Mitochondrial transfer from cancer-associated fibroblasts increases migration in aggressive breast cancer.

Cancer-associated fibroblasts (CAFs) have distinct roles within the tumor microenvironment, which can impact the mode and efficacy of tumor cell migration. CAFs are known to increase invasion of less-aggressive breast cancer cells through matrix remodeling and leader-follower dynamics. Here, we demonstrate that CAFs communicate with breast cancer cells through the formation of contact-dependent tunneling nanotubes (TNTs), which allow for the exchange of cargo between cell types. CAF mitochondria are an integral cargo component and are sufficient to increase the 3D migration of cancer cells. This cargo transfer results in an increase in mitochondrial ATP production in cancer cells, whereas it has a negligible impact on glycolytic ATP production. Manually increasing mitochondrial oxidative phosphorylation (OXPHOS) by providing extra substrates for OXPHOS fails to enhance cancer cell migration unless glycolysis is maintained at a constant level. Together, these data indicate that tumor-stromal cell crosstalk via TNTs and the associated metabolic symbiosis is a finely controlled mechanism by which tumor cells co-opt their microenvironment to promote cancer progression and may become a potential therapeutic target.

Mechanical strain induces phenotypic changes in breast cancer cells and promotes immunosuppression in the tumor microenvironment.

Breast cancer (BCa) proliferates within a complex, three-dimensional microenvironment amid heterogeneous biochemical and biophysical cues. Understanding how mechanical forces within the tumor microenvironment (TME) regulate BCa phenotype is of great interest. We demonstrate that mechanical strain enhanced the proliferation and migration of both estrogen receptor+ and triple-negative (TNBC) human and mouse BCa cells. Furthermore, a critical role for exosomes derived from cells subjected to mechanical strain in these pro-tumorigenic effects was identified. Exosome production by TNBC cells increased upon exposure to oscillatory strain (OS), which correlated with elevated cell proliferation. Using a syngeneic, orthotopic mouse model of TNBC, we identified that preconditioning BCa cells with OS significantly increased tumor growth and myeloid-derived suppressor cells (MDSCs) and M2 macrophages in the TME. This pro-tumorigenic myeloid cell enrichment also correlated with a decrease in CD8+ T cells. An increase in PD-L1+ exosome release from BCa cells following OS supported additive T cell inhibitory functions in the TME. The role of exosomes in MDSC and M2 macrophage was confirmed in vivo by cytotracking fluorescent exosomes, derived from labeled 4T1.2 cells, preconditioned with OS. In addition, in vivo internalization and intratumoral localization of tumor-cell derived exosomes was observed within MDSCs, M2 macrophages, and CD45-negative cell populations following direct injection of fluorescently-labeled exosomes. Our data demonstrate that exposure to mechanical strain promotes invasive and pro-tumorigenic phenotypes in BCa cells, indicating that mechanical strain can impact the growth and proliferation of cancer cell, alter exosome production by BCa, and induce immunosuppression in the TME by dampening anti-tumor immunity.

Gli1 promotes cell survival and is predictive of a poor outcome in ERalpha-negative breast cancer.

Gli1 is a transcription factor and oncogene with documented roles in the progression of several cancer types, including cancers of the skin and pancreas. The contribution of Gli1 to the progression of breast cancer is less established. In order to investigate the functional impact of Gli1 in breast cancer, expression of Gli1 and its contribution to cell growth was assessed in breast cancer cell lines. These in vitro results were compared to expression of Gli1, determined by immunohistochemistry, in 171 breast cancers. In these cancers, the association of Gli1 with expression of estrogen receptor alpha (ERalpha) and progesterone receptor (PR), ErbB2, p53, the rate of proliferation, and clinicopathologic parameters and outcome was assessed. Expression of Gli1 and ERalpha mRNA was strongly correlated in ERalpha-positive cell lines (r = 0.999). Treatment with estrogen increased expression of Gli1 in 2 of 3 ERalpha-positive cell lines; this increase was prevented by treatment with the ERalpha-specific antagonist MPP. Silencing of Gli1 by shRNA markedly reduced the survival of two ERalpha-negative cell lines, but caused only a modest reduction in ERalpha-positive cell lines. In breast cancer tissues, cancers with nuclear localization of Gli1 had a higher ERalpha (P=0.027) and lower p53 expression (P=0.017) than those without nuclear localization of Gli1. However, nuclear localization of Gli1 was predictive of a poorer cancer-specific survival in ERalpha-negative, including triple negative, cancers (P = 0.005), but not ERalpha-positive cancers. In conclusion, we demonstrate a positive association between expression of Gli1 and ERalpha; however, our data indicate a greater functional effect of Gli1 in ERalpha-negative cancers.

