de9dbac77d900c1a0bca42a576359639 pmc_34066490.pdf f03ed1c2f93411e7a12fe26e7447f968e77c7af9 pmc_34066490.pdf ac8ce2faa7edb0bc64e353ab78d04a0154d5815bf1d8839f181fd70be2aab69d pmc_34066490.pdf Title: Spontaneous Cell Detachment and Reattachment in Cancer Cell Lines: An In Vitro Model of Metastasis and Malignancy Subject: There is an unmet need for simplified in vitro models of malignancy and metastasis that facilitate fast, affordable and scalable gene and compound analysis. “Adherent” cancer cell lines frequently release “free-floating” cells into suspension that are viable and can reattach. This, in a simplistic way, mimics the metastatic process. We compared the gene expression profiles of naturally co-existing populations of floating and adherent cells in SW620 (colon), C33a (cervix) and HeLa (cervix) cancer cells. We found that 1227, 1367 and 1333 genes were at least 2-fold differentially expressed in the respective cell lines, of which 122 were shared among the three cell lines. As proof of principle, we focused on the anti-metastatic gene NM23-H1, which was downregulated both at the RNA and protein level in the floating cell populations of all three cell lines. Knockdown of NM23-H1 significantly increased the number of floating (and viable) cells, whereas overexpression of NM23-H1 significantly reduced the proportion of floating cells. Other potential regulators of these cellular states were identified through pathway analysis, including hypoxia, mTOR (mechanistic target of rapamycin), cell adhesion and cell polarity signal transduction pathways. Hypoxia, a condition linked to malignancy and metastasis, reduced NM23-H1 expression and significantly increased the number of free-floating cells. Inhibition of mTOR or Rho-associated protein kinase (ROCK) significantly increased cell death specifically in the floating and not the adherent cell population. In conclusion, our study suggests that dynamic subpopulations of free-floating and adherent cells is a useful model to screen and identify genes, drugs and pathways that regulate the process of cancer metastasis, such as cell detachment and anoikis. Keywords: metastasis models; floating cells; suspension cells; anoikis; NM23; mTOR; cell detachment; cell reattachment Author: Elena Vargas-Accarino, Carlos Herrera-Montávez, Santiago Ramón y Cajal and Trond Aasen Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Sat May 8 03:50:50 2021 CEST ModDate: Sat May 8 04:07:34 2021 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 19 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 4384130 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- DRZUJM+VnURWPalladioL Type 1 Custom yes yes no 10 0 LCRBKJ+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 OIOCFP+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 JWERMT+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 ENXZUH+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 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Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 481 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: Presence of Floating Cells in “Adherent” Cell Lines Destination: subsection.2.1 Item: Title: Differential Gene Expression Comparing Floating vs. Adherent Cell Populations Destination: subsection.2.2 Item: Title: Proof of Principle Validation: Nm23-H1 Destination: subsection.2.3 Item: Title: Pathways and Gene Analysis Destination: subsection.2.4 Children: Item: Title: Hypoxia Destination: subsubsection.2.4.1 Item: Title: Floating Cells Are Specifically Sensitive to Cell Death upon mTOR and ROCK Inhibition Destination: subsubsection.2.4.2 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Cell Culture Destination: subsection.4.1 Item: Title: RNA Isolation and Sequencing Destination: subsection.4.2 Item: Title: Timelapse Imaging Destination: subsection.4.3 Item: Title: Drug Treatments Destination: subsection.4.4 Item: Title: Viability Assays Destination: subsection.4.5 Item: Title: Western Blotting and Antibodies Destination: subsection.4.6 Item: Title: NM23-H1 Knockdown and Overexpression Destination: subsection.4.7 Item: Title: Hypoxia Treatment Destination: subsection.4.8 Item: Title: Flow Cytometry Analysis Destination: subsection.4.9 Item: Title: Statistical Analysis Destination: subsection.4.10 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: section.5 Info: Title: Spontaneous Cell Detachment and Reattachment in Cancer Cell Lines: An In Vitro Model of Metastasis and Malignancy Author: Elena Vargas-Accarino, Carlos Herrera-Montávez, Santiago Ramón y Cajal and Trond Aasen Subject: There is an unmet need for simplified in vitro models of malignancy and metastasis that facilitate fast, affordable and scalable gene and compound analysis. “Adherent” cancer cell lines frequently release “free-floating” cells into suspension that are viable and can reattach. This, in a simplistic way, mimics the metastatic process. We compared the gene expression profiles of naturally co-existing populations of floating and adherent cells in SW620 (colon), C33a (cervix) and HeLa (cervix) cancer cells. We found that 1227, 1367 and 1333 genes were at least 2-fold differentially expressed in the respective cell lines, of which 122 were shared among the three cell lines. As proof of principle, we focused on the anti-metastatic gene NM23-H1, which was downregulated both at the RNA and protein level in the floating cell populations of all three cell lines. Knockdown of NM23-H1 significantly increased the number of floating (and viable) cells, whereas overexpression of NM23-H1 significantly reduced the proportion of floating cells. Other potential regulators of these cellular states were identified through pathway analysis, including hypoxia, mTOR (mechanistic target of rapamycin), cell adhesion and cell polarity signal transduction pathways. Hypoxia, a condition linked to malignancy and metastasis, reduced NM23-H1 expression and significantly increased the number of free-floating cells. Inhibition of mTOR or Rho-associated protein kinase (ROCK) significantly increased cell death specifically in the floating and not the adherent cell population. In conclusion, our study suggests that dynamic subpopulations of free-floating and adherent cells is a useful model to screen and identify genes, drugs and pathways that regulate the process of cancer metastasis, such as cell detachment and anoikis. 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