cca04ec54db3a2578a6daf90fefc7a76 327701.pdf 84bad23e9c9d2c2ac4b75a5ed9a5f4b1370e7ac3 327701.pdf 33e4cf86829983787cb9609e1218118b063c112a94eeb575d984521ee1b9b3d8 327701.pdf Title: Dissecting Breast Cancer Circulating Tumor Cells Competence via Modelling Metastasis in Zebrafish Subject: Background: Cancer metastasis is a deathly process, and a better understanding of the different steps is needed. The shedding of circulating tumor cells (CTCs) and CTC-cluster from the primary tumor, its survival in circulation, and homing are key events of the metastasis cascade. In vitro models of CTCs and in vivo models of metastasis represent an excellent opportunity to delve into the behavior of metastatic cells, to gain understanding on how secondary tumors appear. Methods: Using the zebrafish embryo, in combination with the mouse and in vitro assays, as an in vivo model of the spatiotemporal development of metastases, we study the metastatic competency of breast cancer CTCs and CTC-clusters and the molecular mechanisms. Results: CTC-clusters disseminated at a lower frequency than single CTCs in the zebrafish and showed a reduced capacity to invade. A temporal follow-up of the behavior of disseminated CTCs showed a higher survival and proliferation capacity of CTC-clusters, supported by their increased resistance to fluid shear stress. These data were corroborated in mouse studies. In addition, a differential gene signature was observed, with CTC-clusters upregulating cell cycle and stemness related genes. Conclusions: The zebrafish embryo is a valuable model system to understand the biology of breast cancer CTCs and CTC-clusters. Keywords: breast cancer; metastasis; circulating tumor cells (CTCs); CTC-clusters; zebrafish; in vitro models; in vivo models; cell survival Author: Inés Martínez-Pena, Pablo Hurtado, Nuria Carmona-Ule, Carmen Abuín, Ana Belén Dávila-Ibáñez, Laura Sánchez, Miguel Abal, Anas Chaachou, Javier Hernández-Losa, Santiago Ramón y Cajal, Rafael López-López and Roberto Piñeiro Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Aug 30 13:11:17 2021 CEST ModDate: Mon Aug 30 13:17:51 2021 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 20 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2375150 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- CIQLLF+VnURWPalladioL Type 1 Custom yes yes no 10 0 PATTQN+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 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Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 643 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Clusters of BC Tumor Cell Lines Show Differential In Vitro Behavior in Functional Assays Than Single Tumor Cells Destination: subsection.2.1 Item: Title: Clusters of Tumor Cells Have a Lower Dissemination Capacity in the Zebrafish Embryo Destination: subsection.2.2 Item: Title: Disseminated Clusters of Tumor Cells Survive Better in the Zebrafish Than Single Cells Destination: subsection.2.3 Item: Title: Clusters of Tumor Cells have a Proliferative Advantage over Single CTCs Destination: subsection.2.4 Item: Title: CTC-Clusters Have an Enhanced Survival and Metastatic Potential in Mice Compared to Single CTCs Destination: subsection.2.5 Item: Title: Gene Expression Profiling Supports the Metastatic Features of CTC-Clusters Destination: subsection.2.6 Item: Title: Discussions Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Cell Culture and Generation of Single and CTC-Cluster In Vitro Models Destination: subsection.4.1 Item: Title: Transwell Migration and Invasion Assays Destination: subsection.4.2 Item: Title: Adhesion to Endothelium Assay Destination: subsection.4.3 Item: Title: Soft-Agar Colonogenesis Assay Destination: subsection.4.4 Item: Title: Immunofluorescence Destination: subsection.4.5 Item: Title: Zebrafish Xenograft and Micromanipulation Experiments Destination: subsection.4.6 Item: Title: Analysis of Cell Dissemination Pattern in the Zebrafish Destination: subsection.4.7 Item: Title: Fluid Shear Stress (FSS) Assay Destination: subsection.4.8 Item: Title: Mouse Lung Colonization Assay and Histological Analyses Destination: subsection.4.9 Item: Title: Isolation of Injected Tumor Cells from Zebrafish Embryos Destination: subsection.4.10 Item: Title: Gene Expression Analysis Destination: subsection.4.11 Item: Title: Data Analysis Destination: subsection.4.12 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: Dissecting Breast Cancer Circulating Tumor Cells Competence via Modelling Metastasis in Zebrafish Author: Inés Martínez-Pena, Pablo Hurtado, Nuria Carmona-Ule, Carmen Abuín, Ana Belén Dávila-Ibáñez, Laura Sánchez, Miguel Abal, Anas Chaachou, Javier Hernández-Losa, Santiago Ramón y Cajal, Rafael López-López and Roberto Piñeiro Subject: Background: Cancer metastasis is a deathly process, and a better understanding of the different steps is needed. The shedding of circulating tumor cells (CTCs) and CTC-cluster from the primary tumor, its survival in circulation, and homing are key events of the metastasis cascade. In vitro models of CTCs and in vivo models of metastasis represent an excellent opportunity to delve into the behavior of metastatic cells, to gain understanding on how secondary tumors appear. Methods: Using the zebrafish embryo, in combination with the mouse and in vitro assays, as an in vivo model of the spatiotemporal development of metastases, we study the metastatic competency of breast cancer CTCs and CTC-clusters and the molecular mechanisms. Results: CTC-clusters disseminated at a lower frequency than single CTCs in the zebrafish and showed a reduced capacity to invade. A temporal follow-up of the behavior of disseminated CTCs showed a higher survival and proliferation capacity of CTC-clusters, supported by their increased resistance to fluid shear stress. These data were corroborated in mouse studies. In addition, a differential gene signature was observed, with CTC-clusters upregulating cell cycle and stemness related genes. Conclusions: The zebrafish embryo is a valuable model system to understand the biology of breast cancer CTCs and CTC-clusters. 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