Detection Of Cellular Senescence Research Articles

An Indocyanine Green‐Based Nanoprobe for In Vivo Detection of Cellular Senescence

AbstractThere is an urgent need to improve conventional cancer‐treatments by preventing detrimental side effects, cancer recurrence and metastases. Recent studies have shown that presence of senescent cells in tissues treated with chemo‐ or radiotherapy can be used to predict the effectiveness of cancer treatment. However, although the accumulation of senescent cells is one of the hallmarks of cancer, surprisingly little progress has been made in development of strategies for their detection in vivo. To address a lack of detection tools, we developed a biocompatible, injectable organic nanoprobe (NanoJagg), which is selectively taken up by senescent cells and accumulates in the lysosomes. The NanoJagg probe is obtained by self‐assembly of indocyanine green (ICG) dimers using a scalable manufacturing process and characterized by a unique spectral signature suitable for both photoacoustic tomography (PAT) and fluorescence imaging. In vitro, ex vivo and in vivo studies all indicate that NanoJaggs are a clinically translatable probe for detection of senescence and their PAT signal makes them suitable for longitudinal monitoring of the senescence burden in solid tumors after chemotherapy or radiotherapy.

Rapid and Live-Cell Detection of Senescence in Mesenchymal Stem Cells by Micro Magnetic Resonance Relaxometry.

Detection of cellular senescence is important quality analytics of cell therapy products, including mesenchymal stromal cells (MSCs). However, its detection is critically limited by the lack of specific markers and the destructive assays used to read out these markers. Here, we establish a rapid, live-cell assay for detecting senescent cells in heterogeneous mesenchymal stromal cell (MSC) cultures. We report that the T2 relaxation time measured by microscale Magnetic Resonance Relaxometry, which is related to intracellular iron accumulation, correlates strongly with senescence markers in MSC cultures under diverse conditions, including different passages and donors, size-sorted MSCs by inertial spiral microfluidic device, and drug-induced senescence. In addition, the live-cell and non-destructive method presented here has general applicability to other cells and tissues and can critically advance our understanding of cellular senescence.

Detection of Cellular Senescence in Human Primary Melanocytes and Malignant Melanoma Cells In Vitro.

Detection and quantification of senescent cells remain difficult due to variable phenotypes and the absence of highly specific and reliable biomarkers. It is therefore widely accepted to use a combination of multiple markers and cellular characteristics to define senescent cells in vitro. The exact choice of these markers is a subject of ongoing discussion and usually depends on objective reasons such as cell type and treatment conditions, as well as subjective considerations including feasibility and personal experience. This study aims to provide a comprehensive comparison of biomarkers and cellular characteristics used to detect senescence in melanocytic systems. Each marker was assessed in primary human melanocytes that overexpress mutant BRAFV600E, as it is commonly found in melanocytic nevi, and melanoma cells after treatment with the chemotherapeutic agent etoposide. The combined use of these two experimental settings is thought to allow profound conclusions on the choice of senescence biomarkers when working with melanocytic systems. Further, this study supports the development of standardized senescence detection and quantification by providing a comparative analysis that might also be helpful for other cell types and experimental conditions.

Design and Development of an HBT-Based Ratiometric Fluorescent Probe to Monitor Stress-Induced Premature Senescence.

Stress-induced premature senescence (SIPS) can be induced in tumor cells by reactive oxygen species (ROS) or oncogenes. The antineoplastic drugs cause apoptosis and senescence by damaging the DNA. Although the detection of cellular senescence is important to monitor drug response during anticancer therapy, only a few probes have been studied for imaging SIPS. In this study, we developed 2-(2′-hydroxyphenyl)benzothiazole (HBT)-based fluorescent probes to determine SIPS by monitoring the oxidative stress and β-galactosidase activity. HBT is a commonly used fluorophore because of its luminescence mechanism via excited-state intramolecular proton transfer, and it has attractive properties, such as a four-level photochemical process and large Stokes shift (151 nm). A novel fluorescent probe, (2-(benzo[d]thiazol-2-yl)phenyl)boronic acid, was prepared for the detection of ROS, including H2O2, via the oxidation reaction of arylboronic acids to form the fluorescent phenol, HBT. In addition, to determine the enzymatic activity of β-galactosidase, a 2-(4′-chloro-2′-hydroxyphenyl)benzothiazole (CBT)-based enzymatic turn-on probe (CBT-β-Gal) was designed and synthesized. β-Galactosidase catalyzed the hydrolysis of β-galactopyranoside from CBT-β-Gal to release the fluorescent CBT. These probes were capable of ratiometric imaging the accumulation of H2O2 and the degree of β-galatosidase activity in contrast to H2O2-untreated and H2O2-treated HeLa cells. Furthermore, these probes were successfully employed for imaging the increased levels of ROS and β-galactosidase activity in the doxorubicin-treated HeLa cells.

Real-time imaging of senescence in tumors with DNA damage

Detection of cellular senescence is important not only in the study of senescence in various biological systems, but also in various practical applications such as image-guided surgical removal of senescent cells, as well as the monitoring of drug-responsiveness during cancer therapies. Due to the lack of suitable imaging probes for senescence detection, particularly in living subjects, we have developed an activatable near-infrared (NIR) molecular probe with far-red excitation, NIR emission, and high “turn-on” ratio upon senescence-associated β-galactosidase (SABG) activation. We present here the first successful demonstration of NIR imaging of DNA damage-induced senescence both in vitro and in human tumor xenograft models.

