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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
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
Mortensen, A. (2018). Precision medicine and targeted therapy: Turning the tables on cancer. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Precision medicine and targeted therapy: Turning the tables on cancer
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

An extended understanding of the molecular characteristics of cancer has led to a revolution within the field of precision medicine. This thesis explores the utilization of two targets for precision medicine, namely, CD44v6 and murine double-minute 2 and X (MDM2/X).

A novel mini-antibody construct targeting CD44v6 (AbD19384), was assessed for possible use in radiodiagnostics, while a recombinant full-length anti-CD44v6 antibody based on the same construct, AbN44v6, was evaluated for radio-immunotherapy (RIT) following labeling with 177Lu and 131I. Additionally, normal tissue biodistribution and dosimetry was assessed for radiolabeled AbN44v6. The efficacy and mechanisms behind the observed effects of PM2 therapy, a novel stapled peptide that inhibits MDM2/X, were assessed in vitro and in vivo both as monotherapy and in combination with external beam radiotherapy (EBRT). Lastly, combination therapy using RIT (177Lu-AbN44v6) and PM2 was evaluated in an in vitro 3D tumor spheroid model.

AbD19384 successfully visualized CD44v6-positive xenografts. Similarly, radiolabeled AbN44v6 bound specifically to CD44v6, and RIT resulted in antigen-dependent, activity-dependent growth inhibition of in vitro 3D tumor spheroids. Biodistribution and dosimetry revealed low-level accumulation in normal tissues and low total effective doses of 0.1 mSv/MBq of injected radioconjugate. PM2-based therapy increased pro-apoptotic protein levels and caused growth inhibition of wt p53 cancer cell lines, which was amplified in combination with radiotherapy. In vivo studies of wt p53 and p53-knockout xenografts demonstrated the specificity of PM2 towards wt p53 cancers and established the potency of combining PM2-based therapy with EBRT.

The work presented in this thesis exemplifies the potency of combination treatments based on a precise understanding of the targeted cancer. Utilizing not one but several of the molecular characteristics of a specific cancer will help turn the tables on cancer and improve patient outcomes in the future.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 56
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1430
Keywords
p53 cancers, PM2, radiosensitization, radio-immunotherapy, MDM2/X inhibition, combination therapy, targeted radionuclide therapy, CD44v6
National Category
Medical and Health Sciences
Research subject
Biomedical Radiation Science
Identifiers
urn:nbn:se:uu:diva-342030 (URN)978-91-513-0239-3 (ISBN)
Public defence
2018-04-06, Rudbecksalen, Dag Hammarskjölds Väg 20, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2018-03-13 Created: 2018-02-18 Last updated: 2018-04-24
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
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.

Keywords
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.

Keywords
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
Spiegelberg, D., Lundsten, S., Mortensen, A. C., Abramenkovs, A., Stenerlöw, B. & Nestor, M. (2017). In Vitro and In Vivo Growth Inhibitory and Radiosensitizing Effects of the Anti-HSP90 agent Onalespib. European Journal of Nuclear Medicine and Molecular Imaging, 44, S182-S182
Open this publication in new window or tab >>In Vitro and In Vivo Growth Inhibitory and Radiosensitizing Effects of the Anti-HSP90 agent Onalespib
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2017 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 44, p. S182-S182Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Springer, 2017
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-377089 (URN)000455019400106 ()
Available from: 2019-02-19 Created: 2019-02-19 Last updated: 2019-02-19Bibliographically approved
Mortensen, A. C., Spiegelberg, D., Lundsten, S., Brown, C., Lane, D. P. & Nestor, M. (2017). In Vivo Assessment of p53 Therapy as a Way of Enhancing Therapeutic Effects of Radiation. European Journal of Nuclear Medicine and Molecular Imaging, 44, S181-S181
Open this publication in new window or tab >>In Vivo Assessment of p53 Therapy as a Way of Enhancing Therapeutic Effects of Radiation
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2017 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 44, p. S181-S181Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Springer, 2017
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-377082 (URN)000455019400104 ()
Available from: 2019-02-20 Created: 2019-02-20 Last updated: 2019-02-20Bibliographically approved
Lundsten, S., Mortensen, A., Makiniemi, A., Spiegelberg, D., Stenerlöw, B. & Nestor, M. (2017). The HSP90-inhibitor Onalespib Potentiates Lu-177-Dotatate Treatment of Neuroendocrine Tumors. European Journal of Nuclear Medicine and Molecular Imaging, 44, S326-S326
Open this publication in new window or tab >>The HSP90-inhibitor Onalespib Potentiates Lu-177-Dotatate Treatment of Neuroendocrine Tumors
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2017 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 44, p. S326-S326Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Springer, 2017
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-377078 (URN)000455019400402 ()
Available from: 2019-02-20 Created: 2019-02-20 Last updated: 2019-02-20Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-6771-3289

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