In vitro scratch assay: method for analysis of cell migration in vitro labeled fluorodeoxyglucose (FDG)

Transcription

1 In vitro scratch assay: method for analysis of cell migration in vitro labeled fluorodeoxyglucose (FDG) 1 Dr Saeb Aliwaini 13/11/2015

2 Migration in vivo Primary tumors are responsible for only about 10% of deaths from cancer. The remaining approximately 90% of patients are struck down by cancerous growths that are discovered at sites far removed from the locations in their bodies where their primary tumors first arose. (metastases ) Using body fluids Breast cancer: often spawn metastatic colonies promiscuously in many tissues throughout the body, including the brain, liver, bones, and lungs. 2 Dr Saeb Aliwaini 13/11/2015

3 It depends on a series of complex biological steps Carcinomas : 80% of cancers 3 Dr Saeb Aliwaini 13/11/2015

4 Migration in vivo Local invasion seems to depend invariably on the release of secreted proteases, which are required to remodel the extracellular matrix (ECM), thereby generating space for the advance of cancer cells. In order to acquire motility and invasiveness, carcinoma cells must shed many of their epithelial phenotypes, detach from epithelial sheets, and undergo a drastic alteration the epithelial mesenchymal transition (EMT). 4 Dr Saeb Aliwaini 13/11/2015

5 EMT EMT involves a shedding by epithelial cells of their characteristic morphology and gene expression pattern and the assumption of a shape and transcriptional program characteristic of mesenchymal cells. An EMT can also be seen at the edges of carcinomas that are invading adjacent tissues 5 Dr Saeb Aliwaini 13/11/2015

6 6 Dr Saeb Aliwaini 13/11/2015

7 EMT Expression of E-cadherin and cytokeratins hallmarks of epithelial cell protein expression is repressed, while the expression of vimentin, an intermediate filament component of the mesenchymal cell cytoskeleton, is induced. Epithelial cells that have undergone an EMT often begin to make fibronectin, an extracellular matrix protein that is normally secreted only by mesenchymal cells such as fibroblasts. At the same time, expression of a typical fibroblastic marker N-cadherin is often acquired in place of E-cadherin 7 Dr Saeb Aliwaini 13/11/2015

8 EMT The transmembrane E-cadherin molecule plays the dominant role in influencing epithelial versus mesenchymal cell phenotypes adherens junctions For example, an analysis of 26 human breast cancer cell lines indicated that 16 had mutations in E-cad. The N-cadherin molecules expressed on the surface of a carcinoma cell that has undergone an EMT increase the affinity of this cancer cell for the stromal cells that normally display N-cadherin. 8 Dr Saeb Aliwaini 13/11/2015

9 9 Dr Saeb Aliwaini 13/11/2015

10 In vitro scratch assay The in vitro scratch assay is a straightforward and economical method to study cell migration in vitro. This method is based on the observation that, upon creation of a new artificial gap, so called scratch, on a confluent cell monolayer, the cells on the edge of the newly created gap will move toward the opening to close the scratch until new cell cell contacts are established again. 10 Dr Saeb Aliwaini 13/11/2015

11 In vitro scratch assay It mimics to some extent migration of cells in vivo - Removal of part of the endothelium in the blood vessels will induce migration of endothelial cells (ECs) into the denuded area to close the wound. Can be done for every cell type With specific ECM It is also compatible with microscopy including live cell imaging To study the effect of gene expression, drugs 11 Dr Saeb Aliwaini 13/11/2015

12 Transwell Migration Assay The transwell migration assay is a commonly used test to study the migratory response of endothelial cells to angiogenic inducers or inhibitors. This assay is also known as the Boyden or modified Boyden chamber assay. Cells are placed on the upper layer of a permeable membrane and a solution containing the test agent is placed below the cell permeable membrane. Following an incubation period (3 18 hours), the cells that have migrated through the membrane are stained and counted. The membrane is usually coated with some extracellular matrix 12 Dr Saeb Aliwaini 13/11/2015

13 13 Dr Saeb Aliwaini 13/11/2015

14 Revision cell cycle and apoptosis assays When do you do cell cycle analysis? What will you conclude? What is next? 14 Dr Saeb Aliwaini 13/11/2015

15 Cell Cycle Analysis DNA content analysis - Propidium Iodide S Phase G0/G1 G2/M Or doublets of G0/G1 cells? DNA Content 13/11/2015 Dr Saeb Aliwaini 15

16 Apoptosis Bcl-2 family members (intracellular staining) Fix and permeabilize Add Antibody Analyse by Flow Cytometry 13/11/2015 Dr Saeb Aliwaini 16

17 Apoptosis Bcl-2 family members Bcl-2 13/11/2015 Dr Saeb Aliwaini 17

18 Apoptosis Propidium Iodide (fixed cells) DNA degradation 13/11/2015 Dr Saeb Aliwaini 18

19 Apoptosis Annexin V-fluorochrome plus Propidium Iodide (non-fixed cells) 13/11/2015 Dr Saeb Aliwaini 19

20 Apoptosis Annexin V plus propidium Iodide 13/11/2015 Dr Saeb Aliwaini 20

21 Apoptosis Annexin V plus propidium Iodide 13/11/2015 Dr Saeb Aliwaini 21