Intratumoral Injection of Fusobacterium nucleatum

For this, 7–8-week-old in-house-bred male and female nod scid gamma (M. musculus NSG) mice were subcutaneously injected with 1 × 10⁶ HT-29 tumour cells. When tumours reached a size of approximately 200 mm³, 5 × 10⁷Fn ATCC 25586 or formate (10 mM) was injected intratumorally (three injection sites per tumour) every other day for 10 d (for a total of five injection time points). Xenografts were harvested at endpoint, weighed, subjected to TIF extraction as previously described^{73 (link)} and dissociated (Human Tumour Dissociation Kit, Miltenyi) for further FACS-based protein expression analysis. For serial transplantation of Fn-injected xenografts, primary tumours were explanted, dissociated in culture and reinjected in secondary recipient mice, as previously described^{28 (link)}. Tumour incidence and growth was monitored over time blindly by palpation of the flanks. The maximal total tumour volume of 2,000 or 1,500 mm³ per single tumour permitted by the AEEC was not exceeded.