Challenges and Concluding Thoughts
Since exosomes are released by multiple cell types, an obvious challenge in the use of exosomal cargo or liquid biopsy techniques in general as biomarkers of DLBCL is the enrichment of tumor specific exosomes in order to avoid dilution of information in tumor exosomes by exosomes from non-neoplastic tissue. Enrichment for tumor derived exosomes before subsequent analysis of exosomal cargo would address the issue of background noise from non-neoplastic exosomes. Castillo et al identified specific surface proteins on prostate cancer cell lines derived exosomes and used antibodies to these proteins for immunocapture in plasma of prostate cancer patients to enrich for tumor derived exosomes for downstream analysis of the tumor derived exosomal cargo (107). Mizutani et al also demonstrated isolation of prostate cancer specific exosomes using immunocapture with prostate specific antibodies. (108) Similar approaches can be employed as pre-analytical step in studying exosomes in fluids from DLBCL patients.
Another challenge in routine use of exosomes in cancer management is the difference in detected cargo observed using different methods of isolation. Jeppesen et al used high resolution gradient fractionation to separate small extracellular vesicles from non-vesicular proteins and direct immunoaffinity capture to isolate exosomes from other vesicles (109). Analyses of these exosomes showed absence in the cargo of some nucleic acids and proteins hitherto found to be present in exosomes using other methods. Contamination of exosomal isolates by other vesicles and non-vesicular molecules probably accounts for such variation. Guidelines to confirm purity of isolation would need to be developed to prevent inter-laboratory variation of exosomal content analyses.
Exosomes thus have great potential for not only improving diagnosis, prognostication, and monitoring of DLBCL, but provide a means of understanding the biology of DLBCL with the ultimate goal of improving care for patients.