Northwestern profiling of potential translation-regulatory proteins in human breast epithelial cells and malignant breast tissues: evidence for pathological activation of the IGF1R IRES.

Genes involved in the control of cell proliferation and survival (those genes most important to cancer pathogenesis) are often specifically regulated at the translational level, through RNA-protein interactions involving the 5'-untranslated region of the mRNA. IGF1R is a proto-oncogene strongly implicated in human breast cancer, promoting survival and proliferation of tumor cells, as well as metastasis and chemoresistance. Our lab has focused on the molecular mechanisms regulating IGF1R expression at the translational level. We previously discovered an internal ribosome entry site (IRES) within the 5'-untranslated region of the human IGF1R mRNA, and identified and functionally characterized two individual RNA-binding proteins, HuR and hnRNP C, which bind the IGF1R 5'-UTR and differentially regulate IRES activity. Here we have developed and implemented a high-resolution northwestern profiling strategy to characterize, as a group, the full spectrum of sequence-specific RNA-binding proteins potentially regulating IGF1R translational efficiency through interaction with the 5'-untranslated sequence. The putative IGF1R IRES trans-activating factors (ITAFs) are a heterogeneous group of RNA-binding proteins including hnRNPs originating in the nucleus as well as factors tightly associated with ribosomes in the cytoplasm. The IGF1R ITAFs can be categorized into three distinct groups: (a) high molecular weight external ITAFs, which likely modulate the overall conformation of the 5'-untranslated region of the IGF1R mRNA and thereby the accessibility of the core functional IRES; (b) low molecular weight external ITAFs, which may function as general chaperones to unwind the RNA, and (c) internal ITAFs which may directly facilitate or inhibit the fundamental process of ribosome recruitment to the IRES. We observe dramatic changes in the northwestern profile of non-malignant breast cells downregulating IGF1R expression in association with acinar differentiation in 3-D culture. Most importantly, we are able to assess the RNA-binding activities of these putative translation-regulatory proteins in primary human breast surgical specimens, and begin to discern positive correlations between individual ITAFs and the malignant phenotype. Together with our previous findings, these new data provide further evidence that pathological dysregulation of IGF1R translational control may contribute to development and progression of human breast cancer, and breast metastasis in particular.

Identification of Distinct Heterogenic Subtypes and Molecular Signatures Associated with African Ancestry in Triple Negative Breast Cancer Using Quantified Genetic Ancestry Models in Admixed Race Populations.

Triple negative breast cancers (TNBCs) are molecularly heterogeneous, and the link between their aggressiveness with African ancestry is not established. We investigated primary TNBCs for gene expression among self-reported race (SRR) groups of African American (AA, n = 42) and European American (EA, n = 33) women. RNA sequencing data were analyzed to measure changes in genome-wide expression, and we utilized logistic regressions to identify ancestry-associated gene expression signatures. Using SNVs identified from our RNA sequencing data, global ancestry was estimated. We identified 156 African ancestry-associated genes and found that, compared to SRR, quantitative genetic analysis was a more robust method to identify racial/ethnic-specific genes that were differentially expressed. A subset of African ancestry-specific genes that were upregulated in TNBCs of our AA patients were validated in TCGA data. In AA patients, there was a higher incidence of basal-like two tumors and altered TP53, NFB1, and AKT pathways. The distinct distribution of TNBC subtypes and altered oncologic pathways show that the ethnic variations in TNBCs are driven by shared genetic ancestry. Thus, to appreciate the molecular diversity of TNBCs, tumors from patients of various ancestral origins should be evaluated.

Loss of TGF-beta or Wnt5a results in an increase in Wnt/beta-catenin activity and redirects mammary tumour phenotype.

