scholarly journals Small Extracellular Vesicles from Head and Neck Squamous Cell Carcinoma Cells Carry a Proteomic Signature for Tumor Hypoxia

Cancers ◽  
2021 ◽  
Vol 13 (16) ◽  
pp. 4176
Author(s):  
Alicja Głuszko ◽  
Mirosław J. Szczepański ◽  
Theresa L. Whiteside ◽  
Torsten E. Reichert ◽  
Jacek Siewiera ◽  
...  
Keyword(s):  
Head And Neck ◽  
Extracellular Vesicles ◽  
Squamous Cell ◽  
High Resolution Mass Spectrometry ◽  
Protein Profile ◽  
Tumor Hypoxia ◽  
Size Exclusion ◽  
Hypoxic Conditions ◽  
Angiogenic Proteins ◽  
Exclusion Chromatography

Tissue hypoxia is commonly observed in head and neck squamous cell carcinomas (HNSCCs), resulting in molecular and functional alterations of the tumor cells. The aim of this study was to characterize tumor-derived small extracellular vesicles (sEVs) released under hypoxic vs. normoxic conditions and analyze their proteomic content. HNSCC cells (FaDu, PCI-30, SCC-25) and HaCaT keratinocytes were cultured in 21, 10, 5, and 1% O2. sEVs were isolated from supernatants using size exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis, electron microscopy, immunoblotting, and high-resolution mass spectrometry. Isolated sEVs ranged in size from 125–135 nm and contained CD63 and CD9 but not Grp94. sEVs reflected the hypoxic profile of HNSCC parent cells: about 15% of the total detected proteins were unique for hypoxic cells. Hypoxic sEVs expressed a common signature of seven hypoxia-related proteins (KT33B, DYSF, STON2, MLX, LIPA3, NEK5, P12L1) and were enriched in pro-angiogenic proteins. Protein profiles of sEVs reflected the degree of tumor hypoxia and could serve as potential sEV-based biomarkers for hypoxic conditions. Adaptation of HNSCC cells to hypoxia is associated with increased release of sEVs, which are enriched in a unique protein profile. Thus, tumor-derived sEVs can potentially be useful for evaluating levels of hypoxia in HNSCC.

scholarly journals Tumor hypoxia is associated with resistance to PD-1 blockade in squamous cell carcinoma of the head and neck

2021 ◽  
Vol 9 (5) ◽  
pp. e002088
Author(s):  
Dan P Zandberg ◽  
Ashley V Menk ◽  
Maria Velez ◽  
Daniel Normolle ◽  
Kristin DePeaux ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
T Cells ◽  
Cell Carcinoma ◽  
Head And Neck ◽  
Squamous Cell ◽  
Cd8 T Cells ◽  
Oxidative Metabolism ◽  
Cox Regression ◽  
Tumor Hypoxia ◽  
Parental Line

The majority of patients with recurrent/metastatic squamous cell carcinoma of the head and neck (HNSCC) (R/M) do not benefit from anti-PD-1 therapy. Hypoxia induced immunosuppression may be a barrier to immunotherapy. Therefore, we examined the metabolic effect of anti-PD-1 therapy in a murine MEER HNSCC model as well as intratumoral hypoxia in R/M patients. In order to characterize the tumor microenvironment in PD-1 resistance, a MEER cell line was created from the parental line that are completely resistant to anti-PD-1. These cell lines were then metabolically profiled using seahorse technology and injected into C57/BL6 mice. After tumor growth, mice were pulsed with pimonidazole and immunofluorescent imaging was performed to analyze hypoxia and T cell infiltration. To validate the preclinical results, we analyzed tissues from R/M patients (n=36) treated with anti-PD-1 mAb, via immunofluorescent imaging for number of CD8+ T cells (CD8), Tregs and the percent area (CAIX) and mean intensity (I) of carbonic anhydrase IX in tumor. We analyzed disease control rate (DCR), progression free survival (PFS), and overall survival (OS) using proportional odds and proportional hazards (Cox) regression. We found that anti-PD-1 resistant MEER has significantly higher oxidative metabolism, while there was no difference in glycolytic metabolism. Intratumoral hypoxia was significantly increased and CD8+ T cells decreased in anti-PD-1 resistant tumors compared with parental tumors in the same mouse. In R/M patients, lower tumor hypoxia by CAIX/I was significantly associated with DCR (p=0.007), PFS, and OS, and independently associated with response (p=0.028) and PFS (p=0.04) in a multivariate model including other significant immune factors. During PD-1 resistance, tumor cells developed increased oxidative metabolism leading to increased intratumoral hypoxia and a decrease in CD8+ T cells. Lower tumor hypoxia was independently associated with increased efficacy of anti-PD-1 therapy in patients with R/M HNSCC. To our knowledge this is the first analysis of the effect of hypoxia in this patient population and highlights its importance not only as a predictive biomarker but also as a potential target for therapeutic intervention.

