Supplementary Materials http://advances. suggests that different intracellular compartments contribute to exosome loading, resulting in distinct exosome subpopulations. However, the loading of gDNA and other nuclear contents into exosomes (nExo) remains poorly understood. Here, we identify the relationship between cancer cell micronuclei (MN), which are markers of genomic instability, and nExo formation. Imaging flow cytometry analyses reveal that 10% of exosomes derived from cancer cells and <1% of exosomes derived from blood and ascites from patients with ovarian cancer carry nuclear contents. Treatment with genotoxic drugs resulted in increased MN and nExos both in vitro and in vivo. We observed that multivesicular body precursors and exosomal markers, such as the tetraspanins, directly interact with MN. Collectively, this work provides new insights related to nExos, which have implications for cancer biomarker development. INTRODUCTION Exosomes TFR2 are small extracellular vesicles that mediate biological and cellular functions including cell-to-cell communication (= 62 [kidney chromophobe (KICH)], = 418 [brain low-grade glioma (LGG)], = 7 [pancreatic cancer (PAAD)], = 138 [pheochromocytoma (PCPG)], = 353 [prostate adenocarcinoma (PRAD)], = 184 [thyroid carcinoma (THCA)], = 543 [glioblastoma (GBM)], = 415 [kidney clear cell carcinoma (KIRC)], = Trabectedin 61 [uveal melanoma (UVM)], = 415 [uterine endometrial carcinoma (UCEC)], = 257 [skin cutaneous melanoma (SKCM)], = 501 [head and neck squamous carcinoma (HNSC)], = 155 [kidney papillary carcinoma (KIRP)], = 330 [stomach adenocarcinoma (STAD)], = 940 [breast cancer (BRCA)], = 187 [liver hepatocellular carcinoma (LIHC)], = 396 [colon adenocarcinoma (COAD)], = 34 [cervical cancer (CESC)], = 85 [adrenocortical carcinoma (ACC)], = 158 [renal adenocarcinoma (READ)], Trabectedin = 435 [lung squamous carcinoma (LUSC)], = 544 [ovarian cancer (OV)], = 429 [lung adenocarcinoma (LUAD)], = 144 [bladder cancer (BLCA)], and = 55 [uterine carcinosarcoma (UCS)]. (B) Cryo-EM image of the exosomes isolated from OVCAR-5 cells. Scale bars, 100 nm. (C) NTA for the exosomes isolated from OVCAR-5 cells. (D) Western blot analysis of exosome markers in OVCAR-5. TSG101, Alix, and CD63 are used as exosome markers, and GRP94 is used as a marker of cellular contamination. TCL, total cell lysate. (E) Pie chart of cellular compartment proteins resulting from MS analysis in OVCAR-5 cellCderived exosomes. Nuclear components are highlighted Trabectedin in red: 1, endoplasmic reticulum; 2, endosome; 3, Golgi; 4, cell surface; 5, mitochondrion; 6, proteasome; 7, vacuole; 8, spliceosomal complex. (F) Counts of the cellular compartment origin of proteins resulting from MS analysis in OVCAR-5 cellCderived exosomes. The axis represents the categories of cellular compartments. Nuclear proteins identified in chromosome and nucleus are highlighted in red. (G) CNVs of both the exosomal DNA (inner red circle) and cellular DNA (outer blue circle), both derived from OVCAR-5 cells, are displayed on a chromosome map generated using Circos (v0.69.3). The outermost circle represents human chromosomes with coordinates (megabases). The green and red histograms inside the blue and red inner circles represent copy number alterations identified by cnvkit. The larger the bar on the track, the larger the copy number alteration (log scale). Green bars represent amplification events, and red bars represent deletions. (H) A Venn diagram of all the CNVs overlapping between the exosomal and cellular DNA derived from OVCAR-5 cells. (I) Representative plots of OVCAR-5 exosomes from flow cytometry analysis. Top left: Particles are shown as black dots, and exosomes are in the green area. Right: Each dot indicates single exosomes stained with CellMask Green (Ch02), and the red gate indicates DNA-positive particles stained with DRAQ5 (Ch11). Bottom left: Snapshots of individually stained exosomes. (A) and (B) are the exosomes present in the areas indicated in the Trabectedin right panel. (A) represents the DNA-positive exosomes, and Trabectedin (B) represents the negative exosomes. (J) Representative gate images of OVCAR-5 exosomes from imaging flow cytometry analysis. Left: Each green dot indicates a single exosome, and the blue gate indicates a Lamin.