Breast cancer dormancy is associated with a 4NG1 state and not senescence

Reactivation of dormant cancer cells can lead to cancer relapse, metastasis, and patient death. Dormancy is a nonproliferative state and is linked to late relapse and death. No targeted therapy is currently available to eliminate dormant cells, highlighting the need for a deeper understanding and reliable models. Here, we thoroughly characterize the dormant D2.OR and ZR-75-1, and proliferative D2A1 breast cancer cell line models in vivo and/or in vitro, and assess if there is overlap between a dormant and a senescent phenotype. We show that D2.OR but not D2A1 cells become dormant in the liver of an immunocompetent model. In vitro, we show that D2.OR and ZR-75-1 cells in response to a 3D environment or serum-free conditions are growth-arrested in G1, of which a subpopulation resides in a 4NG1 state. The dormancy state is reversible and not associated with a senescence phenotype. This will aid future research on breast cancer dormancy.

Human Adipose Stem Cells (hASCs) Grown on Biodegradable Microcarriers in Serum- and Xeno-Free Medium Preserve Their Undifferentiated Status

Human adipose stem cells (hASCs) are promising candidates for cell-based therapies, but they need to be efficiently expanded in vitro as they cannot be harvested in sufficient quantities. Recently, dynamic bioreactor systems operated with microcarriers achieved considerable high cell densities. Thus, they are a viable alternative to static planar cultivation systems to obtain high numbers of clinical-grade hASCs. Nevertheless, the production of considerable biomass in a short time must not be achieved to the detriment of the cells’ quality. To facilitate the scalable expansion of hASC, we have developed a new serum- and xeno-free medium (UrSuppe) and a biodegradable microcarrier (BR44). In this study, we investigated whether the culture of hASCs in defined serum-free conditions on microcarriers (3D) or on planar (2D) cell culture vessels may influence the expression of some marker genes linked with the immature degree or the differentiated status of the cells. Furthermore, we investigated whether the biomaterials, which form our biodegradable MCs, may affect cell behavior and differentiation. The results confirmed that the quality and the undifferentiated status of the hASCs are very well preserved when they grow on BR44 MCs in defined serum-free conditions. Indeed, the ASCs showed a gene expression profile more compatible with an undifferentiated status than the same cells grown under standard planar conditions.

Annexin A2 Flop-Out Mediates the Non-Vesicular Release of DAMPs/Alarmins from C6 Glioma Cells Induced by Serum-Free Conditions

Prothymosin alpha (ProTα) and S100A13 are released from C6 glioma cells under serum-free conditions via membrane tethering mediated by Ca2+-dependent interactions between S100A13 and p40 synaptotagmin-1 (Syt-1), which is further associated with plasma membrane syntaxin-1 (Stx-1). The present study revealed that S100A13 interacted with annexin A2 (ANXA2) and this interaction was enhanced by Ca2+ and p40 Syt-1. Amlexanox (Amx) inhibited the association between S100A13 and ANXA2 in C6 glioma cells cultured under serum-free conditions in the in situ proximity ligation assay. In the absence of Amx, however, the serum-free stress results in a flop-out of ANXA2 through the membrane, without the extracellular release. The intracellular delivery of anti-ANXA2 antibody blocked the serum-free stress-induced cellular loss of ProTα, S100A13, and Syt-1. The stress-induced externalization of ANXA2 was inhibited by pretreatment with siRNA for P4-ATPase, ATP8A2, under serum-free conditions, which ablates membrane lipid asymmetry. The stress-induced ProTα release via Stx-1A, ANXA2 and ATP8A2 was also evidenced by the knock-down strategy in the experiments using oxygen glucose deprivation-treated cultured neurons. These findings suggest that starvation stress-induced release of ProTα, S100A13, and p40 Syt-1 from C6 glioma cells is mediated by the ANXA2-flop-out via energy crisis-dependent recovery of membrane lipid asymmetry.

Highly Osmotic Oxidized Sucrose-Crosslinked Polyethylenimine for Gene Delivery Systems

In this work, highly osmotic oxidized sucrose-crosslinked polyethylenimine (SP2K) polymers were developed for gene delivery systems, and the transfection mechanism is examined. First, periodate-oxidized sucrose and polyethylenimine 2K (PEI2K) were crosslinked with various feed ratios via reductive amination. The synthesis was confirmed by 1H NMR and FTIR. The synthesized SP2K polymers could form positively charged (~40 mV zeta-potential) and nano-sized (150–200 nm) spherical polyplexes with plasmid DNA (pDNA). They showed lower cytotoxicity than PEI25K but concentration-dependent cytotoxicity. Among them, SP2K7 and SP2K10 showed higher transfection efficiency than PEI25K in both serum and serum-free conditions, revealing the good serum stability. It was found that SP2K polymers possessed high osmolality and endosome buffering capacity. The transfection experiments with cellular uptake inhibitors suggest that the transfection of SP2K polymers would progress by multiple pathways, including caveolae-mediated endocytosis. It was also thought that caveolae-mediated endocytosis of SP2K polyplexes would be facilitated through cyclooxygenase-2 (COX-2) expression induced by high osmotic pressure of SP2K polymers. Confocal microscopy results also supported that SP2K polyplexes would be internalized into cells via multiple pathways and escape endosomes efficiently via high osmolality and endosome buffering capacity. These results demonstrate the potential of SP2K polymers for gene delivery systems.

