Cell Fate In Response Research Articles

Structural characterization and expression analysis of novel MAPK1 transcript variants with the development of a multiplexed targeted nanopore sequencing approach.

Mitogen-activated protein kinases (MAPKs) represent a protein family firmly involved in many signaling cascades, regulating a vast spectrum of stimulated cellular processes. Studies have shown that alternatively spliced isoforms of MAPKs play a crucial role in determining the desired cell fate in response to specific stimulations. Although the implication of most MAPKs transcript variants in the MAPK signaling cascades has been clarified, the transcriptional profile of a pivotal member, MAPK1, has not been investigated for the existence of additional isoforms. In the current study we developed and implemented targeted long-read and short-read sequencing approaches to identify novel MAPK1 splice variants. The combination of nanopore sequencing and NGS enabled the implementation of a long-read polishing pipeline using error-rate correction algorithms, which empowered the high accuracy of the results and increased the sequencing efficiency. The utilized multiplexing option in the nanopore sequencing approach allowed not only the identification of novel MAPK1 mRNAs, but also elucidated their expression profile in multiple human malignancies and non-cancerous cell lines. Our study highlights for the first time the existence of ten previously undescribed MAPK1 mRNAs (MAPK1 v.3 - v.12) and evaluates their relative expression levels in comparison to the main MAPK1 v.1. The optimization and employment of qPCR assays revealed that MAPK1 v.3 - v.12 can be quantified in a wide spectrum of human cell lines with notable specificity. Finally, our findings suggest that the novel protein-coding mRNAs are highly expected to participate in the regulation of MAPK pathways, demonstrating differential localizations and functionalities.

Seleno-aspirin compound AS-10 promotes histone acetylation ahead of suppressing androgen receptor transcription, G1 arrest, and apoptosis of prostate cancer cells.

The novel selenium-aspirin compound AS-10 was recently reported by us with a cancer cell killing potency three orders of magnitude greater than aspirin in pancreatic cancer cell lines with caspase-mediated apoptosis and a reasonable selectivity against malignant cells. Although we also observed its cytocidal activity against PC-3 and DU145 androgen receptor (AR)-negative and P53-null/mutant aggressive human prostate cancer (PCa) cell lines in NCI-60 screen, the potential involvement and targeting of AR and P53 pathways that are intact in early-stage prostate carcinogenesis has not been examined, nor its primary molecular signaling after exposure. Human LNCaP PCa cells with functional AR and intact P53 were used to examine their cell cycle and cell fate responses to AS-10 exposure and upstream molecular signaling events including histone acetylation as a known aspirin effect. The AR-positive 22Rv1 human PCa cells were used to validate key findings. In addition to confirming AS-10's superior cytocidal potency than aspirin against all four PCa cell lines, we report a rapid (within 5 min) promotion of histone acetylation several hours ahead of the suppression of AR and prostate-specific antigen (PSA, coded by KLK3 gene) in LNCaP and 22Rv1 cells. AS-10 decreased AR and KLK3 mRNA levels without impacting pre-existing AR protein degradation or nuclear translocation in LNCaP cells. Sustained exposure to AS-10 arrested cells predominantly in G1 , and induced caspase-mediated apoptosis without necrosis. The death induced by AS-10 in LNCaP cells was attenuated by nontranscriptional activation of P53 protein or Jun N-terminal Kinase cellular stress signaling and was mitigated modestly by glutathione-boosting antioxidant N-acetylcysteine. AS-10 synergized with histone deacetylase inhibitor SAHA to suppress AR/PSA abundance and kill LNCaP cells. RNA-seq confirmed AR suppression at the transcriptional level and suggested multiple oncogene, cyclin, and CDK/CKI transcriptional actions to contribute to the cellular consequences. AS-10 promotes histone acetylation as its probable primary mechanism of action to induce PCa cell-cycle arrest and apoptosis, regardless of AR and P53 status. Nevertheless, the inhibition of AR signaling through mechanisms distinct from canonical AR antagonists may hold promise for combinatorial use with androgen deprivation therapy regimens or AR-axis targeting drugs.