scholarly journals Kinetics and Topology of DNA Associated with Circulating Extracellular Vesicles Released during Exercise

Genes ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 522
Author(s):  
Elmo W. I. Neuberger ◽  
Barlo Hillen ◽  
Katharina Mayr ◽  
Perikles Simon ◽  
Eva-Maria Krämer-Albers ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Size Exclusion ◽  
Affinity Capture ◽  
Healthy Humans ◽  
Dnase Digestion ◽  
And Topology ◽  
Single Bout ◽  
Exclusion Chromatography ◽  
Free Circulating Dna ◽  
Line Elements

Although it is widely accepted that cancer-derived extracellular vesicles (EVs) carry DNA cargo, the association of cell-free circulating DNA (cfDNA) and EVs in plasma of healthy humans remains elusive. Using a physiological exercise model, where EVs and cfDNA are synchronously released, we aimed to characterize the kinetics and localization of DNA associated with EVs. EVs were separated from human plasma using size exclusion chromatography or immuno-affinity capture for CD9+, CD63+, and CD81+ EVs. DNA was quantified with an ultra-sensitive qPCR assay targeting repetitive LINE elements, with or without DNase digestion. This model shows that a minute part of circulating cell-free DNA is associated with EVs. During rest and following exercise, only 0.12% of the total cfDNA occurs in association with CD9+/CD63+/CD81+EVs. DNase digestion experiments indicate that the largest part of EV associated DNA is sensitive to DNase digestion and only ~20% are protected within the lumen of the separated EVs. A single bout of running or cycling exercise increases the levels of EVs, cfDNA, and EV-associated DNA. While EV surface DNA is increasing, DNAse-resistant DNA remains at resting levels, indicating that EVs released during exercise (ExerVs) do not contain DNA. Consequently, DNA is largely associated with the outer surface of circulating EVs. ExerVs recruit cfDNA to their corona, but do not carry DNA in their lumen.

Microfluidic Size Exclusion Chromatography (μSEC) for Extracellular Vesicles and Plasma Protein Separation

Small ◽  
2022 ◽  
pp. 2104470
Author(s):  
Sheng Yuan Leong ◽  
Hong Boon Ong ◽  
Hui Min Tay ◽  
Fang Kong ◽  
Megha Upadya ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Plasma Protein ◽  
Protein Separation ◽  
Size Exclusion ◽  
Exclusion Chromatography

scholarly journals Cisplatin-Resistance in Oral Squamous Cell Carcinoma: Regulation by Tumor Cell-Derived Extracellular Vesicles

Cancers ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 1166 ◽  
Author(s):  
Xin-Hui Khoo ◽  
Ian C. Paterson ◽  
Bey-Hing Goh ◽  
Wai-Leng Lee
Keyword(s):  
Drug Resistance ◽  
Squamous Cell Carcinoma ◽  
Oral Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Extracellular Vesicles ◽  
Squamous Cell ◽  
Metal Ion ◽  
De Novo ◽  
Protein Profile ◽  
Severe Problem

Drug resistance remains a severe problem in most chemotherapy regimes. Recently, it has been suggested that cancer cell-derived extracellular vesicles (EVs) could mediate drug resistance. In this study, the role of EVs in mediating the response of oral squamous cell carcinoma (OSCC) cells to cisplatin was investigated. We isolated and characterized EVs from OSCC cell lines showing differential sensitivities to cisplatin. Increased EV production was observed in both de novo (H314) and adaptive (H103/cisD2) resistant lines compared to sensitive H103 cells. The protein profiles of these EVs were then analyzed. Differences in the proteome of EVs secreted by H103 and H103/cisD2 indicated that adaptation to cisplatin treatment caused significant changes in the secreted nanovesicles. Intriguingly, both resistant H103/cisD2 and H314 cells shared a highly similar EV protein profile including downregulation of the metal ion transporter, ATP1B3, in the EVs implicating altered drug delivery. ICP-MS analysis revealed that less cisplatin accumulated in the resistant cells, but higher levels were detected in their EVs. Therefore, we inhibited EV secretion from the cells using a proton pump inhibitor and observed an increased drug sensitivity in cisplatin-resistant H314 cells. This finding suggests that control of EV secretion could be a potential strategy to enhance the efficacy of cancer treatment.

