Impact of Radiation on Tumor Derived Exosome Production, Proteomics and Biodistribution

Abstract

Exosomes are cell derived nanoparticles implicated in malignant processes, including the determination of organ specific metastasis. This study aims to test the hypothesis that radiation interferes with the ability of tumor-derived exosomes to establish a pre-metastatic niche by affecting exosome production and proteomics. To understand the effect of radiation across tumors with different radiation sensitivities, breast cancer cell lines with known organotropism (MDA-MB-231 parental and the lung tropic subline, 4175) were compared to melanoma cancer cell line (B16F10) and exposed to 2 Gy vs. 10 Gy radiation. Exosomes were harvested from conditioned media using differential centrifugation and characterized using protein BCA, NanoSight, and electron microscopy. Exosome size was further characterized using a novel asymmetric flow fractionator. DNA packaging was quantified with Qubit ds DNA assay kit and proteomics analyzed through mass spectrometry. Biodistribution was assessed in vivo through retro-orbital sinus injection of fluorescently labeled exosomes and subsequent imaging of organ distribution. Radiation increased the quantity of exosomes produced in a dose dependent manner. Across all cell lines, 2 Gy vs. 10 Gy radiation increased exosome yield (ug/cell) by an average of 2.1 ± 0.4 and 7.8 ± 2.7-fold, respectively. In radioresistant melanoma cells, 2 Gy and 10 Gy radiation increased DNA content per exosome by 1.9 and 8.2-fold, respectively, whereas in more radiosensitive breast cancer cells, 2 Gy and 10 Gy only moderately increased DNA packaging by 1.1 and 1.4-fold. In general, radiation decreased exosome particle size. In breast cancer cells, mean particle size decreased from 135±6.9 nm to 125.7±1.8 and 124.3±3.9 nm for 2 Gy and 10 Gy radiation, respectively. Radiation also decreased the abundance of the smallest exosome subparticles, exomeres, which preferentially traffic to the liver to effect changes in metabolomics. For breast cancer cell lines, radiated exosomes retained the ability to traffic to the lung and liver 24 hours after injection. Pathway analysis of proteomic changes across radiation subgroups implicate significant changes in integrin signaling and remodeling of epithelial adherens junctions, including integrins and metalloproteases. Radiation significantly increased the quantity and DNA packaging of exosomes across all tumor cell lines. Although radiated exosomes trafficked to potential metastatic sites 24 hours after injection, their function in establishing a pre-metastatic niche remains to be explored. Changes in proteomics and exosome subpopulations suggest differences in migration and liver education. These changes trended with radiation dose and intrinsic radiation sensitivity, offering a promising platform for monitoring treatment response and underlying tumor biology in patients.