CytoCy5S™, a compound of many structures. in vitro and in vivo evaluation of four near-infrared fluorescent substrates of nitroreductase (NTR)

CytoCy5S™, a quenched, red-shifted fluorescent probe, has been used to exploit the imaging potential of the nitroreductase (NTR) reporter gene platform. Its use has been reported in a number of publications, however there are discrepancies in both the reported structure and its physicochemical properties. Herein, we aim to highlight these discrepancies and to define the best candidate of the four substrates under study for preclinical work in NTR reporting by optical applications. We report the synthesis, purification and characterisation of four NTR substrates, including alternately described structures currently referred by the name CytoCy5S. A comparative NTR enzymatic assay was performed to assess the spectroscopic characteristics of the different reductively activated probes. The NTR expressing triple-negative breast carcinoma cell line, MDA-MB-231 NTR+, was employed to compare, both in vitro and in vivo, the suitability of these fluorescent probes as reporters of NTR activity. Comparison of the reporting properties was achieved by flow cytometry, fluorescence microscopy and optical imaging, both in vivo and ex vivo. This study evaluated the different spectroscopic and biological characteristics of the four substrates and concluded that substrate 1 presents the best features for oncological in vivo preclinical optical imaging.publishedVersio

The HDACi panobinostat shows growth inhibition both in vitro and in a bioluminescent orthotopic surgical xenograft model of ovarian cancer

Background: In most epithelial ovarian carcinomas (EOC), epigenetic changes are evident, and overexpression of histone deacetylases (HDACs) represents an important manifestation. In this study, we wanted to evaluate the effects of the novel HDAC inhibitor (HDACi) panobinostat, both alone and in combination with carboplatin, on ovarian cancer cell lines and in a murine bioluminescent orthotopic surgical xenograft model for EOC. Methods: The effects of panobinostat, both alone and in combination with carboplatin, on proliferation and apoptosis in ovarian cancer cell lines, were evaluated using colony and WST-1 assays, Hoechst staining and flow cytometry analysis. In addition, mechanisms were characterised by western blotting and phosphoflow analysis. Immuno-deficient mice were engrafted orthotopically with SKOV-3luc+ cells and serial bioluminescence imaging monitored the effects of treatment with panobinostat and/or carboplatin and/or surgery. Survival parameters were also measured. Results: Panobinostat treatment reduced cell growth and diminished cell viability, as shown by the induced cell cycle arrest and apoptosis in vitro. We observed increased levels of cleaved PARP and caspase-3, downregulation of cdc2 protein kinase, acetylation of H2B and higher pH2AX expression. The combined administration of carboplatin and panobinostat synergistically increased the anti-tumour effects compared to panobinostat or carboplatin treatment alone. In our novel ovarian cancer model, the mice showed significantly higher rates of survival when treated with panobinostat, carboplatin or a combination of both, compared to the controls. Panobinostat was as efficient as carboplatin regarding prolongation of survival. No significant additional effect on survival was observed when surgery was combined with carboplatin/panobinostat treatment. Conclusions: Panobinostat demonstrates effective in vitro growth inhibition in ovarian cancer cells. The efficacy of panobinostat and carboplatin was equal in the orthotopic EOC model used. We conclude that panobinostat is a promising therapeutic alternative that needs to be further assessed for the treatment of EOC.publishedVersio

Increased plasma colloid osmotic pressure facilitates the uptake of therapeutic macromolecules in a xenograft tumour model

Elevated tumor interstitial fluid pressure (TIFP) is a characteristic of most solid tumors. Clinically, TIFP may hamper the uptake of chemotherapeutic drugs into the tumor tissue reducing their therapeutic efficacy. In this study, a means of modulating TIFP to increase the flux of macromolecules into tumor tissue is presented, which is based on the rationale that elevated plasma colloid osmotic pressure (COP) pulls water from tumor interstitium lowering the TIFP. Concentrated human serum albumin: (20% HSA), used as an agent to enhance COP, reduced the TIFP time-dependently from 8 to 2 mm Hg in human tumor xenograft models bearing A431 epidermoid vulva carcinomas. To evaluate whether this reduction facilitates the uptake of macromolecules, the intratumoral distribution of fluorescently conjugated dextrans (2.5 mg/ml) and cetuximab (2.0 mg/ml) was probed using novel time domain nearinfrared fluorescence imaging. This method permitted discrimination and semiquantification of tumor-accumulated conjugate from background and unspecific probe fluorescence. The coadministration of 20% HSA together with either dextrans or cetuximab was found to lower the TIFP significantly and increase the concentration of the substances within the tumor tissue in comparison to control tumors. Furthermore, combined administration of 20%HSA plus cetuximab reduced the tumor growth significantly in comparison to standard cetuximab treatment. These data demonstrate that increased COP lowers the TIFP within hours and increases the uptake of therapeutic macromolecules into the tumor interstitium leading to reduced tumor growth. This model represents a novel approach to facilitate the delivery of therapeutics into tumor tissue, particularly monoclonal antibodies