INTRODUCTION: The tumour-suppressive effects of transforming growth factor-beta (TGF-beta) are well documented; however, the mechanistic basis of these effects is not fully understood. Previously, we showed that a non-canonical member of the Wingless-related protein family, Wnt5a, is required for TGF-beta-mediated effects on mammary development. Several lines of evidence support the hypothesis that Wnt5a acts as a tumour suppressor. In addition, it has been shown that Wnt5a can antagonise canonical Wnt/beta-catenin signalling in various cell types. Here we test the hypothesis that TGF-beta and Wnt5a can antagonise Wnt/beta-catenin signalling and redirect mammary tumour phenotype. The results provide a new mechanism for the tumour-suppressive effects of TGF-beta. METHODS: Wnt/beta-catenin signalling was measured in tumours with altered TGF-beta (dominant-negative TGF-beta type II receptor, DNIIR) or Wnt5a (Wnt5a-/-) signalling as the accumulation of nuclear beta-catenin using both confocal microscopy and cell fractionation. RT-PCR was used to measure the expression of Wnt/beta-catenin target genes. Sca1 expression was determined by western blot and keratin (K) 6- and K14-positive populations were determined by immunohistochemistry. RESULTS: Loss of TGF-beta or Wnt5a signalling resulted in stabilisation of nuclear beta-catenin and expression of Wnt/beta-catenin target genes suggesting that TGF-beta and Wnt5a act to inhibit Wnt/beta-catenin signalling in mammary epithelium. Increased expression of Sca-1 was observed in developing DNIIR and Wnt5a-/- mammary glands. DNIIR and Wnt5a-/- tumours demonstrated an expanded population of K6- and K14-expressing cells typically seen in Wnt/beta-catenin-induced tumours. CONCLUSIONS: The key findings here are that: TGF-beta and Wnt5a regulate Wnt/beta-catenin activity; and loss of TGF-beta and Wnt5a redirect the phenotype of tumours so that they resemble tumours induced by activation of Wnt/beta-catenin. The findings suggest a new mechanism for the tumour-suppressive effects of TGF-beta.

Dual effects of TGF-beta on ERalpha-mediated estrogenic transcriptional activity in breast cancer.

BACKGROUND: TGF-beta resistance often develops in breast cancer cells that in turn overproduce this cytokine to create a local immunosuppressive environment that fosters tumor growth and exacerbates the invasive and metastatic behavior of the tumor cells themselves. Smads-mediated cross-talk with the estrogen receptor has been implied to play an important role in development and/or progression of breast cancer. We investigated how TGF-beta regulates ERalpha-induced gene transcription and potential mechanisms of frequent TGF-beta resistance in breast cancer. METHODS: Effect of TGF-beta on ERalpha-mediated gene transcription was investigated in breast cancer cell lines using transient transfection, real-time PCR, sequential DNA precipitation, and small interfering RNA assays. The expression of Smads on both human breast cancer cell lines and ERalpha-positive human breast cancer tissue was evaluated by immunofluorescence and immunohistochemical assays. RESULTS: A complex of Smad3/4 mediates TGF-beta inhibition of ERalpha-mediated estrogenic activity of gene transcription in breast cancer cells, and Smad4 is essential and sufficient for such repression. Either overexpression of Smad3 or inhibition of Smad4 leads to the "switch" of TGF-beta from a repressor to an activator. Down-regulation and abnormal cellular distribution of Smad4 were associated with some ERalpha-positive infiltrating human breast carcinoma. There appears a dynamic change of Smad4 expression from benign breast ductal tissue to infiltrating ductal carcinoma. CONCLUSION: These results suggest that aberrant expression of Smad4 or disruption of Smad4 activity lead to the loss of TGF-beta suppression of ERalpha transactivity in breast cancer cells.

A shift from nuclear to cytoplasmic breast cancer metastasis suppressor 1 expression is associated with highly proliferative estrogen receptor-negative breast cancers.

BACKGROUND/AIMS: To determine breast cancer metastasis suppressor 1 (BRMS1) expression in breast cancers and the efficacy of BRMS1 as a prognostic indicator, BRMS1 expression was assessed in two sets of breast cancer tissues. METHODS: Epithelial cells from 36 frozen samples of breast cancers and corresponding normal breast were collected by laser capture microdissection and assessed for BRMS1 by quantitative RT-PCR and immunohistochemistry. BRMS1 was also evaluated by immunohistochemistry in a tissue microarray of 209 breast cancers and correlated with indicators of prognosis [estrogen receptor (ER), progesterone receptor (PR), ErbB2, p53, p27(Kip1), Bcl2 and Ki-67]. RESULTS: BRMS1 mRNA and protein were higher in 94 and 81%, respectively, of breast cancers than in corresponding normal epithelium. BRMS1 localization was predominantly nuclear, but 60-70% of cancers also exhibited cytoplasmic immunostaining. Breast cancers with lower nuclear than cytoplasmic BRMS1 (nuclear score - cytoplasmic score < or =0; 11% of cancers) had lower ER, lower PR and higher Ki-67 expression. There was also a trend toward poorer overall survival in this group of cancers, but this was only of borderline significance (p = 0.073). In Cox proportional hazards models, loss of nuclear BRMS1 was not a significant predictor of overall survival. CONCLUSIONS: Loss of nuclear BRMS1 was associated with ER-negative cancers and a high rate of proliferation, but was not an independent indicator of prognosis.