Sex of donor cell and reprogramming conditions predict the extent and nature of imprinting defects in mouse iPSCs

ABSTRACTReprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs) is a major leap towards personalized approaches to disease modelling and cell-replacement therapies. However, we still lack the ability to fully control the epigenetic status of iPSCs, which is a major hurdle for their downstream applications. A sensible indicator for epigenetic fidelity is genomic imprinting, a phenomenon dependent on DNA methylation, which is frequently perturbed in iPSCs by yet unidentified reasons. By using a secondary reprogramming system with murine hybrid donor cells, we conducted a thorough imprinting analysis using IMPLICON in multiple female and male iPSCs generated under different culture conditions. Our results show that imprinting defects are remarkably common in mouse iPSCs causing dysregulation of the typical monoallelic expression of imprinted genes. Interestingly, the nature of imprinting defects depends on the sex of the donor cell and their respective response to culture conditions. Under serum-free conditions, male iPSCs show global hypomethylation at imprinted regions, whereas in serum conditions show focal hypermethylation at specific loci. In contrast, female iPSCs always exhibit hypomethylation defects regardless of culture conditions. These imprinting defects are more severe than the global changes in DNA methylation, highlighting the sensitivity of imprinting loci to current iPSC generation protocols. Our results reveal clear predictors underlying different types of imprinting defects in mouse iPSCs. This knowledge is essential to devise novel reprogramming strategies aiming at generating epigenetically faithful iPSCs.

Reduced serum methods for contact-based coculture of human dermal fibroblasts and epidermal keratinocytes

Direct contact-based coculture of human dermal fibroblasts and epidermal keratinocytes has been a long-standing and challenging issue owing to different serum and growth factor requirements of the two cell types. Existing protocols employ high serum concentrations (up to 10% fetal bovine serum), complex feeder systems and a range of supplemental factors. These approaches are technically demanding and labor intensive, and pose scientific and ethical limitations associated with the high concentrations of animal serum. On the other hand, serum-free conditions often fail to support the proliferation of one or both cell types when they are cultured together. We have developed two reduced serum approaches (1–2% serum) that support the contact-based coculture of human dermal fibroblasts and immortalized keratinocytes and enable the study of cell migration and wound closure.

Cannabinoid-Induced Autophagy and Heme Oxygenase-1 Determine the Fate of Adipose Tissue-Derived Mesenchymal Stem Cells under Stressful Conditions

The administration of adipose tissue-derived mesenchymal stem cells (ADMSCs) represents a promising therapeutic option after myocardial ischemia or myocardial infarction. However, their potential is reduced due to the high post-transplant cell mortality probably caused by oxidative stress and mitogen-deficient microenvironments. To identify protection strategies for ADMSCs, this study investigated the influence of the non-psychoactive phytocannabinoid cannabidiol (CBD) and the endocannabinoid analogue R(+)-methanandamide (MA) on the induction of heme oxygenase-1 (HO-1) and autophagy under serum-free conditions. At a concentration of 3 µM, CBD induced an upregulation of HO-1 mRNA and protein within 6 h, whereas for MA only a late and comparatively lower increase in the HO-1 protein could be detected after 48 h. In addition, both cannabinoids induced time- and concentration-dependent increases in LC3A/B-II protein, a marker of autophagy, and in metabolic activity. A participation of several cannabinoid-binding receptors in the effect on metabolic activity and HO-1 was excluded. Similarly, knockdown of HO-1 by siRNA or inhibition of HO-1 activity by tin protoporphyrin IX (SnPPIX) had no effect on CBD-induced autophagy and metabolic activity. On the other hand, the inhibition of autophagy by bafilomycin A1 led to a significant decrease in cannabinoid-induced metabolic activity and to an increase in apoptosis. Under these circumstances, a significant induction of HO-1 expression after 24 h could also be demonstrated for MA. Remarkably, inhibition of HO-1 by SnPPIX under conditions of autophagy deficit led to a significant reversal of apoptosis in cannabinoid-treated cells. In conclusion, the investigated cannabinoids increase metabolic viability of ADMSCs under serum-free conditions by inducing HO-1-independent autophagy but contribute to apoptosis under conditions of additional autophagy deficit via an HO-1-dependent pathway.