Evaluating Translational Methods for Personalized Medicine—A Scoping Review

The introduction of personalized medicine, through the increasing multi-omics characterization of disease, brings new challenges to disease modeling. The scope of this review was a broad evaluation of the relevance, validity, and predictive value of the current preclinical methodologies applied in stratified medicine approaches. Two case models were chosen: oncology and brain disorders. We conducted a scoping review, following the Joanna Briggs Institute guidelines, and searched PubMed, EMBASE, and relevant databases for reports describing preclinical models applied in personalized medicine approaches. A total of 1292 and 1516 records were identified from the oncology and brain disorders search, respectively. Quantitative and qualitative synthesis was performed on a final total of 63 oncology and 94 brain disorder studies. The complexity of personalized approaches highlights the need for more sophisticated biological systems to assess the integrated mechanisms of response. Despite the progress in developing innovative and complex preclinical model systems, the currently available methods need to be further developed and validated before their potential in personalized medicine endeavors can be realized. More importantly, we identified underlying gaps in preclinical research relating to the relevance of experimental models, quality assessment practices, reporting, regulation, and a gap between preclinical and clinical research. To achieve a broad implementation of predictive translational models in personalized medicine, these fundamental deficits must be addressed.publishedVersio

Increased plasma colloid osmotic pressure facilitates the uptake of therapeutic macromolecules in a xenograft tumour model

Elevated tumor interstitial fluid pressure (TIFP) is a characteristic of most solid tumors. Clinically, TIFP may hamper the uptake of chemotherapeutic drugs into the tumor tissue reducing their therapeutic efficacy. In this study, a means of modulating TIFP to increase the flux of macromolecules into tumor tissue is presented, which is based on the rationale that elevated plasma colloid osmotic pressure (COP) pulls water from tumor interstitium lowering the TIFP. Concentrated human serum albumin: (20% HSA), used as an agent to enhance COP, reduced the TIFP time-dependently from 8 to 2 mm Hg in human tumor xenograft models bearing A431 epidermoid vulva carcinomas. To evaluate whether this reduction facilitates the uptake of macromolecules, the intratumoral distribution of fluorescently conjugated dextrans (2.5 mg/ml) and cetuximab (2.0 mg/ml) was probed using novel time domain nearinfrared fluorescence imaging. This method permitted discrimination and semiquantification of tumor-accumulated conjugate from background and unspecific probe fluorescence. The coadministration of 20% HSA together with either dextrans or cetuximab was found to lower the TIFP significantly and increase the concentration of the substances within the tumor tissue in comparison to control tumors. Furthermore, combined administration of 20%HSA plus cetuximab reduced the tumor growth significantly in comparison to standard cetuximab treatment. These data demonstrate that increased COP lowers the TIFP within hours and increases the uptake of therapeutic macromolecules into the tumor interstitium leading to reduced tumor growth. This model represents a novel approach to facilitate the delivery of therapeutics into tumor tissue, particularly monoclonal antibodies.publishedVersio

Up-regulation of anti-apoptotic genes confers resistance to the novel anti-leukaemic compound PEP005 in primary AML cells

We showed previously that PEP005 induced apoptosis in leukaemic cell lines and blasts from patients with acute myeloid leukaemia (AML). Here we assess the anti-leukeamic effects of PEP005 in vivo and determine the mechanism of resistance of PEP005 non-responsive cells. We used 2 human xenograft mouse models of AML to assess the anti-leukaemic effects of PEP005 in vivo. Expression microarray analysis of primary AML blasts following treatment with PEP005 was used to determine patterns of gene expression that conferred resistance. PEP005 significantly reduced tumour burden in two human leukaemia mouse xenograft models. We also assessed responsiveness of 33 AML samples to PEP005, with 78% of the samples entering apoptosis at 100nM. Resistance to PEP005 was not restricted to a particular AML subtype. Expression microarray analysis of resistant samples following treatment with PEP005 revealed a significant up regulation of the anti-apoptotic genes Bcl-2A1, Mcl-1, and PHLDA1 which was verified using RT-PCR. We conclude that PEP005 shows broad efficacy against AML subtypes and that up regulation of anti-apoptotic genes underlies resistance to this agent and could be used to screen for patients unlikely to benefit from a therapeutic regime involving PEP005

CD24-targeted intraoperative fluorescence image-guided surgery leads to improved cytoreduction of ovarian cancer in a preclinical orthotopic surgical model

