Delisha A. Stewart

Interactions with Fibroblasts are Distinct in Basal-like and Luminal Breast Cancers

Basal-like breast cancers have several well-characterized distinguishing molecular features, but most of these are features of the cancer cells themselves. The unique stromal–epithelial interactions, and more generally, microenvironmental features of basal-like breast cancers have not been well characterized. To identify characteristic microenvironment features of basal-like breast cancer, we performed cocultures of several basal-like breast cancer cell lines with fibroblasts and compared these with cocultures of luminal breast cancer cell lines with fibroblasts. Interactions between basal-like cancer cells and fibroblasts induced expression of numerous interleukins and chemokines, including IL-6, IL-8, CXCL1, CXCL3, and TGFβ. Under the influence of fibroblasts, basal-like breast cancer cell lines also showed increased migration in vitro. Migration was less pronounced for luminal lines; but, these lines were more likely to have altered proliferation. These differences were relevant to tumor biology in vivo, as the gene set that distinguished luminal and basal-like stromal interactions in coculture also distinguishes basal-like from luminal tumors with 98% accuracy in 10-fold cross-validation and 100% accuracy in an independent test set. However, comparisons between cocultures where cells were in direct contact and cocultures where interaction was solely through soluble factors suggest that there is an important impact of direct cell-to-cell contact. The phenotypes and gene expression changes invoked by cancer cell interactions with fibroblasts support the microenvironment and cell–cell interactions as intrinsic features of breast cancer subtypes. Mol Cancer Res; 9(1); 3–13 ©2010 AACR.

Basal-like Breast Cancer Cells Induce Phenotypic and Genomic Changes in Macrophages

Basal-like breast cancer (BBC) is an aggressive subtype of breast cancer that has no biologically targeted therapy. The interactions of BBCs with stromal cells are important determinants of tumor biology, with inflammatory cells playing well-recognized roles in cancer progression. Despite the fact that macrophage–BBC communication is bidirectional, important questions remain about how BBCs affect adjacent immune cells. This study investigated monocyte-to-macrophage differentiation and polarization and gene expression in response to coculture with basal-like versus luminal breast cancer cells. Changes induced by coculture were compared with changes observed under classical differentiation and polarization conditions. Monocytes (THP-1 cells) exposed to BBC cells in coculture had altered gene expression with upregulation of both M1 and M2 macrophage markers. Two sets of M1 and M2 markers were selected from the PCR profiles and used for dual immunofluorescent staining of BBC versus luminal cocultured THP-1s, and cancer-adjacent, benign tissue sections from patients diagnosed with BBCs or luminal breast cancer, confirming the differential expression patterns. Relative to luminal breast cancers, BBCs also increased differentiation of monocytes to macrophages and stimulated macrophage migration. Consistent with these changes in cellular phenotype, a distinct pattern of cytokine secretion was evident in macrophage–BBC cocultures, including upregulation of NAP-2, osteoprotegerin, MIG, MCP-1, MCP-3, and interleukin (IL)-1β. Application of IL-1 receptor antagonist (IL-1RA) to cocultures attenuated BBC-induced macrophage migration. These data contribute to an understanding of the BBC-mediated activation of the stromal immune response, implicating specific cytokines that are differentially expressed in basal-like microenvironments and suggesting plausible targets for modulating immune responses to BBCs. Mol Cancer Res; 10(6); 727–38. ©2012 AACR.

Age-Associated Gene Expression in Normal Breast Tissue Mirrors Qualitative Age-at-Incidence Patterns for Breast Cancer

Background: Age is the strongest breast cancer risk factor, with overall breast cancer risk increasing steadily beginning at approximately 30 years of age. However, while breast cancer risk is lower among younger women, young womens breast cancer may be more aggressive. Although, several genomic and epidemiologic studies have shown higher prevalence of aggressive, estrogen-receptor negative breast cancer in younger women, the age-related gene expression that predisposes to these tumors is poorly understood. Characterizing age-related patterns of gene expression in normal breast tissues may provide insights on etiology of distinct breast cancer subtypes that arise from these tissues. Methods: To identify age-related changes in normal breast tissue, 96 tissue specimens from patients with reduction mammoplasty, ages 14 to 70 years, were assayed by gene expression microarray. Results: Significant associations between gene expression levels and age were identified for 802 probes (481 increased, 321 decreased with increasing age). Enriched functions included “aging of cells,” “shape change,” and “chemotaxis,” and enriched pathways included Wnt/beta-catenin signaling, Ephrin receptor signaling, and JAK/Stat signaling. Applying the age-associated genes to publicly available tumor datasets, the age-associated pathways defined two groups of tumors with distinct survival. Conclusion: The hazard rates of young-like tumors mirrored that of high-grade tumors in the Surveillance, Epidemiology, and End Results Program, providing a biologic link between normal aging and age-related tumor aggressiveness. Impact: These data show that studies of normal tissue gene expression can yield important insights about the pathways and biologic pressures that are relevant during tumor etiology and progression. Cancer Epidemiol Biomarkers Prev; 21(10); 1735–44. ©2012 AACR.

