Establishing jadomycin B toxicity in zebrafish larvae. Casper zebrafish larvae (48 h post-fertilization) were treated with 10–80 µmol/L jadomycin B (Jad (b) or vehicle control (1:7 MeOH:H₂O) for 120 h. The number of living versus dead larvae was recorded every 24 h and expressed as the % survival. Error bars represent the mean ± SEM of three to five independent data sets.^**P < 0.01 and ^***P < 0.001 compared to the corresponding vehicle control at the same time point, two-way ANOVA with Bonferroni multiple comparison test.

Jadomycin B inhibits proliferation of MDA-MB-231 cells in zebrafish larvae. MDA-MB-231 breast cancer cells were labelled with CellTracker™ CM-Dil (yellow) fluorescent dye and transplanted into the yolk sacs of 48 h post-fertilization casper zebrafish larvae. CM-Dil positive larvae were treated with vehicle control (1:7 MeOH:H₂O), or jadomycin B at final concentrations of 10, 20, and 40 µmol/L. The fold change in MDA-MB-231 cells as determined by live cell imaging of zebrafish larvae 48 h post-treatment (hpt) (A). Error bars represent the mean ± SEM (n = 20 larvae per group). *P < 0.05 compared to the vehicle control, one-way ANOVA followed by Bonferroni multiple comparison test. Representative brightfield (left) and accompanying fluorescent (right) imaging of live larvae comparing vehicle and 40 µm jadomycin B treatment at 72 hpt and 120 hpt (B). Images are representative of 20 treated larvae per group.

Jadomycin B serum concentration versus time in female Balb/C mice. Adult female Balb/C mice were treated with a single intraperitoneal dose (6 mg/kg) jadomycin B followed by serial sample collections at the indicated time points. Each time point represents the mean serum concentration ± SEM (n = 5).

Jadomycin B reduced 4T1 primary tumor growth. Adult female Balb/C mice were injected with 100 000 4T1 cells into the fourth mammary fat pad on day 0, followed by jadomycin B (Jad B) treatment (12 mg/kg every 12 h) from day 6 to 15 after tumor cell implantation. Tumor volumes were measured every 2 days (A). At the study endpoint, tumors were excised and weighed (B) and the number of lung metastasis were determined by the 6-thioguanine colony forming assay (C). The data in panel A are shown as mean ± SEM (n = 8). ^***P ≤ 0.001, compared to the respective vehicle control, as determined by a two-way repeated measures ANOVA, with Bonferroni multiple comparison test. The data in panels B and C are shown as scatter plots with mean ± SEM. The groups were compared using a non-parametric Mann–Whitney test.

Jadomycin B did not cause significant toxicity in female Balb/C mice with 4T1 tumors. Adult female Balb/C mice were injected with 100 000 4T1 cells into the fourth mammary fat pad on day 0, followed by jadomycin B (Jad B) treatment (12 mg/kg every 12 h) from day 6 to 15 after tumor cell implantation. Beginning on day 6, mouse weights were measured every 2 days (A). At the study endpoint, serum was collected and analyzed for alanine amino transferase (B) and creatinine (C) levels as markers of hepatic and renal toxicity, respectively. The data are shown as mean ± SEM (n = 8). ^***P ≤ 0.001, compared to day 6, as determined by a two-way repeated measures ANOVA, with Bonferroni multiple comparison text (A). There were no significant differences in serum ALT (B) or creatinine (C) between groups, unpaired t-test.

Jadomycin B did not deplete overall T cell numbers but may impact a subset of T cells. Adult female Balb/C mice were injected with 100 000 4T1 cells into the fourth mammary fat pad on day 0, followed by jadomycin B (Jad B) treatment (12 mg/kg every 12 h) from day 6 to 15 after tumor cell implantation. On day 16, animals were euthanized for T cell immunological analysis of the primary tumor, draining lymph nodes and spleen. The frequency of CD4+ TCRβ+ T cells (A), immune-activated CD69+ CD4+ T cells (B), CD8+ TCRβ+ T cells, (C) and immune-activated CD69+ CD8+ T cells (D) in the spleen, draining lymph nodes, and primary tumors were measured. The data are shown as mean ± SEM (n = 8). *P ≤ 0.05 indicates significant difference from vehicle control by unpaired t-test.