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  • br d Engineered MSC did

    2022-05-06


    (d) Engineered MSC did not induce significant tissue damage. Quantifications were done on the H&E staining: percentage of connective tissue to assess spleen fibrosis, number of goblet MK-571 per villus to evaluate intestine damage, and number of leukocytes per bone marrow area to measure toxicity. Bar graph shows the median for each group, and each point represents one animal, n = 4 mice per group. Eng MSC = PSGL-1/SLEX/CD/OPG MSC. (e) Engineered MSC did not lead to significant cell death in the bone marrow. Flow cytometry was performed on bone marrow to analyse the percentages of viable, apoptotic and necrotic cells. 2 to 3 animals were used for each group, and both legs were analysed. As no major differences were observed between the healthy and the tumour leg, data from both legs were pooled. Bar graph shows the median for each group, and each point represents one analysed leg. Statistical analysis: Kruskal-Wallis followed by a Dunn's multiple comparison test among each group (viable, apoptotic and necrotic) to compare all conditions to the control; * p ≤ .05, ** p ≤ .01. (f) Engineered MSC did not significantly alter cell composition of the bone marrow. Flow cytometry was performed on equal numbers of bone marrow cells to analyse the different populations: monocytes/macrophages (Mono/Macro), granulocytes and B lymphocytes (B220low and B220high). 2 to 3 animals were used for each group, and both legs were analysed. As no major differences were observed between the healthy and the tumour leg, data from both legs were pooled. Bar graph shows the median for each group, and each
    point represents one analysed leg. Statistical analysis: Kruskal-Wallis followed by a Dunn's multiple comparison test among each population to compare all conditions to the control; * p ≤ .05, **** p ≤ .0001.
    monotherapy (p b .05, Kruskal-Wallis with Dunn's multiple comparison post hoc), and also tends to be more efficient than OPG MSC therapy (p
    = .0638, Kruskal-Wallis with Dunn's multiple comparison post hoc). Moreover, mice treated by CD/OPG MSC seemed healthier than other 
    groups overall, with less moribund mice, better grooming and ability to move around, at the end-point (Supplementary Video 4). Due to the aggressiveness of this model, mouse condition deteriorated ex-tremely rapidly (paraplegia and diarrhoea due to spine metastasis,
    weight loss, and tumours breaking through the cortical bone). We de-cided to score the paralysis to quantify this effect, and confirmed that CD/OPG MSC treated mice were less paralysed than mice in PBS control group (p b .05, Kruskal-Wallis with Dunn's multiple comparison post hoc), with the majority of mice being free of movement, while the ma-jority of animals in other groups were dragging their hind limbs (Fig. 7c). We then examined treatment effects on tumour-induced bone damage for those animals, and randomly picked 6 animals per groups, which possessed clear bone metastatic signal coming from the leg before the treatment. As a majority of bone metastases found in 
    the hind limbs were located in the hip and femur area, we decided to focus on the femurs, instead of the tibias, for this model (Fig. 7D, Supple-mentary Video 5). For each sample, we asked twenty people (75% of them not being familiar with this study or not doing research) to blindly score the shaft damage and the epiphysis damage. The scoring matched the 3D reconstructions, with significantly less bone damage in the group treated with CD/OPG MSC (p b .05 analysed by Kruskal-Wallis with Dunn's multiple comparison post hoc test, between PBS and CD/OPG groups, Fig. 7e). In addition, we quantified the total bone volume and the remaining trabecular bone in the epiphysis. CD/OPG MSC and OPG
    a  Tumour growth monitoring (IVIS)
    b
    Tumour induction
    PBS or MSC treatment
    Tumourgrowthtobeforetreatment) 1,500
    PBS
    Mock
    CD
    OPG
    CD/OPG
    Time (days)
    PBS
    MOCK MSC
    CD MSC
    OPG MSC
    CD/OPG MSC
    Euthanasia
    score
    Paralysis End-point 1.5
    PBS Mock CD OPG CD/OPG 5-FU
    e
    Bonedamage score(A.U.)
    PBS Mock
    CD OPG CD/OPG
    f
    volume
    Total
    PBS Mock
    CD OPG CD/OPG
    g
    (% body weight) Weight loss 
    90 Mock MSC
    CD MSC
    CD/OPG MSC
    Time (days)
    marrow)
    (% of total bone Apoptotic cells 
    MSC tend to have more total bone volume than the PBS control (al-though the results were not significant) (Fig. 7f). Similarly, animals treated with CD/OPG MSC tended to have more remaining trabecular bone than the PBS control group (p = .0748 analysed by Kruskal-Wallis with Dunn's multiple comparison post hoc test, Supplementary Fig. 16d).