Anja C. Mortensen

The novel HSP90 inhibitor AT13387 potentiates radiation effects in squamous cell carcinoma and adenocarcinoma cells.

Overexpression of heat shock protein 90 (HSP90) is associated with increased tumor cell survival and radioresistance. In this study we explored the efficacy of the novel HSP90 inhibitor AT13387 and examined its radiosensitizing effects in combination with gamma-radiation in 2D and 3D structures as well as mice-xenografts. AT13387 induced effective cytotoxic activity and radiosensitized cancer cells in monolayer and tumor spheroid models, where low drug doses triggered significant synergistic effects on cell survival together with radiation. Furthermore, AT13387 treatment resulted in G2/M-phase arrest and significantly reduced the migration capacity. The expression of selected client proteins involved in DNA repair, cell-signaling and cell growth was downregulated in vitro, though the expression of most investigated proteins recurred after 8–24 h. These results were confirmed in vivo where AT13387 treated tumors displayed effective downregulation of HSP90 and its oncogenic client proteins. In conclusion, our results demonstrate that AT13387 is a potent new cancer drug and effective radiosensitizer in vitro with an excellent in vivo efficacy. AT13387 treatment has the potential to improve external beam therapy and radionuclide therapy outcomes and restore treatment efficacy in cancers that are resistant to initial therapeutic regimes.

Different functions of AKT1 and AKT2 in molecular pathways, cell migration and metabolism in colon cancer cells

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 AKT1/2 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.

Evaluation of a novel type of imaging probe based on a recombinant bivalent mini-antibody construct for detection of CD44v6-expressing squamous cell carcinoma

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 biodis-tribution 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.

Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers

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.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.

Preclinical evaluation of a novel engineered recombinant human anti-CD44v6 antibody for potential use in radio-immunotherapy

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 177Lu or 131I as therapeutic radionuclides. In vitro affinity and specificity assays characterized the binding of the antibody labeled with 177Lu, 125I or 131I. The therapeutic effects of 177Lu-AbN44v6 and 131I-AbN44v6 were investigated using two in vitro 3D tumor models with different CD44v6 expression. Finally, the normal tissue biodistribution and dosimetry for 177Lu-AbN44v6 and 125I-AbN44v6/131I-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 177Lu-AbN44v6 and 131I-AbN44v6, with a greater significant therapeutic effect observed on the cells with a higher CD44v6 expression. Biodistribution experiments demonstrated a greater uptake of 177Lu-AbN44v6 in the liver, spleen and bone, compared to 125I-AbN44v6, whereas 125I-AbN44v6 demonstrated a longer circulation time. In dosimetric calculations, the critical organs for 177Lu-AbN44v6 were the liver and spleen, whereas the kidneys and red marrow were considered the critical organs for 131I-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 177Lu-AbN44v6 and 131I-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.

The MDM2/MDMX-p53 Antagonist PM2 Radiosensitizes Wild-Type p53 Tumors

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 external gamma 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 radiotherapy 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. Cancer Res; 78(17); 5084-93. ©2018 AACR.

Analysis of radiation effects in two irradiated tumor spheroid models

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, G2-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.