RESUMEN

Extracellular vesicles (EVs) participate in cell-stroma crosstalk within the tumor microenvironment and fibroblasts (Fb) contribute to tumor promotion in thyroid cancer. However, the role of tumor-stroma derived EVs still needs to be deciphered. We hypothesized that the interaction of thyroid tumor cells with Fb would liberate EVs with a specific proteomic profile, which would have an impact on EV-functionality in thyroid tumor progression-related events. Tumor (TPC-1, 8505c) and non-tumor (NThyOri) thyroid cells were co-cultured with human Fb. EVs, obtained by ultracentrifugation of conditioned media, were characterized by nanoparticle tracking analysis and western blotting. EV-proteomic analysis was performed by mass-spectrometry, and metalloproteinases (MMPs) were studied by zymography. EV-exchange was evaluated using immunofluorescence, confocal microscopy and FACS. EVs expressed classical exosome markers, with EVs from thyroid tumor cell-Fb co-cultures showing a proteomic profile related to extracellular matrix (ECM) remodeling. Bidirectional crosstalk between Fb and TPC-1 cells produced significantly more EVs than their isolated cells, and potentiated EV-functionality. In line with this, Fb-TPC-1 derived EVs induced MMP2 activation in NThyOri supernatants, and MMP2 activity could be evidenced in Fb and TPC-1 contact-independent co-cultures. Besides, MMP2 interactors allowed us to discriminate between EVs from thyroid tumoral and non-tumoral milieus. Interestingly, Fb internalized more EVs from TPC-1 than from NThyOri producing cells. Fb and thyroid tumor cell crosstalk produces specialized EVs with an ECM remodeling proteomic profile, enabling activation of MMP2 and possibly facilitating ECM-degradation, which is potentially linked with thyroid tumor progression.

Asunto(s)

Vesículas Extracelulares , Neoplasias de la Tiroides , Matriz Extracelular , Vesículas Extracelulares/metabolismo , Humanos , Metaloproteinasa 2 de la Matriz/metabolismo , Proteómica/métodos , Neoplasias de la Tiroides/metabolismo , Microambiente Tumoral

RESUMEN

Hepatocellular carcinoma develops as a multistep process, in which cell cycle deregulation is a central feature, resulting in unscheduled proliferation. The PLAGL1 gene encodes a homonym zinc finger protein that is involved in cell-proliferation control. We determined the genomic profile and the transcription and expression level of PLAGL1, simultaneously with that of its molecular partners p53, PPARγ and p21, in cell-lines derived from patients with liver cancer, during in vitro cell growth. Our investigations revealed that genomic and epigenetic changes of PLAGL1 are also present in hepatoma cell-lines. Transcription of PLAGL1 in tumor cells is significantly lower than in normal fibroblasts, but no significant differences in terms of protein expression were detected between these two cell-types, indicating that there is not a direct relationship between the gene transcriptional activity and protein expression. RT-PCR analyses on normal fibroblasts, used as control, also showed that PLAGL1 and p53 genes transcription occurs as an apparent orchestrated process during normal cells proliferation, which gets disturbed in cancer cells. Furthermore, abnormal trafficking of the PLAGL1 protein may occur in hepatocarcinogenesis.