CFP suppresses breast cancer cell growth by TES-mediated upregulation of the transcription factor DDIT3.

Breast cancer is a heterogeneous genetic disease driven by the accumulation of individual mutations per tumor. Whole-genome sequencing approaches have identified numerous genes with recurrent mutations in primary tumors. Although mutations in well characterized tumor suppressors and oncogenes are overrepresented in these sets, the majority of the genetically altered genes have so far unknown roles in breast cancer progression. To improve the basic understanding of the complex disease breast cancer and to potentially identify novel drug targets or regulators of known cancer-driving pathways, we analyzed 86 wild-type genes and 94 mutated variants for their effect on cell growth using a serially constructed panel of MCF7 cell lines. We demonstrate in subsequent experiments that the metal cation transporter CNNM4 regulates growth by induction of apoptosis and identified a tumor suppressive role of complement factor properdin (CFP) in vitro and in vivo. CFP appears to induce the intracellular upregulation of the pro-apoptotic transcription factor DDIT3 which is associated with endoplasmic reticulum-stress response.

Identification of metastasis driver genes by massive parallel sequencing of successive steps of breast cancer progression.

Cancer results from alterations at essential genomic sites and is characterized by uncontrolled cell proliferation, invasion and metastasis. Identification of driver genes of metastatic progression is essential, as metastases, not primary tumors, are fatal. To gain insight into the mutational concordance between different steps of malignant progression we performed exome sequencing and validation with targeted deep sequencing of successive steps of malignant progression from pre-invasive stages to asynchronous distant metastases in six breast cancer patients. Using the ratio of non-synonymous to synonymous mutations, a surprisingly large number of cancer driver genes, ranging between 3 and 145, were estimated to confer a selective advantage in the studied primary tumors. We report a substantial amount of metastasis specific mutations and a number of novel putative metastasis driver genes. Most notable are the DCC, ABCA13, TIAM2, CREBBP, BCL6B and ZNF185 genes, mainly mutated exclusively in metastases and highly likely driver genes of metastatic progression. We find different genes and pathways to be affected at different steps of malignant progression. The Adherens junction pathway is affected in four of the six studied patients and this pathway most likely plays a vital role in the metastatic process.

One-step FPLC-size-exclusion chromatography procedure for purification of rDMBT1 6 kb with increased biological activity.

Deleted in Malignant Brain Tumor 1 (DMBT1, alias SAG or gp340) is a pattern recognition receptor involved in immune defense, cell polarization, differentiation and regeneration. To investigate the role of the protein in physiological and pathological processes, the protein has often been isolated from saliva or produced in vitro and purified by a multistep affinity purification procedure using bacteria, followed by FPLC. Here, we compared a simple, one-step FPLC-SEC protocol for purification of recombinant DMBT1 6 kb, with that of the standard bacteria affinity purification-based protocol. Our data suggest that our FPLC-SEC protocol yields DMBT1 in a more native conformation.

Human DMBT1-Derived Cell-Penetrating Peptides for Intracellular siRNA Delivery.

Small interfering RNA (siRNA) is a promising molecule for gene therapy, but its therapeutic administration remains problematic. Among the recently proposed vectors, cell-penetrating peptides show great promise in in vivo trials for siRNA delivery. Human protein DMBT1 (deleted in malignant brain tumor 1) is a pattern recognition molecule that interacts with polyanions and recognizes and aggregates bacteria. Taking advantage of these properties, we investigated whether specific synthetic DMBT1-derived peptides could be used to formulate nanoparticles for siRNA administration. Using an electrophoretic mobility shift assay and UV spectra, we identified two DMBT1 peptides that could encapsulate the siRNA with a self- and co-assembly mechanism. The complexes were stable for at least 2 hr in the presence of either fetal bovine serum (FBS) or RNase A, with peptide-dependent time span protection. ζ-potential, circular dichroism, dynamic light scattering, and transmission electron microscopy revealed negatively charged nanoparticles with an average diameter of 10-800 nm, depending on the reaction conditions, and a spherical or rice-shaped morphology, depending on the peptide and ß-helix conformation. We successfully transfected human MCF7 cells with fluorescein isothiocyanate (FITC)-DMBT1-peptide-Cy3-siRNA complexes. Finally, DMBT1 peptides encapsulating an siRNA targeting a fluorescent reporter gene showed efficient gene silencing in MCF7-recombinant cells. These results lay the foundation for a new research line to exploit DMBT1-peptide nanocomplexes for therapeutic siRNA delivery.

Evaluation of somatostatin and nucleolin receptors for therapeutic delivery in non-small cell lung cancer stem cells applying the somatostatin-analog DOTATATE and the nucleolin-targeting aptamer AS1411.

