Laser Micro-dissection of a Novel Cytoplasmic Structure in Cancer Stem Cells
EMSL Project ID
41792
Abstract
Laser Micro-dissection of a Novel Cytoplasmic Structure in Cancer Stem Cells We have identified a peri-nuclear fibrillar-granular structure in breast and colon cancer cell lines by transmission electron microscopy that resembles nuage or germinal granules found in germline cells of many animals (Nuage-Like Structure, NLS). This structure lacks a limiting membrane and is surrounded by immature mitochondria. Furthermore, we have visualized this structure at the light microscopic level by staining with a specific fluorescence dye. Using this technique, approximately 20% of cells in the cancer cell lines examined contain this structure. By flow sorting to enrich for cells with this structure, we demonstrated that cancer stem cell-like (CSC-like) markers CD44 and CD24 are selectively expressed in this subpopulation. NLS+ cells grow in the absence of serum, while NLS- cells are serum-dependent. We hypothesize that the NLS structure, similar to germline nuage, confer 'stemness' attributes on CSC-like cells. To further characterize the NLS structure, we propose to isolate fluorescence-labeled NLS from live Colo-205 cells for proteomic analysis using the Laser Capture Micro-dissection Microscope at EMSL. These studies will provide molecular tools for immunohistochemical detection in tissue sections, knock-out or over-expression experiments to study NLS function, and potential therapeutic targets. LCM technology has rarely been applied to subcellular structures, although a recent publication described the proteomic analysis of intracytoplasmic inclusions in human skeletal muscle biopsies (Schessl J. et al., J. Clin. Invest. 118: 904-12, 2008). Comprehensive proteomic analyses of germinal granules or nuage have not been reported. Although an LCM microscope is available at Fred Hutchinson Cancer Research Center, we lack the expertise required for this investigational study. The capabilities of EMSL in subcellular imaging and molecular level analysis are well suited to this project.
Project Details
Project type
Limited Scope
Start Date
2010-09-20
End Date
2010-11-20
Status
Closed
Released Data Link
Team
Principal Investigator