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Nestor, Marika
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Publications (10 of 56) Show all publications
Erngren, I., Haglöf, J., Engskog, M. K., Nestor, M., Hedeland, M., Arvidsson, T. & Pettersson, C. (2019). Adduct formation in electrospray ionisation-mass spectrometry with hydrophilic interaction liquid chromatography is strongly affected by the inorganic ion concentration of the samples. Journal of Chromatography A, 1600, 174-182
Open this publication in new window or tab >>Adduct formation in electrospray ionisation-mass spectrometry with hydrophilic interaction liquid chromatography is strongly affected by the inorganic ion concentration of the samples
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2019 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1600, p. 174-182Article in journal (Refereed) Published
Abstract [en]

Hydrophilic interaction liquid chromatography (HILIC)/electrospray ionisation-mass spectrometry (ESI-MS) has gained interest for the analysis of polar analytes in bioanalytical applications in recent years. However, ESI-MS is prone to adduct formation of analytes. In contrast to reversed phase chromatography, small inorganic ions have retention in HILIC, i.e. analytes and inorganic ions may co-elute, which could influence the adduct formation. In the present paper, it was demonstrated that the co-elution of sodium ions or potassium ions and analytes in HILIC/ESI-MS affect the adduct formation and that different concentrations of sodium ions and potassium ions in biological samples could have an impact on the quantitative response of the respective adducts as well as the quantitative response of the protonated adduct. The co-elution also lead to cluster formation of analytes and sodium formate or potassium formate, causing extremely complicated spectra. In analytical applications using HILIC/ESI-MS where internal standards are rarely used or not properly matched, great care needs to be taken to ensure minimal variation of inorganic ion concentration between samples. Moreover, the use of alkali metal ion adducts as quantitative target ions in relative quantitative applications should be made with caution if proper internal standards are not used.

Place, publisher, year, edition, pages
ELSEVIER SCIENCE BV, 2019
Keywords
Adduct formation, Hydrophilic interaction liquid chromatography, Mass spectrometry, Screening, Metabolomics, Cluster formation
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-390383 (URN)10.1016/j.chroma.2019.04.049 (DOI)000472687800021 ()31047661 (PubMedID)
Available from: 2019-08-12 Created: 2019-08-12 Last updated: 2019-08-12Bibliographically approved
Lundgren Mortensen, A., Spiegelberg, D., Brown, C. J., Lane, D. P. & Nestor, M. (2019). The Stapled Peptide PM2 Stabilizes p53 Levels and Radiosensitizes Wild-Type p53 Cancer Cells. Frontiers in Oncology, 9, Article ID 923.
Open this publication in new window or tab >>The Stapled Peptide PM2 Stabilizes p53 Levels and Radiosensitizes Wild-Type p53 Cancer Cells
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2019 (English)In: Frontiers in Oncology, ISSN 2234-943X, E-ISSN 2234-943X, Vol. 9, article id 923Article in journal (Refereed) Published
Abstract [en]

The tumor suppressor p53 is a key mediator of cellular stress and DNA damage response cascades and is activated after exposure to ionizing radiation. Amplifying wild-type p53 expression by targeting negative regulators such as MDM2 in combination with external beam radiotherapy (EBRT) may result in increased therapeutic effects. The novel stapled peptide PM2 prevents MDM2 from suppressing wild-type p53, and is thus a promising agent for therapeutic combination with EBRT. Effects of PM2 and potential PM2-induced radiosensitivity were assessed in a panel of cancer cell lines using 2D cell viability assays. Western Blot and flow cytometric analyses were used to investigate the mechanisms behind the observed effects in samples treated with PM2 and EBRT. Finally, PM2-treatment combined with EBRT was evaluated in an in vitro 3D spheroid model. PM2-therapy decreased cell viability in wild-type p53, HPV-negative cell lines. Western Blotting and flow cytometry confirmed upregulation of p53, as well as initiation of p53-mediated apoptosis measured by increased cleaved caspase-3 and Noxa activity. Furthermore, 3D in vitro tumor spheroid experiments confirmed the superior effects of the combination, as the only treatment regime resulting in growth inhibition and complete spheroid disintegration. We conclude that PM2 induces antitumorigenic effects in wt p53 HPV-negative cancer cells and potentiates the effects of EBRT, ultimately resulting in tumor eradication in a 3D spheroid model. This strategy shows great potential as a new wt p53 specific tumor-targeting compound, and the combination of PM2 and EBRT could be a promising strategy to increase therapeutic effects and decrease adverse effects from radiotherapy.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2019
Keywords
p53, wild-type p53, radiosensitization, external beam radiation therapy (EBRT), PM2, spheroid apoptosis
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-395461 (URN)10.3389/fonc.2019.00923 (DOI)000487235800001 ()31616635 (PubMedID)
Funder
Swedish Research Council, 2013-30876-104113-30Swedish Cancer Society, CAN 2018/494Swedish Cancer Society, CAN 2015/1080
Available from: 2019-10-31 Created: 2019-10-31 Last updated: 2019-10-31Bibliographically approved
Al-Ramadan, A., Mortensen, A., Carlsson, J. & Nestor, M. V. (2018). Analysis of radiation effects in two irradiated tumor spheroid models. Oncology Letters, 15(3), 3008-3016
Open this publication in new window or tab >>Analysis of radiation effects in two irradiated tumor spheroid models
2018 (English)In: Oncology Letters, ISSN 1792-1074, E-ISSN 1792-1082, Vol. 15, no 3, p. 3008-3016Article in journal (Refereed) Published
Abstract [en]