O-GlcNAc integrates the proteasome and transcriptome to regulate nuclear hormone receptors.

Mechanisms controlling nuclear hormone receptors are a central question to mammalian developmental and disease processes. Herein, we show that a subtle increase in O-GlcNAc levels inhibits activation of nuclear hormone receptors. In vivo, increased levels of O-GlcNAc impair estrogen receptor activation and cause a decrease in mammary ductal side-branching morphogenesis associated with loss of progesterone receptors. Increased O-GlcNAc levels suppress transcriptional expression of coactivators and of the nuclear hormone receptors themselves. Surprisingly, increased O-GlcNAc levels are also associated with increased transcription of genes encoding corepressor proteins NCoR and SMRT. The association of the enzyme O-GlcNAc transferase with these corepressors contributes to specific regulation of nuclear hormone receptors by O-GlcNAc. Overall, transcriptional inhibition is related to the integrated effect of O-GlcNAc by direct modification of critical elements of the transcriptome and indirectly through O-GlcNAc modification of the proteasome.

Epigenetic silencing contributes to the loss of BRMS1 expression in breast cancer.

Breast Cancer Metastasis Suppressor 1 (BRMS1) suppresses metastasis of human breast cancer, ovarian cancer and melanoma in athymic mice. Studies have also shown that BRMS1 is significantly downregulated in some breast tumors, especially in metastatic disease. However, the mechanisms which regulate BRMS1 expression are currently unknown. Upon examination of the BRMS1 promoter region by methylation specific PCR (MSP) analysis, we discovered a CpG island (-3477 to -2214), which was found to be hypermethylated across breast cancer cell lines. A panel of 20 patient samples analyzed showed that 45% of the primary tumors and 60% of the matched lymph node metastases, displayed hypermethylation of BRMS1 promoter. Furthermore, we found a direct correlation between the methylation status of the BRMS1 promoter in the DNA isolated from tissues, with the loss of BRMS1 expression assessed by immunohistochemistry. There are several studies investigating the mechanism by which BRMS1 suppresses metastasis; however thus far there is no study that reports the cause(s) of loss of BRMS1 expression in aggressive breast cancer. Here we report for the first time that BRMS1 is a novel target of epigenetic silencing; and aberrant methylation in the BRMS1 promoter may serve as a cause of loss of its expression.

Sulfotransferase 2B1b in human breast: differences in subcellular localization in African American and Caucasian women.

Breast cancer (BC) is the most commonly diagnosed cancer among American women; however, the development of post-menopausal BC is significantly lower in African Americans as compared to Caucasians. Hormonal stimulation is important in BC development and differences in the conversion of dehydroepiandrosterone (DHEA) into estrogens may be involved in the lower incidence of post-menopausal BC in African American women. DHEA sulfation by sulfotransferase 2B1b (SULT2B1b) is important in regulating the conversion of DHEA into estrogens in tissues. SULT2B1b is localized in both cytosol and nuclei of some tissues including cancerous and associated-normal breast tissue. Immunohistochemical staining was used to evaluate the total expression and subcellular localization of SULT2B1b in African American and Caucasian breast tissues. Cell fractionation, immunoblot analysis and sulfation assays were used to characterize the subcellular expression and activity of SULT2B1b in BC tissues and T-47D breast adenocarcinoma cells. Immunohistochemical analysis of SULT2B1b showed that African Americans had a significantly greater amount of SULT2B1b in epithelial cells of associated-normal breast tissue as compared to Caucasians. Also, more SULT2B1b in African American associated-normal breast epithelial cells was localized in the nuclei than in Caucasians. Equivalent levels of SULT2B1b were detected in breast adenocarcinoma tissues from both African American and Caucasian women. Nuclei isolation and immunoblot analysis of both BC tissue and human T-47D breast adenocarcinoma cells demonstrated that SULT2B1b is present in nuclei and cytoplasm.

