Patricia Casbas-Hernandez

Gene expression in extratumoral microenvironment predicts clinical outcome in breast cancer patients

IntroductionA gene expression signature indicative of activated wound responses is common to more than 90% of non-neoplastic tissues adjacent to breast cancer, but these tissues also exhibit substantial heterogeneity. We hypothesized that gene expression subtypes of breast cancer microenvironment can be defined and that these microenvironment subtypes have clinical relevance.MethodsGene expression was evaluated in 72 patient-derived breast tissue samples adjacent to invasive breast cancer or ductal carcinoma in situ. Unsupervised clustering identified two distinct gene expression subgroups that differed in expression of genes involved in activation of fibrosis, cellular movement, cell adhesion and cell-cell contact. We evaluated the prognostic relevance of extratumoral subtype (comparing the Active group, defined by high expression of fibrosis and cellular movement genes, to the Inactive group, defined by high expression of claudins and other cellular adhesion and cell-cell contact genes) using clinical data. To establish the biological characteristics of these subtypes, gene expression profiles were compared against published and novel tumor and tumor stroma-derived signatures (Twist-related protein 1 (TWIST1) overexpression, transforming growth factor beta (TGF-β)-induced fibroblast activation, breast fibrosis, claudin-low tumor subtype and estrogen response). Histological and immunohistochemical analyses of tissues representing each microenvironment subtype were performed to evaluate protein expression and compositional differences between microenvironment subtypes.ResultsExtratumoral Active versus Inactive subtypes were not significantly associated with overall survival among all patients (hazard ratio (HR) = 1.4, 95% CI 0.6 to 2.8, P = 0.337), but there was a strong association with overall survival among estrogen receptor (ER) positive patients (HR = 2.5, 95% CI 0.9 to 6.7, P = 0.062) and hormone-treated patients (HR = 2.6, 95% CI 1.0 to 7.0, P = 0.045). The Active subtype of breast microenvironment is correlated with TWIST-overexpression signatures and shares features of claudin-low breast cancers. The Active subtype was also associated with expression of TGF-β induced fibroblast activation signatures, but there was no significant association between Active/Inactive microenvironment and desmoid type fibrosis or estrogen response gene expression signatures. Consistent with the RNA expression profiles, Active cancer-adjacent tissues exhibited higher density of TWIST nuclear staining, predominantly in epithelium, and no evidence of increased fibrosis.ConclusionsThese results document the presence of two distinct subtypes of microenvironment, with Active versus Inactive cancer-adjacent extratumoral microenvironment influencing the aggressiveness and outcome of ER-positive human breast cancers.

Impact of Tumor Microenvironment and Epithelial Phenotypes on Metabolism in Breast Cancer

Purpose: Cancer cells have altered metabolism, with increased glucose uptake, glycolysis, and biomass production. This study conducted genomic and metabolomic analyses to elucidate how tumor and stromal genomic characteristics influence tumor metabolism. Experimental Design: Thirty-three breast tumors and six normal breast tissues were analyzed by gene expression microarray and by mass spectrometry for metabolites. Gene expression data and clinical characteristics were evaluated in association with metabolic phenotype. To evaluate the role of stromal interactions in altered metabolism, cocultures were conducted using breast cancer cells and primary cancer-associated fibroblasts (CAF). Results: Across all metabolites, unsupervised clustering resulted in two main sample clusters. Normal breast tissue and a subset of tumors with less aggressive clinical characteristics had lower levels of nucleic and amino acids and glycolysis byproducts, whereas more aggressive tumors had higher levels of these Warburg-associated metabolites. While tumor-intrinsic subtype did not predict metabolic phenotype, metabolic cluster was significantly associated with expression of a wound response signature. In cocultures, CAFs from basal-like breast cancers increased glucose uptake and basal-like epithelial cells increased glucose oxidation and glycogen synthesis, suggesting interplay of stromal and epithelial phenotypes on metabolism. Cytokine arrays identified hepatocyte growth factor (HGF) as a potential mediator of stromal–epithelial interaction and antibody neutralization of HGF resulted in reduced expression of glucose transporter 1 (GLUT1) and decreased glucose uptake by epithelium. Conclusions: Both tumor/epithelial and stromal characteristics play important roles in metabolism. Warburg-like metabolism is influenced by changes in stromal–epithelial interactions, including altered expression of HGF/Met pathway and GLUT1 expression. Clin Cancer Res; 19(3); 571–85. ©2012 AACR.

