Chronic sleep fragmentation (SF) increases cancer aggressiveness in mice. Exosomes exhibit pleiotropic biological functions, including immune regulatory functions, antigen presentation, intracellular communication and inter-cellular transfer of RNA and proteins. University of Chicago researchers hypothesized that SF-induced alterations in biosynthesis and cargo of plasma exosomes may affect tumor cell properties.
Plasma-derived exosomes from C57/B6 mice exposed to 6 wks of SF or sleep control (SC), and from adult human patients with obstructive sleep apnea (OSA) before (Pre) and after adherent treatment for 6 wks (Post) were co-cultured with mouse lung TC1 or human adenocarcinoma tumor cell lines, respectively. Proliferation, migration, invasion, endothelial barrier integrity and extravasation assays of tumor cells were performed. Plasma mouse exosomal miRNAs were profiled with arrays, and transcriptomic assessments of TC1 cells exposed to SF or SC exosomes were conducted to identify gene targets.
SF-derived exosomes increased tumor cell proliferation (~13%), migration (~2.3-fold) and extravasation (~10%) when compared to exosomes from SC-exposed mice. Similarly, Pre exosomes from OSA patients significantly enhanced proliferation and migration of human adenocarcinoma cells compared to Post. SF-exosomal cargo revealed 3 discrete differentially expressed miRNAs, and exploration of potential mRNA targets in TC1 tumor cells uncovered 132 differentially expressed genes that encode for multiple cancer-related pathways.
Effect of plasma-derived exosomes on Electric Cell-substrate Impedance Sensing (ECIS) and confocal microscopy imaging.
Time-course of normalized endothelial cells resistance (bEnd30 cells and their values were continuously monitored for up to 30 hours after adding exosomes to the confluent endothelial monolayer (Panel A). Plasma-derived exosomes from SF-exposed mice induce increased endothelial cell monolayer disruption compared to exosomes derived from tumor-bearing or non tumor-bearing mice exposed to SC (n = 8). Panel (B) shows values recorded at 30 hours (p-value < 0.001 for SF(−) vs. SC(−); p-value < 0.0001 for SF(+) vs. SC(+)). Panel (C) shows tight junction protein ZO-1 immunohistochemistry in bEnd3 cells following treatment with exosomes. Exosomes from SF and SC mice with (+) and without tumors (−) were applied to mouse bEnd3 cells for 24 hrs. Tight junction protein ZO-1 (green) and nuclei (DAPI blue) were immunostained with corresponding antibodies. Panel (C) is representative for SC(−), SF(−), SC(+), SF(+). Disruption of ZO-1 continuity is particularly apparent in SF mice with and without tumors. Images are representative of n = 6/group. Scale bar represents 25 μm.
Chronic SF induces alterations in exosomal miRNA cargo that alter the biological properties of TC1 lung tumor cells to enhance their proliferative, migratory and extravasation properties, and similar findings occur in OSA patients, in whom SF is a constitutive component of their sleep disorder. Thus, exosomes could participate, at least in part, in the adverse cancer outcomes observed in OSA.