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Nestor, Marika
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Publications (10 of 41) Show all publications
Häggblad Sahlberg, S., Mortensen, A. C., Haglöf, J., Engskog, M. K. R., Arvidsson, T., Pettersson, C., . . . Nestor, M. (2017). Different functions of AKT1 and AKT2 in molecular pathways, cell migration and metabolism in colon cancer cells. International Journal of Oncology, 50(1), 5-14
Open this publication in new window or tab >>Different functions of AKT1 and AKT2 in molecular pathways, cell migration and metabolism in colon cancer cells
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2017 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 50, no 1, p. 5-14Article in journal (Refereed) Published
Abstract [en]

AKT is a central protein in many cellular pathways such as cell survival, proliferation, glucose uptake, metabolism, angiogenesis, as well as radiation and drug response. The three isoforms of AKT (AKT1, AKT2 and AKT3) are proposed to have different physiological functions, properties and expression patterns in a cell type-dependent manner. As of yet, not much is known about the influence of the different AKT isoforms in the genome and their effects in the metabolism of colorectal cancer cells. In the present study, DLD-1 isogenic AKT1, AKT2 and AKT'/2 knockout colon cancer cell lines were used as a model system in conjunction with the parental cell line in order to further elucidate the differences between the AKT isoforms and how they are involved in various cellular pathways. This was done using genome wide expression analyses, metabolic profiling and cell migration assays. In conclusion, downregulation of genes in the cell adhesion, extracellular matrix and Notch-pathways and upregulation of apoptosis and metastasis inhibitory genes in the p53-pathway, confirm that the knockout of both AKT1 and AKT2 will attenuate metastasis and tumor cell growth. This was verified with a reduction in migration rate in the AKT1 KO and AKT2 KO and most explicitly in the AKT1/2 KO. Furthermore, the knockout of AKT1, AKT2 or both, resulted in a reduction in lactate and alanine, suggesting that the metabolism of carbohydrates and glutathione was impaired. This was further verified in gene expression analyses, showing downregulation of genes involved in glucose metabolism. Additionally, both AKT1 KO and AKT2 KO demonstrated an impaired fatty acid metabolism. However, genes were upregulated in the Wnt and cell proliferation pathways, which could oppose this effect. AKT inhibition should therefore be combined with other effectors to attain the best effect.

Keyword
Microarray, metabolism, cell migration AKT1, AKT2, AKT, PKB, gene expression, colon-cancer, DLD-1, metabolomics, CD44, CD133
National Category
Biochemistry and Molecular Biology
Research subject
Biomedical Radiation Science; Biology with specialization in Molecular Cell Biology; Biology with specialization in Molecular Biology
Identifiers
urn:nbn:se:uu:diva-222834 (URN)10.3892/ijo.2016.3771 (DOI)000391419200001 ()
Available from: 2014-04-14 Created: 2014-04-14 Last updated: 2017-12-05Bibliographically approved
Haylock, A.-K., Nilvebrant, J., Mortensen, A., Velikyan, I., Nestor, M. & Falk, R. (2017). Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers. OncoTarget, 8(39), 65152-65170
Open this publication in new window or tab >>Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers
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2017 (English)In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 8, no 39, p. 65152-65170Article in journal (Refereed) Published
Abstract [en]

Aim: The aim of this study was to generate and characterize scFv antibodies directed to human CD44v6, as well as to radiolabel and evaluate top candidates in vitro and in vivo for their potential use in CD44v6-targeted molecular imaging in cancer patients.

Materials and methods: Phage display selections were used to isolate CD44v6-specific scFvs. A chain shuffling strategy was employed for affinity maturation based on a set of CD44v6-specific first-generation clones. Two second-generation scFv clones were then chosen for labeling with 111In or 125I and assessed for CD44v6-specific binding on cultured tumor cells. In vivo uptake and distribution was evaluated in tumor-bearing mice using a dual tumor model. Finally, a proof-of-concept small animal PET-CT study was performed on one of the candidates labeled with 124I.