Multicellular spheroids have proven suitable as three-dimensional in vivo-like models of non-vascularized micrometastases. Unlike monolayer-based models, spheroids mirror the cellular milieu and the pathophysiological gradients inside tumor nodules. However, there is limited knowledge of the radiation effects at the molecular level in spheroids of human origin. The present study is a presentation of selected cell biological processes that may easily be analyzed with methods available at routine pathology laboratories. Using gamma irradiated pancreatic neuroendocrine BON1 and colonic adenocarcinoma HCT116 spheroids as model systems, the present study assessed the radiobiological response in these models. Spheroid growth after irradiation was followed over time and molecular responses were subsequently assessed with immunohistochemistry (IHC) staining for descriptive analyses and semi-automatic grading of apoptosis, G(2)-phase and senescence in thin sections of the spheroids. Growth studies demonstrated the BON1 spheroids were slower growing and less sensitive to radiation compared with the HCT116 spheroids. IHC staining for G2-phase was primarily observed in the outer viable P-cell layers of the spheroids, with the 6 Gy irradiated HCT116 spheroids demonstrating a very clear increase in staining intensity compared with unirradiated spheroids. Apoptosis staining results indicated increased apoptosis with increasing radiation doses. No clear association between senescence and radiation exposure in the spheroids were observed. The present results demonstrate the feasibility of the use of multicellular spheroids of human origin in combination with IHC analyses to unravel radiobiological responses at a molecular level. The present findings inspire further investigations, including other relevant IHC-detectable molecular processes in time-and radiation dose-dependent settings.

Keywords
three-dimensional cell culture, spheroids, irradiation, IHC, pancreatic neuroendocrine cancer, colonic adenocarcinoma
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-354258 (URN)10.3892/ol.2017.7716 (DOI)000430818300039 ()29435031 (PubMedID)
Available from: 2018-06-28 Created: 2018-06-28 Last updated: 2018-06-28Bibliographically approved
Kennedy, P. J., Sousa, F., Ferreira, D., Pereira, C., Nestor, M., Oliveira, C., . . . Sarmento, B. (2018). Fab-conjugated PLGA nanoparticles effectively target cancer cells expressing human CD44v6. Acta Biomaterialia, 81, 208-218
Open this publication in new window or tab >>Fab-conjugated PLGA nanoparticles effectively target cancer cells expressing human CD44v6
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2018 (English)In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 81, p. 208-218Article in journal (Refereed) Published
Abstract [en]

Targeting of CD44 isoforms containing exon v6 (CD44v6) represents a viable strategy for the therapy and/or early diagnosis of metastatic cancers of the epithelium (e.g. gastric and colorectal cancer). We developed and characterized poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) modified with polyethylene glycol (PEG) and engrafted, by site-directed conjugation, with an engineered human Fab that specifically target human CD44v6 (v6 Fab-PLGA NPs). The v6 Fab-PLGA NPs displayed spherical morphology around 300 nm and were negatively charged. They strongly bound to a CD44v6-derived peptide and, more importantly, to cells that endogenously and exogenously express CD44v6, but not to non expressing cells and cells expressing the standard isoform of CD44. The v6 Fab-PLGA NPs also recognized CD44v6 in tumor sections from cells grown subcutaneously within mice. The NPs had nominal cytotoxicity at 50 mu g/mL and withstood simulated intestinal fluid exposure. Interestingly, v6 Fab-PLGA NPs cryopreserved in 10% trehalose and stored maintained specific cell binding. In conclusion, we envision NPs targeting CD44v6 as potential in vivo diagnostic agents and/or as anti-cancer agents in patients previously stratified with CD44v6(+) carcinomas. Statement of Significance The v6 Fab-PLGA NPs displayed many favorable qualities as a potential CD44v6-targeted drug and/or diagnostic delivery agent. The NPs were designed for optimal ligand orientation and for immediate administration into humans. v6 Fab-PLGA NPs strongly bound to cells that endogenously and exogenously express CD44v6, but not to non-expressing cells and cells expressing the standard isoform of CD44. Binding ability was retained after freeze-drying and long-term storage, providing evidences on the stability of Fab-functionalized NPs. These NPs can potentially be used as an in vivo diagnostic from parenteral or oral/rectal administration.

