A hyperactive Mpl-based cell growth switch drives macrophage-associated erythropoiesis through an erythroid-megakaryocytic precursor.

Several approaches for controlling hematopoietic stem and progenitor cell expansion, lineage commitment, and maturation have been investigated for improving clinical interventions. We report here that amino acid substitutions in a thrombopoietin receptor (Mpl)--containing cell growth switch (CGS) extending receptor stability improve the expansion capacity of human cord blood CD34(+) cells in the absence of exogenous cytokines. Activation of this CGS with a chemical inducer of dimerization (CID) expands total cells 99-fold, erythrocytes 70-fold, megakaryocytes 0.5-fold, and CD34(+) stem/progenitor cells 4.4-fold by 21 days of culture. Analysis of cells in these expanded populations identified a CID-dependent bipotent erythrocyte-megakaryocyte precursor (PEM) population, and a CID-independent macrophage population. The CD235a(+)/CD41a(+) PEM population constitutes up to 13% of the expansion cultures, can differentiate into erythrocytes or megakaryocytes, exhibits very little expansion capacity, and exists at very low levels in unexpanded cord blood. The CD206(+) macrophage population constitutes up to 15% of the expansion cultures, exhibits high-expansion capacity, and is physically associated with differentiating erythroblasts. Taken together, these studies describe a fundamental enhancement of the CGS expansion platform, identify a novel precursor population in the erythroid/megakaryocytic differentiation pathway of humans, and implicate an erythropoietin-independent, macrophage-associated pathway supporting terminal erythropoiesis in this expansion system.

Sparse expression bases in cancer reveal tumor drivers.

We define a new category of candidate tumor drivers in cancer genome evolution: 'selected expression regulators' (SERs)-genes driving dysregulated transcriptional programs in cancer evolution. The SERs are identified from genome-wide tumor expression data with a novel method, namely SPARROW ( SPAR: se selected exp R: essi O: n regulators identified W: ith penalized regression). SPARROW uncovers a previously unknown connection between cancer expression variation and driver events, by using a novel sparse regression technique. Our results indicate that SPARROW is a powerful complementary approach to identify candidate genes containing driver events that are hard to detect from sequence data, due to a large number of passenger mutations and lack of comprehensive sequence information from a sufficiently large number of samples. SERs identified by SPARROW reveal known driver mutations in multiple human cancers, along with known cancer-associated processes and survival-associated genes, better than popular methods for inferring gene expression networks. We demonstrate that when applied to acute myeloid leukemia expression data, SPARROW identifies an apoptotic biomarker (PYCARD) for an investigational drug obatoclax. The PYCARD and obatoclax association is validated in 30 AML patient samples.

Signaling crosstalk between tumor endothelial cells and immune cells in the microenvironment of solid tumors.

Tumor-associated endothelial cells (TECs) are crucial mediators of immune surveillance and immune escape in the tumor microenvironment (TME). TECs driven by angiogenic growth factors form an abnormal vasculature which deploys molecular machinery to selectively promote the function and recruitment of immunosuppressive cells while simultaneously blocking the entry and function of anti-tumor immune cells. TECs also utilize a similar set of signaling regulators to promote the metastasis of tumor cells. Meanwhile, the tumor-infiltrating immune cells further induce the TEC anergy by secreting pro-angiogenic factors and prevents further immune cell penetration into the TME. Understanding the complex interactions between TECs and immune cells will be needed to successfully treat cancer patients with combined therapy to achieve vasculature normalization while augmenting antitumor immunity. In this review, we will discuss what is known about the signaling crosstalk between TECs and tumor-infiltrating immune cells to reveal insights and strategies for therapeutic targeting.

Tumor stromal topography promotes chemoresistance in migrating breast cancer cell clusters.

