The Human Fetal and Adult Stem Cell Secretome Can Exert Cardioprotective Paracrine Effects against Cardiotoxicity and Oxidative Stress from Cancer Treatment

Figures

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  Figure 1. hAFS and hMSC secretome formulations exert cardioprotective effects on mNVCM exposed to Dox. (A) Schematic of the experimental design for in vitro experiments. (B) Left panel: representative confocal images of live mNVCM exposed to 1 µM Dox for 16 h with or without (Dox) prior priming with hAFS-CM (hAFS-CM + Dox) or hMSC-CM (hMSC-CM + Dox) for 3 h. Mitochondria are represented in red by the MitoTracker signal; Dox nuclear localization is represented in green by means of the drug auto-fluorescent signal, scale bar: 5 µm. Right panel: evaluation of Dox nuclear fluorescence by arbitrary units (A.U.) in Dox-exposed mNVCM (Dox: 689.67 ± 37.19) versus mNVCM primed with either hAFS-CM (hAFS-CM + Dox: 99.33 ± 36.22; **** p < 0.0001) or hMSC-CM (hMSC-CM + Dox: 345.33 ± 31.15; ** p = 0.0011). Values are expressed as the mean ± s.e.m. of n = 3 independent experiments. hAFS-CM + Dox vs hMSC-CM + Dox ** p = 0.0060. (C) Left panel: representative brightfield microscopy images and immunocytochemistry analysis of the cleaved-caspase 3 enzyme (CC-3) on untreated mNVCM (Ctrl) and on mNVCM exposed for 21 h to 1 µM Dox with or without (Dox) previous priming with hAFS-CM (hAFS-CM + Dox) or hMSC-CM…

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  Figure 2. hAFS and hMSC secretomes preserve mitochondrial transmembrane potential in mNVCM exposed to Dox. (A) Representative confocal images of live mNVCM in control condition (Ctrl), undergoing stimulation with either hAFS-CM (hAFS-CM) or hMSC-CM (hMSC-CM) for 3 h or exposed to 1 µM Dox for 16 h with or without (Dox) prior priming with hAFS-CM (hAFS-CM + Dox) or hMSC-CM (hMSC-CM + Dox) for 3 h. The cell mitochondrial network is represented in red by the MitoTracker signal, scale bar: 5 µm, (B) Mitochondrial transmembrane potential evaluated by MitoTracker fluorescence analysis and expressed by arbitrary units (A.U.) mNVCM treated as follows: control condition (n = 4, Ctrl: 351.85 ± 17.13); after exposure to 1 μM Dox for 16 h (n = 4, Dox: 167.52 ± 23.49); pretreated with hAFS-CM or hMSC-CM for 3 h before 1 μM Dox stimulation for 16 h (n = 4, hAFS-CM + Dox: 286.77 ± 12.94, and hMSC-CM + Dox: 254.57 ± 18.56, respectively), or only pretreated with hAFS-CM or hMSC-CM (n = 3, hAFS-CM: 421.63 ± 9.55 and hMSC-CM: 397.93 ± 9.57, respectively). Values are expressed as the mean ± SEM of the independent experiments. hAFS-CM vs. Ctrl and hMSC-CM vs. Ctrl: not significant; Ctrl vs. Dox; §§§§ p <…

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  Figure 3. Dox-elicited mitochondrial dysfunction is rescued in mNVCM pretreated with either hAFS-CM or hMSC-CM. From the top left corner: Oxygen consumption rate stimulated with pyruvate plus malate; ATP production by Fo-F1 ATP synthase stimulated with pyruvate plus malate; mNVCM P/O ratio after pyruvate plus malate stimulation, as a marker of OxPhos efficiency; Glucose consumption; lactate release; lactate fermentation percentage. All the biochemical measurements were performed in mNVCM treated as follows: control condition (Ctrl), after exposure to 1 µM Dox (Dox), pretreated with hAFS-CM or hMSC-CM before the Dox stimulation (hAFS-CM+Dox and hMSC-CM + Dox, respectively), or only pretreated with hAFS-CM or hMSC-CM (HAFS-CM and hMSC-CM, respectively). In each panel, values are expressed as the mean ± SEM of the independent experiments; hAFS-CM vs. Ctrl and hMSC-CM vs. Ctrl # p = 0.0495 or #### p < 0.0001; Ctrl vs. Dox §§§§ p < 0.0001; hAFS-CM + Dox vs. Dox and hMSC-CM + Dox vs. Dox **** p < 0.0001.

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  Figure 4. hAFS and hMSC secretomes induce pro-survival effects on mNVFib exposed to 1 μM Dox and sustain their mitochondrial metabolism. (A) Left panel: representative images of immunocytochemistry analysis for the expression of cleaved-caspase 3 enzyme (CC-3) on untreated mNVFib (Ctrl) and on mNVFib exposed for 21 h to 1 μM Dox with or without (Dox) previous priming with hAFS-CM (hAFS-CM + Dox) or hMSC-CM (hMSC-CM + Dox) for 3 h, scale bar: 20 µm. Right panel: percentage of mNVFib expressing cleaved-caspase-3 (%CC-3+ cells) in control condition (Ctrl: 5.31 ± 1.75%), after either hAFS-CM (hAFS-CM: 4.65 ± 1.54%) or hMSC-CM (hMSC-CM: 3.14 ± 0.74%) stimulation alone or after exposure to 1 μM Dox with or without (Dox: 24.83 ± 3.88%) pre-incubation with hAFS-CM (hAFS-CM+Dox: 7.15 ± 0.72%) or hMSC-CM (hMSC-CM + Dox: 12.52 ± 0.33%). Values are expressed as the mean ± SEM of n = 3 independent experiments; hAFS-CM vs. Ctrl and hMSC-CM. Ctrl: not significant; Ctrl vs. Dox: ### p = 0.0001; hAFS-CM + Dox vs. Dox: *** p = 0.0003 and hMSC-CM + Dox vs. Dox: ** p = 0.0064. (B) From the top left corner: Oxygen consumption rate stimulated with pyruvate plus malate; ATP production by Fo-F1 ATP synthase…

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  Figure 5. hAFS and hMSC secretomes exert long-term cardioprotective effects in a preclinical mouse model of Dox-induced cardiomyopathy. (A) Schematic of the experimental design for in vivo experiments; a cumulative 12 mg/Kg Dox dose was administered by intra-peritoneal delivery three times, once a week, over 14 days (4 mg/kg on days 0-7-14). The hAFS-CM and hMSC-CM (100 μg/mouse) were administered 3 h before each 4 mg/kg Dox injection. (B) Evaluation of mice body weight over time in sham healthy control animals (n = 3, Sham: day 0: 19.27 ± 1.27 g; day 42: 22.03 ± 1.03 g) and in mice receiving Dox with or without (n = 4, Dox: day 0: 18.85 ± 0.83 g; day 42: 17.90 ± 0.52 g) pre-incubation with hAFS-CM (n = 6, hAFS-CM + Dox: day 0: 19.16 ± 0.15 g; day 42: 20.70 ± 0.47 g) or hMSC-CM (n = 5, hMSC-CM + Dox: day 0: 18.72 ± 0.53 g; day 42: 20.28 ± 0.33 g). Values are expressed as the mean ± SEM of the different animals. Sham vs. Dox at day 42: #### p < 0.0001; hAFS-CM + Dox vs. Dox at day 42: *** p = 0.0008 and hMSC-CM +Dox vs. Dox at day 42: ** p = 0.0083. (C) Ultrasound analysis of cardiac function via evaluation of fractional shortening percentage over time in mice receiving a cumulative dose of…