A successful matching of a PEG group size with the EPR effect for an off-to-on responsive NIR-fluorophore conjugate has been accomplished which allows two distinct in vivo tumor imaging periods, the first being the switch on during the initial tumor uptake via enhanced permeability into the ROI (as background is suppressed) and a second, later, due to enhanced retention within the tumor.Methods: Software simulation (https://mihaitodor.github.io/particle_simulation/index.html),synthetic chemistry, with in vitro and in vivo imaging have been synergistically employed to identify an optimal PEG conjugate of a bio-responsive NIR-AZA fluorophore for in vivo tumor imaging.Results: A bio-responsive NIR-AZA fluorophore conjugated to a 10 kDa PEG group has shown excellent in vivo imaging performance with sustained high tumor to background ratios and peak tumor emission within 24 h.Analysis of fluorescence profiles over 7 days has provided evidence for the EPR effect playing a positive role.Conclusion: Preclinical results show that exploiting the EPR effect by utilizing an optimized PEG substituent on a bio-responsive fluorophore may offer a means for intraoperative tumor margin delineation.The off-to-on responsive nature of the fluorophore makes tumor imaging achievable without waiting for clearance from normal tissue.
Sunitinib is a tyrosine kinase inhibitor approved for the treatment of multiple solid tumors. However, cardiotoxicity is of increasing concern, with a need to develop rational mechanism driven approaches for the early detection of cardiac dysfunction. We sought to interrogate changes in cardiac energy substrate usage during sunitinib treatment, hypothesising that these changes could represent a strategy for the early detection of cardiotoxicity. Balb/CJ mice or Sprague-Dawley rats were treated orally for 4 weeks with 40 or 20 mg/kg/day sunitinib. Cardiac positron emission tomography (PET) was implemented to investigate alterations in myocardial glucose and oxidative metabolism. Following treatment, blood pressure increased, and left ventricular ejection fraction decreased. Cardiac [18F]-fluorodeoxyglucose (FDG)-PET revealed increased glucose uptake after 48 hours. [11C]Acetate-PET showed decreased myocardial perfusion following treatment. Electron microscopy revealed significant lipid accumulation in the myocardium. Proteomic analyses indicated that oxidative metabolism, fatty acid β-oxidation and mitochondrial dysfunction were among the top myocardial signalling pathways perturbed. Sunitinib treatment results in an increased reliance on glycolysis, increased myocardial lipid deposition and perturbed mitochondrial function, indicative of a fundamental energy crisis resulting in compromised myocardial energy metabolism and function. Our findings suggest that a cardiac PET strategy may represent a rational approach to non-invasively monitor metabolic pathway remodeling following sunitinib treatment.
Abstract Colorectal cancer (CRC) is a leading cause of cancer deaths. Molecularly targeted therapies ( e.g . bevacizumab) have improved survival rates but drug resistance ultimately develops and newer therapies are required. We identified quininib as a small molecule drug with anti-angiogenic activity using in vitro , ex vivo and in vivo screening models. Quininib (2-[( E )-2-(Quinolin-2-yl) vinyl] phenol), is a small molecule drug (molecular weight 283.75 g/mol), which significantly inhibited blood vessel development in zebrafish embryos (p < 0.001). In vitro , quininib reduced endothelial tubule formation (p < 0.001), cell migration was unaffected by quininib and cell survival was reduced by quininib (p < 0.001). Using ex vivo human CRC explants, quininib significantly reduced the secretions of IL-6, IL-8, VEGF, ENA-78, GRO-α, TNF, IL-1β and MCP-1 ex vivo (all values p < 0.01). Quininib is well tolerated in mice when administered at 50 mg/kg intraperitoneally every 3 days and significantly reduced tumour growth of HT-29-luc2 CRC tumour xenografts compared to vehicle control. In addition, quininib reduced the signal from a α v β 3 integrin fluorescence probe in tumours 10 days after treatment initiation, indicative of angiogenic inhibition. Furthermore, quininib reduced the expression of angiogenic genes in xenografted tumours. Collectively, these findings support further development of quininib as a novel therapeutic agent for CRC.
