Meningioma animal models: a systematic review and meta-analysis.

BACKGROUND: Animal models are widely used to study pathological processes and drug (side) effects in a controlled environment. There is a wide variety of methods available for establishing animal models depending on the research question. Commonly used methods in tumor research include xenografting cells (established/commercially available or primary patient-derived) or whole tumor pieces either orthotopically or heterotopically and the more recent genetically engineered models-each type with their own advantages and disadvantages. The current systematic review aimed to investigate the meningioma model types used, perform a meta-analysis on tumor take rate (TTR), and perform critical appraisal of the included studies. The study also aimed to assess reproducibility, reliability, means of validation and verification of models, alongside pros and cons and uses of the model types. METHODS: We searched Medline, Embase, and Web of Science for all in vivo meningioma models. The primary outcome was tumor take rate. Meta-analysis was performed on tumor take rate followed by subgroup analyses on the number of cells and duration of incubation. The validity of the tumor models was assessed qualitatively. We performed critical appraisal of the methodological quality and quality of reporting for all included studies. RESULTS: We included 114 unique records (78 using established cell line models (ECLM), 21 using primary patient-derived tumor models (PTM), 10 using genetically engineered models (GEM), and 11 using uncategorized models). TTRs for ECLM were 94% (95% CI 92-96) for orthotopic and 95% (93-96) for heterotopic. PTM showed lower TTRs [orthotopic 53% (33-72) and heterotopic 82% (73-89)] and finally GEM revealed a TTR of 34% (26-43). CONCLUSION: This systematic review shows high consistent TTRs in established cell line models and varying TTRs in primary patient-derived models and genetically engineered models. However, we identified several issues regarding the quality of reporting and the methodological approach that reduce the validity, transparency, and reproducibility of studies and suggest a high risk of publication bias. Finally, each tumor model type has specific roles in research based on their advantages (and disadvantages). SYSTEMATIC REVIEW REGISTRATION: PROSPERO-ID CRD42022308833.

Cisplatin-Resistant CD44+ Lung Cancer Cells Are Sensitive to Auger Electrons.

Cancer stem cells (CSCs) are resistant to conventional therapy and present a major clinical challenge since they are responsible for the relapse of many cancers, including non-small cell lung cancer (NSCLC). Hence, future successful therapy should also eradicate CSCs. Auger electrons have demonstrated promising therapeutic potential and can induce DNA damage while sparing surrounding cells. Here, we sort primary patient-derived NSCLC cells based on their expression of the CSC-marker CD44 and investigate the effects of cisplatin and a thymidine analog (deoxyuridine) labeled with an Auger electron emitter (125I). We show that the CD44+ populations are more resistant to cisplatin than the CD44- populations. Interestingly, incubation with the thymidine analog 5-[125I]iodo-2'-deoxyuridine ([125I]I-UdR) induces equal DNA damage, G2/M cell cycle arrest, and apoptosis in the CD44- and CD44+ populations. Our results suggest that Auger electron emitters can also eradicate resistant lung cancer CD44+ populations.

Preparation and Evaluation of [18F]AlF-NOTA-NOC for PET Imaging of Neuroendocrine Tumors: Comparison to [68Ga]Ga-DOTA/NOTA-NOC.

BACKGROUND: The somatostatin receptors 1-5 are overexpressed on neuroendocrine neoplasms and, as such, represent a favorable target for molecular imaging. This study investigates the potential of [18F]AlF-NOTA-[1-Nal3]-Octreotide and compares it in vivo to DOTA- and NOTA-[1-Nal3]-Octreotide radiolabeled with gallium-68. METHODS: DOTA- and NOTA-NOC were radiolabeled with gallium-68 and NOTA-NOC with [18F]AlF. Biodistributions of the three radioligands were evaluated in AR42J xenografted mice at 1 h p.i and for [18F]AlF at 3 h p.i. Preclinical PET/CT was applied to confirm the general uptake pattern. RESULTS: Gallium-68 was incorporated into DOTA- and NOTA-NOC in yields and radiochemical purities greater than 96.5%. NOTA-NOC was radiolabeled with [18F]AlF in yields of 38 ± 8% and radiochemical purity above 99% after purification. The biodistribution in tumor-bearing mice showed a high uptake in tumors of 26.4 ± 10.8 %ID/g for [68Ga]Ga-DOTA-NOC and 25.7 ± 5.8 %ID/g for [68Ga]Ga-NOTA-NOC. Additionally, [18F]AlF-NOTA-NOC exhibited a tumor uptake of 37.3 ± 10.5 %ID/g for [18F]AlF-NOTA-NOC, which further increased to 42.1 ± 5.3 %ID/g at 3 h p.i. CONCLUSIONS: The high tumor uptake of all radioligands was observed. However, [18F]AlF-NOTA-NOC surpassed the other clinically well-established radiotracers in vivo, especially at 3 h p.i. The tumor-to-blood and -liver ratios increased significantly over three hours for [18F]AlF-NOTA-NOC, making it possible to detect liver metastases. Therefore, [18F]AlF demonstrates promise as a surrogate pseudo-radiometal to gallium-68.

