Subcutaneous Ehrlich Ascites Carcinoma mice model for studying cancer-induced cardiomyopathy

Abstract

Cardiomyopathy is one of the characteristic features of cancer. In this study, we establish a suitable model to study breast cancer-induced cardiomyopathy in mice. We used Ehrlich Ascites Carcinoma cells to induce subcutaneous tumor in 129/SvJ mice and studied its effect on heart function. In Ehrlich Ascites Carcinoma bearing mice, we found significant reduction in left ventricle wall thickness, ejection fraction, and fractional shortening increase in left ventricle internal diameter. We found higher muscle atrophy, degeneration, fibrosis, expression of cell-adhesion molecules and cell death in tumor-bearing mice hearts. As observed in cancer patients, we found that mTOR, a key signalling molecule responsible for maintaining cell growth and autophagy was suppressed in this model. Tumor bearing mice hearts show increased expression and nuclear localization of TFEB and FoxO3a transcription factors, which are involved in the upregulation of muscle atrophy genes, lysosomal biogenesis genes and autophagy genes. We propose that Ehrlich Ascites Carcinoma induced tumor can be used as a model to identify potential therapeutic targets for the treatment of heart failure in patients suffering from cancer-induced cardiomyopathy. This model can also be used to test the adverse consequences of cancer chemotherapy in heart.

Figure 1

Subcutaneous EAC tumor induces cachexia in mice. (a) Schematic diagram of the model proposed to study breast cancer induced cachexia. (b) Graph representing tumor volume in mice. Tumor volume measured at different time points post EAC cells injection. n = 10–11 mice per group. (c) Graph representing percentage change in body weight of non-tumor bearing (NTB) and tumor bearing (TB) mice. n = 15–18 mice per group. (d) Graph depicting the weights of gastrocnemius, quadriceps, triceps and tibialis anterior (TA) muscle of non-tumor bearing (NTB) and tumor bearing (TB) mice. n = 6–10 mice per group. (e) Gross images of spleen (i), liver (ii), kidney (iii) and lungs (iv) of non-tumor bearing (NTB) and tumor bearing (TB) mice. n = 6 mice per group. (f) H&E staining images of lung, liver, spleen and kidney sections of NTB and TB mice demonstrating inflammatory cells infiltration (black arrows) in TB mice sections. n = 6 mice per group. Scale bar = 100 µm.

Figure 2

EAC subcutaneous tumor induces cardiac remodelling and contractile dysfunction in mice. (a) Graph showing posterior wall thickness at diastole of NTB and TB mice as assessed by echocardiography, n = 4–6 mice per group, Mean ± SD, *p < 0.05. (b) Graph showing posterior wall thickness at systole of NTB and TB mice as assessed by echocardiography, n = 3–6 mice per group, Mean ± SD, *p < 0.05. (c) Graph showing anterior wall thickness at diastole of NTB and TB mice as assessed by echocardiography, n = 4–6 mice per group, Mean ± SD, *p < 0.05. (d) Graph showing anterior wall thickness at systole of NTB and TB mice as assessed by echocardiography, n = 3–6 mice per group, Mean ± SD, *p < 0.05. (e) Graph showing left ventricular internal diameter at diastole of NTB and TB mice as assessed by echocardiography, n = 3–6 mice per group, Mean ± SD, *p < 0.05. (f) Graph showing left ventricular internal diameter at systole of NTB and TB mice as assessed by echocardiography, n = 3–6 mice per group, Mean ± SD, *p < 0.05. (g) Graph showing ejection fraction of NTB and TB mice as assessed by echocardiography, n = 5–6 mice per group, Mean ± SD, *p < 0.05. (h) Graph showing fractional shortening of NTB and TB mice as assessed by echocardiography, n = 5–6 mice per group, Mean ± SD, *p < 0.05.