Proteostasis Deregulation in Neurodegeneration and Its Link with Stress Granules: Focus on the Scaffold and Ribosomal Protein RACK1.

The role of protein misfolding, deposition, and clearance has been the dominant topic in the last decades of investigation in the field of neurodegeneration. The impairment of protein synthesis, along with RNA metabolism and RNA granules, however, are significantly emerging as novel potential targets for the comprehension of the molecular events leading to neuronal deficits. Indeed, defects in ribosome activity, ribosome stalling, and PQC—all ribosome-related processes required for proteostasis regulation—can contribute to triggering stress conditions and promoting the formation of stress granules (SGs) that could evolve in the formation of pathological granules, usually occurring during neurodegenerating effects. In this review, the interplay between proteostasis, mRNA metabolism, and SGs has been explored in a neurodegenerative context with a focus on Alzheimer’s disease (AD), although some defects in these same mechanisms can also be found in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), which are discussed here. Finally, we highlight the role of the receptor for activated C kinase 1 (RACK1) in these pathologies and note that, besides its well characterized function as a scaffold protein, it has an important role in translation and can associate to stress granules (SGs) determining cell fate in response to diverse stress stimuli.

Abstract 136: Doxorubicin-induced senescence as a mechanism of resistance in TNBC cell lines

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer which lacks HER2 overexpression, as well estrogen and progesterone receptor expression. TNBC constitutes 10-15% of all breast cancers but has a worse prognosis due to limited treatment options and high rates of metastatic recurrence. Doxorubicin (dox) remains one of the most active chemotherapy agents for the treatment of TNBC, although de novo and acquired resistance to chemotherapy remains a major challenge. The purpose of this work was to characterize dox resistant cell phenotypes and investigate treatment-induced senescence in p53-mutated (Mut) and wildtype (WT) TNBC cell lines. Methods: A panel of 12 TNBC cell lines (p53 WT and p53 Mut) were exposed to dox (0-5 uM) for 72 hours and cellular proliferation was determined using the Cell Titer Glo assay. A subset of sensitive and resistant cell lines were treated with dox (0.1, 0.25 uM) or vehicle control and apoptosis was determined by flow cytometry (Annexin-V) at 24 and 48 hrs. Cells were treated with dox or control for 24 hrs and western blotting was performed for mediators of apoptosis. Senescence was analyzed by ß-galactosidase staining following treatment with dox for 3-14 days. shRNA knockdown (KD) of p53 was performed in the CAL-51 (p53 WT cell line) and cells were subject to investigations above. Results: Doxorubicin treatment resulted in decreased cellular proliferation and increased apoptosis as assessed by Annexin-V expression in a subset of p53 WT and p53 Mut cell lines. Treatment with dox resulted in an increase in the pro-apoptotic proteins BID and p21, as well as a decrease in VAMP in sensitive cell lines. In a subset of cell lines resistant to dox treatment, we observed an increase in cells demonstrating phenotypic features of senescence and ß -galactosidase staining. We observed a decrease in p16 with dox treatment in the CAL-51 (p53 WT cell line) compared to an increase following treatment in p53 Mut cell lines. KD of p53 resulted in an increase in senescent cells following treatment with low dose dox. Conclusions: Treatment with doxorubicin resulted in different terminal cell phenotypes in TNBC cell lines with apoptosis observed in p53 WT and Mut cell lines. Senescence was observed in resistant cells and KD of p53 increased dox-induced senescence, confirming a role for p53 in mediating terminal cell fate. Efforts are ongoing to understand the role of mutant p53 in mediating terminal cell fate in response to dox and rational combinations to overcome dox-induced senescence may be clinically active in metastatic TNBC. Citation Format: Stephen G. Smoots, Anna R. Schreiber, Betelehem Yacob, Adrian Dominguez, Cecilia Levandowski, Todd M. Pitts, Jennifer R. Diamond. Doxorubicin-induced senescence as a mechanism of resistance in TNBC cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 136.

Taking Advantage of the Senescence-Promoting Effect of Olaparib after X-ray and Proton Irradiation Using the Senolytic Drug, ABT-263.

