Stem Cell Death Research Articles

The Gut Microbiome as a Marker of Early Cardiac Allograft Injury

<h3>Purpose</h3> Gut microbial dysbiosis has been correlated to solid organ and stem cell transplant rejection and death. The HeartCare lab test (CareDx) is increasingly used as routine surveillance for heart transplant (HT) injury and rejection. We investigated whether the microbiome can serve as a noninvasive marker of immune activation and allograft injury among HT recipients. <h3>Methods</h3> We conducted a single-center, prospective cohort study of single organ HT recipients from 2020-2021. Shotgun metagenomic sequencing was performed on peri-HT stool samples. Gut microbial diversity and composition were correlated with the highest abnormal AlloSure or AlloMap obtained within 6 months post-HT. <h3>Results</h3> Thirty-eight HT recipients were included. Stool samples were collected 5.3 ± 7.4 days from HT. The highest HeartCare lab values were measured 72 days (IQR [41, 103]) post-HT. Seven HT recipients had high AlloSure (median 0.2%, IQR [0.14, 0.26]) and 7 had high AlloMap (median 32, IQR [30, 33]) scores. The high and low AlloSure and AlloMap groups had similar within sample microbial i.e. α-diversity. Low AlloSure scores significantly correlated with an increased abundance of phylum <i>Bacteroidetes</i> (34.5% vs 14.4%, p=0.04), and class <i>Bacteroidia</i> (34.3% vs 14.3%, p=0.04); and tended to have lower abundance of the phylum <i>Proteobacteria</i> (5.0% vs 23.3%, p=0.06) (Fig. 1A). Higher AlloMap scores correlated with higher abundance of class <i>Deltaproteobacteria</i> (1.3% vs 0.10%, p=0.04) and order <i>Desulfovibrionales</i> (1.25% vs 0.07%, p=0.02), and a trend towards lower abundance of <i>Firmicutes</i> (36.8% vs 57.3%, p=0.08) (Fig. 1B). Intergroup β-diversity analysis showed clustering based on AlloSure but not AlloMap scores (Fig. 1C). <h3>Conclusion</h3> In our cohort, increased abundance of certain gut microbial taxa in the peri-HT period correlated with increased HeartCare values. The gut microbiome may play a role in immune activation and allograft injury or serve as a biomarker. Further investigation into the mechanistic ties between the gut microbiome and alloimmunity is needed.

Dynamic Changes in Reactive Oxygen Species in the Shoot Apex Contribute to Stem Cell Death in Arabidopsis thaliana.

In monocarpic plants, stem cells are fated to die. However, the potential mechanism of stem cell death has remained elusive. Here, we reveal that the levels of two forms of reactive oxygen species (ROS), superoxide anion free radical () and hydrogen peroxide (H2O2), show dynamic changes in the shoot apex during the plant life cycle of Arabidopsis thaliana. We found that the level of decreased and disappeared at four weeks after bolting (WAB), while H2O2 appeared at 3 WAB and showed a burst at 5 WAB. The timing of dynamic changes in and H2O2 was delayed for approximately three weeks in clv3-2, which has a longer lifespan. Moreover, exogenous application of H2O2 inhibited the expression of the stem cell determinant WUSCHEL (WUS) and promoted the expression of the developmentally programmed cell death (dPCD) marker gene ORESARA 1 (ORE1). These results indicate that H2O2 triggers an important signal inducing dPCD in stem cells. Given that plays roles in maintaining WUS expression and stem cell activity, we speculate that the dynamic shift from to H2O2 in the shoot apex results in stem cell death. Our findings provide novel insights for understanding ROS-mediated regulation during plant stem cell death.

The MicroRNA miR-277 Controls Physiology and Pathology of the Adult Drosophila Midgut by Regulating the Expression of Fatty Acid β-Oxidation-Related Genes in Intestinal Stem Cells.