Preclinical Evaluation of the Copper-64 Labeled GRPR-Antagonist RM26 in Comparison with the Cobalt-55 Labeled Counterpart for PET-Imaging of Prostate Cancer.

Gastrin-releasing peptide receptor (GRPR) is overexpressed in the majority of prostate cancers. This study aimed to investigate the potential of 64Cu (radionuclide for late time-point PET-imaging) for imaging of GRPR expression using NOTA-PEG2-RM26 and NODAGA-PEG2-RM26. Methods: NOTA/NODAGA-PEG2-RM26 were labeled with 64Cu and evaluated in GRPR-expressing PC-3 cells. Biodistribution of [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was studied in PC-3 xenografted mice and compared to the biodistribution of [57Co]Co-NOTA/NODAGA-PEG2-RM26 at 3 and 24 h p.i. Preclinical PET/CT imaging was performed in tumor-bearing mice. NOTA/NODAGA-PEG2-RM26 were stably labeled with 64Cu with quantitative yields. In vitro, binding of [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was rapid and GRPR-specific with slow internalization. In vivo, [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 bound specifically to GRPR-expressing tumors with fast clearance from blood and normal organs and displayed generally comparable biodistribution profiles to [57Co]Co-NOTA/NODAGA-PEG2-RM26; tumor uptake exceeded normal tissue uptake 3 h p.i.. Tumor-to-organ ratios did not increase significantly with time. [64Cu]Cu-NOTA-PEG2-RM26 had a significantly higher liver and pancreas uptake compared to other agents. 57Co-labeled radioconjugates showed overall higher tumor-to-non-tumor ratios, compared to the 64Cu-labeled counterparts. [64Cu]Cu-NOTA/NODAGA-PEG2-RM26 was able to visualize GRPR-expression in a murine PC model using PET. However, [55/57Co]Co-NOTA/NODAGA-PEG2-RM26 provided better in vivo stability and overall higher tumor-to-non-tumor ratios compared with the 64Cu-labeled conjugates.

Dual time-point FDG PET/CT and FDG uptake and related enzymes in lymphadenopathies: preliminary results.

PURPOSE: The purpose of this study was to determine the ability of dual time-point (DTP) PET/CT with (18)F-FDG to discriminate between malignant and benign lymphadenopathies. The relationship between DTP FDG uptake and glucose metabolism/hypoxia markers in lymphadenopathies was also assessed. METHODS: Patients with suspected lymphoma or recently diagnosed treatment-naive lymphoma were prospectively enrolled for DTP FDG PET/CT (scans 60 min and 180 min after FDG administration). FDG-avid nodal lesions were segmented to yield volume and standardized uptake values (SUV), including SUVmax, SUVmean, cSUVmean (with partial volume correction), total lesion glycolysis (TLG) and cTLG (with partial volume correction). Expression of glucose transporter-1 (GLUT-1), hexokinase-II (HK-II), glucose-6-phosphatase (G6Pase) and hypoxia-inducible factor-1alpha (HIF-1alpha) were assessed with immunohistochemistry and enzyme activity was determined for HK and G6Pase. RESULTS: FDG uptake was assessed in 203 lesions (146 malignant and 57 benign). Besides volume, there were significant increases over time for all parameters, with generally higher levels in the malignant lesions. The retention index (RI) was not able to discriminate between malignant and benign lesions. Volume, SUVmax, TLG and cTLG for both scans were able to discriminate between the two groups statistically, but without complete separation. Glucose metabolism/hypoxia markers were assessed in 15 lesions. TLG and cTLG were correlated with GLUT-1 expression on the 60-min scan. RI-max and RI-mean and SUVmax, SUVmean and cSUVmean on the 60-min scan were significantly correlated with HK-II expression. CONCLUSION: RI was not able to discriminate between malignant and benign lesions, but some of the SUVs were able to discriminate on the 60-min and 180-min scans. Furthermore, FDG uptake was correlated with GLUT-1 and HK-II expression.

