RESUMO
Mesothelin (MSLN), a glycoprotein normally expressed by mesothelial cells, is overexpressed in ovarian cancer (OvCa) suggesting a role in tumor progression, although the biological function is not fully understood. OvCa has a high mortality rate due to diagnosis at advanced stage disease with intraperitoneal metastasis. Tumor cells detach from the primary tumor as single cells or multicellular aggregates (MCAs) and attach to the mesothelium of organs within the peritoneal cavity producing widely disseminated secondary lesions. To investigate the role of host MSLN in the peritoneal cavity we used a mouse model with a null mutation in the MSLN gene (MSLNKO). The deletion of host MSLN expression modified the peritoneal ultrastructure resulting in abnormal mesothelial cell surface architecture and altered omental collagen fibril organization. Co-culture of murine OvCa cells with primary mesothelial cells regardless of MSLN expression formed compact MCAs. However, co-culture with MSLNKO mesothelial cells resulted in smaller MCAs. An allograft tumor study, using wild-type mice (MSLNWT) or MSLNKO mice injected intraperitoneally with murine OvCa cells demonstrated a significant decrease in peritoneal metastatic tumor burden in MSLNKO mice compared to MSLNWT mice. Together, these data support a role for host MSLN in the progression of OvCa metastasis.
Assuntos
Células Epiteliais/metabolismo , Mesotelina/genética , Neoplasias Ovarianas/genética , Neoplasias Peritoneais/genética , Células Estromais/metabolismo , Microambiente Tumoral/genética , Animais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Técnicas de Cocultura , Células Epiteliais/patologia , Feminino , Expressão Gênica , Xenoenxertos , Humanos , Mesotelina/deficiência , Mesotelina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Células Estromais/patologiaRESUMO
The majority of women with ovarian cancer are diagnosed with metastatic disease, therefore elucidating molecular events that contribute to successful metastatic dissemination may identify additional targets for therapeutic intervention and thereby positively impact survival. Using two human high grade serous ovarian cancer cell lines with inactive TP53 and multiple rounds of serial in vivo passaging, we generated sublines with significantly accelerated intra-peritoneal (IP) growth. Comparative analysis of the parental and IP sublines identified a common panel of differentially expressed genes. The most highly differentially expressed gene, upregulated by 60-65-fold in IP-selected sublines, was the type I transmembrane protein AMIGO2. As the role of AMIGO2 in ovarian cancer metastasis remains unexplored, CRISPR/Cas9 was used to reduce AMIGO2 expression, followed by in vitro and in vivo functional analyses. Knockdown of AMIGO2 modified the sphere-forming potential of ovarian cancer cells, reduced adhesion and invasion in vitro, and significantly attenuated IP metastasis. These data highlight AMIGO2 as a new target for a novel anti-metastatic therapeutic approach aimed at blocking cohesion, survival, and adhesion of metastatic tumorspheres.
Assuntos
Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/patologia , Neoplasias Peritoneais/secundário , Regulação para Cima , Animais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Mutação , Transplante de Neoplasias , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
The majority of women with recurrent ovarian cancer (OvCa) develop malignant ascites with volumes that can reach > 2 L. The resulting elevation in intraperitoneal pressure (IPP), from normal values of 5 mmHg to as high as 22 mmHg, causes striking changes in the loading environment in the peritoneal cavity. The effect of ascites-induced changes in IPP on OvCa progression is largely unknown. Herein we model the functional consequences of ascites-induced compression on ovarian tumor cells and components of the peritoneal microenvironment using a panel of in vitro, ex vivo and in vivo assays. Results show that OvCa cell adhesion to the peritoneum was increased under compression. Moreover, compressive loads stimulated remodeling of peritoneal mesothelial cell surface ultrastructure via induction of tunneling nanotubes (TNT). TNT-mediated interaction between peritoneal mesothelial cells and OvCa cells was enhanced under compression and was accompanied by transport of mitochondria from mesothelial cells to OvCa cells. Additionally, peritoneal collagen fibers adopted a more linear anisotropic alignment under compression, a collagen signature commonly correlated with enhanced invasion in solid tumors. Collectively, these findings elucidate a new role for ascites-induced compression in promoting metastatic OvCa progression.
Assuntos
Ascite/patologia , Neoplasias Ovarianas/patologia , Peritônio/patologia , Microambiente Tumoral , Animais , Anisotropia , Adesão Celular , Linhagem Celular Tumoral , Colágeno/metabolismo , Epitélio/patologia , Epitélio/ultraestrutura , Feminino , Humanos , Camundongos Endogâmicos C57BL , Mitocôndrias/patologia , Modelos Biológicos , Nanotubos/química , Nanotubos/ultraestrutura , Metástase Neoplásica , Neoplasias Ovarianas/ultraestrutura , Peritônio/ultraestruturaRESUMO
The noncanonical Wnt ligand Wnt5a is found in high concentrations in ascites of women with ovarian cancer. In this study, we elucidated the role of Wnt5a in ovarian cancer metastasis. Wnt5a promoted ovarian tumor cell adhesion to peritoneal mesothelial cells as well as migration and invasion, leading to colonization of peritoneal explants. Host components of the ovarian tumor microenvironment, notably peritoneal mesothelial cells and visceral adipose, secreted Wnt5a. Conditional knockout of host WNT5A significantly reduced peritoneal metastatic tumor burden. Tumors formed in WNT5A knockout mice had elevated cytotoxic T cells, increased M1 macrophages, and decreased M2 macrophages, indicating that host Wnt5a promotes an immunosuppressive microenvironment. The Src family kinase Fgr was identified as a downstream effector of Wnt5a. These results highlight a previously unreported role for host-expressed Wnt5a in ovarian cancer metastasis and suggest Fgr as a novel target for inhibition of ovarian cancer metastatic progression.Significance: This study establishes host-derived Wnt5a, expressed by peritoneal mesothelial cells and adipocytes, as a primary regulator of ovarian cancer intraperitoneal metastatic dissemination and identifies Fgr kinase as novel target for inhibition of metastasis.