Keywords
Human CD44v6, Targeted drug delivery, Antibody-conjugated nanoparticles, PLGA nanoparticles, Theranostics
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-372718 (URN)10.1016/j.actbio.2018.09.043 (DOI)000451937500016 ()30267881 (PubMedID)
Available from: 2019-01-08 Created: 2019-01-08 Last updated: 2019-01-08Bibliographically approved
Spiegelberg, D., Mortensen, A. C., Lundsten, S., Brown, C. J., Lane, D. P. & Nestor, M. (2018). First in vivo study of the MDM2/MDMX-p53 antagonist PM2 as potentiator of external radiotherapy in wt p53 cancer. Paper presented at 31st Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 13-17, 2018, Dusseldorf, GERMANY. European Journal of Nuclear Medicine and Molecular Imaging, 45(Supplement: 1), S30-S30
Open this publication in new window or tab >>First in vivo study of the MDM2/MDMX-p53 antagonist PM2 as potentiator of external radiotherapy in wt p53 cancer
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2018 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, no Supplement: 1, p. S30-S30Article in journal, Meeting abstract (Other academic) Published
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-373337 (URN)10.1007/s00259-018-4148-3 (DOI)000449266200037 ()
Conference
31st Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 13-17, 2018, Dusseldorf, GERMANY
Note

Meeting Abstract OP-074

Available from: 2019-01-14 Created: 2019-01-14 Last updated: 2019-01-14Bibliographically approved
Kennedy, P. J., Perreira, I., Ferreira, D., Nestor, M., Oliveira, C., Granja, P. L. & Sarmento, B. (2018). Impact of surfactants on the target recognition of Fab-conjugated PLGA nanoparticles. European journal of pharmaceutics and biopharmaceutics, 127, 366-370
Open this publication in new window or tab >>Impact of surfactants on the target recognition of Fab-conjugated PLGA nanoparticles
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2018 (English)In: European journal of pharmaceutics and biopharmaceutics, ISSN 0939-6411, E-ISSN 1873-3441, Vol. 127, p. 366-370Article in journal (Refereed) Published
Abstract [en]

Targeted drug delivery with nanoparticles (NPs) requires proper surface ligand presentation and availability. Surfactants are often used as stabilizers in the production of targeted NPs. Here, we evaluated the impact of surfactants on ligand functionalization and downstream molecular recognition. Our model system consisted of fluorescent poly(lactic-co-glycolic acid) (PLGA) NPs that were nanoprecipitated in one of a small panel of commonly-used surfactants followed by equivalent washes and conjugation of an engineered Fab antibody fragment. Size, polydispersity index and zeta potential were determined by dynamic light scattering and laser Doppler anemometry, and Fab presence on the NPs was assessed by enzyme-linked immunosorbent assay. Most importantly, Fab-decorated NP binding to the cell surface receptor was monitored by fluorescence-activated cell sorting. 2% polyvinyl alcohol, 1% sodium cholate, 0.5% Pluronic F127 (F127) and 2% Tween-80 were initially tested. Of the four surfactants tested, PLGA NPs in 0.5% F127 and 2% Tween-80 had the highest cell binding. These two surfactants were then retested in two different concentrations, 0.5% and 2%. The Fab-decorated PLGA NPs in 2% F127 had the highest cell binding. This study highlights the impact of common surfactants and their concentrations on the downstream targeting of ligand-decorated NPs. Similar principles should be applied in the development of future targeted nanosystems where surfactants are employed.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
Targeted nanoparticles, PLGA nanoparticles, Surfactant, Fab antibody fragment
National Category
Physical Chemistry
Identifiers
urn:nbn:se:uu:diva-357382 (URN)10.1016/j.ejpb.2018.03.005 (DOI)000433650400039 ()29549023 (PubMedID)
Funder
EU, Horizon 2020, NORTE-01-0145-FEDER-000012EU, Horizon 2020
Available from: 2018-08-24 Created: 2018-08-24 Last updated: 2018-08-31Bibliographically approved
Elmsjö, A., Haglöf, J., Engskog, M. K., Erngren, I., Nestor, M., Arvidsson, T. & Pettersson, C. (2018). Method selectivity evaluation using the co-feature ratio in LC/MS metabolomics: Comparison of HILIC stationary phase performance for the analysis of plasma, urine and cell extracts.. Journal of Chromatography A, 1568, 49-56
Open this publication in new window or tab >>Method selectivity evaluation using the co-feature ratio in LC/MS metabolomics: Comparison of HILIC stationary phase performance for the analysis of plasma, urine and cell extracts.
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2018 (English)In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1568, p. 49-56Article in journal (Refereed) Published
Abstract [en]

