P12 - Cáncer

Isolation and characterization of small extracellular vesicles derived from the human amniotic membrane conditioned medium: an approach onto their anti-tumoral effects.

Pérez, Luciano ¹ - Bonardi, Mabel ¹ -Santa Cruz, Diego ² - Riedel, Rodrigo ¹ -Jaime, Mariana ³ - Casale, Roberto ³ -Varone, Cecilia ¹ - Pacienza, Natalia ² - Maymó, Julieta ¹

1) Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), CONICET-UBA, Ciudad autónoma de Buenos Aires, Argentina
2) Instituto de Medicina Traslacional, Trasplante y Bioingeniería (IMETTyB), CONICET-FUNDACIÓN FAVALORO, Ciudad autónoma de Buenos Aires, Argentina
3) Departamento de Maternidad, Hospital Nacional Profesor Alejandro Posadas, Buenos Aires, Argentina
Contacto: lucianoangelp@gmail.com

Placental stem cells have been widely reported as a promising tool for regenerative medicine. Substantial evidence supports the anti-tumoral effects of human amniotic membrane (hAM) stem cells, which exhibit anti-proliferative, anti-angiogenic, and pro-apoptotic properties. Extracellular vesicles (EVs) are believed to play a crucial role in mediating these anti-cancer effects.
As the third leading cause of cancer mortality, liver cancer poses a serious clinical challenge requiring new treatments beyond conventional therapies. Our research, in line with others, has previously reported the anti-tumour effects of amniotic membrane-conditioned medium (AM-CM). We have demonstrated AM-CM inhibits hepatocarcinoma cell proliferation and promotes apoptosis. Here, we aimed to isolate and characterise EVs derived from AM-CM and assess their ability to inhibit hepatocellular carcinoma cell survival.
EVs were isolated from AM-CM by anion exchange chromatography and eluted with a NaCl solution. The elution profile was characterised by measuring protein and lipid concentration. CD63, a typical EVs marker, was determined by ELISA. EVs morphology was observed by transmission electron microscopy (TEM) and particle concentration was determined by nanoparticle tracking analysis (NTA). EVs anti-proliferative effect was initially evaluated on HepG2 and Huh-7 hepatocarcinoma cell lines using the MTT assay. Chromatography yielded eight fractions, with fraction 4 demonstrating peak concentrations of proteins and lipids consistent with a CD63 peak. TEM analysis revealed a homogeneous population of spherical EVs with an average diameter of 62 nm. NTA analysis showed an average diameter of 165 nm and a particle concentration of approximately 10^10 particles/ml (n=3). These results would suggest AM-CM is enriched in small EVs. At the assessed concentrations, MTT assay did not show changes in cell viability following a 24 h treatment with EVs (0.1-10 μg/ml). Our findings demonstrated the presence of sEVs in AM-CM. Further research would provide new insights into the therapeutic potential of AM-CM EVs in cancer treatment.

Palabras clave: AMNION, EXTRACELLULAR VESICLES, HEPATOCELLULAR CARCINOMA, PLACENTAL STEM CELL THERAPIES, CANCER TREATMENT.