Simple SummaryRadiotherapy is one of the most common treatments for cancer. Overcoming the failure and side effects of radiotherapy are current challenges. It has been recently demonstrated that senescence contributes to radioresistance. Cellular senescence is a permanent arrest in cell proliferation induced by various factors, such as radiation. Here, we aimed to assess the potential of combining the radiation and DNA damage repair inhibitor, Olaparib to a senolytic drug, ABT-263. We demonstrated that combining radiation, Olaparib and ABT-263 successfully targeted the radio-induced senescent cells resulting in increased cell death and reduced senescence-associated secretory phenotype. These results paved the way towards a new therapeutic combination for patients treated with radiotherapy and Olaparib.Radiotherapy (RT) is a key component of cancer treatment. Although improvements have been made over the years, radioresistance remains a challenge. For this reason, a better understanding of cell fates in response to RT could improve therapeutic options to enhance cell death and reduce adverse effects. Here, we showed that combining RT (photons and protons) to noncytotoxic concentration of PARP inhibitor, Olaparib, induced a cell line-dependent senescence-like phenotype. The senescent cells were characterized by morphological changes, an increase in p21 mRNA expression as well as an increase in senescence-associated β-galactosidase activity. We demonstrated that these senescent cells could be specifically targeted by Navitoclax (ABT-263), a Bcl-2 family inhibitor. This senolytic drug led to significant cell death when combined with RT and Olaparib, while limited cytotoxicity was observed when used alone. These results demonstrate that a combination of RT with PARP inhibition and senolytics could be a promising therapeutic approach for cancer patients.

Reprogramming bone progenitor identity and potency through control of collagen density and oxygen tension

SummaryThe biophysical microenvironment of the cell is being increasingly used to control cell signaling and to direct cell function. Herein, engineered 3D tuneable biomimetic scaffolds are used to control the cell microenvironment of Adipose-derived Mesenchymal Stromal Cells (AMSC), which exhibit a collagen density-specific profile for early and late stage bone cell lineage status. Cell potency was enhanced when AMSCs were cultured within low collagen density environments in hypoxic conditions. A transitional culture containing varied collagen densities in hypoxic conditions directed differential cell fate responses. The early skeletal progenitor identity (PDPN+CD146−CD73+CD164+) was rescued in the cells which migrated into low collagen density gels, with cells continuously exposed to the high collagen density gels displaying a transitioned bone-cartilage-stromal phenotype (PDPN+CD146+CD73−CD164-). This study uncovers the significant contributions of the physical and physiological cell environment and highlights a chemically independent methodology for reprogramming and isolating skeletal progenitor cells from an adipose-derived cell population.

Biosensor for deconvolution of individual cell fate in response to ion beam irradiation

SummaryClonogenic survival assay constitutes the gold standard method for quantifying radiobiological effects. However, it neglects cellular radiation response variability and heterogeneous energy deposition by ion beams on the microscopic scale. We introduce “Cell-Fit-HD4D” a biosensor that enables a deconvolution of individual cell fate in response to the microscopic energy deposition as visualized by optical microscopy. Cell-Fit-HD4D enables single-cell dosimetry in clinically relevant complex radiation fields by correlating microscopic beam parameters with biological endpoints. Decrypting the ion beam’s energy deposition and molecular effects at the single-cell level has the potential to improve our understanding of radiobiological dose concepts as well as radiobiological study approaches in general.

YAP/TAZ in Bone and Cartilage Biology.

YAP and TAZ were initially described as the main regulators of organ growth during development and more recently implicated in bone biology. YAP and TAZ are regulated by mechanical and cytoskeletal cues that lead to the control of cell fate in response to the cellular microenvironment. The mechanical component represents a major signal for bone tissue adaptation and remodelling, so YAP/TAZ contributes significantly in bone and cartilage homeostasis. Recently, mice and cellular models have been developed to investigate the precise roles of YAP/TAZ in bone and cartilage cells, and which appear to be crucial. This review provides an overview of YAP/TAZ regulation and function, notably providing new insights into the role of YAP/TAZ in bone biology.

