Characterization of DLBCL-Derived Exosomes and Investigation of Their Biological Properties
Authors: Rutherford Sarah C. , Sawh Seema , Saxena Ashish , Ishii Jennifer , Dominguez Maria D. , Chambwe Nyasha , Lopes Eloisi Caldas , Agirre Xabier , Betel Doron , Shaknovich Rita 
Published: October 2019

Abstract
Exosomes are membrane-bound vesicles that can be exchanged between cells and have been shown to modify the tumor microenvironment and contribute to progression of solid tumors. Little research has been done on exosomes in lymphoma and their potential biological role in tumor initiation and progression remains unknown. We characterized exosomes secreted by six DLBCL cell lines, four primary DLBCL tumors, and two normal control B cell samples. We optimized their purification and studied their nucleic acid content. We then determined that tumor-derived exosomes can be exchanged not only between tumor and immune cells, as has been shown before, but also between lymphoma cells with various biological consequences. Finally, we sequenced exosomal RNAs in order to better understand their function and role in lymphoma biology.
We used serial ultracentrifugation to purify exosomes and microvesicles from six DLBCL cell lines (LY1, LY3, LY7, LY18, HBL1, TMD8). We isolated exosomes from four primary DLBCLs and two control tonsillar samples enriched for CD20+ B cells using the Total Exosome Isolation from Cell Culture Kit (Invitrogen). We found that the six DLBCL cell lines secrete large quantities of CD63+ exosomes. These exosomes also express the B-cell specific marker CD20, but not T cell marker CD3, which could enable tracing of the cell of origin and differential purification. Exosomes are known to contain small RNAs, which can be extracted with different efficiency depending on the isolation method used. We tested three approaches for nucleic acid isolation: Direct-zol™ and TRIzol® methods which were both followed by column purification, and miRCURY™ RNA isolation kit (Exiqon). We detected no traceable quantities of DNA in exosomal contents. The miRCURY™ RNA isolation kit yielded the highest amounts of RNAs of the techniques used and preserved sizes from 4-150 nucleotides (nt) in length with equal efficiency. Exosomes contained predominantly small RNAs with 6-80 nt species which constituted 9-28% of isolated RNA. We then prepared cDNA libraries using TruSeq Stranded Total kit (Illumina) and sequenced libraries using 50 bp Paired End approach on Illumina HiSeq 2000. We developed a small RNA-seq Analysis Pipeline that included QC, removing Illumina TruSeq adapters, mapping the reads, summarizing the data, and assigning reads to annotated genes. Our results indicate that exosomal RNAs contain protein coding RNAs (50-60%) and antisense RNAs (20-30%) as the most common biotypes.
We also evaluated the exchange of exosomal content between DLBCL cell lines (LY1, LY3). The cells were stained with SYTO® RNASelect™ dye (Molecular Probes Inc) and cultured for 48 hours. The supernatant was collected, filtered, and cultured with unstained cells from the alternate DLBCL cell line for 48 hours. We observed uptake of LY1 exosomes by LY3 cells and LY3 exosomes by LY1 cells, as evidenced by SYTO® RNASelect™ dye transfer and CD63 positivity on FACS analysis. In addition, we tested the effect of exosomal exchange on resistance to doxorubicin, given the varied susceptibilities of LY1 and LY3 cell lines to this drug which is a part of standard frontline treatment for DLBCL. After culturing cells with filtered supernatant from the alternate cell line for 48 hours, we treated with doxorubicin and assessed for cell viability at 48 hours. We also studied the effect of exosome uptake on proliferation and cell cycle of recipient cells. Our results indicate that exosomal exchange between tumor cells and from tumor to normal B cells can modulate fundamental biological processes including drug resistance as well as cell proliferation, survival, and induction of apoptosis. We believe this is related to incorporation of exosomal RNA with resulting effects on the transcriptome of recipient cells. RNA sequencing analysis will aid in the design of additional functional studies in order to further elucidate the biological role of exosomes in DLBCL.

Disclosures
No relevant conflicts of interest to declare.