Effects of ADGRF1 overexpression on phospho- and total HER2 proteins by IHC in HER2+ breast tumors in vivo

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2022-04-14

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The adhesion G-protein coupled receptor (ADGRF1, previously known as GPR110) was recently found to represent a novel drug target in HER2+ BC1,2. In the present study, we aimed to investigate the effects of ADGRF1 overexpression on phospho- and total HER2 proteins expression levels by immunohistochemistry (IHC), using the previous in vivo tumor samples. We also aimed to optimize a protocol to quantify the staining intensity of phospho- and total HER2 proteins in the IHC images. The in vivo tumors formed by BT474 cell line (HER2+) with and without ADGRF1 overexpression (induced by the treatment of Doxycycline (Dox) from our previous study2 were used in the current study. IHC for phospho- and total HER2 proteins was performed on tumors (-Dox: n=11; +Dox: n=14). For IHC images analysis, three random images were taken using the Nikon Eclipse Ts2R microscope for each sample at 20x. Each image was then quantified using the FIJI/Image J software. Due to lack of an optimized protocol to quantify IHC images stained for phospho- and total HER2 proteins, we optimized a protocol for this purpose using previous in vivo tumors stained for phospho- and total HER2 proteins by IHC. Applying this protocol, we found no significant difference in phospho- or total HER2 protein expression between -Dox vs. +Dox groups. Ongoing studies aim to validate this protocol in different samples.

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