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

Abstract

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.

TGFβ+ small extracellular vesicles from head and neck squamous cell carcinoma cells reprogram macrophages towards a pro‐angiogenic phenotype

“…Migration of ECs towards TGFβ high TEX was significantly blocked by mRER (p < 0.01; Figure 3G and H). sEVs isolated from normal keratinocytes (HaCaT cells), which were shown to have a less angiogenic profile compared to TEX (Głuszko et al, 2021), did not significantly stimulate EC migration (Figure S3A). Flow cytometry was used to study the uptake of TEX by ECs and macrophages.…”

Exosomal MicroRNA-223, MicroRNA-146, and MicroRNA-21 Profiles and Biochemical Changes in Laryngeal Cancer

“…Identification of tumor-specific extracellular molecules may be used as a valuable diagnostic tool for the early detection of malignancy. − To identify extracellular molecular phenotypes associated with laryngeal tumors, we isolated serum exosomes from LSCC patients and healthy volunteers. First, serum exosomes were characterized by scanning electron microscopy (SEM) analysis, and exosome shape and size were confirmed as shown in Figure .…”

Levels of small extracellular vesicles in patients treated with hyperbaric oxygenation

“…First, fraction #4 obtained from each patient was concentrated with an Amicon Centrifugal Filter for 40 min at 4,000 x g at RT. Similarly to method previously reported [ 17 , 19 ], the protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher) and Multiskan GO (Thermo Fisher Scientific). sEV preparations were then normalized for protein content (20 μl each), and 2 μl of RIPA buffer (Sigma-Aldrich) and protease inhibitor (2 μl) were added to each sample and incubated on ice for 20 min.…”

“…Size distributions and concentrations of sEVs in fraction #4 samples were analyzed using ZetaView (Particle Matrix) [ 19 ]. For method optimization, varying concentrations of fraction #4 were loaded onto the machine, while the final number of particles in 1 ml of serum was calculated taking all the dilutions from the previous step into account.…”