Cancer-driven cytokine immunomodulation ameliorates cardiac function and suppresses fibrosis

Figures

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  Fig. 1. Serum derived from tumor-bearing MDX mice ameliorates cardiac function. (A) Schematic representation of the experimental timeline. MDX male mice (5 months old, n = 6) were tail-vein injected (IV inj.) with 100 μL each with blood serum (BS) derived from tumor-bearing MDX mice (Cancer BS). Echocardiography (US) was performed pre-injection (0 days), 10, 15, and 25 days post-serum injection, and fractional shortening (FS) was calculated. (B) Three-week-old mice (n = 4/group) were injected with either control blood serum (Control BS) or Cancer BS repeatedly every 30 days from three weeks of age until the endpoint (eight months of age). Echocardiography (US) was performed, and FS was calculated at the indicated time points (C). Body weight (BW, gr) measurement of MDX mice (n = 4/group) at 5 months post-injection and at the end-point. Results are presented as mean ± SEM; one-way repeated measures ANOVA followed by (A, C) Tukey posttests or (B) two-way ANOVA followed by Bonferroni post-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Each dot represents one mouse.

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  Fig. 2. Tumor-bearing mice's blood serum ameliorates cardiac dysfunction following TAC surgery. (A) Experimental timeline. Naïve (n = 5) or TAC-operated two-month-old C57Bl/6 mice were injected with either Control BS (n = 7) or Cancer BS (n = 7). Echocardiography was performed prior to sacrifice, and Fractional shortening (FS%) was calculated. (B) Ventricles to body weight ratio (mg/g) at endpoint. (C–D) Levels of hypertrophy (ANP) and fibrosis (TGFβ3) hallmark genes were measured in qRT-PCR using cDNA derived from heart mRNA of each mouse. Results were normalized to Hsp90 housekeeping gene and compared to naïve levels (non-operated mice). Results are presented as mean ± SEM, One-way ANOVA followed by Tukey post-test was performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Each dot represents one mouse.

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  Fig. 3. NK cells are necessary and sufficient for ameliorating the cardiac contractile function of MDX mice. (A) Experimental timeline. Five-month-old MDX mice (n = 5) were tail-vein-injected with serum derived from NK-depleted tumor-bearing mice. Echocardiography was performed pre- and post-injection (14 and 21 days). Fractional shortening (FS%) was calculated. (B) Experimental timeline. C57Bl/6 were injected with Poly dIdC, and serum was withdrawn 18 h later. Subsequently, five-month-old MDX mice (n = 7) were tail-vein-injected with serum derived from Poly dIdC injected mice. Results are presented as mean ± SEM; one-way repeated measures ANOVA followed by (A) Tukey posttests or (B) Student's t-test. **p < 0.01. (ns) No statistical significance. Each dot represents one mouse.

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  Fig. 4. IFNγ and TNFα are responsible for ameliorating cardiac contractile function in MDX mice. (A) Cytokine array proteome profiler membranes were incubated with serum (pooled from 5 mice/cohort) derived from C57Bl/10 control mice (B10) and MDX mice in the presence or absence of a tumor. Serum was extracted 14 days following cancer cell implantation. The expression level of each cytokine present in C57Bl/10 mice was considered 100 %, and all other groups are presented relative to this. (B) Serum derived from tumor-bearing mice was incubated with IgG-neutralizing antibodies against the indicated candidate factors or with an equivalent amount of IgG control. Subsequently, serum was injected into the tail vein of five-month-old MDX mice (n = 4–5/group). Echocardiography was performed pre- (black dots) and 10 days post-serum injection (turquoise dots). Fractional shortening (FS%) was calculated. (C) Serum derived from Poly dIdC-injected mice was incubated with neutralizing antibodies against IFNγ and TNFα. Serum was injected into the tail vein of five-month-old MDX mice (n = 5). Echocardiography was performed pre- and 10 days post-serum injection. Fractional shortening (FS%) was…

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  Fig. 5. Purified IFNγ and TNFα improve cardiac contractile function via macrophages polarization in MDX mice in a CSF1R macrophages-dependent manner. (A). Experimental timeline. Five-month-old MDX mice were tail-vein-injected with serum either Control BS (n = 3) or Control BS supplemented with purified IFNγ and TNFα (n = 6). Echocardiography was performed pre- and post-injection (7 and 14 days). Fractional shortening (FS%) was calculated. (B) Flow cytometry analysis quantifying cardiac macrophages (% F4/80+CD11c+ and F4/80+CD206+ respectively) in control (n = 4) and IFNγ + TNFα-injected (n = 4) MDX mice. (C) qRT-PCR measuring transcription levels of macrophage hallmark gene markers in the heart of MDX mice injected with serum supplemented with IFNγ and TNFα compared with naïve mice serum (n = 5–7/group). Data are presented as the relative expression compared to MDX mice injected with serum only (determined as a mean of 1). (D) Experimental timeline. Five-month-old MDX control (no depletion, n = 4) and CSF1R macrophages-depleted mice (n = 7) were tail vein injected with control BS supplemented with IFNγ and TNFα. CSF1R-depleted mice (n = 4) were left untreated. Echocardiography was…

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  Fig. 6. IFN-γ and TNF-α administration enhance cardiac function and suppress fibrosis following TAC surgery. (A) Experimental timeline. TAC-operated two-month-old C57Bl/6 mice were injected with either Control BS (n = 7) or Cancer BS supplemented with IFN-γ and TNF-α (n = 6). Echocardiography was performed prior to, one, and two weeks post-injection, and fractional shortening (FS%) was calculated. (B) Levels of fibrosis hallmark genes Col1α1, ACTA2 and αSMA were measured in qRT-PCR using cDNA derived from the heart mRNA of each mouse. Results were normalized to Hsp90 housekeeping gene. Results are presented as mean ± SEM, One-way ANOVA followed by Tukey post-test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. p > 0.05 no statistical significance (ns). Each dot represents one mouse.