Hedgehog pathway expression in heterogeneous pancreatic adenocarcinoma: implications for the molecular analysis of clinically available biopsies.

Recent studies suggest that hedgehog (HH)-pathway signaling is required for the initiation and continued growth of pancreatic adenocarcinoma (PAC). Definitive gene expression analysis of PAC remains difficult, owing to the host desmoplastic stromal interaction and subsequent tumor heterogeneity. The primary goal of this study was to evaluate the effect of heterogeneity within a series (n=5) of matched clinical PAC biopsies [snap-frozen, formalin-fixed paraffin-embedded (FPE), endoscopic ultrasound-guided fine-needle aspirate (EUS-FNA)]. Differential expressions, specific to tumor cells, were evaluated by comparisons of uninvolved pancreas (n=9), EUS-FNA (n=14), and macrodissected (tumor-cell-enriched) biopsies (n=16). To determine whether treatment modulates gene expression, a unique (independent) set of synchronous EUS-FNA samples (n=4) was obtained before, and 2 weeks after, chemoradiation. mRNA levels were evaluated using real-time quantitative polymerase chain reaction formatted in a TaqMan low-density array, which was capable of simultaneously quantifying 46 independent genes in the HH pathway. Protein levels for Patched, Smoothened, and glioma-associated oncogene 1 (Gli-1) in FPE tissues were determined, using immunohistochemistry. A significant concordance (P<0.0001) was observed in the HH-pathway mRNA levels between matched surgically resected (both snap-frozen and FPE) and EUS-FNA biopsies. HH-pathway mRNA levels changed (increased) only after macrodissection, suggesting localization to tumor cells. Immunohistochemical staining for Patched, Smoothened, and Gli-1 confirmed the increased (P<0.001) levels of protein in the PAC cells, compared with cells from uninvolved pancreas. EUS-FNA biopsies that were obtained before and during chemoradiation demonstrated no significant changes in HH-pathway gene expression. Collectively, these studies demonstrate presence of HH-pathway expression in all the clinical PAC biopsies examined, suggesting that this is a significant tumor-associated target and offering the possibility that specific molecular profiling might be attempted from these heterogeneous tissues.

Kinetics of metastatic breast cancer cell trafficking in bone.

PURPOSE: In vivo studies have focused on the latter stages of the bone metastatic process (osteolysis), whereas little is known about earlier events, e.g., arrival, localization, and initial colonization. Defining these initial steps may potentially identify the critical points susceptible to therapeutic intervention. EXPERIMENTAL DESIGN: MDA-MB-435 human breast cancer cells engineered with green fluorescent protein were injected into the cardiac left ventricle of athymic mice. Femurs were analyzed by fluorescence microscopy, immunohistochemistry, real-time PCR, flow cytometry, and histomorphometry at times ranging from 1 hour to 6 weeks. RESULTS: Single cells were found in distal metaphyses at 1 hour postinjection and remained as single cells up to 72 hours. Diaphyseal arrest occurred rarely and few cells remained there after 24 hours. At 1 week, numerous foci (2-10 cells) were observed, mostly adjacent to osteoblast-like cells. By 2 weeks, fewer but larger foci (> or =50 cells) were seen. Most bones had a single large mass at 4 weeks (originating from a colony or coalescing foci) which extended into the diaphysis by 4 to 6 weeks. Little change (<20%) in osteoblast or osteoclast numbers was observed at 2 weeks, but at 4 to 6 weeks, osteoblasts were dramatically reduced (8% of control), whereas osteoclasts were reduced modestly (to approximately 60% of control). CONCLUSIONS: Early arrest in metaphysis and minimal retention in diaphysis highlight the importance of the local milieu in determining metastatic potential. These results extend the Seed and Soil hypothesis by demonstrating both intertissue and intratissue differences governing metastatic location. Ours is the first in vivo evidence that tumor cells influence not only osteoclasts, as widely believed, but also eliminate functional osteoblasts, thereby restructuring the bone microenvironment to favor osteolysis. The data may also explain why patients receiving bisphosphonates fail to heal bone despite inhibiting resorption, implying that concurrent strategies that restore osteoblast function are needed to effectively treat osteolytic bone metastases.

Loss of breast cancer metastasis suppressor 1 protein expression predicts reduced disease-free survival in subsets of breast cancer patients.