Background: The completeness of resection is a key prognostic indicator in patients with ovarian cancer, and the application of tumour-targeted fluorescence image-guided surgery (FIGS) has led to improved detection of peritoneal metastases during cytoreductive surgery. CD24 is highly expressed in ovarian cancer and has been shown to be a suit

Hydroxyurea synergizes with valproic acid in wild-type p53 acute myeloid leukaemia

Palliative care in acute myeloid leukaemia (AML) is inadequate. For elderly patients, unfit for intensive chemotherapy, median survival is 2–3 months. As such, there is urgent demand for low-toxic palliative alternatives. We have repositioned two commonly administered anti-leukaemia drugs, valproic acid (VPA) and hydroxyurea (HU), as a combination therapy in AML. The anti-leukemic effect of VPA and HU was assessed in multiple AML cell lines confirming the superior anti-leukemic effect of combination therapy. Mechanistic studies revealed that VPA amplified the ability of HU to slow S-phase progression and this correlated with significantly increased DNA damage. VPA was also shown to reduce expression of the DNA repair protein, Rad51. Interestingly, the tumour suppressor protein p53 was revealed to mitigate cell cycle recovery following combination induced arrest. The efficacy of combination therapy was validated in vivo. Combination treatment increased survival in OCI-AML3 and patient-derived xenograft mouse models of AML. Therapy response was confirmed by optical imaging with multiplexed near-infrared labelled antibodies. The combination of HU and VPA indicates significant potential in preclinical models of AML. Both compounds are widely available and well tolerated. We believe that repositioning this combination could significantly enhance the palliative care of patients unsuited to intensive chemotherapy.publishedVersio

Intracellular Cytidine Deaminase Regulates Gemcitabine Metabolism in Pancreatic Cancer Cell Lines

Cytidine deaminase (CDA) is a determinant of in vivo gemcitabine elimination kinetics and cellular toxicity. The impact of CDA activity in pancreatic ductal adenocarcinoma (PDAC) cell lines has not been elucidated. We hypothesized that CDA regulates gemcitabine flux through its inactivation and activation pathways in PDAC cell lines. Three PDAC cell lines (BxPC-3, MIA PaCa-2, and PANC-1) were incubated with 10 or 100 µM gemcitabine for 60 minutes or 24 hours, with or without tetrahydrouridine, a CDA inhibitor. Extracellular inactive gemcitabine metabolite (dFdU) and intracellular active metabolite (dFdCTP) were quantified with liquid chromatography tandem mass spectrometry. Cellular expression of CDA was assessed with real-time PCR and Western blot. Gemcitabine conversion to dFdU was extensive in BxPC-3 and low in MIA PaCa-2 and PANC-1, in accordance with their respective CDA expression levels. CDA inhibition was associated with low or undetectable dFdU in all three cell lines. After 24 hours gemcitabine incubation, dFdCTP was highest in MIA PaCa-2 and lowest in BxPC-3. CDA inhibition resulted in a profound dFdCTP increase in BxPC-3 but not in MIA PaCa-2 or PANC-1. dFdCTP concentrations were not higher after exposure to 100 versus 10 µM gemcitabine when CDA activities were low (MIA PaCa-2 and PANC-1) or inhibited (BxPC-3). The results suggest a regulatory role of CDA for gemcitabine activation in PDAC cells but within limits related to the capacity in the activation pathway in the cell lines. SIGNIFICANCE STATEMENT The importance of cytidine deaminase (CDA) for cellular gemcitabine toxicity, linking a lower activity to higher toxicity, is well described. An underlying assumption is that CDA, by inactivating gemcitabine, limits the amount available for the intracellular activation pathway. Our study is the first to illustrate this regulatory role of CDA in pancreatic ductal adenocarcinoma cell lines by quantifying intracellular and extracellular gemcitabine metabolite concentrations.publishedVersio

Development of a bioluminescent nitroreductase probe for preclinical imaging

Bacterial nitroreductases (NTRs) have been widely utilized in the development of novel antibiotics, degradation of pollutants, and gene-directed enzyme prodrug therapy (GDEPT) of cancer that reached clinical trials. In case of GDEPT, since NTR is not naturally present in mammalian cells, the prodrug is activated selectively in NTR-transformed cancer cells, allowing high efficiency treatment of tumors. Currently, no bioluminescent probes exist for sensitive, non-invasive imaging of NTR expression. We therefore developed a "NTR caged luciferin" (NCL) probe that is selectively reduced by NTR, producing light proportional to the NTR activity. Here we report successful application of this probe for imaging of NTR in vitro, in bacteria and cancer cells, as well as in vivo in mouse models of bacterial infection and NTR-expressing tumor xenografts. This novel tool should significantly accelerate the development of cancer therapy approaches based on GDEPT and other fields where NTR expression is important.publishedVersio