Results: Two affinity-matured clones, CD44v6-scFv-A11 and CD44v6-scFv-H12, displayed promising binding kinetics. Seven out of eight radiolabeled conjugates demonstrated CD44v6-specific binding. In vivo studies on selected candidates demonstrated very advantageous tumor-to-organ ratios, in particular for iodinated conjugates, where 125I-labeled scFvs exhibited favorable kinetics and tumor-to-blood ratios above five already at 24 hours p. i.. The small animal PET-CT study using 124I-labeled CD44v6-scFv-H12 was in line with the biodistribution data, clearly visualizing the high CD44v6-expressing tumor.

Conclusion: The single chain fragments, CD44v6-scFv-A11 and CD44v6-scFv-H12 specifically bind to CD44v6, and the radiolabeled counterparts provide high tumor-to-blood ratios and fast clearance from organs and blood. We conclude that radioiodinated CD44v6-scFv-A11 and CD44v6-scFv-H12 possess features highly suitable for stringent molecular imaging.

Keyword
scFv, recombinant antibody formats, CD44v6, squamous cell carcinoma, molecular imaging
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Cancer and Oncology Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-335192 (URN)10.18632/oncotarget.17996 (DOI)000410291200039 ()29029420 (PubMedID)
Funder
Swedish Cancer Society, CAN 2015/1080, CAN 2015/385Swedish Research Council, 2013-30876-104113-30, 637-2013-468Swedish Society for Medical Research (SSMF)Knut and Alice Wallenberg Foundation, 2008.0133
Note

Marika Nestor and Ronny Falk shared senior authorship.

Available from: 2017-12-08 Created: 2017-12-08 Last updated: 2017-12-08Bibliographically approved
Bondza, S., Björkelund, H., Nestor, M., Andersson, K. & Buijs, J. (2017). Novel Real-Time Proximity Assay for Characterizing Multiple Receptor Interactions on Living Cells. Analytical Chemistry, 89(24), 13212-13218
Open this publication in new window or tab >>Novel Real-Time Proximity Assay for Characterizing Multiple Receptor Interactions on Living Cells
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2017 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 89, no 24, p. 13212-13218Article in journal (Refereed) Published
Abstract [en]

Cellular receptor activity is often controlled through complex mechanisms involving interactions with multiple molecules, which can be soluble ligands and/or other cell surface molecules. In this study, we combine a fluorescence-based technology for real-time interaction analysis with fluorescence quenching to create a novel time-resolved proximity assay to study protein-receptor interactions on living cells. This assay extracts the binding kinetics and affinity for two proteins if they bind in proximity on the cell surface. One application of real-time proximity interaction analysis is to study relative levels of receptor dimerization. The method was primarily evaluated using the HER2 binding antibodies Trastuzumab and Pertuzumab and two EGFR binding antibodies including Cetuximab. Using Cetuximab and Trastuzumab, proximity of EGFR and HER2 was investigated before and after treatment of cells with the tyrosine-kinase inhibitor Gefitinib. Treated cells displayed 50% increased proximity signal, whereas the binding characteristics of the two antibodies were not significantly affected, implying an increase in the EGFR-HER2 dimer level. These results demonstrate that real-time proximity interaction analysis enables determination of the interaction rate constants and affinity of two ligands while simultaneously quantifying their relative colocalization on living cells.

National Category
Analytical Chemistry Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-339775 (URN)10.1021/acs.analchem.7b02983 (DOI)000418626300025 ()29160688 (PubMedID)
Available from: 2018-02-09 Created: 2018-02-09 Last updated: 2018-02-09Bibliographically approved
Elmsjö, A., Haglöf, J., Engskog, M. K. R., Nestor, M., Arvidsson, T. & Pettersson, C. (2017). The co-feature ratio, a novel method for the measurement of chromatographic and signal selectivity in LC-MS-based metabolomics.. Analytica Chimica Acta, 956, 40-47
Open this publication in new window or tab >>The co-feature ratio, a novel method for the measurement of chromatographic and signal selectivity in LC-MS-based metabolomics.
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2017 (English)In: Analytica Chimica Acta, ISSN 0003-2670, E-ISSN 1873-4324, Vol. 956, p. 40-47Article in journal (Refereed) Published
Abstract [en]