Cell division, growth, and differentiation are energetically costly and dependent processes. In adult stem cell-based epithelia, cellular identity seems to be coupled with a cell’s metabolic profile and vice versa. It is thus tempting to speculate that resident stem cells have a distinct metabolism, different from more committed progenitors and differentiated cells. Although investigated for many stem cell types in vitro, in vivo data of niche-residing stem cell metabolism is scarce. In adult epithelial tissues, stem cells, progenitor cells, and their progeny have very distinct functions and characteristics. In our study, we hypothesized and tested whether stem and progenitor cell types might have a distinctive metabolic profile in the intestinal lineage. Here, taking advantage of the genetically accessible adult Drosophila melanogaster intestine and the availability of ex vivo single cell sequencing data, we tested that hypothesis and investigated the metabolism of the intestinal lineage from stem cell (ISC) to differentiated epithelial cell in their native context under homeostatic conditions. Our initial in silico analysis of single cell RNAseq data and functional experiments identify the microRNA miR-277 as a posttranscriptional regulator of fatty acid β-oxidation (FAO) in the intestinal lineage. Low levels of miR-277 are detected in ISC and progressively rising miR-277 levels are found in progenitors during their growth and differentiation. Supporting this, miR-277-regulated fatty acid β-oxidation enzymes progressively declined from ISC towards more differentiated cells in our pseudotime single-cell RNAseq analysis and in functional assays on RNA and protein level. In addition, in silico clustering of single-cell RNAseq data based on metabolic genes validates that stem cells and progenitors belong to two independent clusters with well-defined metabolic characteristics. Furthermore, studying FAO genes in silico indicates that two populations of ISC exist that can be categorized in mitotically active and quiescent ISC, of which the latter relies on FAO genes. In line with an FAO dependency of ISC, forced expression of miR-277 phenocopies RNAi knockdown of FAO genes by reducing ISC size and subsequently resulting in stem cell death. We also investigated miR-277 effects on ISC in a benign and our newly developed CRISPR-Cas9-based colorectal cancer model and found effects on ISC survival, which as a consequence affects tumor growth, further underlining the importance of FAO in a pathological context. Taken together, our study provides new insights into the basal metabolic requirements of intestinal stem cell on β-oxidation of fatty acids evolutionarily implemented by a sole microRNA. Gaining knowledge about the metabolic differences and dependencies affecting the survival of two central and cancer-relevant cell populations in the fly and human intestine might reveal starting points for targeted combinatorial therapy in the hope for better treatment of colorectal cancer in the future.

Periostin attenuates oxygen and glucose deprivation-induced death of mouse neural stem cells via inhibition of p38 MAPK activation

Promoting neural stem cells (NSCs) survival in the harsh niche is essential to cell replacement therapy for various central nervous system diseases. As an integral component of the extracellular matrix, Periostin (POSTN) has been shown to protect various cell types from hypoxia-ischemia damage. This study aimed to investigate the neuroprotective effects of POSTN on NSCs injury induced by oxygen and glucose deprivation (OGD). Under challenge with OGD, cell viability significantly decreased in cultured mouse NSCs, and supplement POSTN rescued cell viability in a concentration-dependent manner, as shown by CCK-8. TUNEL and propidium iodide/Hoechst staining showed that POSTN pretreatment protected NSCs against OGD-induced apoptosis. Western blot assay demonstrated that POSTN pretreatment inhibited cleavage of caspase-3 and restored the balance of Bcl-2/Bax. And pretreatment with cilengitide (an inhibitor of POSTN receptors) abolished the protective effect of POSTN. Further investigation demonstrated that supplement POSTN inhibited phosphorylation of p38 in a concentration-dependent manner. Moreover, the neuroprotective effect of POSTN was hampered by anisomycin, an activator of p38. We conclude that POSTN pretreatment in cultured mouse NSCs mitigated OGD-induced cell death, and inhibition of the p38 MAPK pathway might be one of the underlying mechanisms. Our findings may provide a novel strategy for enhancing both endogenous and exogenous NSCs survival after ischemia and hypoxia injury.

Nuclear magnetic resonance footprint of Wharton Jelly mesenchymal stem cells death mechanisms and distinctive in-cell biophysical properties in vitro.

The importance of the biophysical characterization of mesenchymal stem cells (MSCs) was recently pointed out for supporting the development of MSC‐based therapies. Among others, tracking MSCs in vivo and a quantitative characterization of their regenerative impact by nuclear magnetic resonance (NMR) demands a full description of MSCs’ MR properties. In the work, Wharton Jelly MSCs are characterized in a low magnetic field (LF) in vitro by using different approaches. They encompass various settings: MSCs cultured in a Petri dish and cell suspensions; experiments‐ 1D‐T 1, 1D‐T 2, 1D diffusion, 2D T 1‐T 2 and D‐T 2; devices‐ with a bore aperture and single‐sided one. Complex NMR analysis with the aid of random walk simulations allows the determination of MSCs T 1 and T 2 relaxation times, cells and nuclei sizes, self‐diffusion coefficients of the nucleus and cytoplasm. In addition, the influence of a single layer of cells on the effective diffusion coefficient of water is detected with the application of a single‐sided NMR device. It also enables the identification of apoptotic and necrotic cell death and changed diffusional properties of cells suspension caused by compressing forces induced by the subsequent cell layers. The study delivers MSCs‐specific MR parameters that may help tracking MSCs in vivo.