Gene expression analysis of in vitro cocultures to study interactions between breast epithelium and stroma

The interactions between breast epithelium and stroma are fundamental to normal tissue homeostasis and for tumor initiation and progression. Gene expression studies of in vitro coculture models demonstrate that in vitro models have relevance for tumor progression in vivo. For example, stromal gene expression has been shown to vary in association with tumor subtype in vivo, and analogous in vitro cocultures recapitulate subtype-specific biological interactions. Cocultures can be used to study cancer cell interactions with specific stromal components (e.g., immune cells, fibroblasts, endothelium) and different representative cell lines (e.g., cancer-associated versus normal-associated fibroblasts versus established, immortalized fibroblasts) can help elucidate the role of stromal variation in tumor phenotypes. Gene expression data can also be combined with cell-based assays to identify cellular phenotypes associated with gene expression changes. Coculture systems are manipulable systems that can yield important insights about cell-cell interactions and the cellular phenotypes that occur as tumor and stroma co-evolve.

Role of HGF in epithelial–stromal cell interactions during progression from benign breast disease to ductal carcinoma in situ

IntroductionBasal-like and luminal breast cancers have distinct stromal–epithelial interactions, which play a role in progression to invasive cancer. However, little is known about how stromal–epithelial interactions evolve in benign and pre-invasive lesions.MethodsTo study epithelial–stromal interactions in basal-like breast cancer progression, we cocultured reduction mammoplasty fibroblasts with the isogenic MCF10 series of cell lines (representing benign/normal, atypical hyperplasia, and ductal carcinoma in situ). We used gene expression microarrays to identify pathways induced by coculture in premalignant cells (MCF10DCIS) compared with normal and benign cells (MCF10A and MCF10AT1). Relevant pathways were then evaluated in vivo for associations with basal-like subtype and were targeted in vitro to evaluate effects on morphogenesis.ResultsOur results show that premalignant MCF10DCIS cells express characteristic gene expression patterns of invasive basal-like microenvironments. Furthermore, while hepatocyte growth factor (HGF) secretion is upregulated (relative to normal, MCF10A levels) when fibroblasts are cocultured with either atypical (MCF10AT1) or premalignant (MCF10DCIS) cells, only MCF10DCIS cells upregulated the HGF receptor MET. In three-dimensional cultures, upregulation of HGF/MET in MCF10DCIS cells induced morphological changes suggestive of invasive potential, and these changes were reversed by antibody-based blocking of HGF signaling. These results are relevant to in vivo progression because high expression of a novel MCF10DCIS-derived HGF signature was correlated with the basal-like subtype, with approximately 86% of basal-like cancers highly expressing the HGF signature, and because high expression of HGF signature was associated with poor survival.ConclusionsCoordinated and complementary changes in HGF/MET expression occur in epithelium and stroma during progression of pre-invasive basal-like lesions. These results suggest that targeting stroma-derived HGF signaling in early carcinogenesis may block progression of basal-like precursor lesions.

Tumor Intrinsic Subtype Is Reflected in Cancer-Adjacent Tissue

Introduction: Overall survival of early-stage breast cancer patients is similar for those who undergo breast-conserving therapy (BCT) and mastectomy; however, 10% to 15% of women undergoing BCT suffer ipsilateral breast tumor recurrence. The risk of recurrence may vary with breast cancer subtype. Understanding the gene expression of the cancer-adjacent tissue and the stromal response to specific tumor subtypes is important for developing clinical strategies to reduce recurrence risk. Methods: We utilized two independent datasets to study gene expression data in cancer-adjacent tissue from invasive breast cancer patients. Complementary in vitro cocultures were used to study cell–cell communication between fibroblasts and specific breast cancer subtypes. Results: Our results suggest that intrinsic tumor subtypes are reflected in histologically normal cancer-adjacent tissue. Gene expression of cancer-adjacent tissues shows that triple-negative (Claudin-low or basal-like) tumors exhibit increased expression of genes involved in inflammation and immune response. Although such changes could reflect distinct immune populations present in the microenvironment, altered immune response gene expression was also observed in cocultures in the absence of immune cell infiltrates, emphasizing that these inflammatory mediators are secreted by breast-specific cells. In addition, although triple-negative breast cancers are associated with upregulated immune response genes, luminal breast cancers are more commonly associated with estrogen-response pathways in adjacent tissues. Conclusions: Specific characteristics of breast cancers are reflected in the surrounding histologically normal tissue. This commonality between tumor and cancer-adjacent tissue may underlie second primaries and local recurrences. Impact: Biomarkers derived from cancer-adjacent tissue may be helpful in defining personalized surgical strategies or in predicting recurrence risk. Cancer Epidemiol Biomarkers Prev; 24(2); 406–14. ©2014 AACR.

