The role of early placenta insulin like growth factor in invasion and metastasis of HER2 positive breast cancer cells in vivo

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Metastasis is the primary cause of death in breast cancer patients and it is therefore necessary to identify and study factors that control metastatic potential. Our recent studies have shown that increased levels of early placenta insulin like growth factor (EPIL) in HER2 positive breast cancer cells lead to an invasive phenotype in an in vitro extravasation assay. Furthermore clinical data indicates that EPIL is highly abundant at the invasive tumor border compared to the rest of the tumor. In addition, EPIL is an independent prognosticator of poor outcome and indicates a poor prognosis subgroup of HER2 positive tumors. To evaluate the role of EPIL expression in tumor cell invasion and metastasis in vivo, we designed a mouse xenograft model. This model is based on the breast cancer cell lines SKBR3 (high endogenous level of HER2) and MDA-MB-468-HER2 (artificially overexpressing HER2) which were demonstrated to show increased invasiveness upon EPIL stimulation in vitro.

To track cells by whole body in vivo imaging and also identify them microscopically in immunohistochemical analyses we labeled these cell lines with a dual reporter encoding Firefly-Luciferase (fLuc) and enhanced green fluorescent protein (eGFP) via retroviral transduction. Control of EPIL expression was conferred on the cells by transfecting them with an EPIL expression plasmid and a vector encoding an EPIL specific shRNA, respectively.

Tumor cells were introduced into the animals at different anatomic sites including inoculation into the mammary fat pads, tail vein and left ventricle. Whole body imaging using bioluminescence was used to follow primary tumor growth and track metastatic foci. Immunohistochemical analysis is used to characterize EPIL expression in the primary tumor and metastases. The in vivo data will be used to support or refute the hypothesis that EPIL plays a role in metastatic disease progression.