Stem Cell Factor SOX9 Interacts with a Cell Death Regulator RIPK1 and Results in Escape of Cancer Stem Cell Death.

High-grade ovarian cancer (HGOC) is the most lethal gynecological cancer, with high metastasis and recurrence. Cancer stem cells (CSCs) are responsible for its apoptosis resistance, cancer metastasis, and recurrence. Thus, targeting CSCs would be a promising strategy for overcoming chemotherapy resistance and improving patient prognosis in HGOC. Among upregulated oncogenic proteins in HGOC, we found that transcription factor SOX9 showed a strong correlation with stemness-regulating ALDH1A1 and was localized predominantly in the cytoplasm of HGOC with lymph node metastasis. In order to address the role of unusual cytoplasmic SOX9 and to explore its underlying mechanism in HGOC malignancy, a Y2H assay was used to identify a necroptotic cell death-associated cytoplasmic protein, receptor-interacting serine/threonine protein kinase 1 (RIPK1), as a novel SOX9-interacting partner and further mapped their respective interacting domains. The C-terminal region containing the transactivation domain of SOX9 interacted with the death domain of R1PK1. Consistent with its stemness-promoting function, SOX9 knockdown in vitro resulted in changes in cell morphology, cell cycle, stem cell marker expression, cell invasion, and sphere formation. Furthermore, in vivo knockdown completely inhibited tumor growth in mouse xenograft model. We propose that cytoplasmic SOX9-mediated cell death suppression would contribute to cancer stem cell survival in HGOC.

Magnetic bioactive glass nano-heterostructures: a deeper insight into magnetic hyperthermia properties in the scope of bone cancer treatment.

Primary bone cancers commonly involve surgery to remove the malignant tumor, complemented with a postoperative treatment to prevent cancer resurgence. Studies on magnetic hyperthermia, used as a single treatment or in synergy with chemo- or radiotherapy, have shown remarkable success in the past few decades. Multifunctional biomaterials with bone healing ability coupled with hyperthermia property could thus be of great interest to repair critical bone defects resulting from tumor resection. For this purpose, we designed superparamagnetic and bioactive nanoparticles (NPs) based on iron oxide cores (γ-Fe2O3) encapsulated in a bioactive glass (SiO2-CaO) shell. Nanometric heterostructures (122 ± 12 nm) were obtained through a two-step process: co-precipitation of 16 nm sized iron oxide NPs, followed by the growth of a bioactive glass shell via a modified Stöber method. Their bioactivity was confirmed by hydroxyapatite growth in simulated body fluid, and cytotoxicity assays showed they induced no significant death of human mesenchymal stem cells after 7 days. Calorimetric measurements were carried out under a wide range of alternating magnetic field amplitudes and frequencies, considering clinically relevant parameters, and some were made in viscous medium (agar) to mimic the implantation conditions. The experimental specific loss power was predictable with respect to the Linear Response Theory, and showed a maximal value of 767 ± 77 W gFe-1 (769 kHz, 23.9 kA m-1 in water). An interesting value of 166 ± 24 W gFe-1 was obtained under clinically relevant conditions (157 kHz, 23.9 kA m-1) for the heterostructures immobilized in agar. The good biocompatibility, bioactivity and heating ability suggest that these γ-Fe2O3@SiO2-CaO NPs are a promising biomaterial to be used as it is or included in a scaffold to heal bone defects resulting from bone tumor resection.

Autophagy-Dependent Ferroptosis-Related Signature is Closely Associated with the Prognosis and Tumor Immune Escape of Patients with Glioma.

BackgroundFerroptosis is an autophagy-dependent form of cell death, sometimes called “ferritinophagy”. Its related pathway has been proven to regulate the programmed death of glioma stem cells. Mining autophagy-dependent ferroptosis-related gene (AD-FRG) signature could facilitate the discovery of mechanisms and therapeutic targets showing drug resistance to chemotherapeutic drugs.MethodsWe exhaustively searched HADB, MSigDB and FerrDb datasets and obtained 25 genes confirmed to exist in autophagy and ferroptosis death pathways. Glioma gene expression and clinicopathological data were collected from TCGA and CGGA datasets.ResultsLasso regression and Cox regression analysis were carried out to construct a nine AD-FRGs signature (SIRT1, MTDH, HSPB1, CISD2, HMOX1, ATG7, MTOR, PRKAA2 and EIF2AK4). ROC curve showed that nine genes signature could effectively predict 1- (AUC = 0.869), 3- (AUC = 0.922) and 5-year (AUC = 0.870) survival rates. Immunohistochemical images confirmed the protein expression level of the gene model. The prognostic nomogram of risk score, age, WHO grade, isocitrate dehydrogenase (IDH) wild-type condition, 1p/19q co-deletion state was built. The calibration curve demonstrated that the prediction of the nomogram is highly consistent with the actual results. Moreover, tumor microenvironment analysis showed that the high-risk group was associated with high immune infiltration status and high tumor purity. Correlation analysis showed that the expression of SIRT1, CISD2 and HSPB1 might be related to macrophage infiltration and immunotolerance in glioma tissues.ConclusionBased on autophagy-dependent ferroptosis-related genes, we established gene signature and nomogram that maybe effectively predict the overall survival rate of glioma and correlate with the immunosuppressive tumor microenvironment (TME).