Paracrine interactions between primary human macrophages and human fibroblasts enhance murine mammary gland humanization in vivo

IntroductionMacrophages comprise an essential component of the mammary microenvironment necessary for normal gland development. However, there is no viable in vivo model to study their role in normal human breast function. We hypothesized that adding primary human macrophages to the murine mammary gland would enhance and provide a novel approach to examine immune-stromal cell interactions during the humanization process.MethodsPrimary human macrophages, in the presence or absence of ectopic estrogen stimulation, were used to humanize mouse mammary glands. Mechanisms of enhanced humanization were identified by cytokine/chemokine ELISAs, zymography, western analysis, invasion and proliferation assays; results were confirmed with immunohistological analysis.ResultsThe combined treatment of macrophages and estrogen stimulation significantly enhanced the percentage of the total gland humanized and the engraftment/outgrowth success rate. Timecourse analysis revealed the disappearance of the human macrophages by two weeks post-injection, suggesting that the improved overall growth and invasiveness of the fibroblasts provided a larger stromal bed for epithelial cell proliferation and structure formation. Confirming their promotion of fibroblasts humanization, estrogen-stimulated macrophages significantly enhanced fibroblast proliferation and invasion in vitro, as well as significantly increased proliferating cell nuclear antigen (PCNA) positive cells in humanized glands. Cytokine/chemokine ELISAs, zymography and western analyses identified TNFα and MMP9 as potential mechanisms by which estrogen-stimulated macrophages enhanced humanization. Specific inhibitors to TNFα and MMP9 validated the effects of these molecules on fibroblast behavior in vitro, as well as by immunohistochemical analysis of humanized glands for human-specific MMP9 expression. Lastly, glands humanized with macrophages had enhanced engraftment and tumor growth compared to glands humanized with fibroblasts alone.ConclusionsHerein, we demonstrate intricate immune and stromal cell paracrine interactions in a humanized in vivo model system. We confirmed our in vivo results with in vitro analyses, highlighting the value of this model to interchangeably substantiate in vitro and in vivo results. It is critical to understand the signaling networks that drive paracrine cell interactions, for tumor cells exploit these signaling mechanisms to support their growth and invasive properties. This report presents a dynamic in vivo model to study primary human immune/fibroblast/epithelial interactions and to advance our knowledge of the stromal-derived signals that promote tumorigenesis.

Metabolomics Analysis of Hormone-Responsive and Triple-Negative Breast Cancer Cell Responses to Paclitaxel Identify Key Metabolic Differences

To date, no targeted therapies are available to treat triple negative breast cancer (TNBC), while other breast cancer subtypes are responsive to current therapeutic treatment. Metabolomics was conducted to reveal differences in two hormone receptor-negative TNBC cell lines and two hormone receptor-positive Luminal A cell lines. Studies were conducted in the presence and absence of paclitaxel (Taxol). TNBC cell lines had higher levels of amino acids, branched-chain amino acids, nucleotides, and nucleotide sugars and lower levels of proliferation-related metabolites like choline compared with Luminal A cell lines. In the presence of paclitaxel, each cell line showed unique metabolic responses, with some similarities by type. For example, in the Luminal A cell lines, levels of lactate and creatine decreased while certain choline metabolites and myo-inositol increased with paclitaxel. In the TNBC cell lines levels of glutamine, glutamate, and glutathione increased, whereas lysine, proline, and valine decreased in the presence of drug. Profiling secreted inflammatory cytokines in the conditioned media demonstrated a greater response to paclitaxel in the hormone-positive Luminal cells compared with a secretion profile that suggested greater drug resistance in the TNBC cells. The most significant differences distinguishing the cell types based on pathway enrichment analyses were related to amino acid, lipid and carbohydrate metabolism pathways, whereas several biological pathways were differentiated between the cell lines following treatment.