Evaluation of analytical procedures, especially in regards to measuring chromatographic and signal selectivity, is highly challenging in untargeted metabolomics. The aim of this study was to suggest a new straightforward approach for a systematic examination of chromatographic and signal selectivity in LC-MS-based metabolomics. By calculating the ratio between each feature and its co-eluting features (the co-features), a measurement of the chromatographic selectivity (i.e. extent of co-elution) as well as the signal selectivity (e.g. amount of adduct formation) of each feature could be acquired, the co-feature ratio. This approach was used to examine possible differences in chromatographic and signal selectivity present in samples exposed to three different sample preparation procedures. The capability of the co-feature ratio was evaluated both in a classical targeted setting using isotope labelled standards as well as without standards in an untargeted setting. For the targeted analysis, several metabolites showed a skewed quantitative signal due to poor chromatographic selectivity and/or poor signal selectivity. Moreover, evaluation of the untargeted approach through multivariate analysis of the co-feature ratios demonstrated the possibility to screen for metabolites displaying poor chromatographic and/or signal selectivity characteristics. We conclude that the co-feature ratio can be a useful tool in the development and evaluation of analytical procedures in LC-MS-based metabolomics investigations. Increased selectivity through proper choice of analytical procedures may decrease the false positive and false negative discovery rate and thereby increase the validity of any metabolomic investigation.

National Category
Analytical Chemistry Pharmaceutical Sciences
Research subject
Analytical Pharmaceutical Chemistry
Identifiers
urn:nbn:se:uu:diva-314239 (URN)10.1016/j.aca.2016.12.022 (DOI)000393252000005 ()28093124 (PubMedID)
Available from: 2017-01-31 Created: 2017-01-31 Last updated: 2018-01-13Bibliographically approved
Spiegelberg, D., Stenberg, J., Haylock, A.-K. & Nestor, M. (2016). A real-time in vitro assay as a potential predictor of in vivo tumor imaging properties. Nuclear Medicine and Biology, 43(1), 12-18
Open this publication in new window or tab >>A real-time in vitro assay as a potential predictor of in vivo tumor imaging properties
2016 (English)In: Nuclear Medicine and Biology, ISSN 0969-8051, E-ISSN 1872-9614, Vol. 43, no 1, p. 12-18Article in journal (Refereed) Published
Abstract [en]

Introduction: Selective tumor targeting strategies based on cell surface molecules enable new personalized diagnosis and treatments, potentially lowering adverse effects and increasing efficacy. Radio-immunotargeting generally relies on a molecule binding to a cancer-specific target. It is therefore important to understand the properties of molecular interactions in their working environment and how to translate these properties measured in vitro into the in vivo molecular imaging situation. Methods: Time resolved interaction analysis in vitro was compared with a corresponding in vivo xenograft mouse model. The antibody fragment AbD15179 was labeled with I-125 or In-111, and analyzed on cell lines with differing CD44v6 expression in vitro, and in a dual tumor xenograft model derived from the same cell lines. In vitro LigandTracer measurements were analyzed with TraceDrawer and Interaction Map. Conjugate sensitivity, kinetics, and signal-to-background ratios were assessed for both tumor cells in vitro and xenograft tumors in vivo. Results: In vitro results revealed a general biphasic appearance of a high- and a low-affinity interaction event. The In-111-labeled fragment displayed the largest proportion of the high-affinity interaction with increased sensitivity and retention compared to I-125-Fab. In vivo results were in agreement with in vitro data, with increased retention, higher sensitivity and better contrast for the In-111-labeled fragment compared to I-125. Conclusions: Time resolved binding characteristics measured in vitro largely matched the in vivo performance for the conjugates, which is promising for future studies. In vitro time-resolved LigandTracer assays are efficient, rapid, and in this study shown to be able to predict in vivo outcomes. Advances in Knowledge and Implications for Patient Care: Further studies are needed to confirm these findings, but the method is promising considering the ethical need to reduce the use of laboratory animals, as well as reducing costs for the development of tumor targeting compounds in the future.

