Science and Technology Production
Congress
Authorship
RAFFO, DIEGO ALEJANDRO
;
Sampayo, R
;
Pontiggia, O
;
Bal de Kier Joffé, E
;
Simian, M
Date
2011
Publishing House and Editing Place
-
Summary
Information provided by the agent in
SIGEVA
The M05 mouse mammary tumor arouse spontaneously in a one year old virgin BALB/c mouse in our animal facility. It is estrogen dependent and tamoxifen sensitive within the first ten in vivo passages (Simian et al., BCRT, 2009). When treated with tamoxifen tumor growth is inhibited and immunofluorescence staining shows a decrease in ER+/E-cadherin+ epithelial tumor cells together with an increase in the stromal compartment and deposition of extracellular matrix components such as laminin, fibrone...
The M05 mouse mammary tumor arouse spontaneously in a one year old virgin BALB/c mouse in our animal facility. It is estrogen dependent and tamoxifen sensitive within the first ten in vivo passages (Simian et al., BCRT, 2009). When treated with tamoxifen tumor growth is inhibited and immunofluorescence staining shows a decrease in ER+/E-cadherin+ epithelial tumor cells together with an increase in the stromal compartment and deposition of extracellular matrix components such as laminin, fibronectin and collagen IV. The aim of this study was to investigate whether treatment with tamoxifen lead to an increase in cells with self renewing capacity. To do so mice bearing the M05 tumor were treated for 30 days with tamoxifen (5 mg pellet) or vehicle (empty pellet). Tumors were subjected to mechanical and enzymatic digestion until a single cell suspension was obtained. Mammosphere assays were carried out by seeding 10.000 cells/ml in ultralow attachment plates in DMEM/F12 supplemented with B27 and 20 ng/ml EGF. Mammosphere quantification was carried out after 7 days in culture. A statistically significant increase in mammosphere formation was achieved in the cultures derived from tamoxifen treated mice. Next we tested whether this observation was also true for tumors derived from tamoxifen treated and untreated animals that were subsequently passaged to untreated mice and allowed to grow. Mammosphere assays revealed that this was the case. Immunofluorescence studies showed that those tumors that derived from the treated mice contained higher levels of beta 1 and alpha 6 integrins as well as increased phopho-ser118-ER staining. Next to further establish a link between tamoxifen treatment and self renewal capacity we treated in vitro LM05-E cells (a continuous epithelial cell line derived from the M05 tumor that is ER+ and tamoxifen sensitive) with 4-OH-tamoxifen 10-6M or vehicle for 5 days and then carried out mammosphere assays. Tamoxifen treatment again led to an increase in mammosphere formation. However, when tamoxifen treatment was added directly to the mammosphere assays no differences were detected between the treated and control groups suggesting that only those cells that resist the treatment are able to grow in suspension. Finally we had previously shown that fibronectin and laminin conferred tamoxifen resistance through beta 1 and alpha 6 integrins respectively. To test whether these factors additionally lead to an increase in cells with self renewing capacity we treated LM05-E cells with these ECM components for 48 hours and then carried out the suspension cultures. Compared to the control group we found a decrease in the % of mammosphere forming cells as well as a decrease in ALDH activity as determined by the ALDFLOUR assay in the treated groups. Our results show that in the M05 model tamoxifen leads to an increase in cells with self renewal capacity both in vitro and in vivo and that microenvironmental factors seem to play a role. Understanding the effects of tamoxifen on the breast cancer stem cell population has important implications for the development of strategies that could lead to a decrease in recurrence in patients with ER+ tumors.
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Key Words
TamoxifenBreast CancerEndocrine resistanceMamospheres