PRP-1, a toll-like receptor ligand, upregulates the unfolded protein response in human chondrosarcoma cells

BackgroundPrevious studies showed that proline-rich polypeptide (PRP-1) is a ligand for innate immunity toll-like receptors (TLR), and an inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) which induces the death of chondrosarcoma cancer stem cells (CSC). The aim of this study was to investigate the effect of PRP-1 on the regulation of unfolded protein response (UPR) in human chondrosarcoma cells. Materials and methodsLysates were prepared from a monolayer (bulk or ALDHhigh population), or spheroids chondrosarcoma cell cultures and treated with PRP-1 or control, followed by protein levels quantification by western blotting and mRNA expression by RT-qPCR of protein-RNA-like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α), activating transcription factor 4 (ATF4), CCAAT-enhancer-binding protein homologous protein (CHOP), activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1α), and X-box binding protein (XBP1). ResultsThe PRP-1 has been shown to increase the expression of PERK, eIF2α, ATF4, CHOP, ATF6, IRE1α, and XBP1, on both protein and mRNA levels. ConclusionPRP-1 activated UPR branches in monolayer, spheroid, and stem cell populations of human chondrosarcoma.

Monitoring Autophagy in Neural Stem and Progenitor Cells.

Autophagy is a critical cellular program that is necessary for cellular survival and adaptation to nutrient and metabolic stress. In addition to homeostatic maintenance and adaptive response functions, autophagy also plays an active role during development and tissue regeneration. Within the neural system, autophagy is important for stem cell maintenance and the ability of neural stem cells to undergo self-renewal. Autophagy also contributes toward neurogenesis and provides neural progenitorcells with sufficient energy to mediate cytoskeleton remodeling during the differentiation process. In differentiated neural cells, autophagy maintains neuronal homeostasis and viability by preventing the accumulation of toxic and pathological intracellular aggregates. However, prolonged autophagy or dysregulated upregulation of autophagy can result in autophagic cell death. Moreover, mutations or defects in autophagy that result in neural stem cell instability and cell death underlie many neurodegenerative disorders, such as Parkinson'sdisease. Thus, autophagy plays a multi-faceted role during neurogenesis from the stem cell to the differentiated neural cell. In this chapter, we describe methods to monitor autophagy at the protein and transcript level to evaluate alterations within the autophagy program in neural stem and progenitor cells. We describe immunoblotting and immunocytochemistry approaches for evaluating autophagy-dependent protein modifications, as well as quantitative real-time PCR to assess transcript levels of autophagy genes. As autophagy is a dynamic process, we highlight the importance of using late-stage inhibitors to be able to assess autophagic flux and quantify the level of autophagy occurring within cells.

FNDC5 overexpression promotes the survival rate of bone marrow mesenchymal stem cells after transplantation in a rat cerebral infarction model.

