Marie-France Penet

Noninvasive Multiparametric Imaging of Metastasis-Permissive Microenvironments in a Human Prostate Cancer Xenograft

Metastasis continues to be one of the major causes of mortality from prostate cancer. Because human malignant cell lines metastasize more readily from orthotopic sites than from heterotopic sites, to identify metastasis-permissive tumor microenvironments, we used noninvasive imaging to compare the in vivo vascular, metabolic, and physiologic characteristics of a human prostate cancer xenograft implanted orthotopically in the prostate or s.c. in the flank. Hypoxia was detected in these xenografts by placing an enhanced green fluorescence protein optical reporter under the control of a hypoxia response element. A multiparametric analysis of hypoxia, extracellular pH, vascularization, and metabolism provided a characterization of environments that are permissive for metastasis to occur. We found that orthotopic tumors, which metastasized more easily, were characterized by higher vascular volume, permeability, and total choline and a more acidic extracellular pH. Interestingly, metastatic deposits in the lymph nodes as well as cancer cells in ascites fluid were found to be hypoxic, explaining, in part, the refractory nature of metastatic disease. These results also provide the basis for clinically translatable noninvasive imaging markers for predicting metastatic risk in prostate cancer.

MRI of metastasis-permissive microenvironments

One of the earliest documented observations of the importance of the microenvironment in metastasis was made by Stephen Paget in 1889. More than a century later, the metastatic cascade remains a major cause of mortality from cancer. Cancer meets the criterion of a successful organization that is able to survive by adapting to changing environments. In fact, the tumor microenvironment and stroma are co-opted and shaped by cancer cells to derive a survival advantage. Cohesive strategies integrating advances in molecular biology and chemistry, with noninvasive multimodality imaging, provide new insights into the role of the tumor microenvironment in promoting metastasis from primary tumors as well as insights into environments that attract and permit cancer cells to establish colonies in distant organs. This article provides an overview of molecular and functional imaging characterization of microenvironments that can promote or permit cancer cells to metastasize and the microenvironmental characteristics of distant metastases.

Abstract 2970: Cyclooxygenase-2 mediates significant changes in the extracellular matrix in triple negative breast cancer

Triple (ER/PR/HER2) negative breast cancer (TNBC) accounts for approximately 20% of all breast cancer diagnoses and is characterized by metastasis that is refractory to treatment. We and others have demonstrated that cyclooxygenase-2 (COX-2) products are important mediators of increased invasion and metastasis in the MDA-MB-231 human xenograft model of TNBC. Increasing evidence suggest that high density of collagen I fibers in the extracellular matrix (ECM) is predictive of increased metastasis and cancer cells have been observed to migrate along these fibers during the metastatic journey. We previously observed that downregulation of COX-2 significantly reduced the expression of degradative enzymes such as matrix metalloproteinase 1 and altered the expression of ECM components such as hyaluronan and lumican that play a role in intra-fibrillar collagen spacing. Tumor growth of MDA-MB-231 and Clone 13 (a MDA-MB-231-derived COX-2 shRNA-expressing clone secreting less than 10% of PGE2 compared to parental) xenografts, studied following Matrigel-supported orthotopic implantation in SCID mice, showed a 3-week delay in tumor growth. Here, we have used non-Matrigel tumors derived from Clone 13 cells were used to investigate the relationship between COX-2 expression, interstitial fluid transport using MRI and collagen fiber density and volume using second harmonic generation (SHG) microscopy. MRI was performed once tumor volumes were approximately 400-500 mm3. Interstitial fluid transport parameters were measured from quantitative T1 maps obtained before and following intravenous administration of the contrast agent albumin-GdDTPA. Interstitial fluid transport parameters calculated included number of draining and pooling voxels, draining and pooling rates, and volumes. SHG microscopy of tissue slices was performed using a 25x lens on a Zeiss 710 LSM NLO confocal microscope. Collagen I fiber distribution was characterized using an in-house 3D analysis software developed to quantify fiber distance distributions and fiber volumes. Significant differences in the ECM structure and function were evident between high COX-2 expressing MDA-MB-231 tumors compared to Clone 13 tumors with low COX-2 expression. Tumors arising from Clone 13 cells demonstrated an increase in vascular volume, a decrease of draining voxels, an increase of pooling voxels, and a decrease of collagen fiber density. Collectively, these data provide direct evidence for COX-2-mediated changes in the ECM. Here, for the first time, we have shown that dowregulating COX-2 in TNBC cells profoundly impacts collagen I fiber density and volume and alters macromolecular transport. Understanding the role of COX-2 in TNBC and its impact on the ECM and macromolecular transport may provide new insights into preventing relapse and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2970. doi:1538-7445.AM2012-2970