Preclinical evaluation of [58mCo]Co-DOTA-PSMA-617 for Auger electron therapy of prostate cancer.

Prostate-specific membrane antigen (PSMA), highly expressed in prostate cancer, is a promising target for radionuclide therapy. Auger electron-emitting radionuclides are well suited for targeted radionuclide therapy if they can be delivered close to the DNA of the targeted cells. This preclinical study evaluated the theranostic pair [55/58mCo]Co-DOTA-PSMA-617 for PET imaging and Auger electron therapy of prostate cancer. [58mCo]Co-DOTA-PSMA-617 was successfully prepared with > 99% radiochemical yield and purity. In vitro, uptake and subcellular distribution assays in PSMA-positive prostate cancer cells showed PSMA-specific uptake with high cell-associated activity in the nucleus. Incubation with [58mCo]Co-DOTA-PSMA-617 reduced cell viability and clonogenic survival in a significant dose-dependent manner (p < 0.05). Biodistribution of xenografted mice showed high specific tumor uptake of the cobalt-labeled PSMA ligand for all time points with rapid clearance from normal tissues, which PET imaging confirmed. In vivo, therapy with [58mCo]Co-DOTA-PSMA-617 in tumor-bearing mice demonstrated significantly increased median survival for treated mice compared to control animals (p = 0.0014). In conclusion, [55/58mCo]Co-DOTA-PSMA-617 displayed excellent in vitro and in vivo properties, offering significant survival benefits in mice with no observed toxicities.

Protein kinase CK2 localizes to sites of DNA double-strand break regulating the cellular response to DNA damage.

BACKGROUND: The DNA-dependent protein kinase (DNA-PK) is a nuclear complex composed of a large catalytic subunit (DNA-PKcs) and a heterodimeric DNA-targeting subunit Ku. DNA-PK is a major component of the non-homologous end-joining (NHEJ) repair mechanism, which is activated in the presence of DNA double-strand breaks induced by ionizing radiation, reactive oxygen species and radiomimetic drugs. We have recently reported that down-regulation of protein kinase CK2 by siRNA interference results in enhanced cell death specifically in DNA-PKcs-proficient human glioblastoma cells, and this event is accompanied by decreased autophosphorylation of DNA-PKcs at S2056 and delayed repair of DNA double-strand breaks. RESULTS: In the present study, we show that CK2 co-localizes with phosphorylated histone H2AX to sites of DNA damage and while CK2 gene knockdown is associated with delayed DNA damage repair, its overexpression accelerates this process. We report for the first time evidence that lack of CK2 destabilizes the interaction of DNA-PKcs with DNA and with Ku80 at sites of genetic lesions. Furthermore, we show that CK2 regulates the phosphorylation levels of DNA-PKcs only in response to direct induction of DNA double-strand breaks. CONCLUSIONS: Taken together, these results strongly indicate that CK2 plays a prominent role in NHEJ by facilitating and/or stabilizing the binding of DNA-PKcs and, possibly other repair proteins, to the DNA ends contributing to efficient DNA damage repair in mammalian cells.

Establishment of patient­derived lung tumorspheres and their response to internal irradiation by Auger electrons.

