Targeting a Newly Established Spontaneous Feline Fibrosarcoma Cell Line by Gene Transfer

Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas

Allopurinol versus usual care in UK patients with ischaemic heart disease (ALL-HEART) : a multicentre, prospective, randomised, open-label, blinded-endpoint trial

Funding Information: ISM reports research grants from Menarini, EMA, Sanofi, Health Data Research UK, the British Heart Foundation, and Innovative Medicines Initiative; institutional consultancy income from AstraZeneca outside the submitted work; and personal income from AstraZeneca and Amgen outside the submitted work. TMM reports grants from Menarini/Ipsen/Teijin and Merck Sharp & Dohme outside the submitted work, and personal income for consultancy from Novartis and AstraZeneca outside the submitted work, and is a trustee of the Scottish Heart Arterial Risk Prevention Society. AGB reports personal income from Novartis, Mylan, AstraZeneca, Bayer, Daiichi-Sankyo, Boehringer, Pfizer, Galderma, Zambon, and Novo-Nordisk outside the submitted work. ADS and the University of Dundee hold a European patent for the use of xanthine oxidase inhibitors in treating chest pain in angina pectoris. AW declares personal income for consultancy from AbbVie, Akcea, Albireo, Alexion, Allergan, Amarin, Apsara, Arena, Astellas, AstraZeneca, Autolus, Bayer, Biocryst, Biogen, Biomarin, Bristol Myers Squibb, Boehringer Ingelheim, Calico, Celgene, Chiesi, Daiichi Sankyo, Diurnal, Elsai, Eli Lilly, Ferring, Galapagos, Gedeon Richter, Gilead, GlaxoSmithKline, GW Pharma, Idorsia, Incyte, Intercept, Ionis, Ipsen, Janssen, Jazz, Jcyte, Kite Gilead, LEK, Leo Pharma, Les Laboratoires Servier, Lundbeck, Merck (Merck Sharp & Dohme), Merck-Serono, Mitenyi, Mundibiopharma, Mustang Bio, Mylan, Myovant, Norgine, Novartis, Novo Nordisk, Orchard, Paion, Pfizer, Pierre Fabre, PTC, RegenXBio, Rhythm, Sanofi, Santen, Sarepta, SeaGen, Shionogi, Sigmatec, SOBI, Takeda, Tanaya, UCB, and Vertex outside the submitted work. JST declares research funding from the UK National Institute for Health and Care Research (NIHR) and NHS England outside the submitted work and membership of a UK National Institute for Health and Care Excellence guideline committee on management of atrial fibrillation. All other authors declare no competing interests. Funding Information: This study was funded by the NIHR Health Technology Assessment programme (HTA 11/36/41 to ISM, IF, CJH, LW, ADS, AGB, AJA, AW, JST, and TMM). The views expressed are those of the authors and not necessarily those of the NIHR or the UK Department of Health and Social Care. The study was supported by the Scottish Primary Care Research Network, Support for Science Scotland (Grampian, Highlands, Tayside, Fife, Forth Valley, Greater Glasgow and Clyde, Lothian, Ayrshire and Arran, Dumfries and Galloway, and Lanarkshire), and the NIHR Local Clinical Research Networks (East Midlands, West Midlands, Eastern, North Thames, Yorkshire and Humber, North East and North Cumbria, North West Coast, Kent, Surrey and Sussex, and South West Peninsula), which assisted with recruitment and other study activities. We thank Public Health Scotland and NHS Digital for providing data linkage. We thank all the participants, physicians, nurses, and other staff who participated in the ALL-HEART study. Funding Information: This study was funded by the NIHR Health Technology Assessment programme (HTA 11/36/41 to ISM, IF, CJH, LW, ADS, AGB, AJA, AW, JST, and TMM). The views expressed are those of the authors and not necessarily those of the NIHR or the UK Department of Health and Social Care. The study was supported by the Scottish Primary Care Research Network, Support for Science Scotland (Grampian, Highlands, Tayside, Fife, Forth Valley, Greater Glasgow and Clyde, Lothian, Ayrshire and Arran, Dumfries and Galloway, and Lanarkshire), and the NIHR Local Clinical Research Networks (East Midlands, West Midlands, Eastern, North Thames, Yorkshire and Humber, North East and North Cumbria, North West Coast, Kent, Surrey and Sussex, and South West Peninsula), which assisted with recruitment and other study activities. We thank Public Health Scotland and NHS Digital for providing data linkage. We thank all the participants, physicians, nurses, and other staff who participated in the ALL-HEART study. Publisher Copyright: © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licensePeer reviewedPublisher PD

