Spontaneous exophytic tumour in <i>Nothobranchius furzeri</i>, an aging research model

Understanding the nature and diversity of spontaneous neoplasias in a model organism is of peculiar interest to toxicologists and biogerontologists who assess their prevalence in relation to experimental treatments (Wolf et al., 2015). In fishes, exophytic neoplasias are frequently found due to their conspicuous appearance on the body (Roberts, 2012; Vergneau-Grosset et al., 2017). Papillomas are the most common exophytic epithelial fish tumours with a predominant proliferation of epidermal cells with no to little presence of connective tissue (Roberts, 2012). Other exophytic tumours are derived from connective tissue as in fibromas, myxomas and fibromyxomas. Myxomas are soft tumours formed by immature connective tissue with extensive myxoid stroma. In contrast, a fibroma is a firm tumour derived from fibrous connective tissue (Roberts, 2012). Fibromyxomas share some traits of myxoma and fibroma and commonly consist of proliferating connective tissue characterized by neoplastic growth of stellate and spindled cells within the loose, collagenous and myxoid matrix with a low number of mitotic figures (Manera et al., 1997; Pittman & Montgomery, 2015; Vergneau-Grosset et al., 2017). They are typically well-vascularized and are connected to the subcutaneous musculature (Mocellin, 2021; Song et al., 2016). The prevalence of neoplasia in teleost fishes, including laboratory model organisms, is disproportionally biased due to frequent confusion with pathogen-induced lesions (Dyková et al., 2021; Harshbarger, 1984). Therefore, it is always important to test for the presence of known pathogens in the examined animal with neoplasia. Here, we report a case of spontaneous fibromyxoma, in senescent laboratory-kept turquoise killifish Nothobranchius furzeri. Turquoise killifish are a rapidly aging species with a life expectancy of several months in the wild because they inhabit seasonally inundated pools in Southeast African savanna (Reichard & Polačik, 2018). In captivity, they live approximately 1 year; therefore, they have become a popular model organism in biogerontology and toxicology (Cellerino et al., 2016; Thoré et al., 2021). A male 12.5 months old Nothobranchius furzeri (50 mm SL, strain MZCS 222; Cellerino et al., 2016) was examined. The fish was housed individually in a 2 L tank in a recirculation system (water temperature 27.5°C, water conductivity 1.5 mS × cm−2) with 188 other fish of the same age at an accredited (12,210/2018-MZE-17214) breeding facility of the Institute of Vertebrate Biology, Czechia. The fish was euthanized by an overdose of clove oil when it was unable to dive (a common symptom in senescent N. furzeri; Naumann et al., 2023). The tumour tissue sample (5 × 8 mm) was stored at −80°C until virological examination. During the fish dissection, internal organs (liver, kidney, heart, digestive tract) had a normal gross appearance and they were processed for paraffin histology (Žák et al., 2022) together with the lesion. Tissues were stained by H&E, Mason's trichrome, periodic acid Schiff reaction (PAS) and alcian blue. To test for possible infectious agents of the neoplastic lesion, tumour tissue was screened by nested PCR for cyprinid herpesviruses (Engelsma et al., 2013) and tested for cultivatable viruses on Common Carp Brain (CCB, Neukirch et al., 1999), Epithelioma Papulosum Cyprini (EPC) and Bluegill Fry-2 (BF-2, Lorenzen et al., 1999) cell lines. Paraffin sections from (a) the neoplasia under study and (b) from the kidneys of four (age 3, 4, 6 and 11 months) sentinel fish sharing the recirculation system with the specimen were stained by Ziehl-Neelsen (ZN), Gram and Grocott stains to detect bacterial and mycotic organisms respectively in the neoplasia. Kidneys were chosen for pathogen diagnostics as they were found to be the most frequently affected organ by histopathological changes in this species (Dyková et al., 2021; Naumann et al., 2023). The research followed all relevant Czech national (nos. 246/1992 and 419/2012) and international (Directive 2010/63/EU) recommendations and legislative regulations in relation to the protection of research animals ensuring their welfare. Daily care and monitoring of health status followed established recommendations for this species (Naumann et al., 2023). The lesion first appeared at the age of 9 months as a 2 × 1 × 1 mm sized pale-grey ellipsoid nodule on the dorsal left side of the fish. Two months later, the nodule reached 9 × 6 × 6 mm in size, felt rigid to the touch like rubber, and displayed visible blood vessels. It had a pale-grey colour and a smooth surface with no visible ulceration. At the age of 12.5 months the mass became irregular in shape with an uneven surface and enlarged to 13 × 12 × 10 mm with a base of approximately 5 × 6 mm (Figure 1a). A single locus of ulceration covering approximately 10% of the mass was present on the tumour surface. The cut surface was homogeneous, rigid and identical in colour to the rest of the tumour. All organs in the body cavity were of normal size. The fish was senescent with light coloured skin, a right-eye cataract, curved spine and loss of muscle mass. At this age, 80% (149/188) of the same age cohort died due to aging. The detailed histological examinations of the lesion supported its neoplastic nature with typical traits of fibromyxoma (Figure 1b–e). Semi-serial sections showed prevailing neoplastic growth of immature myxocollagenous or myxoid connective tissue with abundant alcian blue positive myxoid matrix and stellate or spindled fibroblasts (Figure 1d,e). Islets of epidermal cells were embedded within the mass of non-mature connective tissue and included PAS-positive goblet cells (Figure 1c). In some places, epidermal cells are surrounded foci of necrotic or unidentifiable particulate material (Figure 2a). The periphery of the lesion consisted of a thin connective tissue layer and a discontinuous layer of squamous epidermal cells (Figure 1b,c). The broad base of the neop