Abstract Triple-negative breast cancer (TNBC) is defined by absent expression of estrogen receptor (ER), progesterone receptor (PR) and non-overexpression of human epidermal growth factor receptor 2 (HER2), representing a heterogeneous subgroup of breast cancer with substantial genotypic and phenotypic diversity. TNBC patients commonly exhibit poor prognosis and high relapse rates at early stages after conventional treatments. Currently, there is a lack of biomarkers and targeted therapies for the management of TNBC. During tumour development and progression, alterations in cellular behaviour are frequently linked with kinase expression and activity. Here, we aimed to identify novel kinase targets that may play a pivotal role in the progression of TNBC and, thus, offer new therapeutic vantage points. We initially focused on identifying kinases correlated with differential outcome. Using publicly available transcriptomic data from a collated set of TNBC patients (n = 483), we identified 9 kinases that were significantly associated with survival at the mRNA level. From this in silico screen, CDK7 (cyclin-dependent kinase 7) was found to be correlated with poor recurrence-free survival. CDK7's trait as a marker of poor prognosis was further validated within another TNBC cohort (n=109) via assessment of a tissue microarray generated as part of the RATHER Consortium (www.ratherproject.com). At the protein level, high CDK7 expression was associated with poor breast cancer-specific, recurrence-free and distant recurrence-free survival. To evaluate CDK7 as a therapeutic target in TNBC, two TNBC cell lines (BT-549 and MDA-MB-231) were selected to evaluate phenotypic alterations post shRNA-mediated CDK7 knockdown. CDK7 silencing led to decreased cell proliferation, colony formation and migration in vitro. CDK7 down-regulation also increased TNBC cell sensitivity to doxorubicin. BS-181 and THZ1, two highly specific CDK7 inhibitors, attenuated TNBC tumour growth by inducing G2/M phase cell cycle arrest and apoptosis, as well as down-regulation of RNAPII phosphorylation, an indication of global RNA transcription inhibition. Moreover, the covalent CDK7 inhibitor THZ1 demonstrated 1000-fold higher potency than BS-181. Inhibition of global RNA transcription preferentially affects proteins with short half-lives. Accordingly, we detected a reduction in the expression of the anti-apoptotic protein MCL-1 in both cell lines. Next, we assessed anti-apoptotic dependence in MDA-MB-231 cells following treatment with THZ1 via BH3 profiling technology, and observed an increased response to the BAD and HRK peptides, inferring an elevated survival dependence on BCL-2/BCL-XL. We subsequently evaluated the combination of the BCL-2/BCL-XL inhibitor ABT-263 with THZ1 and discovered a synergistic inhibition of cell growth and apoptosis. Resulting combination index (CI) values demonstrated that synergistic cell death occurred following combined treatment with THZ1 and ABT-263/ABT-199 at various doses in both TNBC cell lines tested. Our data implicate high CDK7 expression as a promising biomarker of poor prognosis in TNBC. Moreover, these findings suggest that targeting CDK7, combined with the BCL-2/BCL-XL inhibitor ABT-263, may be a useful therapeutic strategy for TNBC. Citation Format: Gallagher WM, Li B, Ni Chonghaile T, Fan Y, Klinger R, O'Connor AE, Conroy E, Tarrant F, O'Hurley G, Mallya Udupi G, Gaber A, Chin S-F, Schouten PC, Dubois T, Linn S, Jirstrom K, Caldas C, Bernards R, O'Connor DP. CDK7: A marker of poor prognosis and tractable therapeutic target in triple-negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-01.
Abstract Invasive lobular carcinoma (ILC) is the second most common type of breast cancer accounting for approximately 10-15% of all breast tumours. ILC is characterized by inactivation of E-Cadherin and cancer cells that invade the stroma in a "single-file" pattern. Women with ILC are more likely to have hormone receptor-positive disease. ILCs are three times more likely to metastasize to the peritoneum, gastrointestinal tract, and ovaries. ILC are currently treated in the same manner as all other ER+ breast cancers. Like invasive ductal carcinoma (IDC), anti-estrogen resistance has emerged as a significant problem in the management of ILC. Approximately half of the recurrences of ER+ breast tumors respond to anti-endocrine treatment, while the other half are resistant. Additionally, anti-estrogen-resistant breast tumor cells appear to have/acquire a more aggressive, invasive phenotype compared to their anti-estrogen-responsive counterparts. ILC is considered to be chemo-resistant and ILC patients receive no additional benefit from adjuvant chemotherapy. As such, there is a pressing need to develop tailored therapeutic options for endocrine-resistant ILC patients. The bromodomain and extra-terminal (BET) motif family are epigenetic readers that bind to acetylated histones, recruiting co-factors to regulate transcription. As part of the FP7 RATHER project aimed at identifying rational treatment options for ILC, we discovered that BRD3 (uniquely among the BET family members) is a marker of poor prognosis in ILC but not in ER+ breast cancers as a whole. We subsequently validated this in an independent cohort from the METABRIC study. These data suggest that Brd3 may play a significant role in tumour progression in ILC and may be a rational therapeutic target for lobular tumours. Using the two ILC cell lines SUM44PE (SUM44) and MDA MB 134 VI (MM134) previously shown to be anti-endocrine resistant we assessed the therapeutic potential of BET inhibition. We found that ILC cell lines that do not respond to anti-endocrine therapy are sensitive to a panel of BET inhibitors in both 2D and 3D assays. In particular the BET inhibitors JQ1 and Mivebresib had the highest potency, these BET inhibitors were selected for further research. Next, a multi-omics approach merging RNA-sequencing with mass spectrometry was utilised to dissect the transcriptional networks employed by BET inhibitors in this ILC setting. RNA-sequencing revealed dysregulated pathways in cell cycle division, DNA damage, apoptosis and MAPK signalling following treatment. Further, rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) was carried out to find the binding partners of the BRD proteins. Integrated pathway analysis through Genome Enhancer was used to assess the master regulators (MTRs) which drive BET inhibitor function. There was 142 MTRs found across the two cell lines treated with both inhibitors. Of note the MTR, FGFR3, was shown to regulate the downstream targets of both BET inhibitors. Further analysis of BET inhibition combined with erdafitinib, an FGFR inhibitor, drives increased cell inhibition compared to either agent alone. Finally, the efficacy of JQ1 in targeting ILC was assessed in vivo. We utilised the ILC cell-derived xenograft (CDX) models established by mammary intraductal implantation. The efficacy of BET inhibition alone and in combination with anti-endocrine therapies, tamoxifen and fulvestrant were assessed in the MM134 cell line. We found that JQ1 works synergistically with tamoxifen to significantly decrease tumour burden and metastatic potential in this tamoxifen resistant ILC model. Taken together this data highlights the need for tailored therapeutics in ILC research and highlights the use of BET inhibition in the anti-endocrine resistant ILC setting. Citation Format: Elspeth Ward, Anna Blümel, Emer Conroy, Grainne Cremin, Binbin Gao, William Gallagher, Idalia Cruz, Leena Hilakivi-Clarke, George Sflomos, Cathrin Brisken, Darran O'Connor. Bromodomain and Extra-Terminal motif (BET) inhibitors are a rational therapeutic choice for treatment of invasive lobular carcinoma [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD14-07.
