Quantification of cancer cell extravasation in vivo

Kim, Young S.; Williams, Karla C.; Gavin, Carson T; Jardine, Emily; Chambers, Ann F.; Leong, Hon S.

doi:10.1038/nprot.2016.050

Cited by 62 publications

(73 citation statements)

References 51 publications

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“…Despite drug testing, the CAM model has been utilized for pro- and antiangiogenic studies [10, 12, 18, 47] and for acute toxicological studies on anticancer drugs [48]. Both in ovo and e x ovo (shell-less) model is used to assess metastatic potential of various cancer cell lines [18, 49]. …”

Section: Discussionmentioning

confidence: 99%

3D chick embryo chorioallantoic membrane model as an in vivo model to study morphological and histopathological features of feline fibrosarcomas

et al. 2017

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BackgroundThe chick embryo chorioallantoic membrane (CAM) model is well described in human medicine as a cost-effective, easy to perform preclinical oncological model for observing pro- and antiangiogenic response, tumor biology and metastasis. The main objective of this article was to present the modification of the CAM assay in order to evaluate tumor growth from two feline fibrosarcoma cell lines (FFS1, FFS3) and describe their morphological and histopathological features.ResultsThe authors described morphological and histopathological features of two feline fibrosarcoma cell lines (FFS1 and FFS3) grown on the CAM. Tumors from the FFS1 cell line showed high malignancy (grade III), while tumors from the FFS3 cell line were grade II. Proliferation markers (Ki-67 and PCNA) were determined and the positive correlation between PCNA and tumor grade (r = 0.8247; p < 0.001) was demonstrated, as opposed to Ki-67.ConclusionsThe results obtained indicate that PCNA may be helpful to evaluate the tumor grade, better than Ki-67, for feline fibrosarcomas. However, further investigations of proliferation marker, in bigger number of feline spontaneous fibrosarcomas and feline fibrosarcomas grown on the CAM from different cell lines, are needed to confirm these observations.

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Section: Discussionmentioning

confidence: 99%

3D chick embryo chorioallantoic membrane model as an in vivo model to study morphological and histopathological features of feline fibrosarcomas

et al. 2017

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“…The chicken CAM assay for ex ovo metastasis and extravasation efficiency was performed as previously described (Kim et al , 2016). MDA-MB-231 cells stably expressing GFP and miR-330-3p or the control mimics were grown to ∼80–90% confluency.…”

Section: Methodsmentioning

confidence: 99%

Targeting of CCBE1 by miR-330-3p in human breast cancer promotes metastasis

et al. 2017

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Background:MicroRNAs (miRs) are involved in the regulation of many processes that contribute to malignancy, including cell proliferation, radiation resistance, invasion and metastasis. The role of miR-330-3p, an miR upregulated in breast cancer, remains unclear.Methods:We examine the association of miR-330-3p with distant relapse-free survival in the Oxford cohort of breast cancer patients. We also study miR-330-3p function using in vitro invasion and ex ovo metastasis assays. Using in vitro luciferase assays, we validate a novel target gene for miR-330-3p, Collagen And Calcium Binding EGF Domains 1 (CCBE1). We assess functional consequences of CCBE1 loss by using siRNA-mediated knockdown followed by in vitro invasion assays. Lastly, we examine the expression profile of CCBE1 in breast carcinomas in the Curtis and TCGA Breast Cancer data sets using Oncomine Platform as well as distant relapse-free and overall survival of patients in the Helsinki University breast cancer data set according to CCBE1 expression status.Results:miR-330-3p is enriched in breast cancer, and higher levels of miR-330-3p expression are associated with lower distant relapse-free survival in a cohort of breast cancer patients. Consistent with these observations, overexpression of miR-330-3p in breast cancer cell lines results in greater invasiveness in vitro, and miR-330-3p-overexpressing cells also metastasise more aggressively ex ovo. We identify CCBE1 as a direct target of miR-330-3p, and show that knockdown of CCBE1 results in a greater invasive capacity. Accordingly, in breast cancer patients CCBE1 is frequently downregulated, and its loss is associated with reduced distant relapse-free and overall survival.Conclusions:We show for the first time that miR-330-3p targets CCBE1 to promote invasion and metastasis. miR-330-3p and CCBE1 may represent promising biomarkers in breast cancer.

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“…Additionally, in many monolayer systems (especially Transwell filters), nearly all tumor cells seeded at t=0 will translocate across a monolayer over a 24–48 hour time frame 34 . When compared to much lower extravasation rates of 40–60% at 24 hours that are typically found in mouse 19,35 and zebrafish 36 models for a variety of cell lines, this suggests that endothelial monolayers exhibit much greater permissiveness to tumor cells compared to in vivo microvasculature.…”

Section: Alternative Extravasation Assaysmentioning

confidence: 99%

On-chip human microvasculature assay for visualization and quantification of tumor cell extravasation dynamics

et al. 2017

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Distant metastasis, which results in >90% of cancer related deaths, is enabled by hematogenous dissemination of tumor cells via the circulation. This requires the completion of a sequence of complex steps including transit, initial arrest, extravasation, survival and proliferation. Increased understanding of the cellular and molecular players enabling each of these steps is key in uncovering new opportunities for therapeutic intervention during early metastatic dissemination. Here, we describe an in vitro model of the human microcirculation with the potential to recapitulate discrete steps of early metastatic seeding, including arrest, transendothelial migration and early micrometastases formation. The microdevice features self-organized human microvascular networks formed over 4–5 days, after which tumor can be perfused and extravasation events easily tracked over 72 hours, via standard confocal microscopy. Contrary to most in vivo and in vitro extravasation assays, robust and rapid scoring of extravascular cells combined with high-resolution imaging can be easily achieved due to the confinement of the vascular network to one plane close to the surface of the device. This renders extravascular cells clearly distinct and allows tumor cells of interest to be identified quickly compared to those in thick tissues. The ability to generate large numbers of devices (~36) per experiment coupled with fast quantitation further allows for highly parametric studies, which is required when testing multiple genetic or pharmacological perturbations. This is coupled with the capability for live tracking of single cell extravasation events allowing both tumor and endothelial morphological dynamics to be observed in high detail with a moderate number of data points. This Protocol Extension describes an adaptation of an existing Protocol describing a microfluidic platform that offers additional applications.

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Quantification of cancer cell extravasation in vivo

Cited by 62 publications

References 51 publications

3D chick embryo chorioallantoic membrane model as an in vivo model to study morphological and histopathological features of feline fibrosarcomas

3D chick embryo chorioallantoic membrane model as an in vivo model to study morphological and histopathological features of feline fibrosarcomas

Targeting of CCBE1 by miR-330-3p in human breast cancer promotes metastasis

On-chip human microvasculature assay for visualization and quantification of tumor cell extravasation dynamics

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