In Vitro Validation Of Tumor-Derived Large Extracellular Vesicles Isolation And Characterization As Suitable Tool For Liquid Biopsy.

Abstract Extracellular vesicles (EVs) are involved in intercellular communication mediated by nucleic acid and protein exchange. Their role in cancer has been widely demonstrated and EVs are under investigation as possible tools for liquid biopsy. Large (L)-EVs represent a heterogeneous class of EVs that are characterized by a diameter >200 nm. Those derived by cancer cells show peculiar cargos including oncogenic proteins, lipids and large DNA fragments. Aim of this study was to validate in vitro the isolation, characterization and mutational analysis of tumor-derived L-EVs. L-EVs were isolated from supernatants (50 ml) of either prostate cancer (PC3) and melanoma (SK-Mel28) cell cultures by centrifugation at 10,000x g, and then resuspended in 200 µl of PBS. Tunable resistive pulse sensing (qNANO) investigated both dimensions and concentration of L-EV preparations. Flow cytometry and Western blotting were used to analyze the expression of typical markers of EVs (CD63 and CD81 tetraspanins) and L-EVs (HSPA5 or CK18), as well as of ALIX (cytoplasmic protein) and APOA1 (contaminant) as internal controls. For genomic analysis, L-EVs were initially treated with DNAse I, Exonuclease III and RNAse to disrupt the external nucleic acid coating, then L-EV-DNA was extracted by DNeasy blood and tissue Kit (Qiagen). Quantitative and qualitative analyses of L-EV-DNA have been assessed with Bioanalyzer DNA High Sensitivity Chip Kit (Agilent) and 1% agarose gel electrophoresis, as compared to parental cell DNA. Finally, Sanger sequencing was used to identify TP53 (p.K139fs*31) or BRAF (p.V600E) mutations in L-EV-DNA, respectively form PC3 and SK-Mel28 cells. L-EVs were purified at a mean concentration of 16.4±5.1 x 103 vesicles/µl. Typical characteristics of L-EVs were confirmed, including a diameter ranging from 0.5 µm to 2 µm as well as the expression of CD63/CD81 tetraspanins and HSPA5 or CK18. Moreover, L-EV preparations resulted all negative for APOA1 but positive for ALIX expression, confirming sample purity and vesicle integrity. The DNA content from each sample of L-EVs ranged from 0.36 to 5.21 ng/µl. Qualitative analysis revealed long DNA fragments (range 3,000-10,000 bp) packaged in L-EVs in a fashion almost similar to the genomic DNA from parental cells. Finally, DNA sequencing revealed either the BRAF p.V600E or the TP53 p.K139fs*31 mutations - respectively from SK-Mel28 and PC3 cells - in both L-EV-DNA and genomic DNA. L-EVs are easily obtainable from culture supernatants and represent a biological surrogate of the cell producer. L-EVs contain large DNA fragments that are suitable for molecular testing, including the identification of pathogenic gene variants. These results make L-EVs ideal candidates as liquid biopsy in oncology, thus justifying further efforts to validate their isolation from body fluids and down-stream analysis. Citation Format: Gaetano Pezzicoli, Domenica Lovero, Marco Tucci, Camillo Porta, Francesco Mannavola. In vitro validation of tumor-derived large extracellular vesicles isolation and characterization as suitable tool for liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2012.