Figure 3

EAC subcutaneous tumor induces atrophy, fibrosis and degenerative changes in heart. (a) Graph showing Heart weight to tibia length ratio (HW/TL) of NTB and TB mice, n = 13 mice per group, Mean ± SD, *p < 0.05. (b–i) Confocal images representing WGA staining in heart sections of NTB and TB mice, Scale bar = 20 µm. (b–ii) Graph showing H&E staining in heart sections of NTB and TB mice showing degenerative changes including reduced muscle fibre diameter. (b-iii) Heart sections of NTB and TB mice stained with Masson’s trichrome stain showing cardiac fibrosis. (c) Graph showing relative fibre diameter in the cardiac sections of NTB and TB mice, quantified from Fig. 2(b-i). (d) Graph showing relative fibrosis in the cardiac sections of NTB and TB mice, quantified from Fig. 2(b-iii). (e) qPCR analysis of ANP, BNP and β-MHC in NTB and TB mice hearts, Mean ± SD, n = 4–5 mice per group, *p < 0.05. (f) qPCR analysis of α-SMA, Col1a and fn1 in NTB and TB mice hearts. Mean ± SD, n = 4–6 mice per group, *p < 0.05. (g) qPCR analysis of Bim and TRAIL in NTB and TB mice hearts, Mean ± SD, n = 5 mice per group, *p < 0.05. (h) Western blotting analysis of cleaved PARP-1 in NTB and TB mice heart samples.

Figure 4

EAC subcutaneous tumor upregulates lysosomal genes in mice hearts. (a) Western blot analysis of NTB and TB mice hearts probed for p-mTOR and mTOR. GAPDH is used as loading control. n = 4 mice per group. (b) qPCR analysis of HEXA, LIPA, CTSB, CTSD and ATP6VOA1 in NTB and TB mice hearts, Mean ± SD, n = 4–5 mice per group, *p < 0.05. (c) qPCR analysis of Beclin-1, LC3 and p62 in NTB and TB mice hearts, Mean ± SD, n = 3–5 mice per group, *p < 0.05. (d) Representative confocal images of NTB and TB mice hearts stained for TFEB. Scale bar = 20 µm for left and middle images; n = 4 mice per group, Right side zoom-in images showing localisation of TFEB inside the nucleus in TB mice hearts marked by circles. (e) Graph showing quantification of TFEB levels in NTB and TB mice hearts. (f) Graph showing percentage of nuclei with and without TFEB localisation in NTB and TB mice hearts.

Figure 5

EAC subcutaneous tumor upregulates atrophy genes in mice hearts. (a) Western blot analysis of NTB and TB mice hearts probed for MuRF-1, Atrogin-1 and FoxO3a. GAPDH is used as loading control. n = 7–8 mice per group. (b) Graph representing quantification of MuRF-1/GAPDH levels in NTB and TB mice hearts. n = 7–8 mice per group. (c) Graph representing quantification of Atrogin-1/GAPDH levels in NTB and TB mice hearts. n = 4–5 mice per group. (d) Graph representing quantification of FoxO3a/GAPDH levels in NTB and TB mice hearts. n = 3 mice per group. (e) qPCR analysis of FoxO3a, Atrogin-1 and MuRF-1 in NTB and TB mice hearts. Mean ± SD, n = 5–7 mice per group, *p < 0.05. (f) Representative confocal images of NTB and TB mice hearts stained for MuRF-1, Atrogin-1 and total-ubiquitin levels, Scale bar = 20 µm. (g) Graph showing quantification of MuRF-1 in NTB and TB mice hearts. (h) Graph showing quantification of Atrogin-1 in NTB and TB mice hearts. (i) Graph showing quantification of total cellular ubiquitin levels in NTB and TB mice hearts. (j) Representative confocal images of NTB and TB mice hearts stained for FoxO3a, Scale bar = 20 µm for left and middle images; n = 4 mice per group, Right side zoom-in images showing localisation of FoxO3a inside the nucleus in TB mice hearts marked by white arrows. (k) Graph showing quantification of FoxO3a expression levels in NTB and TB mice hearts. (l) Graph showing percentage of nuclei with and without FoxO3a localisation in NTB and TB mice hearts.