Probing biological nanotopology via diffusion of weakly constrained plasmonic nanorods with optical coherence tomography

Significance Many diseases are characterized by nanostructural changes in connective fibers and soluble proteins, which can indicate or drive disease progression. Noninvasive methods sensitive to nanotopological changes in 3D tissue models can elucidate biophysical changes associated with disease progression. Nanoparticles probe their environment via their diffusion, which is impacted by the size and connectivity of pores into which they freely diffuse. Here, we show that optical coherence tomography provides depth-resolved imaging of gold nanorods (GNRs) to infer local biological nanotopology. We demonstrate the broad potential of this method by sensing changes in diffusion of GNRs in 3D models of mammary ECM and pulmonary mucus. Biological materials exhibit complex nanotopology, i.e., a composite liquid and solid phase structure that is heterogeneous on the nanoscale. The diffusion of nanoparticles in nanotopological environments can elucidate biophysical changes associated with pathogenesis and disease progression. However, there is a lack of methods that characterize nanoprobe diffusion and translate easily to in vivo studies. Here, we demonstrate a method based on optical coherence tomography (OCT) to depth-resolve diffusion of plasmon-resonant gold nanorods (GNRs) that are weakly constrained by the biological tissue. By using GNRs that are on the size scale of the polymeric mesh, their Brownian motion is minimally hindered by intermittent collisions with local macromolecules. OCT depth-resolves the particle-averaged translational diffusion coefficient (DT) of GNRs within each coherence volume, which is separable from the nonequilibrium motile activities of cells based on the unique polarized light-scattering properties of GNRs. We show how this enables minimally invasive imaging and monitoring of nanotopological changes in a variety of biological models, including extracellular matrix (ECM) remodeling as relevant to carcinogenesis, and dehydration of pulmonary mucus as relevant to cystic fibrosis. In 3D ECM models, DT of GNRs decreases with both increasing collagen concentration and cell density. Similarly, DT of GNRs is sensitive to human bronchial-epithelial mucus concentration over a physiologically relevant range. This novel method comprises a broad-based platform for studying heterogeneous nanotopology, as distinct from bulk viscoelasticity, in biological milieu.

Temporal Trends in the Inflammatory Cytokine Profile of Human Breastmilk

A longer lifetime duration of breastfeeding may decrease the risk of breast cancer by reducing breast inflammation and mitigating inflammatory cytokine expression during postlactational involution. However, little is known about how the inflammatory cytokine profile in human breastmilk changes over time. To study temporal trends in breastmilk cytokine expression, we measured 80 human cytokines in the whey fraction of breastmilk samples from 15 mothers at 1, 4, 8, and 12 weeks postpartum. We used mixed models to identify temporal changes in cytokine expression and investigated parity status (multiparous vs. primiparous) as a potential confounder. Nine cytokines (monocyte chemoattractant protein-1, epithelial-derived neutrophil-activating protein-78, hepatocyte growth factor, insulin-like growth factor-binding protein-1, interleukin-16, interleukin-8, macrophage colony-stimulating factor, osteoprotegerin, and tissue inhibitor of metallopeptidase-2) had significantly decreased expression with increasing breastfeeding duration; all nine have known roles in breast involution, inflammation, and cancer and may serve as biomarkers of changing breast microenvironment. No cytokine significantly increased in level over the study period. Total protein concentration significantly decreased over time (p<0.0001), which may mediate the association between length of breastfeeding and inflammatory cytokine expression. Parity status did not confound temporal trends, but levels of several cytokines were significantly higher among multiparous versus primiparous women. Our results suggest that inflammatory cytokine expression during lactation is dynamic, and expressed milk may provide a noninvasive window into the extensive biological changes that occur in the postpartum breast.

OCT-based quantification of the effect of a drug on the motility of mammary organoids

We quantified the effect of doxorubicin on the motility of mammary epithelial cells in 3D cultures by Optical Coherence Tomography. The measured cellular motility decreased in a time-dependent fashion after exposure to doxorubicin.

Abstract 4530: Nanoparticle formulation of KU55933 as a potent radiosensitizer.

