DataSheet_1_CD9- and CD81-positive extracellular vesicles provide a marker to monitor glioblastoma cell response to photon-based and proton-based radiotherapy.docx

Glioblastoma multiforme (GBM) is the most aggressive tumor of the central nervous system with a poor prognosis. In the treatment of GBM tumors, radiotherapy plays a major role. Typically, GBM tumors cannot be cured by irradiation because of intrinsic resistance machanisms. An escalation of the irradiation dose in the GBM tumor is difficult due to the high risk of severe side effects in the brain. In the last decade, the development of new irradiation techniques, including proton-based irradiation, promised new chances in the treatment of brain tumors. In contrast to conventional radiotherapy, irradiation with protons allows a dosimetrically more confined dose deposition in the tumor while better sparing the normal tissue surrounding the tumor. A systematic comparison of both irradiation techniques on glioblastoma cells has not been performed so far. Despite the improvements in radiotherapy, it remains challenging to predict the therapeutical response of GBM tumors. Recent publications suggest extracellular vesicles (EVs) as promising markers predicting tumor response. Being part of an ancient intercellular communication system, virtually all cells release specifically composed EVs. The assembly of EVs varies between cell types and depends on environmental parameters. Here, we compared the impact of photon-based with proton-based radiotherapy on cell viability and phenotype of four different glioblastoma cell lines. Furthermore, we characterized EVs released by different glioblastoma cells and correlated released EVs with the cellular response to radiotherapy. Our results demonstrated that glioblastoma cells reacted more sensitive to irradiation with protons than photons, while radiation-induced cell death 72 h after single dose irradiation was independent of the irradiation modality. Moreover, we detected CD9 and CD81-positive EVs in the supernatant of all glioblastoma cells, although at different concentrations. The amount of released CD9 and CD81-positive EVs increased after irradiation when cells became apoptotic. Although secreted EVs of non-irradiated cells were not predictive for radiosensitivity, their increased EV release after irradiation correlated with the cytotoxic response to radiotherapy 72 h after irradiation. Thus, our data suggest a novel application of EVs in the surveillance of anti-cancer therapies.</p

CRISPR/Cas9-mediated knockout of <i>c-REL</i> in HeLa cells results in profound defects of the cell cycle

<div><p>Cervical cancer is the fourth common cancer in women resulting worldwide in 266,000 deaths per year. Belonging to the carcinomas, new insights into cervical cancer biology may also have great implications for finding new treatment strategies for other kinds of epithelial cancers. Although the transcription factor NF-κB is known as a key player in tumor formation, the relevance of its particular subunits is still underestimated. Here, we applied CRISPR/Cas9n-mediated genome editing to successfully knockout the NF-κB subunit <i>c-REL</i> in HeLa Kyoto cells as a model system for cervical cancers. We successfully generated a homozygous deletion in the <i>c-REL</i> gene, which we validated using sequencing, qPCR, immunocytochemistry, western blot analysis, EMSA and analysis of off-target effects. On the functional level, we observed the deletion of <i>c-REL</i> to result in a significantly decreased cell proliferation in comparison to wildtype (wt) without affecting apoptosis. The impaired proliferative behavior of <i>c-REL</i>^-/- cells was accompanied by a strongly decreased amount of the H2B protein as well as a significant delay in the prometaphase of mitosis compared to <i>c-REL</i>^+/+ HeLa Kyoto cells. <i>c-REL</i>^-/- cells further showed significantly decreased expression levels of <i>c-REL</i> target genes in comparison to wt. In accordance to our proliferation data, we observed the <i>c-REL</i> knockout to result in a significantly increased resistance against the chemotherapeutic agents 5-Fluoro-2’-deoxyuridine (5-FUDR) and cisplatin. In summary, our findings emphasize the importance of c-REL signaling in a cellular model of cervical cancer with direct clinical implications for the development of new treatment strategies.</p></div

CRISPR/Cas9-mediated deletion of <i>c-REL</i> results in a decreased proliferation of HeLa Kyoto cell accompanied by strongly reduced amounts of histone H2B.

<p><b>A:</b> Cell number assessed by Orangu Cell Proliferation Assay Kit (Cell Guidance Systems) set against cultivation time showed a strongly increased population doubling time of <i>c-REL</i> knockout cells compared to wt HeLa Kyoto cells. PDT: Population doubling time. <b>B:</b> Flow cytometric DNA content measurements of DAPI-stained <i>c-REL</i> knockout cells showed a decrease in the amount of mitotic cells in <i>c-REL</i> knockout cells compared to wildtype. <b>C:</b> Flow cytometric analysis of Annexin V-stained <i>c-REL</i>^-/- and wt HeLa Kyoto cells revealed only slightly increased amounts of apoptotic cells upon <i>c</i>-REL deletion in comparison to wt. <b>D:</b> Flow cytometric analysis of histone H2B-mCherry showed a strongly decreased amount of the H2B protein in 41.48% of <i>c-REL</i>^-/- HeLa Kyoto cells, which was observable in only 8.67% of HeLa Kyoto wt cells.</p

Overexpression of <i>REL</i> in human cancers.

<p>Overexpression of <i>REL</i> in human cancers.</p

Successful validation of the <i>c-REL</i> knockout in HeLa Kyoto cells on DNA, mRNA and protein level.

