC-VF, a poxvirus based cancer vaccine currently in phase III clinical trials for advanced prostate cancer. The subjects were divided into a training set (n=28) and a validation set (n=113). A glycan microarray was used to profile pre-vaccination anti-glycan antibody populations in sera as potential biomarkers for PROSTVAC-VF. For both the training set and validation set, the anti-glycan profiles were measured using four variations of serum dilution and isotype specific secondary antibody (IgM at 1:50, IgG at 1:50, total Ig at 1:50, and total Ig at 1:200). IgG levels for the validation set measured at serum dilution of 1:50 are detailed here. The screen for predictive biomarkers identified anti-glycan antibodies that consistently stratified subjects into groups with different Kaplan Meier survival estimates. Due to the potential for overfitting, a permutation test was used to estimate the false discovery rate. Raw data on superSeries GSE42184 record.

Project description:Purpose: There is evidence that therapeutic cancer vaccines can lengthen survival for some cancer patients, but responses vary widely from one person to another. Methods to predict clinical outcomes will advance the field and provide new insights into critical determinants of in vivo efficacy. This study uses a high-throughput glycan microarray to assess correlations between a subject's overall survival after receiving PROSTVAC-VF and his anti-glycan humoral responses occuring in the first months after treatment with PROSTVAC-VF. Results: Humoral responses to the terminal Forssman disaccharide (Fsdi) were found to have a statistically significant correlation with survival. Long-term survival was approximately doubled in subjects with four-fold or larger anti-Fsdi responses relative to subjects with little or no anti-Fsdi response. This survival correlation was specific to vaccine treatment, as no correlation was observed in control patients immunized with wild-type poxviruses lacking the key tumor antigen, prostate specific antigen (PSA). Moreover, anti-Fsdi humoral responses were not correlated with general measures of disease severity, such as PSA levels, Gleason score, or Halabi predicted survival. Conclusion: In addition to reporting a new biomarker for monitoring benefical responses to PROSTVAC-VF, this study highlights the potential of glycan microarray technology for personalized medicine. The overall study was a retrospective analysis of 141 subjects from phase II trials of PROSTVAC-VF, a poxvirus based cancer vaccine currently in phase III clinical trials for advanced prostate cancer. The subjects were divided into a training set (n=28) and a validation set (n=113). A glycan microarray was used to profile pre-vaccination and post-vaccination anti-glycan antibody populations in sera as potential biomarkers for PROSTVAC-VF. For both the training set and validation set, the anti-glycan profiles were measured using four variations of serum dilution and isotype specific secondary antibody (IgM at 1:50, IgG at 1:50, total Ig at 1:50, and total Ig at 1:200). IgG levels for the training set measured at serum dilution of 1:50 are detailed here. The screen for response biomarkers identified anti-glycan humoral responses that consistently stratified subjects into groups with different Kaplan Meier survival estimates. Raw data on SuperSeries GSE50410 record.

Project description:Purpose: There is evidence that therapeutic cancer vaccines can lengthen survival for some cancer patients, but responses vary widely from one person to another. Methods to predict clinical outcomes will advance the field and provide new insights into critical determinants of in vivo efficacy. This study uses a high-throughput glycan microarray to assess correlations between a subject's overall survival after receiving PROSTVAC-VF and his baseline serum anti-glycan antibody levels. Results: Pre-vaccination antibody levels to blood group A trisaccharide (BG-Atri) were found to have a statistically significant correlation with survival. Long-term survival was approximately doubled in subjects with abundant anti-BG-Atri IgM relative to subjects with little or no pre-existing IgM for BG-Atri. This survival correlation was specific to vaccine treatment, as no correlation was observed in control patients immunized with wild-type poxviruses lacking the key tumor antigen, prostate specific antigen (PSA). Moreover, anti-BG-Atri IgM levels were not correlated with general measures of disease severity, such as PSA levels, Gleason score, or Halabi predicted survival. Conclusion: In addition to reporting a new potentially predictive biomarker for PROSTVAC-VF, this study highlights the potential of glycan microarray technology for personalized medicine. The overall study was a retrospective analysis of 141 subjects from phase II trials of PROSTVAC-VF, a poxvirus based cancer vaccine currently in phase III clinical trials for advanced prostate cancer. The subjects were divided into a training set (n=28) and a validation set (n=113). A glycan microarray was used to profile pre-vaccination anti-glycan antibody populations in sera as potential biomarkers for PROSTVAC-VF. For both the training set and validation set, the anti-glycan profiles were measured using four variations of serum dilution and isotype specific secondary antibody (IgM at 1:50, IgG at 1:50, total Ig at 1:50, and total Ig at 1:200). IgM levels for the validation set measured at serum dilution of 1:50 are detailed here. The screen for predictive biomarkers identified anti-glycan antibodies that consistently stratified subjects into groups with different Kaplan Meier survival estimates. Due to the potential for overfitting, a permutation test was used to estimate the false discovery rate. Raw data on superSeries GSE42184 record.

Project description:Purpose: There is evidence that therapeutic cancer vaccines can lengthen survival for some cancer patients, but responses vary widely from one person to another. Methods to predict clinical outcomes will advance the field and provide new insights into critical determinants of in vivo efficacy. This study uses a high-throughput glycan microarray to assess correlations between a subject's overall survival after receiving PROSTVAC-VF and his anti-glycan humoral responses occuring in the first months after treatment with PROSTVAC-VF. Results: Humoral responses to the terminal Forssman disaccharide (Fsdi) were found to have a statistically significant correlation with survival. Long-term survival was approximately doubled in subjects with four-fold or larger anti-Fsdi responses relative to subjects with little or no anti-Fsdi response. This survival correlation was specific to vaccine treatment, as no correlation was observed in control patients immunized with wild-type poxviruses lacking the key tumor antigen, prostate specific antigen (PSA). Moreover, anti-Fsdi humoral responses were not correlated with general measures of disease severity, such as PSA levels, Gleason score, or Halabi predicted survival. Conclusion: In addition to reporting a new biomarker for monitoring benefical responses to PROSTVAC-VF, this study highlights the potential of glycan microarray technology for personalized medicine. The overall study was a retrospective analysis of 141 subjects from phase II trials of PROSTVAC-VF, a poxvirus based cancer vaccine currently in phase III clinical trials for advanced prostate cancer. The subjects were divided into a training set (n=28) and a validation set (n=113). A glycan microarray was used to profile pre-vaccination and post-vaccination anti-glycan antibody populations in sera as potential biomarkers for PROSTVAC-VF. For both the training set and validation set, the anti-glycan profiles were measured using four variations of serum dilution and isotype specific secondary antibody (IgM at 1:50, IgG at 1:50, total Ig at 1:50, and total Ig at 1:200). Total immunoglobin (IgM + IgG + IgA) levels for the training set measured at serum dilution of 1:200 are detailed here. The screen for response biomarkers identified anti-glycan humoral responses that consistently stratified subjects into groups with different Kaplan Meier survival estimates. Raw data on SuperSeries GSE50410 record.

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Glyco gene profiling of two Hela cell lines to help identify NKp30L expressed on tumoral cells

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