Multicellular clustering provides cancer cells with survival advantages and facilitates metastasis. At the tumor migration front, cancer cell clusters are surrounded by an aligned stromal topography. It remains unknown whether aligned stromal topography regulates the resistance of migrating cancer cell clusters to therapeutics. Using a hybrid nanopatterned model to characterize breast cancer cell clusters at the migration front with aligned stromal topography, we demonstrate that topography-induced migrating cancer cell clusters exhibit upregulated cytochrome P450 family 1 (CYP1) drug metabolism and downregulated glycolysis gene signatures, which correlates with unfavorable prognosis. Screening on approved oncology drugs shows that cancer cell clusters on aligned stromal topography are more resistant to diverse chemotherapeutics. Full-dose drug testings further indicate that topography induces drug resistance of hormone receptor-positive breast cancer cell clusters to doxorubicin and tamoxifen and triple-negative breast cancer cell clusters to doxorubicin by activating the aryl hydrocarbon receptor (AhR)/CYP1 pathways. Inhibiting the AhR/CYP1 pathway restores reactive oxygen species-mediated drug sensitivity to migrating cancer cell clusters, suggesting a plausible therapeutic direction for preventing metastatic recurrence.

Therapeutic targeting of tumor spheroids in a 3D microphysiological renal cell carcinoma-on-a-chip system.

Metastatic renal cell carcinoma (RCC) remains an incurable disease for most patients highlighting an urgent need for new treatments. However, the preclinical investigation of new therapies is limited by traditional two-dimensional (2D) cultures which do not recapitulate the properties of tumor cells within a collagen extracellular matrix (ECM), while human tumor xenografts are time-consuming, expensive and lack adaptive immune cells. We report a rapid and economical human microphysiological system ("RCC-on-a-chip") to investigate therapies targeting RCC spheroids in a 3D collagen ECM. We first demonstrate that culture of RCC cell lines A498 and RCC4 in a 3D collagen ECM more faithfully reproduces the gene expression program of primary RCC tumors compared to 2D culture. We next used bortezomib as a cytotoxin to develop automated quantification of dose-dependent tumor spheroid killing. We observed that viable RCC spheroids exhibited collective migration within the ECM and demonstrated that our 3D system can be used to identify compounds that inhibit spheroid collective migration without inducing cell death. Finally, we demonstrate the RCC-on-a-chip as a platform to model the trafficking of tumor-reactive T cells into the ECM and observed antigen-specific A498 spheroid killing by engineered human CD8+ T cells expressing an ROR1-specific chimeric antigen receptor. In summary, the phenotypic differences between the 3D versus 2D environments, rapid imaging-based readout, and the ability to carefully study the impact of individual variables with quantitative rigor will encourage adoption of the RCC-on-a-chip system for testing a wide range of emerging therapies for RCC.

Single cell atlas of kidney cancer endothelial cells reveals distinct expression profiles and phenotypes.

Background: Tumor endothelial cells (TECs) represent the primary interface between the tumor microenvironment and circulating immune cells, however their phenotypes are incompletely understood in highly vascularized clear cell renal cell carcinoma (ccRCC). Methods: We purified tumor and matched normal endothelial cells (NECs) from ccRCC specimens and performed single-cell RNA-sequencing to create a reference-quality atlas available as a searchable web resource for gene expression patterns. We established paired primary TECs and NECs cultures for ex vivo functional testing. Results: TECs from multiple donors shared a common phenotype with increased expression of pathways related to extracellular matrix regulation, cell-cell communication, and insulin-like growth factor signaling that was conserved in comparison to hepatocellular carcinoma associated TECs, suggesting convergent TEC phenotypes between unrelated tumors. Cultured TECs stably maintained a core program of differentially regulated genes, were inherently resistant to apoptosis after vascular endothelial growth factor removal and displayed increased adhesiveness to subsets of immune cells including regulatory T-cells. Conclusions: Our studies delineate unique functional and phenotypic properties of TECs, which may provide insights into their interactions with available and emerging therapies. Functional phenotypes of cultured TECs suggest potential mechanisms of resistance to both antiangiogenic and immune-based therapies.

Integrated TCR repertoire analysis and single-cell transcriptomic profiling of tumor-infiltrating T cells in renal cell carcinoma identifies shared and tumor-restricted expanded clones with unique phenotypes.