Roles of Key Epigenetic Regulators in the Gene Transcription and Progression of Prostate Cancer.

Prostate cancer (PCa) is a top-incidence malignancy, and the second most common cause of death amongst American men and the fifth leading cause of cancer death in men around the world. Androgen receptor (AR), the key transcription factor, is critical for the progression of PCa by regulating a series of target genes by androgen stimulation. A number of co-regulators of AR, including co-activators or co-repressors, have been implicated in AR-mediated gene transcription and PCa progression. Epigenetic regulators, by modifying chromatin integrity and accessibility for transcription regulation without altering DNA sequences, influence the transcriptional activity of AR and further regulate the gene expression of AR target genes in determining cell fate, PCa progression and therapeutic response. In this review, we summarized the structural interaction of AR and epigenetic regulators including histone or DNA methylation, histone acetylation or non-coding RNA, and functional synergy in PCa progression. Importantly, epigenetic regulators have been validated as diagnostic markers and therapeutic targets. A series of epigenetic target drugs have been developed, and have demonstrated the potential to treat PCa alone or in combination with antiandrogens.

Alternative Splicing of MAPKs in the Regulation of Signaling Specificity

The mitogen-activated protein kinase (MAPK) cascades transmit signals from extracellular stimuli to a variety of distinct cellular processes. The MAPKKs in each cascade specifically phosphorylate and activate their cognate MAPKs, indicating that this step funnels various signals into a seemingly linear pathway. Still, the effects of these cascades vary significantly, depending on the identity of the extracellular signals, which gives rise to proper outcomes. Therefore, it is clear that the specificity of the signals transmitted through the cascades is tightly regulated in order to secure the desired cell fate. Indeed, many regulatory components or processes that extend the specificity of the cascades have been identified. Here, we focus on a less discussed mechanism, that is, the role of distinct components in each tier of the cascade in extending the signaling specificity. We cover the role of distinct genes, and the alternatively spliced isoforms of MAPKKs and MAPKs, in the signaling specificity. The alternatively spliced MEK1b and ERK1c, which form an independent signaling route, are used as the main example. Unlike MEK1/2 and ERK1/2, this route’s functions are limited, including mainly the regulation of mitotic Golgi fragmentation. The unique roles of the alternatively spliced isoforms indicate that these components play an essential role in determining the proper cell fate in response to distinct stimulations.

Targeting CAMKK2 and SOC Channels as a Novel Therapeutic Approach for Sensitizing Acute Promyelocytic Leukemia Cells to All-Trans Retinoic Acid.

Calcium ions (Ca2+) play important and diverse roles in the regulation of autophagy, cell death and differentiation. Here, we investigated the impact of Ca2+ in regulating acute promyelocytic leukemia (APL) cell fate in response to the anti-cancer agent all-trans retinoic acid (ATRA). We observed that ATRA promotes calcium entry through store-operated calcium (SOC) channels into acute promyelocytic leukemia (APL) cells. This response is associated with changes in the expression profiles of ORAI1 and STIM1, two proteins involved in SOC channels activation, as well as with a significant upregulation of several key proteins associated to calcium signaling. Moreover, ATRA treatment of APL cells led to a significant activation of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and its downstream effector AMP-activated protein kinase (AMPK), linking Ca2+ signaling to autophagy. Pharmacological inhibition of SOC channels and CAMKK2 enhanced ATRA-induced cell differentiation and death. Altogether, our results unravel an ATRA-elicited signaling pathway that involves SOC channels/CAMKK2 activation, induction of autophagy, inhibition of cellular differentiation and suppression of cell death. We suggest that SOC channels and CAMKK2 may constitute novel drug targets for potentiating the anti-cancer effect of ATRA in APL patients.

Patterns of differentiation of renin lineage cells during nephrogenesis.