Keyword
Radio-immunotargeting, Radio-immunodiagnostics, Molecular imaging, AbD15179, CD44v6, HNSCC
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-274920 (URN)10.1016/j.nucmedbio.2015.09.004 (DOI)000367419400003 ()26702782 (PubMedID)
Available from: 2016-01-27 Created: 2016-01-26 Last updated: 2017-11-30Bibliographically approved
Spiegelberg, D., Mortensen, A. C., Selvaraju, R. K., Eriksson, O., Stenerlöw, B. & Nestor, M. (2016). Molecular imaging of EGFR and CD44v6 for prediction and response monitoring of HSP90 inhibition in an in vivo squamous cell carcinoma model.. European Journal of Nuclear Medicine and Molecular Imaging, 43(5), 974-982
Open this publication in new window or tab >>Molecular imaging of EGFR and CD44v6 for prediction and response monitoring of HSP90 inhibition in an in vivo squamous cell carcinoma model.
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2016 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, no 5, p. 974-982Article in journal (Refereed) Published
Abstract [en]

PURPOSE: Heat shock protein 90 (HSP90) is essential for the activation and stabilization of numerous oncogenic client proteins. AT13387 is a novel HSP90 inhibitor promoting degradation of oncogenic proteins upon binding, and may also act as a radiosensitizer. For optimal treatment there is, however, the need for identification of biomarkers for patient stratification and therapeutic response monitoring, and to find suitable targets for combination treatments. The aim of this study was to assess the response of surface antigens commonly expressed in squamous cell carcinoma to AT13387 treatment, and to find suitable biomarkers for molecular imaging and radioimmunotherapy in combination with HSP90 inhibition.

METHODS: Cancer cell proliferation and radioimmunoassays were used to evaluate the effect of AT13387 on target antigen expression in vitro. Inhibitor effects were then assessed in vivo in mice-xenografts. Animals were treated with AT13387 (5 × 50 mg/kg), and were imaged with PET using either (18)F-FDG or (124)I-labelled tracers for EGFR and CD44v6, and this was followed by ex-vivo biodistribution analysis and immunohistochemical staining.

RESULTS: AT13387 exposure resulted in high cytotoxicity and possible radiosensitization with IC50 values below 4 nM. Both in vitro and in vivo AT13387 effectively downregulated HSP90 client proteins. PET imaging with (124)I-cetuximab showed a significant decrease of EGFR in AT13387-treated animals compared with untreated animals. In contrast, the squamous cell carcinoma-associated biomarker CD44v6, visualized with (124)I-AbD19384 as well as (18)F-FDG uptake, were not significantly altered by AT13387 treatment.

CONCLUSION: We conclude that AT13387 downregulates HSP90 client proteins, and that molecular imaging of these proteins may be a suitable approach for assessing treatment response. Furthermore, radioimmunotherapy targeting CD44v6 in combination with AT13387 may potentiate the radioimmunotherapy outcome due to radiosensitizing effects of the drug, and could potentially lead to a lower dose to normal tissues.

National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-270260 (URN)10.1007/s00259-015-3260-x (DOI)000373306800020 ()26627081 (PubMedID)
Funder
Swedish Cancer Society, CAN 2012/399; CAN 2014/661Swedish Research Council, 2013-30876-104113-30
Available from: 2015-12-22 Created: 2015-12-22 Last updated: 2018-02-18Bibliographically approved
Spiegelberg, D., Stenberg, J., Haylock, A.-K. & Nestor, M. (2015). A real-time in vitro assay as a potential predictor of in vivo tumour imaging properties. Paper presented at 28th Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 10-14, 2015, Hamburg, GERMANY. European Journal of Nuclear Medicine and Molecular Imaging, 42(S1), S306-S307
Open this publication in new window or tab >>A real-time in vitro assay as a potential predictor of in vivo tumour imaging properties
2015 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, no S1, p. S306-S307Article in journal, Meeting abstract (Other academic) Published
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-269147 (URN)000363013202115 ()
Conference
28th Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 10-14, 2015, Hamburg, GERMANY
Note

Meeting Abstract: PW057

Available from: 2015-12-18 Created: 2015-12-14 Last updated: 2017-12-01Bibliographically approved
Mortensen, A. C., Spiegelberg, D., Scott, A. M., Lane, D. P. & Nestor, M. (2015). Enhancing the effect of radiotherapy in cultured tumour cells using p53 therapy. Paper presented at 28th Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 10-14, 2015, Hamburg, GERMANY. European Journal of Nuclear Medicine and Molecular Imaging, 42(S1), S429-S430
Open this publication in new window or tab >>Enhancing the effect of radiotherapy in cultured tumour cells using p53 therapy
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2015 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, no S1, p. S429-S430Article in journal, Meeting abstract (Other academic) Published
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-269142 (URN)000363013202359 ()
Conference
28th Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 10-14, 2015, Hamburg, GERMANY
Note