Allopurinol versus usual care in UK patients with ischaemic heart disease (ALL-HEART): a multicentre, prospective, randomised, open-label, blinded-endpoint trial

BACKGROUND: Allopurinol is a urate-lowering therapy used to treat patients with gout. Previous studies have shown that allopurinol has positive effects on several cardiovascular parameters. The ALL-HEART study aimed to determine whether allopurinol therapy improves major cardiovascular outcomes in patients with ischaemic heart disease. METHODS: ALL-HEART was a multicentre, prospective, randomised, open-label, blinded-endpoint trial done in 18 regional centres in England and Scotland, with patients recruited from 424 primary care practices. Eligible patients were aged 60 years or older, with ischaemic heart disease but no history of gout. Participants were randomly assigned (1:1), using a central web-based randomisation system accessed via a web-based application or an interactive voice response system, to receive oral allopurinol up-titrated to a dose of 600 mg daily (300 mg daily in participants with moderate renal impairment at baseline) or to continue usual care. The primary outcome was the composite cardiovascular endpoint of non-fatal myocardial infarction, non-fatal stroke, or cardiovascular death. The hazard ratio (allopurinol vs usual care) in a Cox proportional hazards model was assessed for superiority in a modified intention-to-treat analysis (excluding randomly assigned patients later found to have met one of the exclusion criteria). The safety analysis population included all patients in the modified intention-to-treat usual care group and those who took at least one dose of randomised medication in the allopurinol group. This study is registered with the EU Clinical Trials Register, EudraCT 2013-003559-39, and ISRCTN, ISRCTN32017426. FINDINGS: Between Feb 7, 2014, and Oct 2, 2017, 5937 participants were enrolled and then randomly assigned to receive allopurinol or usual care. After exclusion of 216 patients after randomisation, 5721 participants (mean age 72·0 years [SD 6·8], 4321 [75·5%] males, and 5676 [99·2%] white) were included in the modified intention-to-treat population, with 2853 in the allopurinol group and 2868 in the usual care group. Mean follow-up time in the study was 4·8 years (1·5). There was no evidence of a difference between the randomised treatment groups in the rates of the primary endpoint. 314 (11·0%) participants in the allopurinol group (2·47 events per 100 patient-years) and 325 (11·3%) in the usual care group (2·37 events per 100 patient-years) had a primary endpoint (hazard ratio [HR] 1·04 [95% CI 0·89–1·21], p=0·65). 288 (10·1%) participants in the allopurinol group and 303 (10·6%) participants in the usual care group died from any cause (HR 1·02 [95% CI 0·87–1·20], p=0·77). INTERPRETATION: In this large, randomised clinical trial in patients aged 60 years or older with ischaemic heart disease but no history of gout, there was no difference in the primary outcome of non-fatal myocardial infarction, non-fatal stroke, or cardiovascular death between participants randomised to allopurinol therapy and those randomised to usual care. FUNDING: UK National Institute for Health and Care Research

Apoptotic rate of Cocca-6A cells following Adenoviral gene transfer measured by Annexin-V assay.

<p>On the ordinate are indicated the percentages of dead cells following adenoviral transductions. On the abscissa are indicated the different adenoviruses used. Cells were stained using an Annexin-V fluorescence kit and were run on a BD scientific Facs-Aria flow cytometer.</p

Karyogram of the feline fibrosarcoma Cocca-6A cells.

