Nxg mice

Eight-week old female NXG mice (Janvier Labs, Le Genest-Saint-Isle, France) were kept in a pathogen free facility with 12 h light/dark cycle and they were daily supervised by husbandry personnel. The mice were randomly designated to experimental groups of five animals. To evaluate in vivo performance of RevCAR T-cells, HT-29 CEA^HIGH Luc⁺ cells (1x10⁶) were injected subcutaneously with or without RevCAR T-cells (0.5x10⁶) and RevTM (200 pmol) in a final volume of 100 µl PBS. Following a similar experimental set-up, the performance of Dual-RevCAR T-cells in vivo was assessed by injecting subcutaneously HT-29 CEA^HIGH Luc⁺ cells (1x10⁶) without or with Dual-RevCAR T-cells (1x10⁶) in the absence, presence or combination of RevTMs (100 pmol) in a final volume of 100 µl PBS. To measure the luciferase-activity of tumor cells, anesthetized mice were subcutaneously injected with 150 µl D-Luciferin and optically measured with the InVivo Xtreme (Bruker, Nehren, Germany) or the IVIS Spectrum In Vivo Imaging System (PerkinElmer, Rodgau, Germany) at several time points as previously described (36 (link)). All animal procedures have been approved by the local ethics committee for animal experiments and were performed at the Helmholtz-Zentrum Dresden-Rossendorf (HZDR) in compliance with the German regulations of animal welfare.

In vivo experiments involving optical imaging of experimental mice were carried out according to the guidelines of the German Regulations for Animal Welfare. The protocol was approved by the local Ethical Committee for Animal Experiments (AZ DD24.1–5131/449/67). To assess anti-tumoral effect of UniCAR T-cells in combination with αFAP TMs, a co-injection experiment was conducted using 9-week old female NXG mice (Janvier). Each group was composed of five mice and a total of four groups were used. All the groups were injected with 0.5 × 10⁶ of HT1080 hFAP target cells. Target cells alone or in combination with UniCAR T-cells (E:T ratio of 1:1) were used as control groups. As treatment groups, mice were injected in addition with 300 pmol of either αFAP-scFv or -IgG4 TMs. Bioluminescence imaging of anesthetized mice was followed for up to 10 days using IVIS Spectrum In Vivo Imaging System (PerkinElmer) and analyzed using the Living Image Software (PerkinElmer).