Abstract Stem cell-based therapies with clinical applications require millions of cells. Therefore, repeated subculture is essential for cellular expansion, which is often complicated by replicative senescence. Cellular senescence contributes to reduced stem cell regenerative potential as it inhibits stem cell proliferation and differentiation as well as the activation of the senescence-associated secretory phenotype (SASP). In this study, we employed MHY-1685, a novel mammalian target of rapamycin (mTOR) inhibitor, and examined its long-term priming effect on the activities of senile human cardiac stem cells (hCSCs) and the functional benefits of primed hCSCs after transplantation. In vitro experiments showed that the MHY-1685‒primed hCSCs exhibited higher viability in response to oxidative stress and an enhanced proliferation potential compared to that of the unprimed senile hCSCs. Interestingly, priming MHY-1685 enhanced the expression of stemness-related markers in senile hCSCs and provided the differentiation potential of hCSCs into vascular lineages. In vivo experiment with echocardiography showed that transplantation of MHY-1685‒primed hCSCs improved cardiac function than that of the unprimed senile hCSCs at 4 weeks post-MI. In addition, hearts transplanted with MHY-1685-primed hCSCs exhibited significantly lower cardiac fibrosis and higher capillary density than that of the unprimed senile hCSCs. In confocal fluorescence imaging, MHY-1685‒primed hCSCs survived for longer durations than that of the unprimed senile hCSCs and had a higher potential to differentiate into endothelial cells (ECs) within the infarcted hearts. These findings suggest that MHY-1685 can rejuvenate senile hCSCs by modulating autophagy and that as a senescence inhibitor, MHY-1685 can provide opportunities to improve hCSC-based myocardial regeneration.
Nanrilkefusp alfa (Nanril, SOT101) is an IL-15Rβγ superagonist that is comprised of the IL15 cytokine fused to the IL-15Rα and has demonstrated a favorable safety profile and encouraging efficacy signals as a monotherapy and in combination with KEYTRUDA® (pembrolizumab) in the Phase 1/1b AURELIO-03 trial. SOTIO's BOXR cell therapy platform is designed to improve the functionality of CAR-T cells by incorporating novel transgenes that are co-expressed with tumor-targeting receptors to overcome resistance and improve the function of respective immune cells in the solid tumor microenvironment. Here we tested the combination of Nanril with CAR-T or BOXR-T cells in vitro and in in vivo efficacy studies.
Methods
BOXR-T cells, CAR-T cells or untransduced (UTD) control T cells were treated with 0.1–1 nM Nanril for 3- 7 days and proliferation and memory phenotype were assessed by flow cytometry; RNAseq analysis was also performed. To assess in vitro cytotoxicity, T cells were pre-treated for three days with 0.1 nM Nanril and were then co-cultured with target cells and cell killing was monitored using Incucyte analysis. CAKI-1 and NCI-H1975 tumor models were used to assess CAR-T and BOXR-T cell anti-tumor activity in combination with Nanril where the Nanril dosing regimen was administered 7 days following CAR-T or BOXR-T cells treatment.
Results
Nanril treatment induced proliferation in UTD, CAR-T and BOXR-T cells in a dose-dependent manner. Shifts in T cell memory populations were also observed with increasing Nanril concentration, resulting in a higher proportion of effector memory cells and subsequently improved in vitro cytotoxicity. RNAseq analysis findings were consistent with increased proliferation and differentiation with Nanril treatment. When tested in vivo, BOXR-T cells had superior anti-tumor activity compared to CAR-T cells and combination treatment with Nanril further improved both BOXR-T and CAR-T cell efficacy and increased peripheral blood expansion.
Conclusions
These data demonstrate that combination of Nanril with BOXR-T and CAR-T cells results in improved T cell function and anti-tumor activity in preclinical models. Combination of Nanril with T cell-based therapies may be a promising approach to increase efficacy in difficult-to-treat solid tumors.
Mitochondrial adenine nucleotide translocator (ANT) plays important roles in the regulation of mitochondrial permeability transition and cell bioenergetics. The mouse has three ANT isoforms (1, 2 and 4) showing tissue-specific expression patterns. Although ANT1 is known to have a pro-apoptotic property, the specific functions of ANT2 have not been well determined. In the present study, ANT2 expression was significantly lower in the aged rat liver and in a liver fibrosis model. To explore the protective role of ANT2 in the liver, we established a hepa1c1c7 cell line overexpressing ANT2. Overexpression of ANT2 caused hepa1c1c7 cells to be more resistant to oxidative stress, and mitochondrial membrane potential (MMP, ∆Ψm) was relatively intact in ANT2-overexpressing cells under oxidative stress. In addition, ANT2 was found to increase ATP production by influencing mitochondrial bioenergetics. These results imply that the hepatoprotective effect of ANT2 is due to the stabilization of MMP and enhanced ATP production, and thus, maintaining ANT2 levels in the liver might be important to enhance resistance to aging and oxidative stress.
Tumor undergo uncontrolled, excessive proliferation leads to hypoxic microenvironment. To fulfill their demand for nutrient, and oxygen, tumor angiogenesis is required. Endothelial progenitor cells (EPCs) have been known to the main source of angiogenesis because of their potential to differentiation into endothelial cells. Therefore, understanding the mechanism of EPC-mediated angiogenesis in hypoxia is critical for development of cancer therapy. Recently, mitochondrial dynamics has emerged as a critical mechanism for cellular function and differentiation under hypoxic conditions. However, the role of mitochondrial dynamics in hypoxia-induced angiogenesis remains to be elucidated. In this study, we demonstrated that hypoxia-induced mitochondrial fission accelerates EPCs bioactivities. We first investigated the effect of hypoxia on EPC-mediated angiogenesis. Cell migration, invasion, and tube formation was significantly increased under hypoxic conditions; expression of EPC surface markers was unchanged. And mitochondrial fission was induced by hypoxia time-dependent manner. We found that hypoxia-induced mitochondrial fission was triggered by dynamin-related protein Drp1, specifically, phosphorylated DRP1 at Ser637, a suppression marker for mitochondrial fission, was impaired in hypoxia time-dependent manner. To confirm the role of DRP1 in EPC-mediated angiogenesis, we analyzed cell bioactivities using Mdivi-1, a selective DRP1 inhibitor, and DRP1 siRNA. DRP1 silencing or Mdivi-1 treatment dramatically reduced cell migration, invasion, and tube formation in EPCs, but the expression of EPC surface markers was unchanged. In conclusion, we uncovered a novel role of mitochondrial fission in hypoxia-induced angiogenesis. Therefore, we suggest that specific modulation of DRP1-mediated mitochondrial dynamics may be a potential therapeutic strategy in EPC-mediated tumor angiogenesis.
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