Objective responses of metastatic renal cell carcinoma (RCC) associated with systemic immunotherapies suggest the potential for T-cell-mediated tumor clearance. Recent analyses associate clonally expanded T cells present in the tumor at diagnosis with responses to immune checkpoint inhibitors (ICIs). To identify and further characterize tumor-associated, clonally expanded T cells, we characterized the density, spatial distribution, T-cell receptor (TCR) repertoire, and transcriptome of tumor-infiltrating T cells from 14 renal tumors at the time of resection and compared them with T cells in peripheral blood and normal adjacent kidney. Multiplex immunohistochemistry revealed that T-cell density was higher in clear cell RCC (ccRCC) than in other renal tumor histologies with spatially nonuniform T-cell hotspots and exclusion zones. TCR repertoire analysis also revealed increased clonal expansion in ccRCC tumors compared with non-clear cell histologies or normal tissues. Expanded T-cell clones were most frequently CD8+ with some detectable in peripheral blood or normal kidney and others found exclusively within the tumor. Divergent expression profiles for chemokine receptors and ligands and the Ki67 proliferation marker distinguished tumor-restricted T-cell clones from those also present in blood suggesting a distinct phenotype for subsets of clonally expanded T cells that also differed for upregulated markers of T-cell activation and exhaustion. Thus, our single-cell level stratification of clonally expanded tumor infiltrating T-cell subpopulations provides a framework for further analysis. Future studies will address the spatial orientation of these clonal subsets within tumors and their association with treatment outcomes for ICIs or other therapeutic modalities.

Current status of antigen-specific T-cell immunotherapy for advanced renal-cell carcinoma.

In renal-cell carcinoma (RCC), tumor-reactive T-cell responses can occur spontaneously or in response to systemic immunotherapy with cytokines and immune checkpoint inhibitors. Cancer vaccines and engineered T-cell therapies are designed to selectively augment tumor antigen-specific CD8+ T-cell responses with the goal to elicit tumor regression and avoid toxicities associated with nonspecific immunotherapies. In this review, we provide an overview of the central role of T-cell immunity in the treatment of advanced RCC. Clinical outcomes for antigen-targeted vaccines or other T-cell-engaging therapies for RCC are summarized and evaluated, and emerging new strategies to enhance the effectiveness of antigen-specific therapy for RCC are discussed.

Evaluating erythropoietin-associated tumor progression using archival tissues from a phase III clinical trial.

Despite the prevalence of anemia in cancer, recombinant erythropoietin (Epo) has declined in use because of recent Phase III trials showing more rapid cancer progression and reduced survival in subjects randomized to Epo. Since Epo receptor (EpoR), Jak2, and Hsp70 are well-characterized mediators of Epo signaling in erythroid cells, we hypothesized that Epo might be especially harmful in patients whose tumors express high levels of these effectors. Because of the insensitivity of immunohistochemistry for detecting low level EpoR protein, we developed assays to measure levels of EpoR, Jak2 and Hsp70 mRNA in formalin-fixed paraffin-embedded (FFPE) tumors. We tested 23 archival breast tumors as well as 136 archival head and neck cancers from ENHANCE, a Phase III trial of 351 patients randomized to Epo versus placebo concomitant with radiotherapy following complete resection, partial resection, or no resection of tumor. EpoR, Jak2, and Hsp70 mRNA levels varied >30-fold, >12-fold, and >13-fold across the breast cancers, and >30-fold, >40-fold, and >30-fold across the head and neck cancers, respectively. Locoregional progression-free survival (LPFS) did not differ among patients whose head and neck cancers expressed above- versus below-median levels of EpoR, Jak2 or Hsp70, except in the subgroup of patients with unresected tumors (n = 28), where above-median EpoR, above-median Jak2, and below-median Hsp70 mRNA levels were all associated with significantly poorer LPFS. Our results provide a framework for exploring the relationship between Epo, cancer progression, and survival using archival tumors from other Phase III clinical trials.

Synthesis of potent, substituted carbazoles as selective androgen receptor modulators (SARMs).

The synthesis and in vitro binding affinity for a novel series of potent androgen receptor modulators is described. One of the more potent compounds (17, RAD35010) was further characterized in vivo where it restored levator ani weight in castrated male rats to near sham level while having no significant effect on prostate weight.

Engineering Microphysiological Immune System Responses on Chips.

Tissues- and organs-on-chips are microphysiological systems (MPSs) that model the architectural and functional complexity of human tissues and organs that is lacking in conventional cell monolayer cultures. While substantial progress has been made in a variety of tissues and organs, chips recapitulating immune responses have not advanced as rapidly. This review discusses recent progress in MPSs for the investigation of immune responses. To illustrate recent developments, we focus on two cases in point: immunocompetent tumor microenvironment-on-a-chip devices that incorporate stromal and immune cell components and pathomimetic modeling of human mucosal immunity and inflammatory crosstalk. More broadly, we discuss the development of systems immunology-on-a-chip devices that integrate microfluidic engineering approaches with high-throughput omics measurements and emerging immunological applications of MPSs.

