br efficient in vivo in a
efficient in vivo, in a breast cancer bone metastasis model, and 2) if such combinatorial delivery is more beneficial than either therapeutic alone. After verifying the presence of the tumour within the tibia of mice (typ-ically 2 weeks post cancer induction), we injected 1 × 105 MSC engineered with CD (PSGL-1/SLEX/CD MSC), OPG (PSGL-1/SLEX/OPG MSC), CD/OPG (PSGL-1/SLEX/CD/OPG MSC) or Mock-transfected MSC into the tumour-bearing tibia (curative model) (Fig. 5a). PBS was injected in the control group (CT), as well as in the healthy legs (mock control for needle-induced inflammation and bone damages). We chose to use a local injection in this set of experiments, to allow for ro-bust comparison between each treatment condition. The primary goal of this intratibial treatment injection was to test the efficacy of OPG and CD treatments, not to determine MSC homing (although the thera-peutically engineered ML-210 were also equipped with PSLG-1/SLEX, which replicates our final product, to be used in the systemic infusion in the following experiments). Mice were treated with 500 mg/kg of 5-FC pro-drug at 48 hours post-implantation, by which time MSC would have been cleared from filter organs if administered systemically as in our intended future clinical use.
DAPI/RFP CD41 P-selectin Merge
Tumour Tumour Tumour Tumour
P-selectin CD41 d
P-selectin Endomucin Merge
Fig. 3. P-selectin is highly expressed in the bone metastatic niche. (a) Both high P-selectin expression and elevated megakaryocytes/platelets number are seen in the bone marrow surrounding the tumour. Red: RFP constitutively expressed by tumour cells. Yellow: P-selectin, magenta: CD41 and blue: nuclei (DAPI staining). Dashed line outlines the tumour. The area designated by a white rectangle is showed at higher magnification in panel B. Scale bar: 100 μm. (b) Platelets and megakaryocytes express high level of P-selectin. Yellow: P-selectin, magenta: CD41 and blue: nuclei (DAPI staining). Scale bar: 50 μm. (c) P-selectin is expressed at the bone marrow endothelium in the breast cancer bone metastatic environment. Yellow: P-selectin, green: endomucin (vascular endothelium) and blue: nuclei (DAPI staining). Scale bar: 50 μm. (d) P-selectin expression is higher around the tumour area than in the rest of the bone marrow. P-selectin expression was quantified from bone marrow sections of 7 mice per area of 100 μm2 from the tumour. r = Pearson correlation coefficient. * p ≤ .05, *** p ≤ .001, **** p ≤ .0001.
A pilot experiment was first performed using n = 4 mice per group. Through this initial experiment, we established techniques to confirm tumour implantation within the tibia by overlaying bioluminescence and X-Ray (Supplementary Fig. 10a) and to characterise the presence of tumour cells within the bone marrow of the tibia by using both im-munofluorescence (RFP expressed by cancer cells) and H&E staining (Supplementary Fig. 10b). From this experiment, we obtained a prelim-inary assessment on tumour killing and bone preservation among dif-ferent treatment groups and determined the minimum number of mice per group required for statistical analysis using a power analysis (see Methods). We then repeated the experiment following the exact same treatment scheme (Fig. 5a) with n = 10 mice per group. Tumour growth within the tibia was monitored using bioluminescence imaging. Animals were randomised in each group and showed comparable
tumour signal across groups before treatment (Supplementary Fig. 11). A tumour decrease was observed for animals treated with MSC engineered with both CD MSC groups, particularly CD/OPG MSC (p b .05 from Kruskal-Wallis with Dunn's multiple comparison post hoc, compared to Mock MSC), immediately following the treatment (Fig. 5b, c), but most of the tumours eventually grew back to a level comparable to control groups (PBS and Mock MSC). At the end-point, mice treated with CD/OPG MSC also exhibited smaller tumours com-pared to control groups (although the difference was not significant, due to high variability between good and bad responders). However, OPG MSC slowed the tumour growth, leading to significantly smaller tu-mours than the Mock MSC group (p b .05, Kruskal-Wallis with Dunn's multiple comparison post hoc) in the longer-term (Fig. 5b, d, Supple-mentary Fig. 11). In addition, we analysed the tumour growth data