D luciferin

As previously described [33 (link)], MNNG-HOS cells (American Type Culture Collection CRL-1547) were used to induce a primary tumor against the periosteum, behind the tibialis anterior muscle, of four-week-old female athymic mice (NMRI nu/nu; Elevages Janvier, Le Genest St Isle, France). They were injected with 1 × 10⁶ MNNG-HOS cells alone, or with 5 × 10⁵ OSDC or MSC in 50 μL PBS. The tumor volume (mm³) was calculated with the formula (l² × L)/2 where l and L represent the smallest and the largest diameters (mm) respectively, measured by using a vernier caliper. Luciferase-expressing MNNG-HOS cells were obtained using lentiviral production and transduction as described by Rousseau et al. and they were injected in vivo as described above [34 (link)]. On the basis of 25  g weight, mice were intraperitoneally injected with 250 μl of a Rompun-Ketalar solution (8% and 13%, respectively, in PBS) before intraperitoneal injection of 3 mg d-luciferin (Interchim, Montluçon, France) dissolved in 250 μL of water. After 7 min, mice were sacrificed for quick extraction of the lungs, which were placed into a photon Imager NightOWL LB 981 (Berthold technologies, Thoiry, France). Bioluminescence acquisition was performed twice for 1.5 min each. Photons released from luciferin degradation were measured as counts per second (cps) per selected area.

Animal housing and surgical procedures were conducted according to the guidelines of the French Agriculture Ministry (APAFIS 8629-2017011915305978) and were approved by the local animal ethics committee (CEEA-PdL n°6 (pour Comité d’éthique en expérimentation animale_Pays de la loire n°6)). BALB/c mice were purchased from Janvier Laboratories and kept in the UTE Nantes SFR Bonamy animal facility. 4T1-luc2 cells were harvested and resuspended in PBS for animal inoculation. Metastasis spreading was monitored using bioluminescence (Biospace Imager) by injecting 100µl of D-luciferin (Interchim) at 33.33mg/ml through intraperitoneal route. 4T1-luc2 cells (0.25x10⁶/mouse in PBS) were injected into the fat pad of mammary gland of 8-week-old BALB/c female mice (day 0). Mice were treated with αChemR23 or hIgG1 mAbs intraperitoneally at 1mg/kg 3 times a week for 3 weeks from day 7 to day 28. At day 13, primary tumors were surgically removed from mammary glands. Tumor spreading was analyzed by overall survival and by in vivo bioluminescence on lungs every week using a bioimager. Mice were euthanized when critical endpoints were reached according to criteria defined by ethical committees and lungs were harvested for further experiments including transcriptomic analysis (Nanostring technology and RT-qPCR) and flow cytometry, as previously described.

Groups of four to five mice were anesthetized with isoflurane. 2 × 10⁵ or 2 × 10⁶ iPSCs in 100 μl ES medium with ROCK inhibitor (Y27632; Sigma‐Aldrich, St. Louis, MO, https://www.sigmaaldrich.com) were injected into the retro‐orbital sinus. Teratoma formation was monitored after 2 weeksand then every week until the experiment was stopped by sacrificing the mice. Luciferase expression was performed as follows: 150 mg/kg of d‐luciferin (Interchim, Montluçon, France, http://www.interchim.com) was injected intraperitonally. After 10 minutes, mice were anesthetized by isoflurane and placed into a photon bioimager (Biospace Laboratory, Paris, France, http://www.biospacelab.com) for approximately 20 minutes to acquire luminescence images. Bioluminescence detection in cells in 96‐well plates was performed after d‐luciferin was added directly in the wells (3 mg/l). Ex vivo organ imaging was carried out after the organs were incubated with d‐luciferin (3 mg/l) in PBS for 5–10 minutes at room temperature.

p16^+/luc mice experienced the physiological aging (n = 3) or irradiation (n = 3–6) protocol as described above. Transgene activation, an index of p16 expression, was visualized by bioluminescence. Briefly, 4.5 mg of luciferin (d‐Luciferin, K⁺ Salt, Interchim) was injected intraperitoneally before the acquisition of emitted bioluminescence (BLI; ph/s/cm²/sr) by a CCD (charge‐coupled device) camera (PhotonIMAGER Optima—Biospace). Bioluminescence was measured at 6, 12, 18, and 24 months of age or once a month from 3 to 13 months after irradiation.