Leukemia-Derived Exosomes Induce Paracrine and Autocrine Cell Proliferation in Pediatric ALL

Introduction: Liquid biopsies are minimal invasive and changing our understanding of tumor cell biology through the exploration of exosomes research in adult oncology. The clinical application of liquid biopsies to study exosomes as disease biomarkers with correlation to disease status is especially appealing in pediatrics. However in childhood cancer, the first exosome studies only begin to emerge in this exciting new field. Exosomes are microvesicles (30-100 nm) produced by normal as well as malignant cells in most biological fluids. Exosomes represent the fingerprint of the parental tumor and are loaded with unique biomarkers such as proteins, RNA, DNA and non-coding RNA, which may regulate tumor growth. One of the unique features of exosomes of interest to oncology research is the horizontal cargo transfer into target cells changing their biological properties. Our data present the first study to investigate the role of exosomes in acute lymphoid leukemia (ALL) in pediatric patients.

Objectives:

1. Demonstrate transfer of ALL-exosomes into target cells with induction of cell proliferation

2. Confirm that leukemic exosome-induced paracrine and autocrine cell proliferation of leukemic cell lines is regulated by expression of proliferative and pro-survival genes with suppression of pro-apoptotic genes.

Methods: Exosomes were isolated from serum of healthy donors (HD) and pediatric ALL (P-ALL) patients by ultracentrifugation. Exosomes were also isolated from conditioned medium (CM) of SUP-B15 (B-ALL), JM1 (B-ALL), and CL-01 (normal B cells) human cell lines. Proliferation assays were designed by exposing all three cell lines to different sources of leukemia-derived exosomes in a paracrine or autocrine fashion. After 24hr incubation, induction of cell proliferation was assessed by cell counting using trypan blue under the microscope and confirmed by colorimetric assay and gene expression analysis. Total RNA was extracted from cultured SUP-B15, JM1, or CL-01 cells and 2-5 µg of total RNA was used for cDNA synthesis. Primers sequences, probe number and gene accession numbers were identified from the universal probe library (UPL) of Roche Applied Science (Indianapolis, IN, USA). Each sample was assayed in duplicates. Proliferation was evaluated at the gene expression level for the following genes: Ki-67, PCNA (proliferation), MCL1, BCL2 (pro-survival) and BAD, BAX (pro-apoptotic). Fold change was calculated by comparing controls (naive) vs. exosome-induced cell lines both in autocrine and paracrine fashion. Data were analyzed with RQ manager version 1.2.1 (Applied Biosystems, Foster City, CA). The data were expressed as relative mRNA expression with reference to control samples and normalized to an endogenous reference gene (GAPDH).

Results: Exosomes were characterized by NanoSight Tracking Analysis (NTA-Malvern) and western blot. Exosomal uptake and internalization into the cells was visualized by exosome labeling with a PKH67 fluorescent dye. We elucidated that CM-derived exosomes from SUP-B15 and JM1 cell lines induce cell proliferation in SUP-B15, JM1 (autocrine and paracrine for both cell lines) and CL-01 cells (paracrine) (control vs. exosomes, p<0.01). Moreover, P-ALL serum-derived exosomes promote paracrine cell proliferation in SUP-B15, JM1 and CL-01 cell lines compared to HD-derived exosomes (HD vs P-ALL, p<0.0001). At a molecular level, we found that exosomes from JM1 and SUP-B15 cells enhance expression of proliferation genes (PCNA, Ki-67) and pro-survival genes (MCL-1, BCL2), and suppress pro-apoptotic genes (BAD, BAX) in JM1 cells.

Conclusions: Our data suggest that human ALL-derived exosomes induce cell proliferation in both paracrine and autocrine fashion of leukemic and non-leukemic B cell lines. It appears that exosomes regulates this phenomena in a highly orchestrated way, by the induction of proliferative genes and pro-survival genes, with suppression of pro-apoptotic genes.