Cardioprotection and Suppression of Fibrosis by Diverse Cancer and Non-Cancer Cell Lines in a Murine Model of Duchenne Muscular Dystrophy

Abstract

The dynamic relationship between heart failure and cancer poses a dual challenge. While cardiac remodeling can promote cancer growth and metastasis, tumor development can ameliorate cardiac dysfunction and suppress fibrosis. However, the precise mechanism through which cancer influences the heart and fibrosis is yet to be uncovered. To further explore the interaction between heart failure and cancer, we used the MDX mouse model, which suffers from cardiac fibrosis and cardiac dysfunction. A previous study from our lab demonstrated that tumor growth improves cardiac dysfunction and dampens fibrosis in the heart and diaphragm muscles of MDX mice. We used breast Polyoma middle T (PyMT) and Lewis lung carcinoma (LLC) cancer cell lines that developed into large tumors. To explore whether the aggressiveness of the cancer cell line is crucial for the beneficial phenotype, we employed a PyMT breast cancer cell line lacking integrin β1, representing a less aggressive cell line compared to the original PyMT cells. In addition, we examined immortalized and primary MEF cells. The injection of integrin β1 KO PyMT cancer cells and Mouse Embryo Fibroblasts cells (MEF) resulted in the improvement of cardiac function and decreased fibrosis in the heart, diaphragm, and skeletal muscles of MDX mice. Collectively, our data demonstrate that the cancer line aggressiveness as well as primary MEF cells are sufficient to impose the beneficial phenotype. These discoveries present potential novel clinical therapeutic approaches with beneficial outcome for patients with fibrotic diseases and cardiac dysfunction that do not require tumor growth.

Keywords: Duchenne muscular dystrophy; cardiac dysfunction; cardiac remodeling; fibrosis.

Figure 1

Injection of less aggressive PyMT ITGB1 KO cells and non-cancerous MEF^I and MEF^P cells resulted in a significant improvement of cardiac contractile function in MDX mice. (A) Schematic representation of the experimental timeline. MDX male mice (5 months old) were injected in the flanks with PyMT ITGB1 KO (red), MEF^P (blue), or MEF^P (green) cells (10⁶ cells per mouse) or left non-injected (MDX) (black). Echocardiography (US) was performed 14 days after injection. (B) The measured fractional shortening (FS) of control MDX and injected MDX mice group (ITGB1 KO, MEF^P, and MEF^I). FS was assessed by echocardiography and calculated using the formula: FS (%) = [(LVDd − LVDs)/LVDd]. (C) Percent of interstitial fibrosis of all injected groups of MDX mice compared with control MDX mice, quantified using ImageJ 1.53t software, based on at least five fields from each mouse in each cohort. Data are presented as mean ± SE. One-way ANOVA followed by Tukey post-test (B,C). * p < 0.05; ** p < 0.01; **** p < 0.0001. Each dot represents one mouse.

Figure 2

Injection of less aggressive PyMT ITGB1 KO cells and non-cancerous MEF^I and MEF^P cells led to a reduction in the expression of fibrosis hallmark gene markers in the hearts of MDX mice. (A–C) qRT-PCR measuring transcription mRNA levels of fibrosis hallmark gene markers in the hearts of MDX mice injected with (A) PyMT ITGB1 KO, (B) MEF^I, and (C) MEF^P, as compared with control MDX. Data are presented as mean ± SE. One-way ANOVA followed by Tukey post-test (A–C). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Each dot represents one mouse.

Figure 3

Injection of less aggressive cancer cell line PyMT ITGB1 KO cells and non-cancerous MEF^I and MEF^P cells induces the expression of M2-polarizing hallmark gene markers in the hearts of MDX mice. (A–F) qRT-PCR measures the transcription levels of macrophage hallmark gene markers in the hearts of injected MDX mice as compared with control MDX (A–C) mRNA levels of different M2 macrophage markers: IL13, GCSF, CCL2, CD163, and ARG1, (D–F) mRNA levels of different M1 macrophage markers: IL1, TNFα, INFγ and GPR18. Measurements were obtained using qRT-PCR, normalized to the housekeeping gene mB2M. The results are presented as mean ± SE, one-way ANOVA followed by Tukey post-test * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Each dot represents one mouse.

Figure 4

M2 polarizing cytokines IL-13 is elevated in the serum of injected mice (PyMT ITGB1 KO, MEF^I, and MEF^P) compared with control MDX mice. Serum levels are obtained by ELISA for IL-13. Pooled blood sera of control MDX (n = 3 each), tumor-bearing (ITGB1 KO) MDX mice (n = 4), MEF^I-injected MDX mice (n = 3) and MEF^P-injected MDX mice (n = 4) were used. The results are presented as mean ± SE. One-way ANOVA followed by Tukey post-test. * p < 0.05, ** p < 0.01.

Figure 5

Graphical abstract summarizing the conclusions of this study. Tumor progression is not necessary for the beneficial phenotype and reduced fibrosis in the cardiac, skeletal, and diaphragm muscles. These effects are attributed, at least in part, to the M2 macrophage switch.