Evaluation of the chromatographic separation in metabolomics studies has primarily been done using preselected sets of standards or by counting the number of detected features. An alternative approach is to calculate each feature's co-feature ratio, which is a combined selectivity measurement for the separation (i.e. extent of co-elution) and the MS-signal (i.e. adduct formation and in-source fragmentation). The aim of this study was to demonstrate how the selectivity of different HILIC stationary phases can be evaluated using the co-feature ratio approach. The study was based on three sample types; plasma, urine and cell extracts. Samples were analyzed on an UHPLC-ESI-Q-ToF system using an amide, a bare silica and a sulfobetaine stationary phase. For each feature, a co-feature ratio was calculated and used for multivariate analysis of the selectivity differences between the three stationary phases. Unsupervised PCA models indicated that the co-feature ratios were highly dependent on type of stationary phase. For several metabolites a 15-30 fold difference in the co-feature ratio were observed between the stationary phases. Observed selectivity differences related primarily to the retention patterns of unwanted matrix components such as inorganic salts (detected as salt clusters), glycerophospholipids, and polyethylene glycols. These matrix components affected the signal intensity of co-eluting metabolites by interfering with the ionization efficiency and/or their adduct formation. Furthermore, the retention pattern of these matrix components had huge influence on the number of detected features. The co-feature ratio approach has successfully been applied for evaluation of the selectivity performance of three HILIC stationary phases. The co-feature ratio could therefore be used in metabolomics for developing selective methods fit for their purpose, thereby avoiding generic analytical approaches, which are often biased, as type and amount of interfering matrix components are metabolome dependent.

Keywords
Co-feature ratio (CFR), Hydrophilic interaction chromatography, Mass spectrometry, Metabolomics, Salt clusters
National Category
Analytical Chemistry Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-364208 (URN)10.1016/j.chroma.2018.05.007 (DOI)000443669600006 ()29789170 (PubMedID)
Available from: 2018-10-24 Created: 2018-10-24 Last updated: 2018-10-29Bibliographically approved
Lundsten, S., Spiegelberg, D., Raval, N., Stenerlöw, B. & Nestor, M. (2018). Potentiating Lu-177-DOTATATE Therapy By HSP90 Inhibition - First In Vivo Study. Paper presented at 31st Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 13-17, 2018, Dusseldorf, GERMANY. European Journal of Nuclear Medicine and Molecular Imaging, 45, S12-S13
Open this publication in new window or tab >>Potentiating Lu-177-DOTATATE Therapy By HSP90 Inhibition - First In Vivo Study
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2018 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, p. S12-S13Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Springer, 2018
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-372968 (URN)000449266200003 ()
Conference
31st Annual Congress of the European-Association-of-Nuclear-Medicine (EANM), OCT 13-17, 2018, Dusseldorf, GERMANY
Available from: 2019-01-11 Created: 2019-01-11 Last updated: 2019-01-11Bibliographically approved
Mortensen, A., Spiegelberg, D., Haylock, A.-K., Lundqvist, H. & Nestor, M. (2018). Preclinical evaluation of a novel engineered recombinant human anti-CD44v6 antibody for potential use in radio-immunotherapy. International Journal of Oncology, 52(6), 1875-1885
Open this publication in new window or tab >>Preclinical evaluation of a novel engineered recombinant human anti-CD44v6 antibody for potential use in radio-immunotherapy
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2018 (English)In: International Journal of Oncology, ISSN 1019-6439, Vol. 52, no 6, p. 1875-1885Article in journal (Refereed) Published
Abstract [en]