The high recurrence rate of lung cancer is a major clinical challenge associated with therapy­resistant cancer stem cells (CSCs), which are rare subpopulations. Future successful treatment is required to also eradicate these subpopulations. Furthermore, the majority of anti­cancer treatments are being tested in adherent monolayer cultures with the limitations this entails in the translation of results into clinical practice. The present study aimed to establish and characterize patient­derived long­term primary lung cancer tumorspheres enriched in CSCs and evaluate the effects of Auger electrons on them. These electrons are emitted from radionuclides that decay by electron capture or internal conversion and have demonstrated promising therapeutic potential. Their low energy (<1 keV) is sufficiently potent to induce DNA double­strand breaks and eventually cell death while minimizing irradiation of non­targeted surrounding cells. Labeling a thymidine analog (deoxyuridine) with the Auger electron­emitting radionuclide [125I], which is exclusively incorporated into the DNA of proliferating cells during the S­phase, ensures a close distance to the DNA. Primary cell cultures grown as tumorspheres were established and characterized. The tumorspheres were morphologically distinct and differed concerning their proliferation rate and fraction of CSCs. Surface markers associated with CSCs were upregulated and 5­[125I]iodo­2'­deoxyuridine was incorporated in the tumorspheres. The Auger electrons induced DNA double­strand breaks, G2/M arrest and apoptosis in the tumorspheres; however, the tumorspheres derived from different patients exhibited heterogeneities in their sensitivity to Auger electron irradiation.

Surgical resection of glioblastomas induces pleiotrophin-mediated self-renewal of glioblastoma stem cells in recurrent tumors.

BACKGROUND: Glioblastomas are highly resistant to therapy, and virtually all patients experience tumor recurrence after standard-of-care treatment. Surgical tumor resection is a cornerstone in glioblastoma therapy, but its impact on cellular phenotypes in the local postsurgical microenvironment has yet to be fully elucidated. METHODS: We developed a preclinical orthotopic xenograft tumor resection model in rats with integrated 18F-FET PET/CT imaging. Primary and recurrent tumors were subject to bulk and single-cell RNA sequencing. Differentially expressed genes and pathways were investigated and validated using tissue specimens from the xenograft model, 23 patients with matched primary/recurrent tumors, and a cohort including 190 glioblastoma patients. Functional investigations were performed in vitro with multiple patient-derived cell cultures. RESULTS: Tumor resection induced microglia/macrophage infiltration, angiogenesis as well as proliferation and upregulation of several stem cell-related genes in recurrent tumor cells. Expression changes of selected genes SOX2, POU3F2, OLIG2, and NOTCH1 were validated at the protein level in xenografts and early recurrent patient tumors. Single-cell transcriptomics revealed the presence of distinct phenotypic cell clusters in recurrent tumors which deviated from clusters found in primary tumors. Recurrent tumors expressed elevated levels of pleiotrophin (PTN), secreted by both tumor cells and tumor-associated microglia/macrophages. Mechanistically, PTN could induce tumor cell proliferation, self-renewal, and the stem cell program. In glioblastoma patients, high PTN expression was associated with poor overall survival and identified as an independent prognostic factor. CONCLUSION: Surgical tumor resection is an iatrogenic driver of PTN-mediated self-renewal in glioblastoma tumor cells that promotes therapeutic resistance and tumor recurrence.

Interaction between CK2α and CK2ß, the subunits of protein kinase CK2: thermodynamic contributions of key residues on the CK2α surface.

The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) bound to a dimer of noncatalytic subunits (CK2ß). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2α and CK2ß as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2α interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2ß. Additionally, these CK2α mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2α, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2ß-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2ß interactions is important for the future generation of small molecule drug design.

Enzymatic activity with an incomplete catalytic spine: insights from a comparative structural analysis of human CK2α and its paralogous isoform CK2α'.

Eukaryotic protein kinases are fundamental factors for cellular regulation and therefore subject of strict control mechanisms. For full activity a kinase molecule must be penetrated by two stacks of hydrophobic residues, the regulatory and the catalytic spine that are normally well conserved among active protein kinases. We apply this novel spine concept here on CK2α, the catalytic subunit of protein kinase CK2. Homo sapiens disposes of two paralog isoforms of CK2α (hsCK2α and hsCK2α'). We describe two new structures of hsCK2α constructs one of which in complex with the ATP-analog adenylyl imidodiphosphate and the other with the ATP-competitive inhibitor 3-(4,5,6,7-tetrabromo-1H-benzotriazol-1-yl)propan-1-ol. The former is the first hsCK2α structure with a well defined cosubstrate/magnesium complex and the second with an open ß4/ß5-loop. Comparisons of these structures with existing CK2α/CK2α' and cAMP-dependent protein kinase (PKA) structures reveal: in hsCK2α' an open conformation of the interdomain hinge/helix αD region that is critical for ATP-binding is found corresponding to an incomplete catalytic spine. In contrast hsCK2α often adopts the canonical, PKA-like version of the catalytic spine which correlates with a closed conformation of the hinge region. HsCK2α can switch to the incomplete, non-canonical, hsCK2α'-like state of the catalytic spine, but this transition apparently depends on binding of either ATP or of the regulatory subunit CK2ß. Thus, ATP looks like an activator of hsCK2α rather than a pure cosubstrate.