Abstract. Elevated suspended sediment concentrations in fluvial environments have important implications for system ecology and even small concentrations may have serious consequences for sensitive ecosystems or organisms, such as freshwater pearl mussels (Margaritifera margaritifera). Informed decision making is therefore required for land managers to understand and control soil erosion and sediment delivery to the river network. However, given that monitoring of sediment fluxes requires financial and human resources which are often limited at a national scale, sediment mobilisation and delivery models are commonly used for sediment yield estimation and management. The Revised Universal Soil Loss Equation (RUSLE) is the most widely used model for overland flow erosion and can, when combined with a sediment delivery ratio (SDR), provide reasonable sediment load estimations for a catchment. This paper presents RUSLE factors established from extant GIS and rainfall datasets that are incorporated into a flexible catchment modelling approach. We believe that this is the first time that results from a RUSLE application at a national scale are tested against measured sediment yield values available from Ireland. An initial assessment of RUSLE applied to Irish conditions indicates an overestimation of modelled sediment yield values for most of the selected catchments. Improved methods for model and SDR factors estimation are needed to account for Irish conditions and catchment characteristics. Nonetheless, validation and testing of the model in this study using observed values is an important step towards more effective sediment yield modelling tools for nationwide applications.
The back-illuminated Electron Multiplying Charge Coupled Device (EMCCD) camera stands to be one of the most revolutionary contributions ever to the burgeoning fields of low-light dynamic cellular microscopy and single molecule detection, combining extremely high photon conversion efficiency with the ability to eliminate the readout noise detection limit. Here, we present some preliminary measurements recorded by a vary rapid frame rate version of this camera technology, incorporated into a spinning disk confocal microscopy set-up that is used for fast intracellular calcium flux measurements. The results presented demonstrate the united effects of (1) EMCCD technology in amplifying the very weak signal from these fluorescently labelled cells above the readout noise detection limit, that they would otherwise be completely lost in; (2) back-thinned CCD technology in maximizing the singal/shot noise ratio from such weak photon fluxes. It has also been shown how this innovative development can offer significant signal improvements over that afforded by ICCD technology. Practially, this marked advancement in detector sensitivity affords benefits such as shorter exposure times (therefore faster frame rates), lower dye concentrations and reduced excitation powers and will remove some of the barriers that have been restricting the development of new innovative low-light microscopy techniques.
Abstract Bioresponsive NIR-fluorophores offer the possibility for continual visualization of dynamic cellular processes with added potential for direct translation to in vivo imaging. Here we show the design, synthesis and lysosome-responsive emission properties of a new NIR fluorophore. The NIR fluorescent probe design differs from typical amine functionalized lysosomotropic stains with off/on fluorescence switching controlled by a reversible phenol/phenolate interconversion. Emission from the probe is shown to be highly selective for the lysosomes in co-imaging experiments using a HeLa cell line expressing the lysosomal-associated membrane protein 1 fused to green fluorescent protein. The responsive probe is capable of real-time continuous imaging of fundamental cellular processes such as endocytosis, lysosomal trafficking and efflux in 3D and 4D. The advantage of the NIR emission allows for direct translation to in vivo tumour imaging, which is successfully demonstrated using an MDA-MB-231 subcutaneous tumour model. This bioresponsive NIR fluorophore offers significant potential for use in live cellular and in vivo imaging, for which currently there is a deficit of suitable molecular fluorescent tools.
'/%3e%3cpath d='M200 752.362h200v300H200z' style='fill:%23fff;fill-opacity:1;stroke:none' transform='translate(0 -752.362)'/%3e%3cpath d='M400 752.362h200v300H400z' style='fill:%23ff883e;fill-opacity:1;stroke:none' transform='translate(0 -752.362)'/%3e%3c/svg%3e)