Developmentally heterogeneous renin-expressing cells serve as progenitors for mural, glomerular, and tubular cells during nephrogenesis and are collectively termed renin lineage cells (RLCs). In this study, we quantified different renal vascular and tubular cell types based on specific markers and assessed proliferation and de novo differentiation in the RLC population. We used kidney sections of mRenCre-mT/mG mice throughout nephrogenesis. Marker positivity was evaluated in whole digitalized sections. At embryonic day 16, RLCs appeared in the developing kidney, and the expression of all stained markers in RLCs was observed. The proliferation rate of RLCs did not differ from the proliferation rate of non-RLCs. RLCs expanded mainly by de novo differentiation (neogenesis). Fractions of RLCs originating from the stromal progenitors of the metanephric mesenchyme (renin-producing cells, vascular smooth muscle cells, and mesangial cells) decreased during nephrogenesis. In contrast, aquaporin-2-positive RLCs in the collecting duct system, which embryonically emerges almost exclusively from the ureteric bud, expanded postpartum. The cubilin-positive RLC fraction in the proximal tubule, deriving from the cap mesenchyme, remained constant. In summary, RLCs were continuously detectable in the vascular and tubular compartments of the kidney during nephrogenesis. Therein, various patterns of RLC differentiation that depend on the embryonic origin of the cells were identified.NEW & NOTEWORTHY The unifying feature of the renal renin lineage cells (RLCs) is their origin from renin-expressing progenitors. RLCs evolve to an embryologically heterogeneous large population in structures with different ancestry. RLCs are also targets for the widely used renin-angiotensin-system blockers, which modulate their phenotype. Unveiling the different differentiation patterns of RLCs in the developing kidney contributes to understanding changes in their cell fate in response to homeostatic challenges and the use of antihypertensive drugs.

CDKN2A Determines Mesothelioma Cell Fate to EZH2 Inhibition.

Malignant pleural mesothelioma is an aggressive cancer, heterogeneous in its presentation and behaviour. Despite an increasing knowledge about molecular markers and their diagnostic and prognostic value, they are not used as much as they might be for treatment allocation. It has been recently reported that mesothelioma cells that lack BAP1 (BRCA1 Associated Protein) are sensitive to inhibition of the EZH2 (Enhancer of Zeste Homolog 2) histone methyltransferase. Since we observed strong H3K27me3 (histone H3 lysine 27 trimetylation) immunoreactivity in BAP1 wild-type mesothelioma biopsies, we decided to characterize in vitro the response/resistance of BAP1 wild-type mesothelioma cells to the EZH2 selective inhibitor, EPZ-6438. Here we demonstrate that BAP1 wild-type mesothelioma cells were rendered sensitive to EPZ-6438 upon SIRT1 (Sirtuin 1) silencing/inhibition or when cultured as multicellular spheroids, in which SIRT1 expression was lower compared to cells grown in monolayers. Notably, treatment of spheroids with EPZ-6438 abolished H3K27me3 and induced the expression of CDKN2A (Cyclin-Dependent Kinase Inhibitor 2A), causing cell growth arrest. EPZ-6438 treatment also resulted in a rapid and sustained induction of the genes encoding HIF2α (Hypoxia Inducible Factor 2α), TG2 (Transglutaminase 2) and IL-6 (Interleukin 6). Loss of CDKN2 is a common event in mesothelioma. CDKN2A silencing in combination with EPZ-6438 treatment induced apoptotic death in mesothelioma spheroids. In a CDKN2A wild-type setting apoptosis was induced by combining EPZ-6438 with 1-155, a TG2 selective and irreversible inhibitor. In conclusion, our data suggests that the expression of CDKN2A predicts cell fate in response to EZH2 inhibition and could potentially stratify tumors likely to undergo apoptosis.

Dying to Survive-The p53 Paradox.