Role of polyamines in determining the cellular response to chemotherapeutic agents: modulation of protein kinase CK2 expression and activity.

Numerous studies have shown that platinum compounds stimulate the expression of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase resulting in anti-proliferative activity and apoptosis. As many cancer cell types including pancreatic cancer cells express high levels of polyamines, the possibility to develop anti-tumor strategies to deplete polyamine pools has drawn considerable attention in recent years. This has been effectively accomplished by treating cells with platinum drugs in combination with polyamine analogs such as N(1),N(11)-diethylnorspermine (DENSPM). The present study, examined the cytotoxic effects of oxaliplatin in combination with stimulators of polyamine catabolism in human pancreatic cancer cells, that are notoriously resistant to chemotherapeutic treatment, and colorectal cancer cells. Additionally, as protein kinase CK2 has been shown to be an anti-apoptotic and pro-survival enzyme regulated by the intracellular polyamine pools, we aimed to investigate the effect of combined DENSPM and oxaliplatin treatment on CK2-mRNA and -protein levels. Results reported here show that treatment with oxaliplatin and DENSPM in combination impairs cell viability particularly in the case of colorectal cancer cells. The analysis of CK2 expression and activity indicates that the response to a specific treatment may depend on the impact that individual compounds exert on pro-survival and pro-death proteins at the transcription and translation levels that should be carefully evaluated in view of subsequent clinical studies.

Lack of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is accompanied by increased CK2α' levels.

DNA-PKcs is the catalytic subunit of DNA-dependent protein kinase, an enzyme necessary for non-homologous end-joining (NHEJ) and hence repair of DNA double strand breaks. Characterization of two isogenic cell lines, M059K and M059J, which are DNA-PKcs-proficient and -deficient, respectively, revealed that lack of DNA-PKcs is accompanied by an increase in the protein level of one of the catalytic isozymes of protein kinase CK2, i.e., CK2α' and a concomitant increase in CK2 activity. The increase was also detectable at the mRNA level as measured by quantitative real time PCR. However, no increase at the DNA level was observed either by comparative PCR or fluorescent in situ hybridization indicating that gene amplification is not involved. Interestingly, only CK2α' was increased and not the other two subunits of CK2, i.e., CK2ß or CK2α. In addition, the increase in CK2α' protein level was also observed in a DNA-PKcs-deficient mouse cell line.

Cannabidiol Induces Cell Death in Human Lung Cancer Cells and Cancer Stem Cells.

Currently, there is no effective therapy against lung cancer due to the development of resistance. Resistance contributes to disease progression, recurrence, and mortality. The presence of so-called cancer stem cells could explain the ineffectiveness of conventional treatment, and the development of successful cancer treatment depends on the targeting also of cancer stem cells. Cannabidiol (CBD) is a cannabinoid with anti-tumor properties. However, the effects on cancer stem cells are not well understood. The effects of CBD were evaluated in spheres enriched in lung cancer stem cells and adherent lung cancer cells. We found that CBD decreased viability and induced cell death in both cell populations. Furthermore, we found that CBD activated the effector caspases 3/7, increased the expression of pro-apoptotic proteins, increased the levels of reactive oxygen species, as well as a leading to a loss of mitochondrial membrane potential in both populations. We also found that CBD decreased self-renewal, a hallmark of cancer stem cells. Overall, our results suggest that CBD is effective against the otherwise treatment-resistant cancer stem cells and joins a growing list of compounds effective against cancer stem cells. The effects and mechanisms of CBD in cancer stem cells should be further explored to find their Achilles heel.

Auger electron therapy of glioblastoma using [125I]5-iodo-2'-deoxyuridine and concomitant chemotherapy - Evaluation of a potential treatment strategy.

