Method of detecting new cancer stem cell-like enrichment in development front assay (DFA).

Abstract

Unlabelled: Wound healing is an inherent property of injured tissue or a group of cells. The healing front is always developed by new cells which are progenitor of differentiated parental cells. In cancer tissues we aim to study the healing front and observed an enriched population of stem cell like properties in the developing front when compared to the other areas of the cell matrix.

Method: In vitro scratch assays with special focus on stem cell expression was used to analyze metastatic potential of the tumor cell, epithelial to mesenchymal transition and rate of cell migration to get an insight into the genes and the proteins getting expressed at the developing front. In this protocol we describe a fluorescence dependent method to document stem cell like enrichment at the developing front of a given wound in drug treated and untreated control cells under the same culture conditions in a time lag manner. We have tried to compare the rate of cell migration and the expression levels of stem cell markers between the treated and untreated cells.

Results: CD44 being a cell surface protein and being involved in cell migration and proliferation, higher intensity of CD44 was observed at the developing front with increasing time. The rate of cell migration differed with different treatments and so did the CD44 expression with expression being higher in 0.6mM concentration of bleomycin when compared to 0.4mM. Similar expression was observed for ALDH1 stem cell marker. This particular technique can not only be used for studying expression of CSC markers (like CD44, ALDH1) but also in assaying the expression profile of several proteins involved in cellular processes like EMT (Epithelial to Mesenchymal Transition), cell migration, tumorigeneisis and rate of proliferation.

Conclusion: Would healing is an integral property of solid tissues and in solid tumors properties of solid tissue wound are important characteristics of tumor development. Therefore combining the properties of stem cell like enrichment in the development front would be an important and fast assay to study migratory and metastatic properties of an invitro culture.