BackgroundMost bone marrow mesenchymal stem cell (BMSC) death is caused by the harsh ischemia and hypoxic microenvironment, which impacts the therapeutic effects of transplanted BMSCs. Fibronectin type III domain-containing protein 5 (FNDC5) and its cleaved product, irisin, are reportedly involved in cerebral protective effect. Research into whether FNDC5 plays a key role in the survival rate of BMSCs and cerebral infarction (CI) remains inadequate. The present study aimed to clarify the protective role of FNDC5 on the low viability of transplanted BMSCs and improve CI treatment outcomes.MethodsA lentivirus vector, which drives the expression of FNDC5, was constructed and used to transfect BMSCs. Cell Counting Kit-8 (CCK8), flow cytometry, immunofluorescence, and western blot were performed to evaluate the function of FNDC5-overexpressing BMSCs (BMSCs-OE-FNDC5) exposed to hypoxic and serum deprivation (H/SD) stress. Transmission electron microscopy (TEM) was used to monitor autophagy. In addition, BMSCs were engrafted into a middle cerebral artery occlusion (MCAO) rat model with or without FNDC5-overexpression (OE-FNDC5). The survival rate of transplanted BMSCs was evaluated by 5-ethynyl-2'-deoxyuridine (EdU) labeling. The CI volume was assessed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining.ResultsH/SD stress caused increased cell autophagy, apoptosis, and decreased cell viability of BMSCs, while OE-FNDC5 alleviated these injuries. The in vivo results showed that transplantation of BMSCs-OE-FNDC5 reduced the infarct volume in the rat MCAO model. Furthermore, OE-FNDC5 decreased neuronal apoptosis. The improved therapeutic efficacy of BMSCs-OE-FNDC5 may be attributable to the obviously increased cell survival number after transplantation.ConclusionsThese results indicated that FNDC5 overexpression promotes BMSC survival in a CI model, which might provide a potential therapeutic target.

Combined Inhibition of Bcl2 and Bcr-Abl1 Exercises Anti-Leukemia Activity but Does Not Eradicate the Primitive Leukemic Cells.

Background: The management of Philadelphia Chromosome-positive (Ph+) hematological malignancies is strictly correlated to the use of BCR-ABL1 tyrosine kinase inhibitors (TKIs). However, these drugs do not induce leukemic stem cells death and their persistence may generate a disease relapse. Published reports indicated that Venetoclax, a selective BCL2 inhibitor, could be effective in Ph+ diseases, as BCL2 anti-apoptotic activity is modulated by BCR-ABL1 kinase. We, therefore, investigated if BCL2 inhibition, alone or combined with Nilotinib, a BCR-ABL1 inhibitor, affects the primitive and committed Ph+ cells survival. Methods: We used Ph+ cells isolated from leukemic patients at diagnosis. To estimate the therapeutic efficacy of BCL2 and BCR-ABL1 inhibition we employed long-term culture, proliferation and apoptosis assay. Immunoblot was used to evaluate the ability of treatment to interfere with the down-stream targets of BCR-ABL1. Results: Blocking BCL2, we observed reduced proliferation and clonogenic potential of CML CD34-positive cells and this cytotoxicity was improved by combination with BCR-ABL1 inhibitor. However, BCL2 inhibition, alone or in combination regiment with BCR-ABL1 inhibitor, did not reduce the self-renewal of primitive leukemic cells, while strongly induced cell death on primary Ph+ Acute Lymphoblastic Leukemia (ALL). Conclusion: Our results suggest that primitive CML leukemic cells are not dependent on BCL2 for their persistence and support that committed CML and Ph + ALL cells are dependent by BCL2 and BCR-ABL1 cooperation for their survival. The antileukemic activity of BCL2 and BCR-ABL1 dual targeting may be a useful therapeutic strategy for Ph+ ALL patients.

The lupus autoantigen La/Ssb is an Xist-binding protein involved in Xist folding and cloud formation.

Using the programmable RNA-sequence binding domain of the Pumilio protein, we FLAG-tagged Xist (inactivated X chromosome specific transcript) in live mouse cells. Affinity pulldown coupled to mass spectrometry was employed to identify a list of 138 candidate Xist-binding proteins, from which, Ssb (also known as the lupus autoantigen La) was validated as a protein functionally critical for X chromosome inactivation (XCI). Extensive XCI defects were detected in Ssb knockdown cells, including chromatin compaction, death of female mouse embryonic stem cells during in vitro differentiation and chromosome-wide monoallelic gene expression pattern. Live-cell imaging of Xist RNA reveals the defining XCI defect: Xist cloud formation. Ssb is a ubiquitous and versatile RNA-binding protein with RNA chaperone and RNA helicase activities. Functional dissection of Ssb shows that the RNA chaperone domain plays critical roles in XCI. In Ssb knockdown cells, Xist transcripts are unstable and misfolded. These results show that Ssb is critically involved in XCI, possibly as a protein regulating the in-cell structure of Xist.

Lipid raft-disrupting miltefosine preferentially induces the death of colorectal cancer stem-like cells.