INTRODUCTION: Treatment of glioblastomas (GBM) using the Auger electron emitting compound [125I]5-Iodo-2'-deoxyuridine ([125I]I-UdR), combined with the thymidylate synthase inhibitor methotrexate (MTX) and concomitant chemotherapy with temozolomide (TMZ) has recently shown very promising therapeutic effects in vitro and in vivo in animals. The aim of the current study was to investigate if the therapeutic effects of this multimodal treatment strategy could be further increased by the thymidylate synthase inhibitor, 5-fluoro-2'-deoxyuridine (F-UdR), in comparison to MTX, and if the co-treatment should be given in a neoadjuvant or adjuvant setting. METHODS: A patient-derived GBM cancer stem cell (CSC)-enriched cell line, grown as neurospheres, was employed to evaluate DNA-incorporation of [125I]I-UdR, determined by a DNA precipitation assay, using either pre-treatment or co-treatment with MTX or F-UdR. The therapeutic effects in the CSC-enriched cell line after exposure to various combinations of MTX, F-UdR, TMZ and [125I]I-UdR were also investigated by a CellTiter-Blue assay. RESULTS: The highest general increase in [125I]I-UdR incorporation was observed with F-UdR co-treatment, which resulted in approx. 2.5-fold increase in the DNA-associated activity. Also the cell viability was significantly decreased when F-UdR was combined with [125I]I-UdR compared to [125I]I-UdR alone at all activity concentrations tested. MTX was redundant when combined with 400 and 500 Bq/ml [125I]I-UdR. TMZ was effective in combination with either [125I]I-UdR alone or with both thymidylate synthase inhibitors combined with 50-100 Bq/ml [125I]I-UdR. CONCLUSIONS: Overall, our study revealed a higher incorporation and therapeutic effect of [125I]I-UdR when GBM cells were co-treated with F-UdR compared to MTX. The therapeutic effects were further increased when TMZ was combined with [125I]I-UdR in combination with the thymidylate synthase inhibitors. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Auger electron therapy in combination with thymidylate synthase inhibition and concomitant chemotherapy has the potential to become a future therapeutic treatment option for patients with glioblastoma.

Improving Contrast and Detectability: Imaging with [55Co]Co-DOTATATE in Comparison with [64Cu]Cu-DOTATATE and [68Ga]Ga-DOTATATE.

PET imaging at late time points after injection may allow tracer clearance from normal tissue and hence improve image contrast and detectability. 55Co is a promising isotope with high positron yield and a long half-life suitable for imaging at delayed time points. Here, we compared the 3 radioconjugates [68Ga]Ga-DOTATATE, [64Cu]Cu-DOTATATE, and [55Co]Co-DOTATATE by PET/CT imaging in NOD-SCID mice bearing subcutaneous somatostatin receptor-expressing AR42J tumors. Methods:55Co and 64Cu were produced by the 54Fe(d,n)55Co and 64Ni(p,n)64Cu nuclear reactions, whereas 68Ga was obtained from a 68Ge/68Ga generator. 55Co and 64Cu were labeled with DOTATATE by heating in a sodium acetate buffer and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, respectively. AR42J tumor-bearing mice were dynamically scanned 0-1 h after injection. For 64Cu and 55Co, additional imaging was also performed at late time points after 4 and 24 h. Dose calculations were based on a known biodistribution. The cumulated disintegrations in each organ were calculated by integration of a fitted exponential function to the biodistribution of each respective organ. Equivalent doses were calculated by OLINDA/EXM using the MIRD formalism. Results: Tumor uptake was rapid from 0 to 1 h after injection for all 3 radioconjugates. Normal-tissue ratios as represented by tumor-to-liver, tumor-to-kidney, and tumor-to-muscle ratios increased significantly over time, with [55Co]Co-DOTATATE reaching the highest ratio of all radioconjugates. For [55Co]Co-DOTATATE, the tumor-to-liver ratio increased to 65 ± 16 at 4 h and 50 ± 6 at 24 h, which were 15 (P < 0.001) and 30 (P < 0.001) times higher, respectively, than the corresponding ratios for [64Cu]Cu-DOTATATE and 5 (P < 0.001) times higher than that of [68Ga]Ga-DOTATATE at 1 h. Correspondingly, tumor-to-kidney and tumor-to-muscle ratios for [55Co]Co-DOTATATE were 4 (P < 0.001) and 11 (P < 0.001) times higher than that of [64Cu]Cu-DOTATATE at 24 h. An equivalent dose was calculated as 9.6E-02 mSv/MBq for [55Co]Co-DOTATATE. Conclusion: [55Co]Co-DOTATATE demonstrated superior image contrast compared with [64Cu]Cu-DOTATATE and [68Ga]Ga-DOTATATE for PET imaging of somatostatin receptor-expressing tumors, warranting translation into clinical trials. Dosimetry calculations found that effective doses for [55Co]Co-DOTATATE were comparable to those for both [64Cu]Cu-DOTATATE and [68Ga]Ga-DOTATATE.

