TY - JOUR
T1 - A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis
AU - Shadaloey, Arman Alberto Sorin
AU - Karz, Alcida
AU - Moubarak, Rana S.
AU - Agrawal, Praveen
AU - Levinson, Grace
AU - Kleffman, Kevin
AU - Aristizabal, Orlando
AU - Osman, Iman
AU - Wadghiri, Youssef Z.
AU - Hernando, Eva
N1 - Publisher Copyright:
© 2022 JoVE Journal of Visualized Experiments.
PY - 2022/3
Y1 - 2022/3
N2 - Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/ or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.
AB - Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/ or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.
UR - http://www.scopus.com/inward/record.url?scp=85127299740&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85127299740&partnerID=8YFLogxK
U2 - 10.3791/63186
DO - 10.3791/63186
M3 - Article
C2 - 35343960
AN - SCOPUS:85127299740
SN - 1940-087X
VL - 2022
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
IS - 181
M1 - e63186
ER -