Structure and function of a prostate cancer dissemination-permissive extracellular matrix

Purpose: The poor prognosis of metastatic prostate cancer continues to present a major challenge in prostate cancer treatment. The tumor extracellular matrix (ECM) plays an important role in facilitating metastasis. Here, we investigated the structure and function of an ECM that facilitates prostate cancer metastasis by comparing orthotopic tumors that frequently metastasize to poorly metastatic subcutaneous tumors. Experimental Design: Both tumors were derived from a human prostate cancer PC3 cell line engineered to fluoresce under hypoxia. Second harmonic generation (SHG) microscopy was used to characterize collagen 1 (Col1) fiber patterns in the xenografts as well as in human samples. MRI was used to determine albumin-Gd-diethylenetriaminepenta-acetate (alb-GdDTPA) transport through the ECM using a saturation recovery MR method combined with fast T1 SNAPSHOT-FLASH imaging. Cancer-associated fibroblasts (CAF) were also quantified in these tumors. Results: Significant structural and functional differences were identified in the prometastatic orthotopic tumor ECM compared to the less metastatic subcutaneous tumor ECM. The significantly higher number of CAFs in orthotopic tumors may explain the higher Col1 fiber volumes in these tumors. In vivo, alb-GdDTPA pooling was significantly elevated in metastatic orthotopic tumors, consistent with the increased Col1 fibers. Conclusions: Developing noninvasive MRI indices of macromolecular transport, together with characterization of Col1 fiber patterns and CAFs can assist in stratifying prostate cancers for aggressive treatments or active surveillance. These results highlight the role of CAFs in supporting or creating aggressive cancers, and the importance of depleting CAFs to prevent metastatic dissemination in prostate cancer. Clin Cancer Res; 23(9); 2245–54. ©2016 AACR.

Abstract 2191: Multiple imaging reporter labeled acid-degradable dextran nanopolymer as a COX-2 siRNA carrier for COX-2 specific downregulation

Cyclooxygenase-2 (COX-2) as a target has attracted interest for multiple degenerative diseases and cancers. COX-2 siRNA provides specific and effective down-regulation of COX-2. However, because artificial cationic polymers can induce proinflammatory signals that increase COX-2 expression, there are few if any reports about the use of artificial cationic polymers as siRNA carrier to downregulate COX-2 in cancer cells. Here we developed a method to efficiently synthesize a multiple imaging reporter labeled biodegradable dextran to use as an efficient cationic polymer carrier for COX-2 siRNA delivery. Amine function groups were conjugated to the dextran platform through acetal bonds. Acetal bonds were broken at acidic conditions that occur in cancer and endocytosis compartments. Rhodamine was labeled to the amine groups to detect degradation and removal of these groups from the cell, while the dextran scaffold was labeled with Cy5.5. The rapid cleavage and release of amine groups minimized the proinflammatory side effects of the positively charged amine groups.Colorimetric assay of Cy5.5 and rhodamine in different pH buffer indicated that the amine group was cleaved at pH 5.5 buffer but was stable in pH 7.4 buffer. Fluorescence imaging showed that the dextran siRNA nanoplex entered the cells through endocytosis that provided acidic conditions for the breaking of the amine group. For the first time, the intracellular breaking of acetal bonds was clearly visualized by multiple imaging reporters. Because the rapid cleavage and release of amine groups minimized the proinflammatory side-effects, quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay indicated that this dextran COX-2 siRNA nanoplex could downregulate COX-2 expression efficiently. Prostaglandin E2 level decreased significantly with COX-2 siRNA/dextran treatment. In vivo imaging demonstrated that the COX-2 siRNA/dextran nanoplex accumulated in MDA-MB-231 tumors. QRT-PCR and western blot assays of COX-2 levels in tumor tissue demonstrated that this nanoplex significantly downregulated COX-2 expression in vivo efficiently and within 24h. This dextran nanopolymer can be used as a safe, reproducible, and biocompatible siRNA carrier to effectively reduce COX-2 expression in cancer cells and tumors. Citation Format: Zhihang Chen, Balaji Krishnamachary, Marie-France Penet, Zaver M. Bhujwalla. Multiple imaging reporter labeled acid-degradable dextran nanopolymer as a COX-2 siRNA carrier for COX-2 specific downregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2191. doi:10.1158/1538-7445.AM2017-2191