Integrated epigenomic profiling reveals endogenous retrovirus reactivation in renal cell carcinoma.

BACKGROUND: Transcriptional dysregulation drives cancer formation but the underlying mechanisms are still poorly understood. Renal cell carcinoma (RCC) is the most common malignant kidney tumor which canonically activates the hypoxia-inducible transcription factor (HIF) pathway. Despite intensive study, novel therapeutic strategies to target RCC have been difficult to develop. Since the RCC epigenome is relatively understudied, we sought to elucidate key mechanisms underpinning the tumor phenotype and its clinical behavior. METHODS: We performed genome-wide chromatin accessibility (DNase-seq) and transcriptome profiling (RNA-seq) on paired tumor/normal samples from 3 patients undergoing nephrectomy for removal of RCC. We incorporated publicly available data on HIF binding (ChIP-seq) in a RCC cell line. We performed integrated analyses of these high-resolution, genome-scale datasets together with larger transcriptomic data available through The Cancer Genome Atlas (TCGA). FINDINGS: Though HIF transcription factors play a cardinal role in RCC oncogenesis, we found that numerous transcription factors with a RCC-selective expression pattern also demonstrated evidence of HIF binding near their gene body. Examination of chromatin accessibility profiles revealed that some of these transcription factors influenced the tumor's regulatory landscape, notably the stem cell transcription factor POU5F1 (OCT4). Elevated POU5F1 transcript levels were correlated with advanced tumor stage and poorer overall survival in RCC patients. Unexpectedly, we discovered a HIF-pathway-responsive promoter embedded within a endogenous retroviral long terminal repeat (LTR) element at the transcriptional start site of the PSOR1C3 long non-coding RNA gene upstream of POU5F1. RNA transcripts are induced from this promoter and read through PSOR1C3 into POU5F1 producing a novel POU5F1 transcript isoform. Rather than being unique to the POU5F1 locus, we found that HIF binds to several other transcriptionally active LTR elements genome-wide correlating with broad gene expression changes in RCC. INTERPRETATION: Integrated transcriptomic and epigenomic analysis of matched tumor and normal tissues from even a small number of primary patient samples revealed remarkably convergent shared regulatory landscapes. Several transcription factors appear to act downstream of HIF including the potent stem cell transcription factor POU5F1. Dysregulated expression of POU5F1 is part of a larger pattern of gene expression changes in RCC that may be induced by HIF-dependent reactivation of dormant promoters embedded within endogenous retroviral LTRs.

A consideration of the patentability of enantiomers in the pharmaceutical industry in the United States.

During the last thirty years, concern over stereoselectivity of drug action has drawn a great deal of interest within the pharmaceutical field due to an improved understanding of the pharmacology and pharmacokinetics of enantiomers. Developing single enantiomers versus racemates or introducing a single enantiomer following the development of the racemic mixture appears to be the new trend. The intellectual property status of single enantiomers from racemates may be unclear. Drug discoverers and patent attorneys must examine the examples of the past to establish an appropriate pathway towards the development and intellectual property protection of chiral drugs. The review will focus on the patenting of an enantiomer in view of the prior art disclosure for the racemic mixture.

MicroRNA expression dynamics during murine and human erythroid differentiation.

OBJECTIVE: MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs that regulate diverse cellular functions by sequence-specific inhibition of gene expression. We determined miRNA expression profile during erythroid differentiation and putative roles in erythroid differentiation. METHODS: The expression profile of 295 miRNAs before and after their erythroid differentiation induction was analyzed using microarray. Fluorescein-activated cell sorting analysis was used to isolate mouse spleen erythroblasts at different differentiation stages. Human cord blood CD34+ progenitors were differentiated in vitro. Real-time reverse transcriptase polymerase chain reaction was used to confirm the results of miRNA microarray. Synthetic oligonucleotides for miR-451 overexpression or knockdown were transfected into MEL cells. RESULTS: More than 100 miRNAs were found to be expressed in erythroid cells. The majority of them showed changes in their expression levels with progression of erythroid differentiation. Further analysis revealed that overall miRNA expression levels are increased upon erythroid differentiation. Of the miRNAs analyzed, miR-451 was most significantly upregulated during erythroid maturation. Functional studies using gain of function and loss of function approaches showed that miR-451 is associated with erythroid maturation. CONCLUSIONS: Dynamic changes in miRNA expression occurred during erythroid differentiation, with an overall increase in the levels of miRNAs upon terminal differentiation of erythroid cells. MiR-451 may play a role in promoting erythroid differentiation.

