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Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs
Published on: May 24, 2016
Anoikis and metastatic potential of cloudman S91 melanoma cells
Z Zhu1, O Sanchez-Sweatman, X Huang
1University of Pittsburgh Cancer Institute, Pennsylvania 15213, USA.
Abstract:
Anoikis is a form of apoptosis induced in normal cells as a result of loss of their adhesion to substrate. In the present study, we have tested whether tumor cells are also sensitive to anoikis and whether selection of tumor cells for resistance to anoikis could increase their metastatic ability. In vitro cultured Cloudman S91 melanoma cells are strongly adherent to the plastic. Prevention of their adherence by rocking or by covering culture plates with polyhydroxyethylmethacrylate resulted in induction of anoikis and death of almost all cells. Their death was prevented in the presence of caspase inhibitor Z-Val-Ala-Asp-fluoromethyl ketone. To select anoikis-resistant cells, S91 cells floating in the culture medium were sequentially isolated and transferred for seven generations. As a result, a new subline of S91 cells capable of growing in free cell suspension was selected. These S91 nonadherent (S91Nadh) cells were completely resistant to anoikis and manifested higher metastatic ability than S91Adh cells. Anoikis resistance of S91Nadh cells was not attributable to their resistance to other apoptotic signals in vitro, and they showed no increase in their survival in vivo in the lungs after i.v. inoculation. Increased metastatic potential of the anoikis-resistant S91Nadh cells was associated with various phenotypic changes, including increased proliferation and loss of VLA-4 integrin expression because of down-regulation of the VLA-49alpha (CLD49d) gene. In parallel, they showed a reduction in homotypic aggregation and binding to endothelial cells, increased Matrigel invasiveness, and decreased matrix metalloproteinase-2 and matrix metalloproteinase-9 activity that paralleled up-regulation of the TIMP-1 gene. S91Nadh cells also manifested changes in cell surface carbohydrates, such as appearance of alpha-galactosyl epitopes as a result of up-regulation of the alpha1,3-galactosyltransferase gene and concomitant reduction in cell membrane sialylation. Thus, selection of S91 melanoma cells for anoikis resistance resulted in an increase in their metastatic potential in parallel with multiple alterations in their phenotypic properties.
Insights
Tumor cells can develop resistance to anoikis, a form of apoptosis triggered by cell detachment. Selecting for anoikis resistance in melanoma cells enhanced their metastatic potential and caused significant phenotypic changes.
Area of Science:
- Cell Biology
- Cancer Research
- Molecular Oncology
Background:
- Anoikis is programmed cell death occurring when normal cells lose substrate adhesion.
- Tumor cells often exhibit resistance to anoikis, a trait potentially linked to metastasis.
- Cloudman S91 melanoma cells are typically substrate-dependent and undergo anoikis upon detachment.
Purpose of the Study:
- To investigate if tumor cells are sensitive to anoikis.
- To determine if selecting for anoikis resistance increases melanoma cell metastatic ability.
- To characterize the phenotypic changes associated with anoikis resistance in melanoma cells.
Main Methods:
- Cultured Cloudman S91 melanoma cells under conditions preventing substrate adherence (rocking, polyhydroxyethylmethacrylate coating).
- Used a caspase inhibitor (Z-Val-Ala-Asp-fluoromethyl ketone) to confirm anoikis induction.
- Selected for anoikis-resistant cells (S91Nadh) through serial subculture of nonadherent cells over seven generations.
- Analyzed phenotypic alterations including proliferation, integrin expression, cell aggregation, invasiveness, protease activity, and cell surface carbohydrates.
Main Results:
- S91 melanoma cells induced anoikis upon loss of substrate adhesion, preventable by caspase inhibition.
- Selected S91Nadh cells demonstrated complete anoikis resistance and significantly higher metastatic ability compared to parental S91Adh cells.
- Anoikis resistance was not linked to broader apoptotic resistance or increased in vivo survival.
- S91Nadh cells exhibited increased proliferation, reduced VLA-4 integrin expression (due to VLA-49alpha gene down-regulation), decreased homotypic aggregation and endothelial cell binding, enhanced Matrigel invasiveness, and altered matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) activity.
- Cell surface changes included increased alpha-galactosyl epitopes (up-regulated alpha1,3-galactosyltransferase gene) and reduced sialylation.
Conclusions:
- Selection for anoikis resistance in melanoma cells promotes a more metastatic phenotype.
- Anoikis resistance is associated with multiple, coordinated phenotypic alterations, including changes in adhesion molecules, invasiveness, and cell surface glycosylation.
- These findings highlight the role of anoikis resistance in tumor progression and metastasis.