Abstract 5364: Delayed progression of lung metastases in a triple-negative breast cancer model following delivery of cytosine deaminase that converts chemotherapeutic prodrug 5-fluorocytosine to 5-fluorouracil

Triple negative breast cancer (TNBC) is aggressive, has a very poor prognosis with a high rate of reoccurrence and is refractory to standard chemo- and radiotherapy. Still, chemotherapy is an effective option to treat recurrent or metastatic cancers if the debilitating side effects limiting the dose and time of exposure can be diminished. The use of pro-drugs that can be activated locally by a prodrug enzyme can minimize collateral damage since the presence of the enzyme in the tumor and clearance from normal tissue can be verified noninvasively with imaging, which allows for optimal timing of prodrug administration (1). We previously demonstrated the efficacy of the prodrug 5-fluorocytosine (5-FC) in primary triple negative MDA-MB-231 xenografts following administration of a poly-L-lysine based theranostic nanoplex containing bacterial cytosine deaminase (bCD) that converts 5-FC to the chemotherapeutic agent 5-fluorouracil. The size of the bCD-nanoplex of ∼ 300 kD allowed its delivery into the tumor interstitium through leaky tumor vasculature but not through normal vasculature (1). Here we used a more effective variant of bCD (2) to target metastatic MDA-MB-231 cells. We injected 2 × 106 td-tomato fluorescent protein expressing MDA-MB-231 cells intravenously and monitored metastases formation in the lungs for 5 weeks by which time metastatic nodules were detected. We then treated the animals with the bCD-nanoplex (300 mg/kg i.v.). Twenty-four hours later, we injected a first dose of 5-FC (200 mg/kg i.v. and 250 mg/kg i.p.), repeated at 72 hours. The animals were followed for 2 weeks with weekly optical imaging at the end of which mice were euthanized and the lungs excised. We histologically evaluated metastatic burden (area occupied by metastatic cells/total lung area) in mice treated with bCD-nanoplex + 5-FC (n = 5) and in mice injected with the bCD-nanoplex only (n = 6). We observed a 32% decrease in metastatic burden in the pro-drug treated group vs the control group (14.8% + 1.5% in treated mice vs 22.0% + 2.6% in control mice, p = 0.03). These results are a first step towards the longitudinal evaluation of such a strategy with multiple doses. Additionally, the nanoplex can be coupled to multimodal imaging reporters (1) to time prodrug administration and improve the detection and treatment of triple negative, hormone refractory metastatic cancers. References. 1. Li et al., Clin Can Res., 2008; 2. Fuchita et al., Can Res., 2009. This work was supported by NIH R01CA138515 and P50 CA103175. Citation Format: Louis Dore-Savard, Zhihang Chen, Paul T. Winnard, Balaji Krishnamachary, Marie-France Penet, Venu Raman, Margaret E. Black, Zaver Bhujwalla. Delayed progression of lung metastases in a triple-negative breast cancer model following delivery of cytosine deaminase that converts chemotherapeutic prodrug 5-fluorocytosine to 5-fluorouracil. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5364. doi:10.1158/1538-7445.AM2015-5364

Abstract 4021: Cyclooxygenase-2 downregulation reduces activated fibroblasts and modifies the extracellular matrix in MDA-MB-231 breast cancer xenograft

