Since Kupffer cells represent only a small portion of the entire liver cell population, greatly outnumbered by the parenchymal cells, Kupffer cell isolation faces major technical obstacles. Laser capture microdissection (LCM) offers a method of isolating a single cell type from specific regions of tissue sections.
A research team led by Dr Stephen H Gregory from United States outlines isolation of Kupffer using LCM. This will be published on April 14, 2009 in the World Journal of Gastroenterology.
LCM is an essential approach used in conjunction with molecular analysis to study the functional interaction of cells in their native tissue environment. The process of labeling and acquiring cells by LCM prior to mRNA isolation can be elaborate, thereby subjecting the RNA to considerable degradation. Kupffer cell labeling was achieved by injecting India ink intravenously, thus circumventing the need for in vitro staining. The significance of this novel approach was validated using a cholestatic liver injury model.
They found that mRNA extracted from the microdissected cell population displayed marked increases in colony stimulating factor-1 receptor and Kupffer cell receptor message expression, which demonstrated Kupffer cell enrichment. Gene expression by Kupffer cells derived from bile-duct-ligated, versus sham-operated, mice was compared. Microarray analysis revealed a significant (2.5-fold, q value < 10) change in 493 genes. Based on this fold-change and a standardized PubMed search, 10 genes were identified that were relevant to the ability of Kupffer cells to suppress liver injury
The methodology outlined herein provides an approach to isolating high quality RNA from Kupffer cells, without altering the tissue integrity.
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"We found that the addition of dasatinib to standard therapy in the three cell lines with significant SRC pathway deregulation “ IGROV1, OVCAR3 and A2780 “ enhanced the response of the cancer cells to therapy," Teoh said. "Conversely, in SKOV3, which has minimal SRC protein expression and pathway deregulation, we saw the least amount of anti-cancer activity when we added dasatinib."
It's possible that by blocking the SRC activity with the dasatinib, we are enhancing the effect of the other chemotherapeutic agents, Teoh said.
The results of this study support the further investigation of targeted biologic therapy using a SRC inhibitor in some ovarian cancers, she said. Currently a phase I trial of a combination of dasatinib, paclitaxel and carboplatin is available for women with advanced or recurrent ovarian, tubal and peritoneal cancers.
Dasatinib is a chemotherapeutic that is currently FDA-approved for use in leukemia. It is manufactured by Bristol-Myers Squibb and is sold under the brand name Sprycel. Bristol-Myers Squibb provided the dasatinib used in this study.
Other researchers involved in this study include Tina Ayeni, Jennifer Rubatt, Regina Whitaker, Holly Dressman and Andrew Berchuck.
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