Simple Summaryp53 is best known for its tumour suppressive functions mediated through regulation of an extensive-gene regulatory network. Here we review progress in our understanding of the complex role that p53 plays in controlling expression of cell-death regulatory pathways; how paradoxically, this may be important for cellular and organismal survival in response to homeostatic stress; the potential impact on the response to p53 activating therapies; and the ways that this might be exploited in cancer types for which maintaining wild-type p53 is beneficial.The p53 tumour suppressor is best known for its canonical role as “guardian of the genome”, activating cell cycle arrest and DNA repair in response to DNA damage which, if irreparable or sustained, triggers activation of cell death. However, despite an enormous amount of work identifying the breadth of the gene regulatory networks activated directly and indirectly in response to p53 activation, how p53 activation results in different cell fates in response to different stress signals in homeostasis and in response to p53 activating anti-cancer treatments remains relatively poorly understood. This is likely due to the complex interaction between cell death mechanisms in which p53 has been activated, their neighbouring stressed or unstressed cells and the local stromal and immune microenvironment in which they reside. In this review, we evaluate our understanding of the burgeoning number of cell death pathways affected by p53 activation and how these may paradoxically suppress cell death to ensure tissue integrity and organismal survival. We also discuss how these functions may be advantageous to tumours that maintain wild-type p53, the understanding of which may provide novel opportunity to enhance treatment efficacy.

Stress effects on the reactive oxygen species-dependent regulation of plant growth and development.

Plant growth is mediated by cell proliferation and expansion. Both processes are controlled by a network of endogenous factors such as phytohormones, reactive oxygen species (ROS), sugars, and other signals, which influence gene expression and post-translational regulation of proteins. Stress resilience requires rapid and appropriate responses in plant growth and development as well as defence. Regulation of ROS accumulation in different cellular compartments influences growth responses to abiotic and biotic stresses. While ROS are essential for growth, they are also implicated in the stress-induced cessation of growth and, in some cases, programmed cell death. It is widely accepted that redox post-translational modifications of key proteins determine the growth changes and cell fate responses to stress, but the molecular pathways and factors involved remain poorly characterized. Here we discuss ROS as a signalling molecule, the mechanisms of ROS-dependent regulation that influence protein-protein interactions, protein function, and turnover, together with the relocation of key proteins to different intracellular compartments in a manner that can alter cell fate. Understanding how the redox interactome responds to stress-induced increases in ROS may provide a road map to tailoring the dynamic ROS interactions that determine growth and cell fate in order to enhance stress resilience.

Cdkn1a transcript variant 2 is a marker of aging and cellular senescence.

Cellular senescence is a cell fate response characterized by a permanent cell cycle arrest driven primarily the by cell cycle inhibitor and tumor suppressor proteins p16Ink4a and p21Cip1/Waf1. In mice, the p21Cip1/Waf1 encoding locus, Cdkn1a, is known to generate two transcripts that produce identical proteins, but one of these transcript variants is poorly characterized. We show that the Cdkn1a transcript variant 2, but not the better-studied variant 1, is selectively elevated during natural aging across multiple mouse tissues. Importantly, mouse cells induced to senescence in culture by genotoxic stress (ionizing radiation or doxorubicin) upregulated both transcripts, but with different temporal dynamics: variant 1 responded nearly immediately to genotoxic stress, whereas variant 2 increased much more slowly as cells acquired senescent characteristics. Upon treating mice systemically with doxorubicin, which induces widespread cellular senescence in vivo, variant 2 increased to a larger extent than variant 1. Variant 2 levels were also more sensitive to the senolytic drug ABT-263 in naturally aged mice. Thus, variant 2 is a novel and more sensitive marker than variant 1 or total p21Cip1/Waf1 protein for assessing the senescent cell burden and clearance in mice.

The functions and regulation of heat shock proteins; key orchestrators of proteostasis and the heat shock response.

Cells respond to protein-damaging (proteotoxic) stress by activation of the Heat Shock Response (HSR). The HSR provides cells with an enhanced ability to endure proteotoxic insults and plays a crucial role in determining subsequent cell death or survival. The HSR is, therefore, a critical factor that influences the toxicity of protein stress. While named for its vital role in the cellular response to heat stress, various components of the HSR system and the molecular chaperone network execute essential physiological functions as well as responses to other diverse toxic insults. The effector molecules of the HSR, the Heat Shock Factors (HSFs) and Heat Shock Proteins (HSPs), are also important regulatory targets in the progression of neurodegenerative diseases and cancers. Modulation of the HSR and/or its extended network have, therefore, become attractive treatment strategies for these diseases. Development of effective therapies will, however, require a detailed understanding of the HSR, important features of which continue to be uncovered and are yet to be completely understood. We review recently described and hallmark mechanistic principles of the HSR, the regulation and functions of HSPs, and contexts in which the HSR is activated and influences cell fate in response to various toxic conditions.