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnCOX-2 is an important mediator of inflammation that significantly influences tumor angiogenesis, invasion and metastasis. Here, we have investigated the role of COX-2 in modifying the number of activated cancer associated fibroblasts (CAFs) and in altering the extracellular matrix (ECM) in a breast cancer model.nnTo investigate the role of COX-2 in modulating the ECM, we used an MDA-MB-231 cell clone (Clone 13) expressing a short hairpin RNA (shRNA) to downregulate COX-2 [1]. Clone 13 cells were characterized for significantly lower basal and TPA-induced COX-2 and PGE2 expression compared to parental MDA-MB-231 cells using ELISA (PGE2), western blot (COX-2 protein) and q-RT-PCR (COX-2 mRNA). Tumors were derived from parental (n = 5) and Clone 13 (n = 6) MDA-MB-231 cells following inoculation in the mammary fat pad in SCID mice. Tumors were excised at ∼ 500 mm3 and immunohistochemically stained to quantify vessel density (CD31) and activated CAFs (α-smooth muscle actin (SMA)) in 5 μm thick formalin fixed sections. Stained sections were digitally scanned and positive staining quantified using manufacturer supplied software (Aperio Technologies, CA).nnClone 13 tumors showed delayed tumor growth compared to parental MDA-MB-231 tumors. We have previously observed that collagen 1 (Col1) fiber density and fiber volume were significantly lower in COX-2 reduced Clone 13 tumors compared to parental tumors [2]. While cancer cells shape Col1 fiber patterns through the secretion of various enzymes, Col1 fiber is laid down by activated CAFs within or around the tumor. Quantification of activated CAFs by immunohistochemistry for α-SMA in the tumors, and immunoblotting for α-SMA of crude protein extracted from the tumors, revealed significantly fewer CAFs and significantly reduced levels of α-SMA protein in Clone 13 tumors compared to parental MDA-MB-231 tumors. We previously observed a significant decrease in permeability as well as reduced influx and efflux of macromolecular transport in Clone 13 tumors compared to parental tumors, but no difference in vascular volume [2]. Immunohistochemistry for CD31 staining of endothelial cells did not detect a significant difference in CD31 density between Clone 13 and parental tumors further confirming our previous observations about vascular volume. These data reveal the multi-faceted effects of COX-2 in modifying the structure and function of the ECM, and identify the ability to attract and activate fibroblasts as one mechanism by which COX-2 modifies the ECM.nnAcknowledgements: We thank Mr. Gary Cromwell for technical assistance. This work was supported by NIH R01CA82337 and P50 CA103175.nnReferences: [1] Stasinopoulos, I., et al., Mol Cancer Res, 2007; [2], Stasinopoulos, I., et al., AACR, 2013 Chicago.nnCitation Format: Balaji Krishnamachary, Ioannis Stassinopoulos, Samata M. Kakkad, Marie-France Penet, Desmond Jacob, Flonne Wildes, Yelena Mironchik, Arvind Pathak, Meiyappan Solaiyappan, Zaver M. Bhujwalla. Cyclooxygenase-2 downregulation reduces activated fibroblasts and modifies the extracellular matrix in MDA-MB-231 breast cancer xenograft. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4021. doi:10.1158/1538-7445.AM2015-4021

Abstract 5110: Initial characterization of an optical reporter myoblast cell line for non-invasive imaging in a cancer cachexia model in mice

Cancer associated cachexia affects a majority of patients during cancer progression, compromising therapeutic interventions and contributing to decreased survival rates. Identifying factors involved in the onset of cachexia will provide a better understanding of early treatment strategies. To this end, we developed a mouse model system that allows for real time longitudinal monitoring of cancer induced wasting and has the potential of identifying early cachectic events. Several attributes of our system are new. 1) The construction of a dual optical reporter vector with green fluorescence protein (GFP) expression constitutively driven from an EF1α promoter and red fluorescence protein (tdTomato) expression driven by an engineered skeletal muscle specific inducible promoter. The latter is a synthetic sequence of a triple-tandem repeat of the glucocorticoid-FOXO1 response element region from the proximal promoter of the human MuRF1 gene. 2) Generation of a rat L6 myoblast optical reporter cell line (To3B cells) with stable integration of the dual reporter vector construct, which provides living reporter grafts within mouse muscle. 3) A human pancreatic cancer cell line (Pa04C) that as an orthotopic or subcutaneous xenograft causes weight loss in male SCID mice. In preliminary studies, we tested several human pancreatic cancer cell lines as orthotopic xenografts in male SCID mice. We found that red fluorescence signals were reproducibly detected in live mice only from To3B grafts in mice undergoing weight loss, while graft size and viability were readily monitored by imaging GFP fluorescence in all animals. In addition, mice bearing Pa04C tumors lost the most weight while mice bearing Panc1 tumors gained weight. Therefore, Pa04C and Panc1 cells were used for subcutaneous xenografts in male SCID mice and weight loss was followed with optical monitoring of To3B grafts. Importantly, in weight losing mice, we found that red fluorescence could be detected and quantified at a nascent stage of the syndrome; e.g., unambiguous red fluorescent signals were quantified at weight losses of only 1.2 to 2.7% at very low tumor burdens of only ∼0.079 to ∼0.170 cm3. Red fluorescence remained very low to undetectable in mice that gained weight. Tumor sizes were comparable between groups, which was an indication that factors independent of tumor growth were involved in switching on red fluorescence. Ex vivo fluorescence microscopy confirmed a robust presence of red fluorescence only in To3B grafts in skeletal muscle from Pa04C tumor bearing mice. The evidence from this initial development of a unique optical reporter myoblast cell line indicates the potential to detect the onset of cancer cachexia. These studies set the ground work for future research aimed at identifying initiating systemic as well as local molecular events in the muscle of cachectic mice. Supported by NIH P50CA103175 Citation Format: Paul T. Winnard, Marie-France Penet, Yelena Mironchik, Flonne Wildes, Anirban Maitra, Zaver M. Bhujwalla. Initial characterization of an optical reporter myoblast cell line for non-invasive imaging in a cancer cachexia model in mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5110. doi:10.1158/1538-7445.AM2015-5110