Omics Technologies Used in Systems Biology

Abstract In this chapter, we provide information about a number of the leading and innovative “omics” approaches used in various disciplines including biology, environmental health sciences, and toxicological research. This chapter is divided into sections that cover technologies that enable genomics, transcriptomics, proteomics, and metabolomics-based approaches. Within each section we cover aspects of sample handling and the types of molecular and analytical methods used in capturing and preprocessing data. We briefly discuss data analysis as Chapter 5 covers data analysis in more detail. With changes in technology occurring rapidly, each section provides information that can be used as a starting point for further investigation.

Correlated metabolomic, genomic, and histologic phenotypes in histologically normal breast tissue

Breast carcinogenesis is a multistep process accompanied by widespread molecular and genomic alterations, both in tumor and in surrounding microenvironment. It is known that tumors have altered metabolism, but the metabolic changes in normal or cancer-adjacent, nonmalignant normal tissues and how these changes relate to alterations in gene expression and histological composition are not well understood. Normal or cancer-adjacent normal breast tissues from 99 women of the Normal Breast Study (NBS) were evaluated. Data of metabolomics, gene expression and histological composition was collected by mass spectrometry, whole genome microarray, and digital image, respectively. Unsupervised clustering analysis determined metabolomics-derived subtypes. Their association with genomic and histological features, as well as other breast cancer risk factors, genomic and histological features were evaluated using logistic regression. Unsupervised clustering of metabolites resulted in two main clusters. The metabolite differences between the two clusters suggested enrichment of pathways involved in lipid metabolism, cell growth and proliferation, and migration. Compared with Cluster 1, subjects in Cluster 2 were more likely to be obese (body mass index ≥30 kg/m2, p<0.05), have increased adipose proportion (p<0.01) and associated with a previously defined Active genomic subtype (p<0.01). By the integrated analyses of histological, metabolomics and transcriptional data, we characterized two distinct subtypes of non-malignant breast tissue. Further research is needed to validate our findings, and understand the potential role of these alternations in breast cancer initiation, progression and recurrence.

Stable Isotope-Resolved Metabolomic Differences between Hormone-Responsive and Triple-Negative Breast Cancer Cell Lines

Purpose To conduct an exploratory study to identify mechanisms that differentiate Luminal A (BT474 and MCF-7) and triple-negative (MDA-MB-231 and MDA-MB-468) breast cancer (BCa) cell lines to potentially provide novel therapeutic targets based on differences in energy utilization. Methods Cells were cultured in media containing either [U-13C]-glucose or [U-13C]-glutamine for 48 hours. Conditioned media and cellular extracts were analyzed by 1H and 13C NMR spectroscopy. Results MCF-7 cells consumed the most glucose, producing the most lactate, demonstrating the greatest Warburg effect-associated energy utilization. BT474 cells had the highest tricarboxylic acid cycle (TCA) activity. The majority of energy utilization patterns in MCF-7 cells were more similar to MDA-MB-468 cells, while the patterns for BT474 cells were more similar to MDA-MB-231 cells. Compared to the Luminal A cell lines, TNBC cell lines consumed more glutamine and less glucose. BT474 and MDA-MB-468 cells produced high amounts of 13C-glycine from media [U-13C]-glucose which was integrated into glutathione, indicating de novo synthesis. Conclusions Stable isotopic resolved metabolomics using 13C substrates provided mechanistic information about energy utilization that was difficult to interpret using 1H data alone. Overall, cell lines that have different hormone receptor status have different energy utilization requirements, even if they are classified by the same clinical BCa subtype; and these differences offer clues about optimizing treatment strategies.

Abstract 3603: Metabolic profiles distinguish breast cancer progression in African American women

