Anthracycline-free tumor elimination in mice leads to functional and molecular cardiac recovery from cancer-induced alterations in contrast to long-lasting doxorubicin treatment effects

Figures

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  Fig. 1. Establishing a melanoma cancer model in mice enabling cancer cell specific and anthracycline-free tumor elimination. A B16F10 melanoma cells stably expressing herpes simplex thymidine kinase and luciferase (B16F10HSVtk-c) were treated with Ganciclovir (GCV, 10 µM) for 72 h in vitro. B Exemplary images from male C57BL6/N injected with B16F10HSVtk-c; tumor growth was observed via in vivo imaging system (IVIS). Mice were treated with ganciclovir (GCV) starting around day 16 after B16F10HSVtk-c injection and treatment was continued until signal loss in IVIS. C, D Cardiomyocyte cross-sectional area (CSA) after WGA staining and hematoxilin/eosin staining in left ventricular cryosections in age-matched controls without cancer cell injection and without GCV treatment (PBS-m, n = 6), healthy age-matched controls without cancer cell injection and with GCV treatment (GCV-m, n = 6), tumor-bearing mice with advanced tumor burden (HSVtk-m, n = 6), mice recovered from B16F10HSVtk-c-induced tumor burden after GCV treatment (Rec-m, n = 6) and mice recovered from B16F10HSVtk-c-induced tumor burden after GCV treatment. E Survival of mice after injection with B16F10HSVtk-c with (Rec-m, n = 15) or…

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  Fig. 2. Cardiac metabolic phenotype after anthracycline-free tumor elimination and recovery from cancer. A Representative whole body and long-axis myocardial 18F-FDG PET/CT images, with graph summarizing quantitative uptake (percentage injected dose per gram tissue [%ID/g]) from healthy control mice (PBS-m, n = 9) and mice recovered from B16F10HSVtk-c induced tumor burden after GCV treatment (Rec-m, n = 7). B Determination of plasma insulin levels in PBS-m (n = 12), GCV-m (n = 5), HSVtk-m (n = 12) and Rec-m (n = 7). C, D Representative western blot images of phospho-(Ser473) and total AKT from LV tissue of PBS-m (n = 6), GCV-m (n = 7), HSVtk-m (n = 5) and Rec-m (n = 8) and corresponding quantification normalized on Ponceau staining. E Quantitative mRNA levels (qRT-PCR) of Irs2 in LV tissue from PBS-m (n = 13), GCV-m (n = 7), HSVtk-m (n = 11) and Rec-m (n = 7) normalized to 18S. F, G Representative western blot images of CD36 from LV tissue of PBS-m (n = 11), GCV-m (n = 7), HSVtk-m (n = 9) and Rec-m (n = 8) and corresponding quantification normalized on Ponceau staining. Data are depicted as mean ± SD, *P < 0.05, **P < 0.01 vs. respective non-tumor group and #P < 0.05, ##P < 0.01 vs.…

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  Fig. 3. Physiological and pathophysiological stress tests after recovery from cancer. A Grip strength comparison between HSVtk-m (n = 11) at high tumor burden timepoint and accordingly timed PBS-m (n = 11). B Grip strength comparison between PBS-m (n = 15), GCV-m (n = 7) and Rec-m (n = 9). C Systolic blood pressure at chronic acetic acid (n = 9) and AngII stimulation in PBS-m (n = 7), GCV-m (n = 6) and Rec-m (n = 4). D Cardiac 18F-FDG quantitative uptake (percentage injected dose per gram tissue [%ID/g]) in GCV-m (n = 6) and Rec-m (n = 7) after AngII stimulation for 14 days. E Representative whole body and short and long-axis myocardial 18F-FDG PET/CT images from in GCV-m and Rec-m after AngII stimulation for 14 days. Data are depicted as mean ± SD, *P < 0.05, **P < 0.01 vs. respective PBS-m control group using either 1-way ANOVA followed by Bonferroni post hoc tests or two-tailed Student’s t test as required

