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. 2023 Sep 23;15(19):4693. doi: 10.3390/cancers15194693

Figure 2.

Figure 2

In vivo characterization of M1, M2, and M1-derived novel transplantable mouse HR-NB cell lines. (A) Efficiency of cell lines in forming primary tumors. C57Bl6 mice were injected subcutaneously (0.5 million cells/mouse, n = 4 animals per group), and their tumor sizes were measured twice a week until they were euthanized due to their tumor burden (statistical test—non-linear regression model, F-test, 9464D vs. M1-2, p-value <0.0001, and 9464D vs. M1-6, p-value <0.0001, 9464D vs. M1, M1-1, M1-3, M1-5, and M-7, p-value—ns (0.692), 9464D vs. M2 p-value—ns (0.199)). (B) Tumor sizes at day 30 post-cell-injections (statistical test—Welch’s ANOVA test, mean ± SEM, p-values: 9464D vs. M1—ns (0.232), 9464D vs. M1-1—ns (0.999), 9464D vs. M1-2— ** (0.002), 9464D vs. M1-3—ns (0.989), 9464D vs. M1-5—ns (0.999), 9464D vs. M1-6— * (0.030), 9464D vs. M1-7—ns (0.999) and 9464D vs. M2—ns (0.899)). (C) Survival affected by primary tumors—Kaplan–Meier analysis, log-rank (Mantel–Cox) test, 9464D vs. M1-2: p-value—0.010, 9464D vs. M1-6: p-value—0.010, 9464D vs. M1, M1-1, M1-3, M1-5, M1-7, and M2: p-value—ns (0.4380). (D) Ability of cells to form metastasis using an experimental metastasis approach. Metastasis monitored via bioluminescence in vivo imaging (in vivo bioluminescence image—week 4 post-cell-injection, 1 million cells/mouse IV-injected (the tail vein), n = 5 animals/group). Although some of the mice lost luciferase expression (luminescence), all of them developed metastases. (E) Mortality due to metastasis for the mice shown in (D)—Kaplan–Meier analysis, log-rank (Mantel–Cox) test, p-value <0.0001. (F) Tumor sites, fraction of total mice IV-injected with NB cells, with subsequent metastasis in the indicated location, represented as percentages (M1 cells: bone—20%, liver—60%, and lung—20%; M2 cells: bone—20%, liver—80%, and lung—0%; and M1-2 cells: bone—73%, liver—9%, and lung—18%).