Abstract Stem-like breast cancer cells (SBCCs) are drug resistant, invasive, and likely to lead to tumor recurrence and repopulation. High expression of the adhesion molecule CD44, the drug transporter ABCG2, and of the enzyme ALDH1A1 are well-established markers associated with SBCC-enriched tumor populations [1]. Hypoxic tumor microenvironments are frequently associated with increased aggressiveness and resistance to chemo and radiation therapy. Hypoxia results in the stabilization of the hypoxia inducible factor -1 (HIF-1), a transcription factor that activates a battery of genes, including those associated with SBCCs, that help cancer cells to survive, repopulate and finally metastasize to distant location. Recently, we reported the role of hypoxia and HIF-1α in regulating the expression of CD44 and its variant isoforms in triple negative breast cancer [2]. Here we have validated the association between hypoxia and CD44 expression in these tumors. We used tumors derived from MDA-MB-231 cells genetically engineered to express red fluorescent protein (tdtomato) under the control of hypoxia response element (231-HRE-RFP). Optical imaging (Nikon fluorescence microscope) was performed to detect hypoxia in fresh tissue slices, followed by immunohistochemistry (IHC) staining for HIF-1α, CD44 and ABCG2 expression in 5μm thickness adjacent sections from paraffin embedded 231-HRE-RFP tumors. Slides were scanned on an Image Scope digital scanner. Analysis for HIF-1 α nuclear staining was performed by drawing regions of interest (ROI) on scanned images using manufacturer supplied macro (Aperio Technologies Inc. CA, USA). For co-registration and quantification studies, ROI drawn images of HIF-1α and CD44 were co-registered to the bright field and fluorescent optical images using an in-house program developed in MATLAB (Mathworks Inc.). Statistical analysis (t-test) was performed using Microsoft Excel 2010 (Microsoft Inc. Seattle, USA). Following co-registration, intensely fluorescing regions of 231-HRE-RFP tumors were found to be associated with elevated nuclear HIF-1α expression and higher CD44 membrane expression. A trend of increased optical intensity (p≤0.09) and significantly increased CD44 pixel intensity (p≤0.05) was observed in the high HIF-1α ROI compared to the low HIF-1α ROI. Work is under way to co-register other breast cancer stem cell markers such as ABCG2 and ALDH1A1 in these tumors. These data further highlight the role of hypoxia in engendering a stem-like phenotype, and the potential importance of targeting hypoxia to minimize the burden of cells with stem-like characteristics in tumors. All animal protocols were approved by the JHU animal care and use committee. This work was supported by NIH R01CA136576 and P50 CA103175. 1. Al-Hajj, M et al., Proc Natl Acad Sci U S A, 2003. 2. Krishnamachary.B. et al., PLoS One 2012;7(8)e44078- Citation Format: Balaji Krishnamachary, Samata Kakkad, Marie-France Penet, Keve Zoltani, Venu Raman, Mayur Gadiya, Yelena Mironchik, Flonne Wildes, Zaver M. Bhujwalla. Validation of the co-expression of breast cancer stem cell markers with HIF-1α in tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3745. doi:10.1158/1538-7445.AM2013-3745