BackgroundLipid rafts (LRs), cholesterol‐enriched microdomains on cell membranes, are increasingly viewed as signalling platforms governing critical facets of cancer progression. The phenotype of cancer stem‐like cells (CSCs) presents significant hurdles for successful cancer treatment, and the expression of several CSC markers is associated with LR integrity. However, LR implications in CSCs remain unclear.MethodsThis study evaluated the biological and molecular functions of LRs in colorectal cancer (CRC) by using an LR‐disrupting alkylphospholipid (APL) drug, miltefosine. The mechanistic role of miltefosine in CSC inhibition was examined through normal or tumour intestinal mouse organoid, human CRC cell, CRC xenograft and miltefosine treatment gene expression profile analyses.ResultsMiltefosine suppresses CSC populations and their self‐renewal activities in CRC cells, a CSC‐targeting effect leading to irreversible disruption of tumour‐initiating potential in vivo. Mechanistically, miltefosine reduced the expression of a set of genes, leading to stem cell death. Among them, miltefosine transcriptionally inhibited checkpoint kinase 1 (CHEK1), indicating that LR integrity is essential for CHEK1 expression regulation. In isolated CD44high CSCs, we found that CSCs exhibited stronger therapy resistance than non‐CSC counterparts by preventing cell death through CHEK1‐mediated cell cycle checkpoints. However, inhibition of the LR/CHEK1 axis by miltefosine released cell cycle checkpoints, forcing CSCs to enter inappropriate mitosis with accumulated DNA damage and resulting in catastrophic cell death.ConclusionOur findings underscore the therapeutic potential of LR‐targeting APLs for CRC treatment that overcomes the therapy‐resistant phenotype of CSCs, highlighting the importance of the LR/CHEK1 axis as a novel mechanism of APLs.

Genome Maintenance Mechanisms at the Chromatin Level.

Genome integrity is constantly threatened by internal and external stressors, in both animals and plants. As plants are sessile, a variety of environment stressors can damage their DNA. In the nucleus, DNA twines around histone proteins to form the higher-order structure “chromatin”. Unraveling how chromatin transforms on sensing genotoxic stress is, thus, key to understanding plant strategies to cope with fluctuating environments. In recent years, accumulating evidence in plant research has suggested that chromatin plays a crucial role in protecting DNA from genotoxic stress in three ways: (1) changes in chromatin modifications around damaged sites enhance DNA repair by providing a scaffold and/or easy access to DNA repair machinery; (2) DNA damage triggers genome-wide alterations in chromatin modifications, globally modulating gene expression required for DNA damage response, such as stem cell death, cell-cycle arrest, and an early onset of endoreplication; and (3) condensed chromatin functions as a physical barrier against genotoxic stressors to protect DNA. In this review, we highlight the chromatin-level control of genome stability and compare the regulatory systems in plants and animals to find out unique mechanisms maintaining genome integrity under genotoxic stress.

3D Confinement Regulates Cell Life and Death

AbstractBiophysical properties of the cellular microenvironment, including stiffness and geometry, have been shown to influence cell function. Recent findings have implicated 3D confinement as an important regulator of cell behavior. The understanding of how mechanical signals direct cell function is based primarily on 2D studies. To investigate how the extent of 3D confinement affects cell function, a single cell culture platform is fabricated with geometrically defined and fully enclosed microwells and it is applied to investigate how niche volume and stiffness affect human mesenchymal stem cells (hMSC) life and death. The viability and proliferation of hMSCs in confined 3D microniches are compared with unconfined cells in 2D. Confinement biases hMSC viability and proliferation, and this influence depends on the niche volume and stiffness. The rate of cell death increases and proliferation markedly decreases upon 3D confinement. The observed differences in hMSC behavior are correlated to changes in nuclear morphology and YES‐associated protein (YAP) localization. In smaller 3D microniches, hMSCs display smaller and more rounded nuclei and primarily cytoplasmic YAP localization, indicating reduced mechanical activation upon confinement. Interestingly, these effects scale with the extent of 3D confinement. These results demonstrate that the extent of confinement in 3D can be an important regulator of cell function.

Impairment of Autophagic Flux Participates in the Antitumor Effects of TAT-Cx43266-283 in Glioblastoma Stem Cells.