Abstract P5-10-01: Cellular senescence within HER2-amplified breast cancer: Potential implications for breast cancer immune surveillance and HER2-targeted therapy resistance

Abstract Background:Oncogene-induced senescence is considered as a barrier to tumor progression that arrests cells at risk for malignant transformation. Nevertheless, numerous findings demonstrate that senescent cells may also have the opposite function and promote tumor progression through the release of multiple factors called the senescence-associated secretory phenotype or senescence secretome. It is likely that the composition and the physiological consequences mediated by the senescence secretome are dependent on the oncogenes that trigger the senescence program. Breast cancer represents a heterogenous disease that can be divided into breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities. Therefore, tumor initiation and progression of breast cancer subtypes is triggered by variable oncogenic stimuli, and differences in the senescence secretomes within breast tumors might be responsible for tumor initiation, progression, metastasis and therapeutic response. Beside many studies concerning the role of senescence as a barrier to tumor progression using murine xenograft models very few investigations have been performed to elucidate how often senescent tumor cells appear within untreated human tumors, and if present whether these senescent tumor cells may play a role in disease progression, cancer immunosurveillance and therapy resistance. Methode: In the present study we analysed the appearance of senescent cells within invasive human breast cancers from 129 untreated patients. Cellular senescence was detected by the use of SAβ-gal staining and by immunohistochemical detection of p16, p21, p53, Ki67 and lamin B1. Results: Detection of cellular senescence by the use of SAβ-gal staining and detection of p16, p53, Ki67 and lamin B1 within invasive breast carcinomas indicate that senescent tumor cells varies strongly according to the breast cancer subtype. The highest percentages of senescent tumor cells exist within in the HER2-positive and luminal A breast carcinomas whereas no or very few senescent tumor cells were found in triple negative breast tumors. Based on these findings we suggest that the composition of secretomes released by senescent tumor cells from different breast cancer subtypes might be very distinct in respect to their ability to recruit immune cells, which can eliminate senescent tumor cells on one hand and regulate tumor growth, immune surveillance and therapy resistance on the other. Conclusion: Further characterization of senescent secretomes from HER2 and other breast cancer subtypes and their potential role in tumor progression, immune surveillance and therapy response might be warranted for the understanding of cancer biology as well as prognostic and therapeutic applications. Citation Format: Thaler S, Schad A, Kirkpatrick CJ, Sleeman JP, Springer E, Schmidt M, Cotarelo CL. Cellular senescence within HER2-amplified breast cancer: Potential implications for breast cancer immune surveillance and HER2-targeted therapy resistance [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-10-01.

Induction and Detection of Oncogene-Induced Cellular Senescence in Drosophila.

Cellular senescence is induced by various cellular stresses, including activation of the Ras oncogene. In Drosophila imaginal epithelia, clones of cells expressing oncogenic Ras (RasV12) show several markers of cellular senescence, such as elevation of SA-β-gal activity, upregulation of the Cdk inhibitor Dacapo (Dap), and heterochromatinization. However, these cells do not undergo cell cycle arrest or exhibit a DNA damage response (DDR), cellular hypertrophy, or a senescence-associated secretory phenotype (SASP), other essential markers of cellular senescence. However, we found that inducing mitochondrial dysfunction within RasV12-expressing cells caused all above-mentioned aspects of cellular senescence. This provided the first evidence that cellular senescence occurs in invertebrates and is intriguing because mitochondrial dysfunction is frequently observed in human cancers. Here, we describe the procedures for the induction and detection of cellular senescence in Drosophila epithelia.

Detection of cellular senescence within human invasive breast carcinomas distinguishes different breast tumor subtypes.

Oncogene-induced senescence is thought to act as a barrier to tumorigenesis by arresting cells at risk of malignant transformation. Nevertheless, numerous findings suggest that senescent cells may conversely promote tumor progression through the development of the senescence-associated secretome they produce. It is likely that the composition and the physiological consequences mediated by the senescence secretome are dependent on the oncogenes that trigger the senescence program. Breast cancer represents a heterogenous disease that can be divided into breast cancer subtypes due to different subsets of genetic and epigenetic abnormalities. As tumor initiation and progression of these breast cancer subtypes is triggered by diverse oncogenic stimuli, differences in the senescence secretomes within breast tumors might be responsible for tumor initiation, progression, metastasis and therapeutic response. Many studies have addressed the role of senescence as a barrier to tumor progression using murine xenograft models. However, few investigations have been performed to elucidate the degree to which senescent tumor cells are present within untreated human tumors, and if present, whether these senescent tumor cells may play a role in disease progression. In the present study we analysed the appearance of senescent cells within invasive breast cancers. Detection of cellular senescence by the use of SAβ-galactosidase (SAβ-gal) staining within invasive breast carcinoms from 129 untreated patients revealed differences in the amount of SAβ-gal+ tumor cells between breast cancer subtypes. The highest percentages of SAβ-gal+ tumor cells were found in HER2-positive and luminal A breast carcinomas whereas triple negative tumors showed either little or no positivity.