KU55933 is a potent and specific inhibitor of the Ataxia Telangiectasia Mutated (ATM) protein. Given ATM9s critical role in DNA damage repair, specifically double-strand break repair, KU55933 holds high potential as a radiosensitizer. Indeed, previous preclinical studies have demonstrated the radiosentization effect of KU55933. However, clinical translation of KU55933 as a radiosensitizer has been prevented due to concerns of its potential toxicity. Systemic administration of KU55933 would sensitize normal cells as well as cancer cells to the effects of radiotherapy. Such sensitization would not improve the therapeutic index of radiotherapy. Therefore, the key challenge in the translation of KU55933 is to identify methods that can selectively deliver KU55933 to tumors while minimizing normal tissue dose. While traditional drug delivery methods cannot overcome this challenge, the development of nanoparticle (NP) drug delivery vehicles offers a unique opportunity. NPs are known to preferentially accumulate in tumors while provide low drug dose to nearby normal tissues. We hypothesize that NP delivery of KU55933 can not only improve the therapeutic efficacy of KU55933, but also lower its toxicity. In this study, we engineered a NP formulation of KU55933 using a biodegradable lipid-polymer NP. We then compared the radiosensitization effect of NP KU55933 to that of KU55933 in vitro using the non-small cell lung cancer (NSCLC) cell lines H23, A549 and H460. Clonogenic survival assays showed that NP KU55933 is a more potent radiosensitizer than KU55933. The differential radiosensitization effect of both KU55933 formulations was also evaluated in a xenograft mouse model of NSCLC. We validated our in vitro results by demonstrating that NP KU55933 is more effective than KU55933 as radiosensitizer in flank xenograft models of NSCLC. To compare the toxicity of NP KU55933 and KU55933, we determined the skin toxicity after radiotherapy with the two drug formulations. We found lower skin toxicity in mice treated with the NP KU55933 than that for KU55933. In conclusion, our studies demonstrate that NP formulation of KU55933 can enhance the radiosensitization effect and decreases the toxicity in vitro and in vivo. Our results suggest that NP KU55933 holds potential for clinical translation. Citation Format: Haydee Lara, Michael B. Foote, Edina C. Wang, Manish Sethi, Patricia Casbas-Hernandez, Andrew Z. Wang. Nanoparticle formulation of KU55933 as a potent radiosensitizer. [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 4530. doi:10.1158/1538-7445.AM2013-4530

Abstract LB-501: Role of HGF in epithelial-stromal cell interactions during progression from benign breast disease to ductal carcinoma in situ

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Role of HGF in epithelial-stromal cell interactions during progression from benign breast disease to ductal carcinoma in situ. Introduction: Tumor-stroma interactions create a ‘permissive microenvironment’ during cancer progression. Basal and luminal breast cancers (BC) have distinct tumor-stroma interactions, but few studies show how gene expression evolves in premalignant cells. We observed that hepatic fibrosis/HGF signaling is induced in basal-like microenvironments, and previous studies have demonstrated that overexpression of HGF is a predictor of breast cancer recurrence and survival. However it is not known whether HGF signaling is altered in early (premalignant and DCIS) lesions. Methods: To study tumor-stroma interactions during cancer progression, we cocultured breast fibroblasts with a panel of isogenic cell lines, the MCF10 series. The MCF10 series represents different stages of cancer progression (benign, atypical hyperplasia and ductal carcinoma in situ). RNA from cocultures was used to perform gene expression microarrays and proteins secreted into media were analyzed by antibody arrays. Differentially expressed genes were identified using supervised analysis, followed by gene ontology analysis. In addition, a signature of basal-like microenvironment was evaluated to test whether the stroma-epithelial interactions observed in basal-like cancers were also present in premalignant cells. Finally, HGF signaling was inhibited (using an HGF-antibody) to assess effects of this pathway on migration and gene expression. Results: Cocultures with MCF10DCIS express higher levels of RNA involved in immune response processes and connective tissue disorders, as reported previously for basal-like BC. In contrast, premalignant MCF10AT and MCF10A cells showed weak expression of these profiles. In cytokine arrays, striking overexpression of HGF was observed in both MCF10AT1 and MCF10DCIS cocultures relative to cocultures with the benign MCF10A cell line, with corresponding increases in c-MET levels. These changes correlated with increased migration, which was blocked by adding HGF-specific antibodies to the media. Conclusions: Inhibition of HGF alters stromal-epithelial interactions, rendering the premalignant microenvironment more similar to normal microenvironments. HGF signaling is necessary for stroma-induced migratory phenotypes in vitro and is induces widespread gene expression changes. Targeting HGF signaling in early carcinogenesis may have value in preventing progression of basal-like precursor lesions. 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 LB-501. doi:1538-7445.AM2012-LB-501