Simple SummaryAutophagy is a process in which the cell recycles components that are not needed at that moment and uses the resulting elements to satisfy more urgent needs. Depending on the specific context, this can be beneficial or detrimental for tumor development. We found that in glioblastoma, the most lethal brain tumor, autophagy is upregulated and contributes to glioblastoma stem cell survival under starvation. Importantly, the antitumor peptide TAT-Cx43266-283 blocks autophagy flux, contributing to the death of glioblastoma stem cells. This peptide induces glioblastoma stem cell death in nutrient-deprived and complete environments, while the effect of other unsuccessful drugs for glioblastoma depends on nutrient context, supporting the potential of TAT-Cx43266-283 as a treatment to improve the lives of glioblastoma patients.Autophagy is a physiological process by which various damaged or non-essential cytosolic components are recycled, contributing to cell survival under stress conditions. In cancer, autophagy can have antitumor or protumor effects depending on the developmental stage. Here, we use Western blotting, immunochemistry, and transmission electron microscopy to demonstrate that the antitumor peptide TAT-Cx43266-283, a c-Src inhibitor, blocks autophagic flux in glioblastoma stem cells (GSCs) under basal and nutrient-deprived conditions. Upon nutrient deprivation, GSCs acquired a dormant-like phenotype that was disrupted by inhibition of autophagy with TAT-Cx43266-283 or chloroquine (a classic autophagy inhibitor), leading to GSC death. Remarkably, dasatinib, a clinically available c-Src inhibitor, could not replicate TAT-Cx43266-283 effect on dormant GSCs, revealing for the first time the possible involvement of pathways other than c-Src in TAT-Cx43266-283 effect. TAT-Cx43266-283 exerts an antitumor effect both in nutrient-complete and nutrient-deprived environments, which constitutes an advantage over chloroquine and dasatinib, whose effects depend on nutrient environment. Finally, our analysis of the levels of autophagy-related proteins in healthy and glioma donors suggests that autophagy is upregulated in glioblastoma, further supporting the interest in inhibiting this process in the most aggressive brain tumor and the potential use of TAT-Cx43266-283 as a therapy for this type of cancer.

Maximum Tolerated Dose and Anti-Tumor Activity of Intraperitoneal Cantrixil (TRX-E-002-1) in Patients with Persistent or Recurrent Ovarian Cancer, Fallopian Tube Cancer, or Primary Peritoneal Cancer: Phase I Study Results.

Simple SummarySurvival outcomes with standard cytotoxic chemotherapy are poor, and most patients with ovarian cancer will die with platinum-resistant disease. This may reflect the existence of drug-resistant ovarian cancer stem cells. Cantrixil is a novel third-generation benzopyran molecule, with potent cytotoxicity against chemoresistant ovarian cancer stem cells and chemosensitive ovarian cancer cell lines. The aims of this Phase I study were to define the maximum tolerated dose, tolerability, and antitumor activity of Cantrixil when administered via an intraperitoneal (IP) port. Cantrixil was tolerable even in patients with heavily pretreated disease. This first in-human study has demonstrated the potential for prolonged survival in advanced ovarian cancer by inducing ovarian cancer stem cells’ death and sensitizing cells to standard chemotherapy with IP-administered Cantrixil. Future studies should focus on confirming its mechanism of action alongside further assessment of cancer stem cell biomarkers, and determining optimal clinical settings to maximize survival outcomes in ovarian cancer.Survival outcomes in ovarian cancer are poor. The aims of this Phase I progressive design study (NCT02903771) were to evaluate the maximum tolerated dose (MTD), tolerability, and antitumor activity of Cantrixil—a novel third-generation benzopyran molecule—in patients (n = 25) with advanced, recurrent/persistent epithelial ovarian, primary peritoneal, or fallopian tube cancer. All had completed ≥ 2 prior regimens; 3 (12%) had platinum-refractory disease, and 17 (68%) had platinum-resistant disease. Following intraperitoneal (IP) port placement, patients received weekly IP Cantrixil in 3-week cycles as monotherapy (Cycles 1–2), and then in combination with intravenous (IV) chemotherapy (Cycles 3–8). Part A (dose escalation) enrolled 11 patients in 6 dose-level cohorts. An MTD of 5 mg/kg was established with dose-limiting toxicity of ileus. Most treatment-related adverse events were gastrointestinal. Across Parts A and B (dose expansion), 16 (64%) patients received ≥ 1 3-week Cantrixil cycle, and had ≥ 1 post-baseline efficacy measurement available. The results show promising anti-tumor activity in monotherapy (stable disease rate of 56%) and in combination with IV chemotherapy (objective response rate of 19%, disease control rate of 56%, and median progression-free survival of 13.1 weeks). The molecular target and mechanism of action of Cantrixil are yet to be confirmed. Preliminary analysis of stem cell markers suggests that IP Cantrixil might induce ovarian cancer stem cell death and sensitize cells to standard chemotherapy, warranting further evaluation.

