Fischer f344 rats

Fischer F344 rats were purchased from Charles River Laboratories (L’Arbresle, France), and maintained under SPF status and standard conditions in the UTE-IRS UN animal facility of the University of Nantes in compliance with the European Union guidelines for the care and use of laboratory animals in research protocols (Agreement #01257.03). All experiments were approved by the ethics committee for animal experiments of the Pays de la Loire Region, France (CEEA.2011.38 and CEEA.2013.7.). In order to follow the recommendations on the welfare and use of animals in cancer research, particular attention was given to incorporating the objectives of the 3Rs (replacement, reduction, and refinement) [67 (link)]. The four neoplastic cell lines (M5-T2, F4-T2, F5-T1, and M5-T1) were injected into syngeneic rats and tumors were collected and fixed as previously described [16 (link),17 (link)]. The rats were anesthetized in an isoflurane chamber (Forene^®, Abbott, France) and euthanized with a rate of 30% volume displacement per minute of CO₂ into their home cage.

A total of 56 female Fischer F344 rats (Charles River, L’Arbresle Cedex, France) were acclimatized for at least 1 week before start of the experiments. The rats, weighing 170 ± 10 g, were housed in cages (Techniplast, Milan, Italy) with goldflakes bedding (SPPS, Frasne, France) and environmental enrichment, in a temperature-controlled environment with a 12-hour light/dark cycle. Rats had free access to standard chow and water. Daily, the rats were weighed and monitored for well-being. Animal procedures were performed according to protocols, approved by the Institutional Animal Care and Use Committee (IACUC), committee for animal experiments (Radboud University Nijmegen Medical Centre, The Netherlands) and were in compliance with Dutch and European regulations. Group size was calculated using an α of 0.05, a power of 80 and 80% tumor development. The expected therapy effect was estimated to be 50% considering a pathological complete response (pT0) as primary endpoint. This resulted in a minimal group size of 14 rats.

Fischer F344 rats were obtained from Charles River Laboratories (L’Arbresle, 69, France) and maintained under standard conditions in the UTE IRS-UN animal-holding area, in agreement with European Union guidelines for the care and use of laboratory animals in research protocols. The experiments were approved by the regional ethical committee for animal experiments (CEEA.2011.38, CEEA.2013.7, and project n° 01257.03). Rats were fed a pelleted standard diet (RM1, Special Diet Services, Witham, Essex, UK) with tap water ad libitum, anesthetized via an isoflurane chamber (Forene^®, Abbott France) and euthanized with Dolethal^® (Centravet, Pluduno, Plancoët, France) for experiments E2-E3.
The M5-T1 mesothelioma cell line used in this study was selected from a biocollection established in 2011 [
https://migratech.inserm-transfert.fr/srv/tech/2/index100.asp?cl=CL] after 136 to 415 days of induction with 10 mg crocidolite fibers suspended in 0.5 ml 0.9% NaCl, administered intraperitoneally to rats (UICC analytical sample, ref. 02704A, Neyco, 75017 Paris, France). After 378 days of induction, one male rat was necropsied about one hour after death, presenting signs of hemorrhage, widespread neoplastic implants and nodules in the peritoneal cavity, as previously described [30 (link)], which, when dissociated with a scalpel and cultured, led to the M5-T1 cell line.

F98 Glioma cells (ATCC, 20 000 in 5 μl cell suspension) were inoculated 2 mm posterior and 2.5 mm lateral to the bregma in the right frontal hemisphere of female Fischer F344 rats (Charles River^®) (n = 10, body weight 173 ± 11 g, mean ± SD). Full details of the protocol can be found in our previous publications [43 (link)–45 (link)]. For inoculation, rats were anesthetized with ketamine/xylazine (4/3; 0.13 ml/100 g). Post-surgery, a close follow-up of the animals was performed (body temperature, wound healing and behavior). Animals were kept separately post-inoculation.

Guidelines for Care and Use of Laboratory Animals were followed during the investigation. All animal procedures were approved by the Ethical Commission of the University of Cagliari and the Italian Ministry of Health. Male Fischer F-344 rats (100-125 g) purchased from Charles River (Milan, Italy) and NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice (γNull) were originally by Jackson Laboratories (Bar Harbor, Maine, USA). Animals were kept on a laboratory diet (Ditta Mucedola, Milan, Italy) and given food and water ad libitum with a 12-hour light/dark daily cycle.
HCC was induced according to the Resistant Hepatocytes (R-H) model [7 (link)]. Rats were injected intraperitoneally with the chemical carcinogen diethylnitrosamine (DENA, Sigma, MO) at a dose of 150 mg/kg body weight. After a 2-week recovery, rats were fed a diet containing 0.02% 2-acetylaminofluorene (2-AAF, Sigma, MO) for 1 week followed by a two-thirds partial hepatectomy (2/3 PH), and an additional week of 2-acetylaminofluorene diet. The animals were then returned to the basal diet and euthanized at 14 months (Supplementary Figure 1). Rats exposed to 2-AAF and 2/3 PH, but in the absence of carcinogen, were used as controls.