Observations of actin dynamics in living cells using fluorescence microscopy have been foundational in the exploration of the mechanisms underlying cell migration. We used CRISPR/Cas9 gene editing to generate neutrophil‐like HL‐60 cell lines expressing GFP‐β‐actin from the endogenous locus (ACTB). In light of many previous reports outlining functional deficiencies of labeled actin, we anticipated that HL‐60 cells would only tolerate a monoallelic edit, as biallelic edited cells would produce no normal β‐actin. Surprisingly, we recovered viable monoallelic GFP‐β‐actin cells as well as biallelic edited GFP‐β‐actin cells, in which one copy of the ACTB gene is silenced and the other contains the GFP tag. Furthermore, the edited cells migrate with similar speeds and persistence as unmodified cells in a variety of motility assays, and have nearly normal cell shapes. These results might partially be explained by our observation that GFP‐β‐actin incorporates into the F‐actin network in biallelic edited cells at similar efficiencies as normal β‐actin in unedited cells. Additionally, the edited cells significantly upregulate γ‐actin, perhaps helping to compensate for the loss of normal β‐actin. Interestingly, biallelic edited cells have only modest changes in global gene expression relative to the monoallelic line, as measured by RNA sequencing. While monoallelic edited cells downregulate expression of the tagged allele and are thus only weakly fluorescent, biallelic edited cells are quite bright and well‐suited for live cell microscopy. The nondisruptive phenotype and direct interpretability of this fluorescent tagging approach make it a promising tool for studying actin dynamics in these rapidly migrating and highly phagocytic cells.
Cell Communication Network Factor 4 (CCN4/WISP1) Shifts Melanoma Cells from a Fragile Proliferative State to a Resilient Metastatic State
Introduction—Cellular communication network factor 4
(CCN4/WISP1) is a secreted matricellular protein that
stimulates metastasis in multiple malignancies but has an
unclear impact on phenotypic changes in melanoma. Recent
data using cells edited via a double-nickase CRISPR/Cas9
approach suggest that CCN4/WISP1 stimulates invasion and
metastasis of melanoma cells. While these data also suggest
that loss of CCN4/WISP1 increases cell proliferative, the
CRISPR approach used may be an alternative explanation
rather than the loss of gene function.
Methods—To test whether CCN4/WISP1 also influences the
proliferative phenotype of melanoma cells, we used mouse
melanoma models and knocked out Ccn4 using a homologydirected
repair CRISPR/Cas9 system to generate pools of
Ccn4-knockout cells. The resulting edited cell pools were
compared to parental cell lines using an ensemble of in vitro
and in vivo assays.
Results—In vitro assays using knockout pools supported
previous findings that CCN4/WISP1 promoted an epithelial–
mesenchymal-like transition in melanoma cells and stimulated
invasion and metastasis. While Ccn4 knockout also
enhanced cell growth in optimal 2D culture conditions, the
knockout suppressed certain cell survival signaling pathways
and rendered cells less resistant to stress conditions. Tumor
cell growth assays at sub-optimal conditions in vitro, quantitative
analysis of tumor growth assays in vivo, and
transcriptomics analysis of human melanoma cell lines were
also used to quantify changes in phenotype and generalize
the findings.
Conclusions—In addition to stimulating invasion and metastasis
of melanoma cells, the results suggested that CCN4/WISP1 repressed cell growth and simultaneously enhanced
cell survival.
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- Award ID(s):
- 1644932
- NSF-PAR ID:
- 10126856
- Date Published:
- Journal Name:
- Cellular and Molecular Bioengineering
- ISSN:
- 1865-5025
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract -
Modeling metastasis in vivo with animals is a priority for both revealing mechanisms of tumor dissemination and developing therapeutic methods. While conventional intravenous injection of tumor cells provides an efficient and consistent system for studying tumor cell extravasation and colonization, studying spontaneous metastasis derived from orthotopic tumor sites has the advantage of modeling more aspects of the metastatic cascade, but is challenging as it is difficult to detect small numbers of metastatic cells. In this work, we developed an approach for quantifying spontaneous metastasis in the syngeneic mouse B16 system using real time PCR. We first transduced B16 cells with lentivirus expressing firefly luciferase Luc2 gene for bioluminescence imaging. Next, we developed a real time quantitative PCR (qPCR) method for the detection of luciferase-expressing, metastatic tumor cells in mouse lungs and other organs. To illustrate the approach, we quantified lung metastasis in both spontaneous and experimental scenarios using B16F0 and B16F10 cells in C57BL/6Ncrl and NOD-Scid Gamma (NSG) mice. We tracked B16 melanoma metastasis with both bioluminescence imaging and qPCR, which were found to be self-consistent. Using this assay, we can quantitatively detect one Luc2 positive tumor cell out of 10 4 tissue cells, which corresponds to a metastatic burden of 1.8 × 10 4 metastatic cells per whole mouse lung. More importantly, the qPCR method was at least a factor of 10 more sensitive in detecting metastatic cell dissemination and should be combined with bioluminescence imaging as a high-resolution, end-point method for final metastatic cell quantitation. Given the rapid growth of primary tumors in many mouse models, assays with improved sensitivity can provide better insight into biological mechanisms that underpin tumor metastasis.more » « less
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- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1007/s12195-019-00602-2