Assuntos
Carcinoma Epitelial do Ovário/patologia , Neoplasias Ovarianas/patologia , Neoplasias Peritoneais/imunologia , Peritônio/patologia , Proteínas Proto-Oncogênicas/metabolismo , Proteína Wnt-5a/metabolismo , Quinases da Família src/metabolismo , Animais , Carcinoma Epitelial do Ovário/imunologia , Adesão Celular/imunologia , Linhagem Celular Tumoral , Movimento Celular/imunologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Metástase Neoplásica/imunologia , Metástase Neoplásica/patologia , Neoplasias Ovarianas/imunologia , Neoplasias Peritoneais/secundário , Peritônio/citologia , Peritônio/imunologia , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/imunologia , Microambiente Tumoral/imunologia , Proteína Wnt-5a/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders.
Assuntos
Colágeno Tipo I/ultraestrutura , Matriz Extracelular/ultraestrutura , Imagem Molecular/métodos , Proteólise , Coloração e Rotulagem/métodos , Animais , Colágeno Tipo I/química , Doenças do Tecido Conjuntivo/etiologia , Doenças do Tecido Conjuntivo/patologia , Matriz Extracelular/química , Humanos , Imuno-Histoquímica/métodos , Microscopia Eletrônica de Varredura/instrumentação , Microscopia Eletrônica de Varredura/métodos , Imagem Molecular/instrumentação , Coloração e Rotulagem/instrumentaçãoRESUMO
Ovarian cancer, the most deadly gynecological malignancy in U.S. women, metastasizes uniquely, spreading through the peritoneal cavity and often generating widespread metastatic sites before diagnosis. The vast majority of ovarian cancer cases occur in women over 40 and the median age at diagnosis is 63. Additionally, elderly women receive poorer prognoses when diagnosed with ovarian cancer. Despite age being a significant risk factor for the development of this cancer, there are little published data which address the impact of aging on ovarian cancer metastasis. Here we report that the aged host is more susceptible to metastatic success using two murine syngeneic allograft models of ovarian cancer metastasis. This age-related increase in metastatic tumor burden corresponds with an increase in tumor infiltrating lymphocytes (TILs) in tumor-bearing mice and alteration of B cell-related pathways in gonadal adipose tissue. Based on this work, further studies elucidating the status of B cell TILs in mouse models of metastasis and human tumors in the context of aging are warranted.
Assuntos
Tecido Adiposo/patologia , Envelhecimento/patologia , Aloenxertos/patologia , Metástase Neoplásica/patologia , Neoplasias Ovarianas/patologia , Adulto , Idoso , Animais , Linhagem Celular , Feminino , Humanos , Linfócitos do Interstício Tumoral/patologia , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Prognóstico , Fatores de Risco , Carga Tumoral/fisiologia , Adulto JovemRESUMO
Ovarian cancer is the fifth leading cause of cancer deaths in U.S. women and the deadliest gynecologic malignancy. This lethality is largely due to the fact that most cases are diagnosed at metastatic stages of the disease when the prognosis is poor. Epidemiologic studies consistently demonstrate that parous women have a reduced risk of developing ovarian cancer, with a greater number of births affording greater protection; however little is known about the impact of parity on ovarian cancer metastasis. Here we report that multiparous mice are less susceptible to ovarian cancer metastasis in an age-matched syngeneic murine allograft model. Interferon pathways were found to be upregulated in healthy adipose tissue of multiparous mice, suggesting a possible mechanism for the multiparous-related protective effect against metastasis. This protective effect was found to be lost with age. Based on this work, future studies exploring therapeutic strategies which harness the multiparity-associated protective effect demonstrated here are warranted.
Assuntos
Tecido Adiposo/metabolismo , Interferons/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Paridade , Peritônio/metabolismo , Tecido Adiposo/patologia , Aloenxertos , Animais , Carcinoma Epitelial do Ovário , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/prevenção & controle , Neoplasias Peritoneais/secundário , Peritônio/patologia , Gravidez , Fatores de RiscoRESUMO
Wnt5a is involved in activating several non-canonical WNT signaling pathways, through binding to different members of the Frizzled- and Ror-family receptors. Wnt5a signaling is critical for regulating normal developmental processes, including proliferation, differentiation, migration, adhesion and polarity. However, the aberrant activation or inhibition of Wnt5a signaling is emerging as an important event in cancer progression, exerting both oncogenic and tumor suppressive effects. Recent studies show the involvement of Wnt5a in regulating cancer cell invasion, metastasis, metabolism and inflammation. In this article, we review findings regarding the molecular mechanisms and roles of Wnt5a signaling in various cancer types, and highlight Wnt5a in ovarian cancer.