Resorufin: a lead for a new protein kinase CK2 inhibitor.

Screening a natural compound library led to the identification of resorufin as a highly selective and potent inhibitor of protein kinase CK2. Out of 52 kinases tested, only CK2 was inhibited, in contrast to emodin, a structurally related, known CK2 inhibitor that, in addition to CK2, inhibited ten other kinases by 90%. The IC50 values determined for the CK2 holoenzymes were 1.5 mol/l and for the free catalytic subunits ca. 4 mol/l. Altogether four cell lines were subjected to resorufin and emodin treatment. In the case of the three prostate carcinoma cell lines (PC-3, DU-145, LNCaP), 24 h treatment with 40 mol/l resorufin led to 15-20% dead cells; however, no caspase-mediated apoptosis was observed. In the case of the colorectal carcinoma HCT116 cell line, a similar picture was obtained, yet, when resorufin was administered to cells treated with doxorubicin, apoptosis was strongly induced within 24 h. Endogenous protein kinase CK2 was inhibited by resorufin by ca. 80% in the three prostate cell lines. In the case of the HCT116 cells, the inhibition was only 40% supporting the notion of cell line-specific selectivity. Moreover, we analysed the effect of resorufin and emodin on selected signalling molecules in the cell lines under investigation.

Linked Hexokinase and Glucose-6-Phosphatase Activities Reflect Grade of Ovarian Malignancy.

PURPOSE: Malignant cells exhibit increased rates of aerobic glycolysis. Here, we tested whether the accumulation of fluoro-deoxyglucose-6-phosphate (FDG6P) in ovarian cancers of differential malignancy reflects inversely correlated elevations of hexokinase (HK) and glucose-6-phosphatase (G6Pase) activities. PROCEDURES: Twenty-nine women with suspected ovarian cancer had positron emission tomography (PET) prior to surgery. From fresh-frozen tissue, we determined the activities of HK and G6Pase, and from the PET images, we determined the tumor maximum standardized uptake value (SUVmax) of 2-deoxy-2-[18F]fluoro-D-glucose. RESULTS: The SUVmax of malignant lesions significantly exceeded the SUVmax of benign (p < 0.005) and borderline lesions (p < 0.0005) that did not differ significantly. We found no significant correlation between measured HK or G6Pase activities and histological tumor type or SUVmax except that G6Pase activities were higher in malignant than borderline lesions (p < 0.05). Measured HK and G6Pase activities correlated inversely (p < 0.05). The slopes from the regression lines of the three correlations yielded positively correlated abscissa and ordinate intercepts, designated HKmax and G6Pasemax, respectively (r = 0.67, p < 0.0001). The positive correlations between the abscissa and ordinate intercepts with SUVmax had regression coefficients of r = 0.44, p < 0.05; and r = 0.39, p < 0.05, respectively. CONCLUSIONS: The results distinguished two ovarian cancer phenotypes, one with elevated HK activity and low G6Pase activity, and another with the opposite characteristics.

Exploring the intramolecular phosphorylation sites in human Chk2.

