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 neoplasia contained hyperchromatic thick collagen fibres (Figure 1d) and was connected to subcutaneous and muscle tissues which exhibited pronounced atrophy of muscle fibres substituted with connective tissue (Figure 2c). No cell types contained mitotic figures and the tumour appeared to be benign, as there was no indication of dysregulated cellular proliferation in muscle, liver and kidney. At the surface ulceration, an inflammatory reaction with exclusive presence of lymphocytes and monocytic phagocytes was present (Figure 2b). All molecular, cultivation and histological-staining diagnostic procedures in all examined samples were negative for virus (PCR and cultivation, negative), bacterial (ZN, negative) and mycotic (Grocott's, negative) organisms. This suggests that the tumour was not pathogen-induced. Spontaneous neoplasia reports in turquoise killifish Nothobranchius furzeri, a key model in biogerontology and toxicology, are exceptionally rare. A first report of an exophytic mesenchymal tumour – fibromyxoma from turquoise killifish is described here. The diagnosis is based on morphological criteria. In contrast to a papilloma, the tumour was predominantly formed by connective tissue. The tumour was firm and not soft as a myxoma would be. Namely, the tumour mass contained mostly myxoid matrix with spindle and star-shaped cells with varying degrees of pleomorphism which is in accordance with the characteristics of a fibromyxoma (Manera & Biavati, 1995). Our observations matched closely with findings from Tinca tinca (Manera & Biavati, 1995) and mullet Mugil cephalus (Lopez & Raibaut, 1981). The presence of epidermal cell islets within proliferating connective tissue distinguishes our fibromyxoma from previous reports (Singaravel et al., 2016; Stevens et al., 2017). The neoplastic connective tissue can enclose other cell types due to its fast growth, as was reported for pigment cells in fibroma of mullet (Lopez & Raibaut, 1981). We speculate that proliferating epidermal cells gradually covered connective tissue, and during ongoing myxocollagenous stroma proliferation, epidermal cells ended up inside the neoplastic tissue. This opinion is supported by the regular presence of goblet cells (rare in papillomas) and deposits of particulate material surrounded with epidermal cells. To our knowledge, this is the first report of an exophytic tumour in the Nothobranchius fishes. It has never been observed in the 15-year history of killifish breeding at the Institute of Vertebrate Biology nor during 10 field sampling campaigns. In contrast to previous assumptions (Baumgart et al., 2015; Blažek et al., 2017; Di Cicco et al., 2011), spontaneous neoplasias are rare in Nothobranchius fishes even at advanced age (Dyková et al., 2021) and there are only a few reports of indisputably spontaneous neoplasia in this genus (Cooper et al., 1983; Dyková et al., 2020; Markofsky & Milstoc, 1979a, 1979b; Stolk, 1959). The spontaneous origin of the neoplasia reported here is supported by morphological diagnosis, by the normal appearance of visceral organs (which would have changed colour or size when infected) and by the negative results of molecular, cultivation and staining diagnostic methods for pathogens. Similarly, an injury caused by the environment is unlikely due to constant health monitoring and innocuous breeding conditions. A lifespan of 12.5 months is among the longest lifespans for this species and the survival of 20% of the cohort to this age (together with negative results for pathogen presence in sentinel fish) suggests the absence of background infection in the colony. The tumour reported on here is rare, however, we cannot exclude that it may be more common in relation to chemical exposure as seen for various tumours in other fishes (e.g. Chen et al., 1996). Reporting spontaneous lesions as comparative material is of particular interest for toxicologists. The tumour grew rapidly and reached the size of the specimen's head within 3 months. It did not affect the specimen's ability to swim and eat, which is in accord with other fibromyxoma reports (Shokrpoor et al., 2017). During the tumour's growth, it changed its smooth oval shape to a more irregular shape with a small ulceration on the surface. Ulcerations are common in large tumours (Manera & Biavati, 1995; Singaravel et al., 2016). The fibromyxoma was benign as in other reports (Manera & Biavati, 1995; Shokrpoor et al., 2017). The tumour formation was likely a consequence of senescence. Organisms living in captivity beyond their natural lifespan in the wild, are more prone to neoplasia (Hochberg & Noble, 2017). This has been demonstrated in mice (Gardner et al., 1973; Pompei et al., 2001). Here, fibromyxoma started to develop at an advanced age corresponding to three times the maximum lifespan of wild N. furzeri (Žák et al., 2021) when fish had a senescent phenotype. The diagnosed skeletal muscle atrophy may have been a senescence symptom, and the fish had (likely senescence-related) signs of low body condition including lack of liver vacuolization and no visceral adipose stores. To date, undisputably spontaneous neoplasia in Nothobranchius spp. have been found in advanced age (e.g. Dyková et al., 2020; Markofsky & Milstoc, 1979a, 1979b) but are not as frequently as previously assumed (e.g. Blažek et al., 2017). Fibromyxoma in humans is frequently diagnosed by immunohistochemical methods (Mocellin, 2021; Pittman & Montgomery, 2015; Song et al., 2016). We did not employ immunohistochemical analysis as this method is rarely suitable for tumours in teleost fishes and currently cannot be compared to mammalian findings (Groff, 2004; Vergneau-Grosset et al., 2017). On the other hand, the microanatomical appearance of the tumour clearly supports our diagnosis of fibromyxoma. To our knowledge, this report describes the first known example of an exophytic fibromyxoma in turquoise killifish. We assume that tumour formation was attributed to senescence because it developed at an advanced age and was unassociated with pathogens. These findings provide new evidence of spontaneous neoplasia in Nothobranchius furzeri, a species previously suspected to be tumour-prone. Conceptualization: JZ; Methodology, Validation, Formal analysis, Investigation, Resources: JZ, ID, LP; Data Curation: JZ, Writing-Original Draft: JZ, ID; Writing-Review and Editing: JZ, ID, LP; Project Administration: JZ, ID. We thank Ingrid Steenebergen for the English correction. Open access publishing facilitated by Masarykova univerzita, as part of the Wiley - CzechELib agreement. This research was supported by Masaryk University Specific Research (MUNI/A/1602/2023) and the Ministry of Agriculture of the Czech Republic MZE-RO0523. The authors declare that they have no competing interests. All data are presented in this work; therefore, no data are stored in a repository. For more details, please contact the corresponding author. Full size and resolution pictures from this paper can be found at Figshare repository 10.6084/m9.figshare.26833831.