ABSTRACT
Os sarcomas de partes moles (SPM) são tumores malignos derivados de células mesenquimais, com grande capacidade de formar metástases e geralmente apresentam elevada resistência à terapia. Formam um grupo extremamente heterogêneo com mais de 100 subtipos tumorais, como os sarcomas pleomórficos indiferenciados (SPI) e os sarcomas fusocelulares (SF). Em conjunto, esses dois subgrupos não compartilham semelhanças morfológicas com os outros subtipos. Adicionalmente, carecem de informações moleculares que auxiliem tanto no diagnóstico quanto no prognóstico da doença. O objetivo deste trabalho foi estabelecer e caracterizar modelos pré-clínicos de SPI e SF baseados em Patient-derived xenografts (PDX) e organoids (PDO) para definir uma plataforma de estudos destes tumores e avaliar o potencial de alvos moleculares específicos. Utilizando amostras de 11 pacientes, foi possível estabelecer oito PDX de SPI ou SF. O estabelecimento de organóides de sarcoma se mostrou desafiador para amostras tumorais oriundas diretamente de pacientes ou de PDX e após testarmos diferentes protocolos, conseguimos obter organóides de culturas primárias tumorais. Os organoides e os PDXs gerados foram caracterizados por IHQ e foi possível observar a preservação das características moleculares. Para a caracterização genética, realizamos o sequenciamento de exoma de um conjunto de oito amostras (paciente/PDX/cultura celular) para identificação das principais alterações somáticas. Por fim, padronizamos o ensaio de citotoxicidade à doxorrubicina em três culturas primárias de SPI e SF.
Soft tissue sarcomas (STS) are malignant tumors derived from mesenchymal cells, with a great capacity to form metastases and generally show high resistance to therapy. They form an extremely heterogeneous group with more than 100 tumor subtypes, such as undifferentiated pleomorphic sarcoma (UPS) and fusocellular sarcomas (SS). Taken together, these two subgroups do not share morphological similarities with the other subtypes. Additionally, they lack molecular information that helps both in the diagnosis and prognosis of the disease. The objective of this work was to establish and characterize preclinical models of UPS and SS based on Patient-derived xenografts (PDX) and organoids (PDO) to define a platform for studies of these tumors and to evaluate the potential of specific molecular targets. Using samples from eleven patients, it was possible to establish eight PDX of UPS and SS. The establishment of sarcoma organoids proved to be challenging for tumor samples derived directly from patients or from PDX and after testing different protocols, we were able to obtain organoids from primary tumor cultures. The generated organoids and PDXs were also characterized by IHC and immunofluorescence with sarcoma marker proteins and it was also possible to observe the preservation of molecular characteristics. For genetic characterization, we performed exome sequencing of a set of eight samples (patient/PDX/cell culture) to identify the main somatic alterations. Finally, we standardized the doxorubicin cytotoxicity assay in three primary cultures of SPI and SF.
Subject(s)
Humans , Animals , Sarcoma , Heterografts , Soft Tissue Neoplasms , OrganoidsABSTRACT
Gallbladder cancer is an aggressive disease with late diagnosis and no efficacious treatment. The Hippo-Yes-associated protein 1 (YAP1) signaling pathway has emerged as a target for the development of new therapeutic interventions in cancers. However, the role of the Hippo-targeted therapy has not been addressed in advanced gallbladder cancer (GBC). This study aimed to evaluate the expression of the major Hippo pathway components mammalian Ste20-like protein kinase 1 (MST1), YAP1 and transcriptional coactivator with PDZ-binding motif (TAZ) and examined the effects of Verteporfin (VP), a small molecular inhibitor of YAP1-TEA domain transcription factor (TEAD) protein interaction, in metastatic GBC cell lines and patient-derived organoids (PDOs). Immunohistochemical analysis revealed that advanced GBC patients had high nuclear expression of YAP1. High nuclear expression of YAP1 was associated with poor survival in GBC patients with subserosal invasion (pT2). Additionally, advanced GBC cases showed reduced expression of MST1 compared to chronic cholecystitis. Both VP treatment and YAP1 siRNA inhibited the migration ability in GBC cell lines. Interestingly, gemcitabine resistant PDOs with high nuclear expression of YAP1 were sensitive to VP treatment. Taken together, our results suggest that key components of the Hippo-YAP1 signaling pathway are dysregulated in advanced gallbladder cancer and reveal that the inhibition YAP1 may be a candidate for targeted therapy.