<p><b>A:</b> Genomic PCR depicting a profound deletion of the <i>c-REL</i> gene in the <i>c-REL</i> knockout clone (band at 300 bp) compared to the wt clone (band at 700 bp). <b>B:</b> Sequencing analysis confirmed the knockout in exon 2 of <i>c-REL</i>. <b>C:</b> qPCR with specific primers in targeted deletion of exon 2 showed no expression of <i>c-REL</i> on mRNA level in the <i>c-REL</i> knockout clone in comparison to wt. <b>D:</b> Western blot analysis validated the knockout of <i>c-REL</i> on protein level. <b>E:</b> Electrophoretic mobility shift assays (EMSA) showed DNA-binding of c-REL in HeLa Kyoto wt cells (arrow), which was not observable in the <i>c-REL</i> KO clone. <b>F:</b> Immunocytochemistry depicted a nearly complete loss of c-REL-protein in <i>c-REL</i> knockout clone compared to HeLa Kyoto wt cells.</p

Assessment of <i>c-REL</i> overexpression in human cancers and target design of CRISPR/Cas9n-mediated <i>c-REL</i> knockout.

<p><b>A</b>: Database mining revealed more profound overexpression of <b><i>c-</i></b><i>REL</i> in cancers from human ovary, cervix and endometrium compared to oesophagus ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182373#pone.0182373.ref035" target="_blank">35</a>], sancer.sanger.ac.uk; 02-14-2017 16:00; 02-21-2017 15:10). <b>B</b>: Target design showing the proposed <b><i>c-</i></b><i>REL</i> knockout with an expected deletion around 450 bp targeting the intron 1-exon 2-boundary of the <b><i>c-</i></b><i>REL</i> gene. The design was done with the CRISPR/Cas9n Target Online Predictor from the University of Heidelberg [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182373#pone.0182373.ref036" target="_blank">36</a>], crispr.cos.uni-heidelberg.de) and the gene sequence was taken from Ensembl Genome Browser (ensembl.org).</p

<i>c-REL</i> knockout leads to significantly decreased expression levels of NF-κB family member and cell cycle-associated genes.

<p><b>A:</b> qPCR analysis showing significantly decreased mRNA levels of NF-κB family members <i>RELA</i>, <i>NFKB1 (p50)</i>, <i>NFKB2</i> (<i>p52)</i>, <i>IKBKE</i> and <i>TBK1</i> in <i>c-REL</i> knockout cells compared to wildtype cells. <b>B-C:</b> Expression levels of cell cycle-related <i>c-REL</i> target genes <i>A20</i>, <i>BCL2</i>, <i>BCL-XL</i> and <i>TGFB1</i> and <i>c-REL</i> target genes <i>MYC</i> and <i>ICAM-1</i> were significantly decreased in <i>c-REL</i> knockout cells in comparison to HeLa Kyoto wildtype cells. <b>D:</b> Western blot analysis validated the reduced expression levels of RELA and A20 in <i>c-REL</i>^-/- cells in comparison to wt on protein level. WB were performed after TNFα-dependent stimulation of <i>c-REL</i>^-/- and <i>c-REL</i>^+/+ cells. <b>E:</b> Immunocytochemistry revealed a strongly decreased protein amount of ICAM in <i>c-REL</i>^-/- cells in comparison to wt.</p

Supplementary information files for Cell culture-derived extracellular vesicles: Considerations for reporting cell culturing parameters

Supplementary files for article Cell culture-derived extracellular vesicles: Considerations for reporting cell culturing parametersCell culture‐conditioned medium (CCM) is a valuable source of extracellular vesicles (EVs) for basic scientific, therapeutic and diagnostic applications. Cell culturing parameters affect the biochemical composition, release and possibly the function of CCM‐derived EVs (CCM‐EV). The CCM‐EV task force of the Rigor and Standardization Subcommittee of the International Society for Extracellular Vesicles aims to identify relevant cell culturing parameters, describe their effects based on current knowledge, recommend reporting parameters and identify outstanding questions. While some recommendations are valid for all cell types, cell‐specific recommendations may need to be established for non‐mammalian sources, such as bacteria, yeast and plant cells. Current progress towards these goals is summarized in this perspective paper, along with a checklist to facilitate transparent reporting of cell culturing parameters to improve the reproducibility of CCM‐EV research.</p

Cell culture‐derived extracellular vesicles: Considerations for reporting cell culturing parameters

Cell culture‐conditioned medium (CCM) is a valuable source of extracellular vesicles (EVs) for basic scientific, therapeutic and diagnostic applications. Cell culturing parameters affect the biochemical composition, release and possibly the function of CCM‐derived EVs (CCM‐EV). The CCM‐EV task force of the Rigor and Standardization Subcommittee of the International Society for Extracellular Vesicles aims to identify relevant cell culturing parameters, describe their effects based on current knowledge, recommend reporting parameters and identify outstanding questions. While some recommendations are valid for all cell types, cell‐specific recommendations may need to be established for non‐mammalian sources, such as bacteria, yeast and plant cells. Current progress towards these goals is summarized in this perspective paper, along with a checklist to facilitate transparent reporting of cell culturing parameters to improve the reproducibility of CCM‐EV research.</p