*2.19. Immunodeficient Mice Study*

We generated HCC1954 cells expressing luciferase in order to implement bioluminescent imaging analysis to follow breast tumour growth in small animal models in vivo. Briefly, HCC1954 cells were transduced at multiplicity of infection MOI 10 with a third-generation self-inactivating lentiviral vector expressing firefly luciferase [46]. Six-week-old CB17SCID female mice were purchased from Charles River (Calco, Italy) and housed with laboratory chow and water available ad libitum.

A cell-line derived orthotopic xenograft model of breast cancer was established by mammary gland implantation of 5 × 10<sup>5</sup> HCC1954 luciferase-expressing cells. Mice were regularly palpated and tumour dimensions were measured once a week using a digital calliper. Moreover, tumour cell engraftment and early detection of tumour growth was assessed by longitudinal bioluminescent analysis (BLI). BLI analysis has been performed using the IVIS® Lumina II equipped with the Living Image® software for data quantification (PerkinElmer). Animals were sedated and D-luciferin (PerkinElmer) dissolved in PBS (150 mg/kg body weight) was administered i.p. 10 min before analysis [47]. Photons emitted from luciferase expressing HCC1954 cells implanted into the animals were collected with final accumulation times ranging from of 1 s to 1 min, depending on the intensity of the bioluminescence emission. All animal experiments were conducted in accordance with institutional guidelines, in the full observation of the Directive 2010/63/UE.