Development of Next Generation Biomedical Sensor for Single Cell Four Dimensional Tracing of Cellular Response to Ion Beam Irradiation

Clonogenic survival of few colonies out of a population of cells that have received a defined amount of energy (J) per mass (kg) represented by the dose unit Gray (Gy) constitutes the gold standard method for quantifying radiobiological effects. We aimed to engineer the next generation biophysical hybrid detector technology using ion beam irradiation with clinical high linear energy transfer (LET) carbon ions combined with 4D single cell fate imaging. Development of a novel biomedical sensor (Cell-Fit-HD4D) and the first successful deconvolution of individual cell fate in response to microscopic ion beam deposition visualized by optical microscopy is reported. Cell-Fit-HD4D enabled single-cell dosimetry in clinically relevant complex radiation fields. A spectrum of experimentally derived physical parameter such as the quantity and the sum of LETs (ΣLET) of primary carbon ion particles traversing cell nuclei were correlated with biological endpoints i.e., DNA-Damage repair kinetic, chronic cell cycle arrest (senescence) and clonogenic survival. Decrypting the physical dose and molecular effects at single cell resolution via Cell-Fit-HD 4D has the potential to revolutionize our comprehension of radiobiological dose concept.

The Art of Engineering Biomimetic Cellular Microenvironments.

Cells and their surrounding microenvironment exist in dynamic reciprocity, where bidirectional feedback and feedforward crosstalk drives essential processes in development, homeostasis, and disease. With the ongoing explosion of customizable biomaterial innovation for dynamic cell culture, an ever-expanding suite of user-programmable scaffolds now exists to probe cell fate in response to spatiotemporally controlled biophysical and biochemical cues. Here, we highlight emerging trends in these efforts, emphasizing strategies that offer tunability over complex network mechanics, present biomolecular cues anisotropically, and harness cells as physiochemical actuators of the pericellular niche. Altogether, these material advances will lead to breakthroughs in our basic understanding of how cells interact with, integrate signals from, and influence their surrounding microenvironment.

Senolytic Targeting of Bcl-2 Anti-Apoptotic Family Increases Cell Death in Irradiated Sarcoma Cells.

Simple SummaryLimited volumetric change after pre-operative radiotherapy (RT) suggests that sarcomas generally do not undergo cell death. Senolytic drugs represent a highly promising field as a new therapy approach to drive senescent cancer cells towards cell death to enhance treatment response. Here, we demonstrate that the Bcl-2 family of anti-apoptotic proteins in irradiated senescent sarcoma cells represents a senotherapeutic target to improve the cell death response in RT. This study paves the way for new treatment options in soft tissue sarcoma management.Radiotherapy (RT) is a key component of cancer treatment. Most of the time, radiation is given after surgery but for soft-tissue sarcomas (STS), pre-surgical radiation is commonly utilized. However, despite improvements in RT accuracy, the rate of local recurrence remains high and is the major cause of death for patients with STS. A better understanding of cell fates in response to RT could provide new therapeutic options to enhance tumour cell killing by RT and facilitate surgical resection. Here, we showed that irradiated STS cell cultures do not die but instead undergo therapy-induced senescence (TIS), which is characterized by proliferation arrest, senescence-associated β-galactosidase activity, secretion of inflammatory cytokines and persistent DNA damage. STS-TIS was also associated with increased levels of the anti-apoptotic Bcl-2 family of proteins which rendered cells targetable using senolytic Bcl-2 inhibitors. As oppose to radiation alone, the addition of senolytic agents Venetoclax (ABT-199) or Navitoclax (ABT-263) after irradiation induced a rapid apoptotic cell death in STS monolayer cultures and in a more complex three-dimensional culture model. Together, these data suggest a new promising therapeutic approach for sarcoma patients who receive neoadjuvant RT. The addition of senolytic agents to radiation treatments may significantly reduce tumour volume prior to surgery and thereby improve the clinical outcome of patients.