A 3D Human Renal Cell Carcinoma-on-a-Chip for the Study of Tumor Angiogenesis.

Tractable human tissue-engineered 3D models of cancer that enable fine control of tumor growth, metabolism, and reciprocal interactions between different cell types in the tumor microenvironment promise to accelerate cancer research and pharmacologic testing. Progress to date mostly reflects the use of immortalized cancer cell lines, and progression to primary patient-derived tumor cells is needed to realize the full potential of these platforms. For the first time, we report endothelial sprouting induced by primary patient tumor cells in a 3D microfluidic system. Specifically, we have combined primary human clear cell renal cell carcinoma (ccRCC) cells from six independent donors with human endothelial cells in a vascularized, flow-directed, 3D culture system ("ccRCC-on-a-chip"). The upregulation of key angiogenic factors in primary human ccRCC cells, which exhibited unique patterns of donor variation, was further enhanced when they were cultured in 3D clusters. When embedded in the matrix surrounding engineered human vessels, these ccRCC tumor clusters drove potent endothelial cell sprouting under continuous flow, thus recapitulating the critical angiogenic signaling axis between human ccRCC cells and endothelial cells. Importantly, this phenotype was driven by a primary tumor cell-derived biochemical gradient of angiogenic growth factor accumulation that was subject to pharmacological blockade. Our novel 3D system represents a vascularized tumor model that is easy to image and quantify and is fully tunable in terms of input cells, perfusate, and matrices. We envision that this ccRCC-on-a-chip will be valuable for mechanistic studies, for studying tumor-vascular cell interactions, and for developing novel and personalized antitumor therapies.

A machine learning approach to integrate big data for precision medicine in acute myeloid leukemia.

Cancers that appear pathologically similar often respond differently to the same drug regimens. Methods to better match patients to drugs are in high demand. We demonstrate a promising approach to identify robust molecular markers for targeted treatment of acute myeloid leukemia (AML) by introducing: data from 30 AML patients including genome-wide gene expression profiles and in vitro sensitivity to 160 chemotherapy drugs, a computational method to identify reliable gene expression markers for drug sensitivity by incorporating multi-omic prior information relevant to each gene's potential to drive cancer. We show that our method outperforms several state-of-the-art approaches in identifying molecular markers replicated in validation data and predicting drug sensitivity accurately. Finally, we identify SMARCA4 as a marker and driver of sensitivity to topoisomerase II inhibitors, mitoxantrone, and etoposide, in AML by showing that cell lines transduced to have high SMARCA4 expression reveal dramatically increased sensitivity to these agents.

Selective Androgen Receptor Modulator RAD140 Inhibits the Growth of Androgen/Estrogen Receptor-Positive Breast Cancer Models with a Distinct Mechanism of Action.

Purpose: Steroidal androgens suppress androgen receptor and estrogen receptor positive (AR/ER+) breast cancer cells and were used to treat breast cancer, eliciting favorable response. The current study evaluates the activity and efficacy of the oral selective AR modulator RAD140 in in vivo and in vitro models of AR/ER+ breast cancer.Experimental Design: A series of in vitro assays were used to determine the affinity of RAD140 to 4 nuclear receptors and evaluate its tissue-selective AR activity. The efficacy and pharmacodynamics of RAD140 as monotherapy or in combination with palbociclib were evaluated in AR/ER+ breast cancer xenograft models.Results: RAD140 bound AR with high affinity and specificity and activated AR in breast cancer but not prostate cancer cells. Oral administration of RAD140 substantially inhibited the growth of AR/ER+ breast cancer patient-derived xenografts (PDX). Activation of AR and suppression of ER pathway, including the ESR1 gene, were seen with RAD140 treatment. Coadministration of RAD140 and palbociclib showed improved efficacy in the AR/ER+ PDX models. In line with efficacy, a subset of AR-repressed genes associated with DNA replication was suppressed with RAD140 treatment, an effect apparently enhanced by concurrent administration of palbociclib.Conclusions: RAD140 is a potent AR agonist in breast cancer cells with a distinct mechanism of action, including the AR-mediated repression of ESR1 It inhibits the growth of multiple AR/ER+ breast cancer PDX models as a single agent, and in combination with palbociclib. The preclinical data presented here support further clinical investigation of RAD140 in AR/ER+ breast cancer patients. Clin Cancer Res; 23(24); 7608-20. ©2017 AACR.