Breast cancer (BCa) is one of the most common malignancies in women and the incidence, distribution, clinical outcome and mortality rates vary widely among women of different ethnic backgrounds. Because tissue from African American (AA) women is difficult to obtain for biomarker studies, new population-relevant biomarkers that enable earlier detection and novel therapeutic intervention development are critical. To identify new biomarkers and targets that have the potential to be leveraged for earlier detection, classification of disease progression and development of improved therapeutics for AA patients, plasma and tissue samples were selected from two AA BCa case and control cohorts at the Tissue, Plasma and Clinical Bank at the Howard University Cancer Center (HUCC). Samples were from women either diagnosed with BCa, screened for potential BCa lesions or undergoing reduction mammoplasty surgery at both Howard University and Providence Hospitals. Samples were analyzed by untargeted metabolomics using 1 H nuclear magnetic resonance (NMR) spectroscopy. Multivariate and statistical analyses determined bins important to differentiating BCa by progressive stage and grade from control reduction mammoplasty tissues and fibrocystic fibroadenoma. Significant bins were library-matched to identify corresponding metabolites and distinguish common and unique metabolites between tissue groups and compare tissue profiles to plasma samples. Metabolites from each study group were also correlated with other known clinicopathologic BCa risk factors, including age, BMI, and smoking status, to determine their influence on disease progression. Several metabolites were found to distinguish nonmalignant reduction mammoplasty tissues from fibrocystic fibroadenomas, and from Grade (G) I-II estrogen receptor (ER)-positive or GI-II ER-negative tumors and GIII ER-positive or GIII ER-negative tumors. For example, in three comparisons using orthogonal partial least squares discriminant analyses (OPLS-DA) between reduction mammoplasty, fibrocystic fibroadenomas and the malignant tissues, we found 8 unique metabolites when comparing reduction mammoplasty versus fibrocystic fibroadenomas (4-hydroxybenzoate, dimethylamine, formate, glutamine, glutathione, histidine, methionine and UDP-N-acetylglucosamine); 2 unique metabolites comparing reduction mammoplasty versus GI-II (ER-positive and -negative) tumors (2-phenylpropionate and succinate); and 6 unique metabolites comparing reduction mammoplasty versus GIII (ethanolamine, glycine, hypoxanthine, maltose, sucrose and uridine). Our results demonstrate the continued usefulness of metabolomics-based research and the potential for these findings to identify early detection or disease staging biomarkers in a population that experiences a disparate burden of this disease. Citation Format: Delisha A. Stewart, Wimal W. Pathmasiri, Susan L. McRitchie, Lance Buckley, Tammey J. Naab, Robert L. DeWitty, Vikisha T. Fripp, Estelle Cooke-Sampson, Desta A. Beyene, Luisel Ricks-Santi, Robert L. Copeland, Susan J. Sumner, Yasmine M. Kanaan. Metabolic profiles distinguish breast cancer progression in African American women [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3603.

Abstract 502: Characterizing breast cancer subtype-specific responses to macrophages.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Breast cancer is a multi-faceted disease giving rise to five distinct subtypes. Emerging data show that each of these subtypes has distinct interactions with the surrounding stroma and immune cell populations present in the microenvironment. We hypothesized that macrophages have distinct interactions with breast cancer in a subtype-dependent manner. Using a coculture system, we evaluated changes in THP-1 differentiation, morphology and gene expression during the monocyte-to-macrophage transition in response to cell lines representing the basal-like (HCC1937, MDA-MB-468, SUM149), luminal (MCF-7, T47D, ZR-75-1), epidermal growth factor receptor/HER2-enriched (BT474, SKBR3) and claudin-low (Hs578T, MDA-MB-231, SUM159) breast cancer subtypes. Macrophage polarization phenotypes were validated by immunofluorescence staining for M1 (CD163) and M2 (CD36) macrophage markers in normal tissue sections adjacent to representative breast tumors. We further investigated differences in genomic and cytokine expression profiles, proliferation, and chemosensitivity (to Doxorubicin) of the breast cancer cell lines in response to THP-1 coculturing. Greatest levels of THP-1 differentiation was induced in basal-like cocultures (mean differentiation 79%, compared to differentiation <42% for all other cell lines). Additionally, all basal-like breast cancers strongly induced macrophage polarization to a mixed M1 and M2 phenotype, however, one cell line from both the claudin-low (SUM159) and HER2-enriched (SKBR3) subtypes also elicited this response. The impact on THP-1 differentiation was clearly visualized by morphological changes after 48 hours of coculturing with these cell lines. Similar macrophage polarization phenotypes were predominantly observed in the triple negative breast cancer, normal-adjacent human tissues. Basal-like breast cancers underwent equally dramatic gene expression changes in response to macrophages compared to luminal cell lines, and demonstrated a more highly differential milieu of secreted cytokines. Ingenuity Pathway Analysis (IPA) revealed significant upregulation of DNA damage and repair pathways in the basal-like subtype under coculture conditions. Interestingly, luminal breast cancer cell lines exhibited increased proliferation as a result of macrophage interaction, while the growth of all basal-like lines was diminished in the presence of THP-1 macrophages. Consistent with alterations in DNA damage response, basal-like breast cancers (SUM149) displayed reduced chemosensitivity after coculturing. These results define subtype-specific interactions between breast cancers and macrophages, and suggest a possible mechanism by which macrophages may promote chemoresistance in more aggressive basal-like breast cancers. Citation Format: Delisha A. Stewart, Yinmeng Yang, Xuezheng Sun, Liza Makowski, Kristen Brantley, Jean Gowen Cook, Melissa A. Troester. Characterizing breast cancer subtype-specific responses to macrophages. [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 502. doi:10.1158/1538-7445.AM2013-502