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  Fig. 4. Changes in LV RNA expression at high tumor burden and after recovery from cancer. A Volcano plot of log2 fold change vs. adj. P value data from RNA-Seq. of LV tissue of PBS-m compared to HSVtk-m with adj. P value ≤ 0.01 highlighted in red, (filter of a base mean read count of ≥ 100, an adjusted P value ≤ 0.01 vs. healthy PBS-m; n = 2–4 individuals were pooled resulting in 3 pool samples per group). B, C Quantitative mRNA levels (qRT-PCR) of Atrogin1 and Myh7 in LV tissue from PBS-m (n = 13), GCV-m (n = 7), HSVtk-m (n = 11) and Rec-m (n = 7) normalized to 18S. D–F Quantitative mRNA levels (qRT-PCR) of Gadd45a, Mbnl2 and Hnrnpa1 in LV tissue from PBS-m (n = 8) and HSVtk-m (n = 8) normalized to 18S. G, H Representative western blot images of P-H2AX from LV tissue of PBS-m (n = 6) vs. (n = 6) and corresponding quantification normalized on Ponceau staining. I Volcano plot of log2 fold change vs. adj. P value data from RNA-Seq. of LV tissue of GCV-m compared to Rec-m with adj. P value ≤ 0.01 highlighted in red (filter of a base mean read count of ≥ 100, an adjusted P value ≤ 0.01 vs. healthy GCV-m; n = 2–4 individuals were pooled resulting in 3 pool samples per group). Data are…

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  Fig. 5. Cardiac phenotype after recovery from cancer and doxorubicin therapy. A Percent survival comparison of Rec-m (n = 15) and Rec-m + Dox (n = 20) from HSVtk-c injection until day of tissue harvesting. B-E) Fractional shortening, cardiac output, heart rate and stroke volume from echocardiographic analyses of Rec-m (n = 9) and Rec-m + Dox (n = 9). F–J Body weight and heart weight, gastrocnemius weight and soleus weight per tibia length from Rec-m (n = 8) and Rec-m + Dox (n = 8). K GS-Score per tibia length from Rec-m (n = 8) and Rec-m + Dox (n = 5). Data are depicted as mean ± SD, *P < 0.05, **P < 0.01 vs. Rec-m using Log-rank Mantel-Cox test, 2-tailed-Student’s t test or Mann–Whitney test as required

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  Fig. 6. Doxorubicin-induced changes in cardiac circadian rhythm. A Volcano plot of log2 fold change vs. adj. P value data from RNA-Seq. of LV tissue of Rec-m compared to Rec-m + Dox with adj. P value ≤ 0.05 highlighted in red. (filter of a base mean read count of ≥ 100, an adjusted P value ≤ 0.05 vs. healthy Rec-m; n = 2–4 individuals were pooled resulting in 3 pool samples per group) B DAVID pathway analyses of significantly different expressed genes in RNA-Seq. data comparison of Rec-m and Rec-m + Dox. C Regulation percentage of circadian rhythm pathway components in Rec-m + Dox in comparison to Rec-m

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  Fig. 7. Doxorubicin-induced increased apoptosis in NRCM with siRNA-mediated knockdown of BMAL1. A Representative images of TMRE in NRCM treated with Dox for 24 h after pre-treatment with Bmal1-si-RNA or scrambled siR control for 48 h, scale bar indicates 50 μm. FCCP (carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone) served as negative control and untreated NRCM as positive control. B Quantification of relative TMRE-mitochondrial membrane potential (MOMP) in living NRCM after treatment with Bmal1-si-RNA or scrambled siR-control for 48 h followed by 24 h Dox normalized to total protein concentration, n = 4–5 per group. FCCP served as negative control and untreated NRCM as positive control. C, D Representative western blot images of cytochrome C and GAPDH protein from the cytosolic protein fraction of NRCM after treatment with Bmal1-si-RNA or scrambled siR-control for 48 h followed by 24 h Dox and corresponding quantification normalized on Ponceau staining from n = 4 pooled samples per group (each pool consists of 2 individual samples). E–I Representative western blot images of BMAL1, PARP, cleaved PARP, p-H2AX, Caspase 3 and cleaved Caspase 3 protein from NRCM transfected with…