A Multicenter Comparison of Rates of Central Venous Catheter-Associated Bloodstream Infections By Central Venous Catheter Type Among Pediatric Patients Receiving Treatment for Newly Diagnosed Acute Myeloid Leukemia

▪Therapy for pediatric acute myeloid leukemia (AML) requires long term central vascular access, which puts patients at risk for central line-associated bloodstream infections and their associated morbidity and mortality. Options for central venous catheter (CVC) access include tunneled external catheters, implanted ports, or peripherally inserted central catheters (PICC). Multiple studies provide conflicting reports on which CVC type is least likely to be associated with bloodstream infections in pediatric hematology-oncology patients. This multicenter cohort of pediatric AML patients is the first to investigate the course-specific incidence of positive CVC blood cultures by line type.The study included patients receiving frontline chemotherapy for AML between 1/1/2012 and 6/30/2017 at 12 US institutions. Trained personnel completed manual chart abstraction from 12/1/2015 to 07/15/2017 to obtain information on demographics, diagnosis, treatment, antibacterial prophylaxis, CVC type at the start of each course, neutropenia duration, and blood culture results obtained from CVC. Abstractors followed a standardized protocol and were required to achieve >95% concordance with trainer before starting formal chart review.Course-specific follow-up began the first day absolute neutrophil count (ANC) fell below 200 cells/uL and continued to the earliest of first positive CVC culture (overall and separately for gram-positive and gram-negative species), ANC recovery, start of conditioning for stem cell transplant, relapse, death or start of next course. Per National Healthcare Safety Network guidance, common commensals were not considered true events with the exception of viridans group streptococci (VGS). Nonbacterial pathogens were also excluded. Covariate distributions were compared by CVC type using chi-square tests. Variability in CVC type by site was examined graphically. Incidence rates for CVC positive blood cultures were reported by line type as per 1000 neutropenic days. Multivariate Poisson regression with log of neutropenia duration as offset was used to compute adjusted rate ratios (aIRR) comparing rates of positive CVC blood cultures by line type, controlling for covariates itemized in Table 1. General estimating equation methods were used to account for non-independence of observations from patients from the same institution.The study population included 357 patients who received 1,164 chemotherapy courses. Median course duration was 38 days (IQR: 34-44) with a median neutropenia duration of 19 days (IQR: 14-25). CVC were present in all patients at the start of each course, with the exception of 11 (3.3%) patients lacking a CVC at the start of Induction I. While tunneled CVC were most prevalent representing 59.1% of courses, PICC and implanted ports were utilized in 28.9% and 11.6% of courses, respectively. There was considerable variation in the distribution of CVC type by treating institution (Figure 1). Age, race, and ethnicity distributions varied by line type (Table 1). Antibacterial prophylaxis was utilized in 47% of courses.In total, there were 300 (25.8%) courses in 199 (55.7%) patients complicated by a positive CVC blood culture during the neutropenic period. The overall incidence rate of positive CVC blood cultures was 6.5 per 1000 neutropenic days. Rates increased over the course of frontline treatment from 3.1 per 1000 neutropenic days in Induction I to 9.6 per 1000 neutropenic days in Intensification II. The rate of gram-positive events (4.2 per 1000 neutropenic days) was higher than gram-negative events (2.0 per 1000 neutropenic days). The most common isolated organisms were VGS at 57.3% and less than 1% of events were polymicrobial infections. Overall rates of positive CVC blood cultures were higher for PICC relative to tunneled CVC (aIRR=1.19, 95% CI: 1.02, 1.38), while rates for implanted ports and tunneled CVC were similar (aIRR=1.06, 95% CI: 0.75, 1.51).While bacteremia is an expected complication during post-chemotherapy neutropenia and not always due to the presence of a CVC, the risk for a positive CVC blood culture is significantly increased for children with PICC compared with those with tunneled CVC or implanted port. Also, the risk was higher after Induction I and increased with age. Given the notable practice variation in line choice, these data highlight an opportunity for standardization with a goal to reduce infectious complications. [Display omitted] DisclosuresFisher:Pfizer: Research Funding; Merck: Research Funding. Gramatges:Bristol Meyer Squibb: Research Funding.

Mechanisms of stress response in the root stem cell niche.

As plants are sessile organisms unable to escape from environmental hazards, they need to adapt for survival. The stem cell niche in the root apical meristem is particularly sensitive to DNA damage induced by environmental stresses such as chilling, flooding, wounding, UV, and irradiation. DNA damage has been proven to cause stem cell death, with stele stem cells being the most vulnerable. Stress also induces the division of quiescent center cells. Both reactions disturb the structure and activity of the root stem cell niche temporarily; however, this preserves root meristem integrity and function in the long term. Plants have evolved many mechanisms that ensure stem cell niche maintenance, recovery, and acclimation, allowing them to survive in a changing environment. Here, we provide an overview of the cellular and molecular aspects of stress responses in the root stem cell niche.