A comparative biochemical analysis was performed using recombinant human protein kinase Chk2 (checkpoint kinase 2) expressed in bacteria and insect cells. Dephosphorylated, inactive, recombinant human Chk2 could be reactivated in a concentration-dependent manner. Despite distinct time-dependent autophosphorylation kinetics by monitoring the phosphorylation of amino acid residues T68, S19, S33/35, T432, in Chk2 wildtype and Chk2 mutants (T68A, T68D and Q69E) they gave identical specific activities. However, upon gel filtration of Chk2 wildtype and the mutants, only Chk2 wildtype and the T68D mutant led to the formation of a 'pure' dimer; dephosphorylated wildtype Chk2 eluted as a monomer. Transfection of HEK293 cells with Chk2 wildtype and Chk2 mutants in the absence or presence of DNA damage showed significant T68 phosphorylation already in the absence of DNA damaging reagents. Upon DNA damage, phosphorylation of additional Chk2 sites was observed (S19, S33/35). A comparison of ATM+/+ and ATM-/- cells with respect to phosphorylation of residues T68, S19, S33/35 in the absence and presence of DNA damage showed in all cases phosphorylation of T68, although signal intensity was increased ca. three-fold after DNA damage. Mass spectrometric analyses of human recombinant Chk2 isolated from bacteria and insect cells showed distinct differences. The number of phosphorylated residues in human recombinant Chk2 isolated from bacteria was 16, whereas in the case of the recombinant human Chk2 from insect cells it was 8. Except for phosphorylated amino acid T378 which was not found in the Chk2 isolated from bacteria, all other phosphorylated residues identified in human Chk2 from insect cells were present also in Chk2 from bacteria.

Chelation, formulation, encapsulation, retention, and in vivo biodistribution of hydrophobic nanoparticles labelled with 57Co-porphyrin: Oleylamine ensures stable chelation of cobalt in nanoparticles that accumulate in tumors.

BACKGROUND AND MOTIVATION: While small molecules can be used in cancer diagnosis there is a need for imageable diagnostic NanoParticles (NPs) that act as surrogates for the therapeutic NPs. Many NPs are composed of hydrophobic materials so the challenge is to formulate hydrophobic imaging agents. To develop individualized medical treatments based on NP, a first step should be the selection of patients who are likely responders to the treatment as judged by imaging tumor accumulation of NPs. This requires NPs with the same size and structure as the subsequent therapeutic NPs but labelled with a long-lived radionuclide. Cobalt isotopes are good candidates for NP labelling since 55Co has half-life of 17.5 h and positron energy of 570 keV while 57Co (t1/2 271.6 d) is an isotope suited for preclinical single photon emission tomography (SPECT) to visualize biodistribution and pharmacokinetics of NPs. We used the hydrophobic octaethyl porphyrin (OEP) to chelate cobalt and to encapsulate it inside hydrophobic liquid NPs (LNPs). We hypothesized that at least two additional hydrophobic axial ligands (oleylamine, OA) must be provided to the OEP-Co complex in order to encapsulate and retain Co inside LNP. RESULTS: 1. Cobalt chelation by OEP and OA. The association constant of cobalt to OEP was 2.49 × 105 M-1 and the formation of the hexacoordinate complex OEP-Co-4OA was measured by spectroscopy. 2. NP formulation and characterization: LNPs were prepared by the fast ethanol injection method and were composed of a liquid core (triolein) surrounded by a lipid monolayer (DSPC:Cholesterol:DSPE-PEG2000). The size of the LNPs loaded with the cobalt complex was 40 ±â€¯5 nm, 3. Encapsulation of OEP-Co-OA: The loading capacity of OEP-Co-OA in LNP was 5 mol%. 4. Retention of OEP-57Co-4OA complex in the LNPs: the positive effect of the OA ligands was demonstrated on the stability of the OEP-57Co-4OA complex, providing a half-life for retention in PBS of 170 h (7 days) while in the absence of the axial OA ligands was only 22 h. 5 Biodistribution Study: the in vivo biodistribution of LNP was studied in AR42J pancreatic tumor-bearing mice. The estimated half-life of LNPs in blood was about 7.2 h. Remarkably, the accumulation of LNPs in the tumor was as high as 9.4% ID/g 24 h after injection with a doubling time for tumor accumulation of 3.22 h. The most important result was that the nanoparticles could indeed accumulate in the AR42J tumors up to levels greater than those of other NPs previously measured in the same tumor model, and at about half the values reported for the molecular agent 57Co-DOTATATE. CONCLUSIONS: The additional hydrophobic chelator OA was indeed needed to obtain a stable octahedral OEP-Co-4OA. Cobalt was actually well-retained inside LNP in the OEP-Co-4OA complex. The method described in the present work for the core-labelling of LNPs with cobalt is now ready for labeling of NPs with 55Co, or indeed other hexadentate radionuclides of interest for preclinical in vivo PET-imaging and radio-therapeutics.