Bazedoxifene acetate: a selective estrogen receptor modulator with improved selectivity.

We assessed the preclinical characteristics of a novel, stringently screened selective estrogen receptor modulator, bazedoxifene acetate, including its ability to bind to and activate estrogen receptors and promote increased bone mineral density and bone strength in rats, and the effects impacting the uterine endometrium, breast cancer cell proliferation, and central nervous system-associated vasomotor responses in an animal model. Bazedoxifene bound to estrogen receptor-alpha with an IC50 of 26 nm, an affinity similar to that of raloxifene. Bazedoxifene did not stimulate proliferation of MCF-7 cells but did inhibit 17beta-estradiol-induced proliferation with an IC50 of 0.19 nm. In an immature rat uterine model, bazedoxifene (0.5 and 5.0 mg/kg) was associated with less increase in uterine wet weight than either ethinyl estradiol (10 microg/kg) or raloxifene (0.5 and 5.0 mg/kg). Histological analysis revealed that coadministration of bazedoxifene also appeared to reduce raloxifene-stimulated endometrial luminal epithelial cell and myometrial cell hypertrophy. In ovariectomized rats, bazedoxifene was associated with significant increases in bone mineral density at 6 wk, compared with control, and better compressive strength of bone samples from the L4 vertebrae, compared with samples from ovariectomized animals. In the morphine-addicted rat model of vasomotor activity, bone-sparing doses of bazedoxifene alone were not associated with 17beta-estradiol inhibition of increased vasomotor activity. Bazedoxifene acetate represents a promising new treatment for osteoporosis, with a potential for less uterine and vasomotor effects than selective estrogen receptor modulators currently used in clinical practice. Controlled clinical trial data will be needed to confirm these effects.

The proteomic landscape of triple-negative breast cancer.

Triple-negative breast cancer is a heterogeneous disease characterized by poor clinical outcomes and a shortage of targeted treatment options. To discover molecular features of triple-negative breast cancer, we performed quantitative proteomics analysis of twenty human-derived breast cell lines and four primary breast tumors to a depth of more than 12,000 distinct proteins. We used this data to identify breast cancer subtypes at the protein level and demonstrate the precise quantification of biomarkers, signaling proteins, and biological pathways by mass spectrometry. We integrated proteomics data with exome sequence resources to identify genomic aberrations that affect protein expression. We performed a high-throughput drug screen to identify protein markers of drug sensitivity and understand the mechanisms of drug resistance. The genome and proteome provide complementary information that, when combined, yield a powerful engine for therapeutic discovery. This resource is available to the cancer research community to catalyze further analysis and investigation.

Estrogen receptors alpha and beta have similar activities in multiple endothelial cell pathways.

The presence of both estrogen receptor alpha (ERalpha) and ERbeta in vascular cells has greatly increased the complexity of potential estrogen regulatory pathways in the cardiovascular system. Here, human umbilical vein endothelial cells were engineered using adenovirus vectors to express either ERalpha or ERbeta. The activities of ERalpha and ERbeta were compared in three distinct gene regulatory pathways, including inhibition of IL-1beta induction of E-selectin expression, inhibition of basal endothelin-1 production, and the ability to induce two matrix-stabilizing enzymes: tissue transglutaminase and a novel member of the lysyl oxidase family. Both ERs were active on these end points, although ERbeta was typically less efficacious than ERalpha. As no class of gene regulation could differentiate ERalpha from ERbeta activity, we characterized a novel steroid (7alpha-thiophenyl-E2) that bound with similar affinities to ERalpha and ERbeta, but functioned as an ERalpha agonist and ERbeta antagonist for all of these endothelial responses. This pattern of receptor subtype-selective activity was not unique to endothelial cells, but was also seen in metabolically active HepG2 cells, suggesting potential in vivo utility. The panel of endothelial responses coupled with a selective modulator should provide a means to characterize the roles of ERalpha and ERbeta in endothelial cells in vivo.