Expression of Erythropoietin and Erythropoietin Receptor in Squamous Cell Carcinomas of the Head and Neck: Levels of Erythropoietin Expression Correlate with Tumor Hypoxia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2908-2908
Author(s):  
Murat O. Arcasoy ◽  
Khalid Amin ◽  
Shu-Chuan Chou ◽  
Zishan A. Haroon ◽  
Mahesh Varia ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Head And Neck Cancer ◽  
Cell Carcinoma ◽  
Head And Neck ◽  
Tumor Cells ◽  
Neck Cancer ◽  
Squamous Cell ◽  
Tumor Hypoxia ◽  
Squamous Cell Carcinomas ◽  
Glycoprotein Hormone

Abstract Erythropoietin (EPO), an oxygen-regulated glycoprotein hormone, is a hematopoietic cytokine that stimulates erythropoiesis by binding to its cellular receptor EPOR. The recombinant form of human EPO is widely used in clinical practice for the prevention or treatment of anemia associated with cancer and chemo-radiation therapy. However, in a recent randomized, placebo-controlled trial involving patients receiving curative radiotherapy for squamous cell carcinoma of the head and neck, EPO treatment was associated with poorer loco-regional progression-free survival. The purpose of this study was to determine whether EPOR and its ligand EPO are expressed in primary squamous cell carcinomas of the head and neck. We also investigated the hypothesis that EPO expression in malignant cells may be associated with the presence of tumor hypoxia, an important factor involved in resistance to radiation treatment, tumor aggressiveness and poor prognosis. Twenty-one patients with squamous cell carcinoma of the head and neck were enrolled in a tumor hypoxia study under a research protocol approved by the Institutional Review Board at the University of North Carolina Hospitals. All patients provided signed informed consent. The patients received an intravenous infusion of the hypoxia marker pimonidazole hydrochloride (Hypoxyprobe-1™) prior to multiple tumor biopsies. Two or more biopsies were available from all except one primary tumor. The tissue specimen from one patient with laryngeal carcinoma was excluded because of availability of only a single, small and fragmented biopsy. Contiguous sections from 74 biopsies were analyzed by immunohistochemistry for expression of EPOR and EPO as well as pimonidazole binding. We found EPOR expression in tumor cells in 97% of the biopsies. The pattern of EPOR immunoreactivity was predominantly cytoplasmic but was found to be localized to the membrane in some sections. Co-expression of EPO was observed in 90% of biopsies. Qualitative and semi-quantitative analyes for EPO staining and tumor hypoxia on a section-by-section basis revealed that EPO and pimonidazole adduct staining did not always co-localize within tumors but there was a significant positive correlation between levels of micro-regional EPO expression and pimonidazole binding (r = 0.736, P < 0.001, n=20 by two-tailed Spearman’s rank correlation analysis). These data demonstrate the co-expression of EPOR and its ligand EPO in squamous carcinoma cells suggesting that EPO may play a novel role as a potential autocrine or paracrine growth factor in head and neck cancer. Furthermore, EPO expression in tumor cells may be modulated, at least in part, by tumor hypoxia. The expression of EPOR needs to be taken into consideration in the design of future clinical trials investigating the role of recombinant human EPO in head and neck cancer.

scholarly journals Size-Exclusion Chromatography-based isolation minimally alters Extracellular Vesicles’ characteristics compared to precipitating agents

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Ana Gámez-Valero ◽  
Marta Monguió-Tortajada ◽  
Laura Carreras-Planella ◽  
Marcel·la Franquesa ◽  
Katrin Beyer ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Size Exclusion ◽  
Exclusion Chromatography

scholarly journals Proteomic analysis of extracellular vesicles in cerebrospinal fluid of patients with Alzheimer’s disease

2020 ◽  
Author(s):  
Davide Chiasserini ◽  
Irene Bijnsdorp ◽  
Giovanni Bellomo ◽  
Pier Luigi Orvietani ◽  
Sander R. Piersma ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Cerebrospinal Fluid ◽  
Extracellular Vesicles ◽  
Biomarker Discovery ◽  
Neurodegenerative Disorder ◽  
High Resolution Mass Spectrometry ◽  
Biological Fluids ◽  
Protein Profile ◽  
Protein Markers

AbstractCerebrospinal fluid (CSF) contains different types of extracellular vesicles (EVs) with undisclosed biomarker potential for neurodegenerative diseases. The aims of the present study were: i) to compare the proteome EVs isolated using different ultracentrifugation speed ii) to preliminary explore the EVs proteome in a common neurodegenerative disorder, Alzheimer’s disease (AD) compared to neurological controls. CSF samples from control subjects and AD patients were pooled separately (15 mL) and subjected to ultracentrifugation (UC) at different speeds (20,000g and 100,000g) to isolate separate EV fractions (P20 and P100). The proteome was analysed using high-resolution mass spectrometry (LC-MS/MS) and comparisons were made using bioinformatic analysis. EVs isolated at 100,000g (P100) had a proteome consistent with vesicles secreted via an ESCRT-dependent mechanism, being highly enriched in alix (PDCD6IP), syntenin-1 (SDCBP) and TSG101. EVs isolated at 20,000g were substantially different, showing enrichment in cytoskeletal and cell adhesion molecules. The pools from patients diagnosed with AD showed a distinct protein profile of CSF EVs, with increased levels of ADAM10, SPON1, CH3IL1 and MDK in the P100 fraction. CSF EV offer a new potential biosource of protein markers for AD detection and a complementary framework to the analysis of whole biological fluids for biomarker discovery.

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