PURPOSE: This study aims to determine the effect of loss of breast cancer metastasis suppressor 1 (BRMS1) protein expression on disease-free survival in breast cancer patients stratified by estrogen receptor (ER), progesterone receptor (PR), or HER2 status, and to determine whether loss of BRMS1 protein expression correlated with genomic copy number changes. EXPERIMENTAL DESIGN: A tissue microarray immunohistochemical analysis was done on tumors of 238 newly diagnosed breast cancer patients who underwent surgery at the Cleveland Clinic between January 1, 1995 and December 31, 1996, and a comparison was made with 5-year clinical follow-up data. Genomic copy number changes were determined by array-based comparative genomic hybridization in 47 breast cancer cases from this population and compared with BRMS1 staining. RESULTS: BRMS1 protein expression was lost in nearly 25% of cases. Patients with tumors that were PR negative (P=0.006) or HER2 positive (P=0.039) and <50 years old at diagnosis (P=0.02) were more likely to be BRMS1 negative. No overall correlation between BRMS1 staining and disease-free survival was observed. A significant correlation, however, was seen between loss of BRMS1 protein expression and reduced disease-free survival when stratified by either loss of ER (P=0.008) or PR (P=0.029) or HER2 overexpression (P=0.026). Overall, there was poor correlation between BRMS1 protein staining and copy number status. CONCLUSIONS: These data suggest a mechanistic relationship between BRMS1 expression, hormone receptor status, and HER2 growth factor. BRMS1 staining could potentially be used in patient stratification in conjunction with other prognostic markers. Further, mechanisms other than genomic deletion account for loss of BRMS1 gene expression in breast tumors.

Hypersialylation of beta1 integrins, observed in colon adenocarcinoma, may contribute to cancer progression by up-regulating cell motility.

Colon adenocarcinomas are known to express elevated levels of alpha2-6 sialylation and increased activity of ST6Gal-I, the Golgi glycosyltransferase that creates alpha2-6 linkages. Elevated ST6Gal-I positively correlates with metastasis and poor survival, and therefore ST6Gal-I-mediated hypersialylation likely plays a role in colorectal tumor invasion. Previously we found that oncogenic ras (present in roughly 50% of colon adenocarcinomas) up-regulates ST6Gal-I and, in turn, increases sialylation of beta1 integrin adhesion receptors in colon epithelial cells. However, we wanted to know if this pattern held true in vivo and, if so, how beta1 hypersialylation might contribute to colon tumor progression. In the present study, we find that beta1 integrins from colon adenocarcinomas consistently carry higher levels of alpha2-6 sialic acid. To explore the effects of increased alpha2-6 sialylation on beta1-integrin function, we stably expressed ST6Gal-I in a colon epithelial cell line lacking endogenous ST6Gal-I. ST6Gal-I expressors (with alpha2-6 sialylated beta1 integrins) exhibited up-regulated attachment to collagen I and laminin and increased haptotactic migration toward collagen I, relative to parental cells (with completely unsialylated beta1 integrins). Blockade of ST6Gal-I expression with short interfering RNA reversed collagen binding back to the level of ST6Gal-I nonexpressors, confirming that alpha2-6 sialylation regulates beta1 integrin function. Finally, we show that beta1 integrins from ST6Gal-I expressors have increased association with talin, a marker for integrin activation. Collectively, these findings suggest that beta1 hypersialylation may augment colon tumor progression by altering cell preference for certain extracellular matrix milieus, as well as by stimulating cell migration.

Computational and Experimental Analysis of Fluid Transport Through Three-Dimensional Collagen-Matrigel Hydrogels.

A preclinical testing model for cancer therapeutics that replicates in vivo physiology is needed to accurately describe drug delivery and efficacy prior to clinical trials. To develop an in vitro model of breast cancer that mimics in vivo drug/nutrient delivery as well as physiological size and bio-composition, it is essential to describe the mass transport quantitatively. The objective of the present study was to develop in vitro and computational models to measure mass transport from a perfusion system into a 3D extracellular matrix (ECM). A perfusion-flow bioreactor system was used to control and quantify the mass transport of a macromolecule within an ECM hydrogel with embedded through-channels. The material properties, fluid mechanics, and structure of the construct quantified in the in vitro model were input into, and served as validation of, the computational fluid dynamics (CFD) simulation. Results showed that advection and diffusion played a complementary role in mass transport. As the CFD simulation becomes more complex with embedded blood vessels and cancer cells, it will become more recapitulative of in vivo breast cancers. This study is a step toward development of a preclinical testing platform that will be more predictive of patient response to therapeutics than two-dimensional cell culture.