<p>(A) Oil immersion microphotograph (1000× magnification) of Giemsa block stained spotted metaphases from a representative colchicine treatment of the Cocca-6A cells. The Cocca-6A cells show the absence of one of the E1 chromosomes. (B) Semi-quantitative PCR of p53 gene in human HEK-293, feline FSkMC and Cocca 6A cells.</p

Western blot analysis of HEK-293, FSkMC and Cocca-6A cell lysates.

<p>On the left lane are loaded control human embryonic kidney HEK-293 cells and the central lane are loaded normal feline muscle skeletal cells. In the lane on right are loaded the Cocca-6A cells. Anti beta-actin was used as a loading control. 50 µg of total lysates were run in SDS polyacrylamide gels.</p

ANOVA (Analysis of Variance) significance table with a Post Hoc Dunnett’s T3 test for Annexin-V assay experiment of Cocca-6A cells following adenoviral gene transfer.

*<p>The mean difference is significant at the 0.05 level.</p><p>The statistical analysis was run using IBM SPSS software.</p

Western blot analysis of Cocca-6A cell lysates following adenoviral transduction.

<p>On the left lane are loaded control Cocca-6A cells. In the lane on right are loaded the transduced Cocca-6A cells. Anti beta-actin was used as a loading control. 50 µg of total lysates were run in SDS polyacrylamide gels. On the left side are indicated the different adenoviral transductions.</p

The Effects of the combination of Adenoviral gene transfer on the feline fibrosarcoma cells Cocca-6A.

<p>Every panel shows the phase contrast imaging (200× magnification, 24-hours post transduction) and the propidium iodide flow cytometry analysis of Cocca-6A cells transduced with 50 MOIs of various combinations of double or triple transductions using Ads carrying Ad.<i>RB/p105</i>, Ad.<i>Ras</i>-DN, Ad.<i>mda-7/IL24</i>, CTV.<i>mda-7/IL24</i> or CTV.<i>IFNγ.</i> (a–g) Single adenoviral transduction and controls: (a) Control untransduced Cocca-6A cells. (b) Ad.GFP, (c) Ad.Rb/p105, (d) Ad.Ras-DN, (e) Ad.Mda7/IL-24, (f) CTV.Mda7/IL-24, (g) CTV.IFNγ transduced Cocca-6A cells. (h–q) Combinations of double or triple adenoviral transductions: (h) Ad.Rb/p105+ Ad.Ras-DN, (i) Ad.Rb/p105+ Ad.Mda7/IL-24, (j) Ad.Rb/p105+ CTV.Mda7/IL-24, (k) Ad.Rb/p105+ CTV.<i>IFNγ,</i> (l) Ad.Ras-DN + Ad.Mda7/IL-24, (m) Ad.Ras-DN + CTV.Mda7/IL-24, (n) Ad.Ras-DN + CTV.<i>IFNγ,</i> (o) Ad.Rb/p105+ Ad.Ras-DN + Ad.mda7/IL-24, <i>(p)</i> Ad.Rb/p105+ Ad.Ras-DN + CTV.mda7/IL-24, (q) Ad.Rb/p105+ Ad.Ras-DN + Ad.CTV.<i>IFNγ</i> transduced Cocca-6A cells.</p

Establishment of a feline fibrosarcoma cell line.

<p>(A) Hematoxylin & Eosin staining of the pathologic specimen. Optical microscope 100× magnification of the neoplastic tissue shows cells that appear to have indistinct cell borders, round to oval nuclei with finely stippled chromatin, and small single or multiple nucleoli. (B–F) Phase contrast imaging of the Cocca-1A, -2A, -3A, -6A, and -3B clones after 48 hours of culture (200× magnification). (G) Phase contrast, 200× magnification of the clone 6A after 3 months of continuous culture. (H–J) Phase contrast, 200× magnification of the clone 6A after 6-, 12-, and 24 months of continuous culture.</p