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. 2019 Jul 4;45:39–57. doi: 10.1016/j.ebiom.2019.06.047

Fig. 7.

Fig. 7

CD/OPG MSC exhibit therapeutic effects and minimal toxicity in a syngeneic mouse model of spontaneous bone metastasis. (a) Timeline of the therapeutic treatment. Spontaneous bone metastases were induced by caudal artery injection of a 4T1 bone metastatic clone (CLL1) to BALB/cJ mice. Once bone metastases were detected, engineered MSC or PBS were systemically administered to animals via i.v. injection, and 5-FC was injected i.p. for 5 consecutive days 48 h post transplantation. 12.5 mg/kg 5-FU was used as a reference group for treatment. MSC were engineered as follows: Mock group (Mock transfected), CD group (PSGL-1/SLEX/CD), OPG group (PSGL-1/SLEX/OPG) and CD/OPG group (PSGL-1/SLEX/CD/OPG). PBS, Mock and 5-FU groups: n = 10 animals per group. CD, OPG and CD/OPG groups: n = 13 per group. (b) CD/OPG MSC inhibits tumour growth compared to PBS control group. Bioluminescence imaging was performed over time and signal was quantified in the lower body to measure bone metastases development in legs and spine. The median of the tumour growth ratio (total photon flux over time normalised to total photon flux before treatment) was plotted for each group. Statistical analysis done at the end-point: Kruskal-Wallis with Dunn's multiple comparison post hoc, * p ≤ .05 between PBS and CD/OPG MSC. (c) CD/OPG treatment improves mouse mobility. Paralysis of the animals was scored at the end-point (see Methods). Bar graph shows the median score for each group, and each point represents one animal. Statistical analysis: Kruskal-Wallis with Dunn's multiple comparison post hoc, * p ≤ .05 between PBS and CD/OPG MSC. (d) Femurs bearing bone metastases are less damaged in CD/OPG MSC treated group. Micro-CT analysis was done on mouse femurs exhibiting clear leg metastases (usually around the hip area) before treatment from 6 mice per group. 3D reconstructions were made from the whole femurs. (e) Bone damage was blindly scored by a panel of 20 unbiased persons (shaft damage from 0 to 3 and epiphysis damage from 0 to 5 based on importance of damage). The average scoring was shown for each animal. Bar graph represents the median score for each group, and each point represents one animal. Statistical analysis: Kruskal-Wallis with Dunn's multiple comparison post hoc, * p ≤ .05 between PBS and CD/OPG MSC. (f) CD/OPG MSC and OPG MSC treatments seem to protect against bone loss. The total bone volume was quantified for each femur. Bar graph shows the median bone volume for each group, and each point represents one animal. Statistical analysis: Kruskal-Wallis with Dunn's multiple comparison post hoc. (g) 5-FU treatment, but not MSC groups, induces significant body weight loss. Graph shows the median body weight loss for each group over time. Statistical analysis done at the end-point: Kruskal-Wallis with Dunn's multiple comparison post hoc, ** p ≤ .01 between PBS and 5-FU. (h) Only 5-FU treatment induces additional apoptosis in the bone marrow. At the end-point, the bone marrow of the healthier leg was isolated and cell apoptosis was analysed using flow-cytometry (AnnexinV+/7-AAD-) for n = 10 animals per group. Bar graph shows the median percent of apoptosis for each group, and each point represents one animal. Statistical analysis: Kruskal-Wallis with Dunn's multiple comparison post hoc, * p ≤ .05 between 5-FU and CD/OPG MSC, and ** p ≤ .01 between PBS and 5-FU.