Flow-perfusion bioreactor system for engineered breast cancer surrogates to be used in preclinical testing.

There is a need for preclinical testing systems that predict the efficacy, safety and pharmacokinetics of cancer therapies better than existing in vitro and in vivo animal models. An approach to the development of predictive in vitro systems is to more closely recapitulate the cellular and spatial complexity of human cancers. One limitation of using current in vitro systems to model cancers is the lack of an appropriately large volume to accommodate the development of this complexity over time. To address this limitation, we have designed and constructed a novel flow-perfusion bioreactor system that can support large-volume, engineered tissue comprised of multicellular cancer surrogates by modifying current microfluidic devices. Key features of this technology are a three-dimensional (3D) volume (1.2 cm3 ) that has greater tissue thickness than is utilized in existing microfluidic systems and the ability to perfuse the volume, enabling the development of realistic tumour geometry. The constructs were fabricated by infiltrating porous carbon foams with an extracellular matrix (ECM) hydrogel and engineering through-microchannels. The carbon foam structurally supported the hydrogel and microchannel patency for up to 161 h. The ECM hydrogel was shown to adhere to the carbon foam and polydimethylsiloxane flow chamber, which housed the hydrogel-foam construct, when surfaces were coated with glutaraldehyde (carbon foam) and nitric acid (polydimethylsiloxane). Additionally, the viability of breast cancer cells and fibroblasts was higher in the presence of perfused microchannels in comparison to similar preparations without microchannels or perfusion. Therefore, the flow-perfusion bioreactor system supports cell viability in volume and stromal contexts that are physiologically-relevant. Copyright © 2015 John Wiley & Sons, Ltd.

Methods to Evaluate Cell Growth, Viability, and Response to Treatment in a Tissue Engineered Breast Cancer Model.

The use of in vitro, engineered surrogates in the field of cancer research is of interest for studies involving mechanisms of growth and metastasis, and response to therapeutic intervention. While biomimetic surrogates better model human disease, their complex composition and dimensionality make them challenging to evaluate in a real-time manner. This feature has hindered the broad implementation of these models, particularly in drug discovery. Herein, several methods and approaches for the real-time, non-invasive analysis of cell growth and response to treatment in tissue-engineered, three-dimensional models of breast cancer are presented. The tissue-engineered surrogates used to demonstrate these methods consist of breast cancer epithelial cells and fibroblasts within a three dimensional volume of extracellular matrix and are continuously perfused with nutrients via a bioreactor system. Growth of the surrogates over time was measured using optical in vivo (IVIS) imaging. Morphologic changes in specific cell populations were evaluated by multi-photon confocal microscopy. Response of the surrogates to treatment with paclitaxel was measured by optical imaging and by analysis of lactate dehydrogenase and caspase-cleaved cytokeratin 18 in the perfused medium. Each method described can be repeatedly performed during culture, allowing for real-time, longitudinal analysis of cell populations within engineered tumor models.

Induction of KLF4 in basal keratinocytes blocks the proliferation-differentiation switch and initiates squamous epithelial dysplasia.

KLF4/GKLF normally functions in differentiating epithelial cells, but also acts as a transforming oncogene in vitro. To examine the role of this zinc finger protein in skin, we expressed the wild-type human allele from inducible and constitutive promoters. When induced in basal keratinocytes, KLF4 rapidly abolished the distinctive properties of basal and parabasal epithelial cells. KLF4 caused a transitory apoptotic response and the skin progressed through phases of hyperplasia and dysplasia. By 6 weeks, lesions exhibited nuclear KLF4 and other morphologic and molecular similarities to squamous cell carcinoma in situ. p53 determined the patch size sufficient to establish lesions, as induction in a mosaic pattern produced skin lesions only when p53 was deficient. Compared with p53 wild-type animals, p53 hemizygous animals had early onset of lesions and a pronounced fibrovascular response that included outgrowth of subcutaneous sarcoma. A KLF4-estrogen receptor fusion protein showed tamoxifen-dependent nuclear localization and conditional transformation in vitro. The results suggest that KLF4 can function in the nucleus to induce squamous epithelial dysplasia, and indicate roles for p53 and epithelial-mesenchymal signaling in these early neoplastic lesions.