dianas 7(1) > Yllera-García etal

dianas | Vol 7 Num 1 | marzo 2018 | e201803p08

Exosomes as new tools for searching relevant proteins in colorectal cancer metastasis.

Beatriz Yllera García^a, Vivian de los Rios Benítez, José Ignacio Casal Álvarez

Centro de Investigaciones Biológicas, CSIC.

a. beatriz.yllera@gmail.com

III Congreso de Señalización Celular, SECUAH 2018.
20-22 de marzo, 2018. Universidad de Alcalá. Alcalá de Henares, Madrid. España.
Sesión de paneles

Resumen

Exosomes are extracellular vesicles with a wide range of functions, including metastasis seeding, in most types of cancer. The aim of this study was to find proteins critical for colorectal cancer metastasis. A non-metastatic murine model of colorectal cancer was compared with the metastatic mouse cell line CT-26 to investigate the exosomal protein content. To optimize the recovery of exosomes, cells were incubated in serum-free DMEM for 48h. Colon tissues were incubated for 2, 4 and 8h in serum-free DMEM. The exosomes were isolated by ultracentrifugation and lysated with different buffers: 7M urea/2M thiourea, methanol-chloroform and RIPA buffer. Exosomal lysates were analyzed in the Q-Exactive using a gradient: 5–35% (90min), 35%-100% (4min) and 100% (8min) ACN with 0.1% formic acid. Mass spectra were acquired using a “top 15” method and searched against SwissProt using Sequest HT search engine through Proteome Discoverer. Identified peptides were filtered using Percolator with a q-value threshold of 0.01 at a FDR of 1%. The largest amount of mouse exosomes from colon was recovered at 2h. The 7M urea / 2M thiourea buffer was the most effective lysis method, with 35% more exosomal proteins detected by mass spectrometry. The identified proteins correlated with: remodelling of the ECM, organotropism, angiogenesis and chemo-attraction, EMT, cellular metabolism, etc. Significant differences were observed between metastatic and non-metastatic exosomes. Proteins of metastatic exosomes are largely related to the interaction and modulation of the extracellular matrix and cell migration, including α3 and α5 integrins, in contrast to the non-metastatic exosomes. Laminins α5, β1 and ɣ1, which form laminin-511, are major candidates for further studies. Methods for extracting and lysing exosomes from mouse colon and metastatic cells were optimised. A preliminary protein profile involved in metastasis has been obtained and will be further analysed using functional experiments. Keywords: exosomes, metastasis, colorectal cancer, extracellular matrix, mouse models.