Meeting Abstract: P184

Available from: 2015-12-18 Created: 2015-12-14 Last updated: 2017-12-01Bibliographically approved
Haylock, A.-K., Spiegelberg, D., Mortensen, A. C., Selvaraju, R. K., Nilvebrant, J., Eriksson, O., . . . Nestor, M. V. (2015). Evaluation of a novel type of imaging probe based on a recombinant bivalent mini-antibody construct for detection of CD44v6-expressing squamous cell carcinoma. International journal of oncology, 48(2), 461-470
Open this publication in new window or tab >>Evaluation of a novel type of imaging probe based on a recombinant bivalent mini-antibody construct for detection of CD44v6-expressing squamous cell carcinoma
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2015 (English)In: International journal of oncology, ISSN 1791-2423, Vol. 48, no 2, p. 461-470Article in journal (Refereed) Published
Abstract [en]

We have developed the CD44v6-targeting human bivalent antibody fragment AbD19384, an engineered recombinant human bivalent Fab antibody formed via dimerization of dHLX (synthetic double helix loop helix motif) domains, for potential use in antibody-based molecular imaging of squamous cell carcinoma in the head and neck region. This is a unique construct that has, to the best of our knowledge, never been assessed for molecular imaging in vivo before. The objective of the present study was to evaluate for the first time the in vitro and in vivo binding properties of radio-iodinated AbD19384, and to assess its utility as a targeting agent for molecular imaging of CD44v6-expressing tumors. Antigen specificity and binding properties were assessed in vitro. In vivo specificity and biodistribution of 125I-AbD19384 were next evaluated in tumor-bearing mice using a dual-tumor setup. Finally, AbD19384 was labeled with 124I, and its imaging properties were assessed by small animal PET/CT in tumor bearing mice, and compared with 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG). In vitro studies demonstrated CD44v6-specific binding with slow off-rate for AbD19384. A favorable biodistribution profile was seen in vivo, with tumor-specific uptake. Small animal PET/CT images of 124I-AbD19384 supported the results through clearly visible high CD44v6-expressing tumors and faintly visible low expressing tumors, with superior imaging properties compared to 18F-FDG. Tumor-to-blood ratios increased with time for the conjugate (assessed up to 72 h p.i.), although 48 h p.i. proved best for imaging. Biodistribution and small-animal PET studies demonstrated that the recombinant Fab-dHLX construct AbD19384 is a promising tracer for imaging of CD44v6 antigen expression in vivo, with the future aim to be used for individualized diagnosis and early detection of squamous cell carcinomas in the head and neck region. Furthermore, this proof-of-concept research established the feasibility of using recombinant Fab-dHLX constructs for in vivo imaging of tumor biomarkers.

Keyword
molecular imaging, CD44v6; Fab-dHLX; F(ab')(2), recombinant antibodies, immuno-PET, head and neck squamous cell carcinoma
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-270259 (URN)10.3892/ijo.2015.3290 (DOI)000366897500003 ()26676731 (PubMedID)
Available from: 2015-12-22 Created: 2015-12-22 Last updated: 2017-11-15Bibliographically approved
Spiegelberg, D., Mortensen, A. C., Selvaraju, R. K., Stenberg, J., Scott, A. & Nestor, M. (2015). Improving EGFR imaging by use of an anti-EGFRvIII monoclonal antibody: characterizations in an in vivo squamous cell carcinoma model. Paper presented at 28th Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 10-14, 2015, Hamburg, GERMANY. European Journal of Nuclear Medicine and Molecular Imaging, 42(S1), S467-S467
Open this publication in new window or tab >>Improving EGFR imaging by use of an anti-EGFRvIII monoclonal antibody: characterizations in an in vivo squamous cell carcinoma model
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2015 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 42, no S1, p. S467-S467Article in journal, Meeting abstract (Other academic) Published
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-269146 (URN)000363013202435 ()
Conference
28th Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 10-14, 2015, Hamburg, GERMANY
Note

Meeting Abstract: P262

Available from: 2015-12-18 Created: 2015-12-14 Last updated: 2017-12-01Bibliographically approved
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