Abstract 509: Hypoxia and HIF silencing mediated dysregulation of total choline, CD44 expression, and metastatic burden in MDA-MB-231 human breast cancers

Hypoxic tumors frequently exhibit an aggressive phenotype due to dysregulated gene expression and metabolic changes. Hypoxia results in the stabilization of hypoxia inducible factors (HIF-1/2) that transcriptionally activate genes involved in invasion, metastasis, metabolism, and in the adaptation of cancer cells to their microenvironment. In breast cancer, stem-like breast cancer cells that survive, repopulate and metastasize to distant locations, have elevated expression of CD44. In a previous study, we observed elevated expression of CD44 in hypoxic tumor regions, and identified HIF-1α as a regulator of CD44 expression in breast cancer cells under hypoxic conditions [1]. Hypoxia has also been implicated in increasing the activity of choline kinase (Chk)-alpha, the enzyme responsible for elevated phosphocholine (PC) and total choline (tCho) consistently observed in cancers [2]. In previous studies, lentiviral transduction of MDA-MB-231 breast cancer cells (231 cells) with shRNA against Chk-alpha and the in vivo delivery of the Chk-shRNA virus into tumor bearing mice resulted in decreased CD44 message and expression together with effective silencing of Chk message and expression [3]. Here, using non-invasive proton magnetic resonance spectroscopic imaging (1H MRSI), we have established the importance of HIF in reducing total choline and metastatic tumor burden, and have identified a role for CD44 in establishing lung metastasis. HIF silencing in MDA-MB-231 cells significantly delayed tumor growth in mice. Both, the in vitro 1H and 31P MR spectra and in vivo 1H MRS images of tumors derived from engineered cells showed decreased tCho levels and distribution. This decrease of tCho was statistically significant in tumors derived from double silenced cells. Western blot analysis of tumors detected a decrease in Chk expression in double silenced (HIF-1 and 2) tumors. Silencing HIF-1α, -2α or both resulted in a significant reduction of metastatic lung burden in mice. Additionally, HIF-2α silencing was more effective at reducing lung colonization than HIF-1α, while silencing both was the most effective. Although metastatic burden decreased in HIF-1α silenced cells, the percentage of cells with high CD44 expression in the metastatic foci was comparable to that in the wild type or empty vector foci. These data identify the importance of targeting HIF and CD44 to prevent lung colonization and disrupt the metastatic cascade. This work was supported by NIH R01CA136576 and P50 CA103175. We thank Mr. Gary Cromwell for valuable technical assistance. References: 1. Krishnamachary B. et al., PLoS One, 2012; 2. Glunde, K., et al., Cancer Res, 2008; 3. Ackerstaff E. et al., Neoplasia. 2007. Citation Format: Balaji Krishnamachary, Santosh Kumar Bharti, Marie-France Penet, Samata M. Kakkad, Flonne Wildes, Keve Zoltani, Yelena Mironchik, Zaver M. Bhujwalla. Hypoxia and HIF silencing mediated dysregulation of total choline, CD44 expression, and metastatic burden in MDA-MB-231 human breast cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 509. doi:10.1158/1538-7445.AM2014-509

Abstract 3745: Validation of the co-expression of breast cancer stem cell markers with HIF-1α in tumors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnStem-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.nnThis work was supported by NIH R01CA136576 and P50 CA103175.nn1. Al-Hajj, M et al., Proc Natl Acad Sci U S A, 2003.nn2. Krishnamachary.B. et al., PLoS One 2012;7(8)e44078-nnCitation 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