Time-lapse analysis and microdissection of living 3D melanoma cell cultures for genomics and proteomics.
A novel technique is presented for the monitoring and morphological characterization of 3D cell cultures targeted for laser capture microdissection (LCM). A custom-made chamber enables time-lapse topography and pre-selection of cell targets in order to minimize microdissection time, optimizing the quality of biomolecules for downstream analyses. The method complements the recently presented novel application of LCM in living 3D cultures, whose compatibility with standard genomics and proteomics assays such as microarrays, real-time PCR, and 2D gel electrophoresis is further corroborated here. Specifically, the above techniques are employed in tandem to study, as a proof of principle, the dynamics of in vitro vasculogenic mimicry. It was shown previously that aggressive melanoma cells spontaneously differentiate on collagen gels into vascular-like networks with strong endogenous angiogenic potential. Here the evolution of vasculogenic mimicry was quantified by three time-dependent variables: the distribution of the vascular-like network lengths, widths, and area coverage. Based on these morphological descriptors the networks were locally classified over time as "early" or "mature" stage. LCM of networks and randomly oriented cells followed by real-time PCR for select genes revealed that differential expression was time-dependent and increased with network maturity. The method is widely applicable for microgenomics and microproteomics analyses in phenotypically evolving 3D cultures (i.e., of stem cells), under spontaneous or directed differentiation. Therefore beyond enabling future rigorous analyses on the mechanistics of vasculogenic mimicry, it provides a practical discovery engine for a range of developmental studies and tissue regenerative engineering applications.
Demou
Zoe N
ZN
Children's Memorial Research Center, Department of Pediatrics, Northwestern University Feinberg School of Medicine, 2300 Children's Plaza, Box 204, Chicago, Illinois 60614-4314, USA. zdemou@childrensmemorial.org
D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D020869:Gene Expression Profiling; D023281:Genomics; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D021621:Imaging, Three-Dimensional; D007834:Lasers; D008545:Melanoma; D042282:Microdissection / Q000379:methods*; D008954:Models, Biological; D040901:Proteomics; D013997:Time Factors