CD44v6 is overexpressed in a variety of cancers, rendering it a promising target for radio-immunotherapy (RIT). In this study, we have characterized a novel engineered recombinant monoclonal anti-CD44v6 antibody, AbN44v6, and assessed its potential for use in RIT using either Lu-177 or I-131 as therapeutic radionuclides. In vitro affinity and specificity assays characterized the binding of the antibody labeled with Lu-177, I-125 or I-131. The therapeutic effects of Lu-177-AbN44v6 and I-131-AbN44v6 were investigated using two in vitro 3D tumor models with different CD44v6 expression. Finally, the normal tissue biodistribution and dosimetry for Lu-177-AbN44v6 and I-125-AbN44v6/I-131-AbN44v6 were assessed in vivo using a mouse model. All AbN44v6 radioconjugates demonstrated CD44v6-specific binding in vitro. In the in vitro 3D tumor models, dose-dependent therapeutic effects were observed with both Lu-177-AbN44v6 and I-131-AbN44v6, with a greater significant therapeutic effect observed on the cells with a higher CD44v6 expression. Biodistribution experiments demonstrated a greater uptake of Lu-177-AbN44v6 in the liver, spleen and bone, compared to I-125-AbN44v6, whereas I-125-AbN44v6 demonstrated a longer circulation time. In dosimetric calculations, the critical organs for Lu-177-AbN44v6 were the liver and spleen, whereas the kidneys and red marrow were considered the critical organs for I-131-AbN44v6. The effective dose was in the order of 0.1 mSv/MBq for both labels. In conclusion, AbN44v6 bound specifically and with high affinity to CD44v6. Furthermore, in vitro RIT demonstrated growth inhibition in a CD44v6-specific activity-dependent manner for both radioconjugates, demonstrating that both Lu-177-AbN44v6 and I-131-AbN44v6 may be promising RIT candidates. Furthermore, biodistribution and dosimetric analysis supported the applicability of both conjugates for RIT. The CD44v6-specific therapeutic effects observed with radiolabeled AbN44v6 in the 3D tumor models in vitro, combined with the beneficial dosimetry in vivo, render AbN44v6 a potential candidate for RIT.

Place, publisher, year, edition, pages
SPANDIDOS PUBL LTD, 2018
Keywords
radio-immunotherapy, 3D tumor models, dosimetry, biodistribution, Lu-177, I-131
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-356443 (URN)10.3892/ijo.2018.4364 (DOI)000432241200010 ()29658563 (PubMedID)
Funder
Swedish Research CouncilSwedish Cancer Society
Available from: 2018-07-31 Created: 2018-07-31 Last updated: 2018-07-31Bibliographically approved
Spiegelberg, D., Mortensen, A., Lundsten, S., Brown, C. J., Lane, D. P. & Nestor, M. (2018). The MDM2/MDMX-p53 Antagonist PM2 Radiosensitizes Wild-Type p53 Tumors. Cancer Research, 78(17), 5084-5093
Open this publication in new window or tab >>The MDM2/MDMX-p53 Antagonist PM2 Radiosensitizes Wild-Type p53 Tumors
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2018 (English)In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 78, no 17, p. 5084-5093Article in journal (Refereed) Published
Abstract [en]

Radiotherapy amplifies p53 expression in cancer cells with wild-type (wt) p53. Blocking the negative regulators MDM2 and MDMX stabilizes p53 and may therefore potentiate radiotherapy outcomes. In this study, we investigate the efficacy of the novel anti-MDM2/X stapled peptide PM2 alone and in combination with externalgamma radiation in vitro and in vivo. PM2 therapy combined with radiotherapy elicited synergistic therapeutic effects compared with monotherapy in cells with wt p53 in both in vitro and in vivo assays, whereas these effects did not manifest in p53(-/-) cells. Biodistribution and autoradiography of 125I-PM2 revealed high and retained uptake homogenously distributed throughout the tumor. In mice carrying wt p53 tumors, PM2 combined with radiother-apy significantly prolonged the median survival by 50%, whereas effects of PM2 therapy on mutant and p53(-/-) tumors were negligible. PM2-dependent stabilization of p53 was confirmed with ex vivo immunohistochemistry. These data demonstrate the potential of the stapled peptide PM2 as a radiotherapy potentiator in vivo and suggest that clinical application of PM2 with radiotherapy in wt p53 cancers might improve tumor control.

Significance: These findings contribute advances to cancer radiotherapy by using novel p53-reactivating stapled peptides as radiosensitizers in wild-type p53 cancers.

Place, publisher, year, edition, pages
AMER ASSOC CANCER RESEARCH, 2018
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-365821 (URN)10.1158/0008-5472.CAN-18-0440 (DOI)000443753700024 ()30026328 (PubMedID)
Funder
Swedish Research Council, 2013-30876-104113-30Swedish Research Council, 2013-8807Swedish Cancer Society, CAN 2015/1080Swedish Cancer Society, CAN 2015/385
Available from: 2018-11-26 Created: 2018-11-26 Last updated: 2018-11-26Bibliographically approved
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