Cancer stem cells represent the putative tumor-driving subpopulation thought to account for drug resistance, relapse, and metastatic spread of epithelial and other cancer types. Accordingly, cell surface markers for therapeutic delivery to cancer stem cells are subject of intense research. Somatostatin receptor 2 and nucleolin are known to be overexpressed by various cancer types, which have elicited comprehensive efforts to explore their therapeutic utilization. Here, we evaluated somatostatin receptor 2 targeting and nucleolin targeting for therapeutic delivery to cancer stem cells from lung cancer. Nucleolin is expressed highly but not selectively, while somatostatin receptor 2 is expressed selectively but not highly by cancer cells. The non-small cell lung cancer cell lines A549 and H1299, displayed average levels of both surface molecules as judged based on analysis of a larger cell line panel. H1299 compared to A549 cells showed significantly elevated sphere-forming capacity, indicating higher cancer stem cell content, thus qualifying as suitable test system. Nucleolin-targeting 57Co-DOTA-AS1411 aptamer showed efficient internalization by cancer cells and, remarkably, at even higher efficiency by cancer stem cells. In contrast, somatostatin receptor 2 expression levels were not sufficiently high in H1299 cells to confer efficient uptake by either non-cancer stem cells or cancer stem cells. The data provides indication that the nucleolin-targeting AS1411 aptamer might be used for therapeutic delivery to non-small cell lung cancer stem cells.

The scavenging capacity of DMBT1 is impaired by germline deletions.

The Scavenger Receptor Cysteine-Rich (SRCR) proteins are an archaic group of proteins characterized by the presence of multiple SRCR domains. They are membrane-bound or secreted proteins, which are generally related to host defense systems in animals. Deleted in Malignant Brain Tumors 1 (DMBT1) is a SRCR protein which is secreted in mucosal fluids and involved in host defense by pathogen binding by its SRCR domains. Genetic polymorphism within DMBT1 leads to DMBT1-alleles giving rise to polypeptides with interindividually different numbers of SRCR domains, ranging from 8 SRCR domains (encoded by 6 kb DMBT1 variant) to 13 SRCR domains (encoded by the 8 kb DMBT1 variant). In the present study, we have investigated whether reduction from 13 to 8 amino-terminal SRCR domains leads to reduction of bacterial binding. The 6 kb variant bound ~20-45% less bacteria compared to the 8 kb variant. These results support the hypothesis that genetic variation in DMBT1 may influence microbial defense.

Systematic screening of isogenic cancer cells identifies DUSP6 as context-specific synthetic lethal target in melanoma.

Next-generation sequencing has dramatically increased genome-wide profiling options and conceptually initiates the possibility for personalized cancer therapy. State-of-the-art sequencing studies yield large candidate gene sets comprising dozens or hundreds of mutated genes. However, few technologies are available for the systematic downstream evaluation of these results to identify novel starting points of future cancer therapies.We improved and extended a site-specific recombination-based system for systematic analysis of the individual functions of a large number of candidate genes. This was facilitated by a novel system for the construction of isogenic constitutive and inducible gain- and loss-of-function cell lines. Additionally, we demonstrate the construction of isogenic cell lines with combinations of the traits for advanced functional in vitro analyses. In a proof-of-concept experiment, a library of 108 isogenic melanoma cell lines was constructed and 8 genes were identified that significantly reduced viability in a discovery screen and in an independent validation screen. Here, we demonstrate the broad applicability of this recombination-based method and we proved its potential to identify new drug targets via the identification of the tumor suppressor DUSP6 as potential synthetic lethal target in melanoma cell lines with BRAF V600E mutations and high DUSP6 expression.

Genomic Analyses of Breast Cancer Progression Reveal Distinct Routes of Metastasis Emergence.

A main controversy in cancer research is whether metastatic abilities are present in the most advanced clone of the primary tumor or result from independently acquired aberrations in early disseminated cancer cells as suggested by the linear and the parallel progression models, respectively. The genetic concordance between different steps of malignant progression is mostly unexplored as very few studies have included cancer samples separated by both space and time. We applied whole exome sequencing and targeted deep sequencing to 26 successive samples from six patients with metastatic estrogen receptor (ER)-positive breast cancer. Our data provide support for both linear and parallel progression towards metastasis. We report for the first time evidence of metastasis-to-metastasis seeding in breast cancer. Our results point to three distinct routes of metastasis emergence. This may have profound clinical implications and provides substantial novel molecular insights into the timing and mutational evolution of breast cancer metastasis.

DMBT1 expression in biliary carcinogenesis with correlation of clinicopathological data.

AIMS: Deleted in malignant brain tumours 1 (DMBT1) exerts functions in the regulation of epithelial differentiation and inflammation and has been proposed as a tumour suppressor. Because chronic inflammation is a hallmark of cholangiocarcinogenesis, the aim of this study was to investigate the expression of DMBT1 in biliary tract cancer (BTC) and to correlate this expression with clinicopathological data. METHODS AND RESULTS: The expression of DMBT1 protein was examined immunohistochemically in 157 BTC patients [41 intrahepatic (ICC), 60 extrahepatic cholangiocarcinomas (ECC) and 56 adenocarcinomas of the gallbladder (GBAC)]. Additionally, 56 samples of high-grade biliary intraepithelial neoplasia (BilIN 3) and 92 corresponding samples of histological non-neoplastic biliary tract tissues were included. DMBT1 expression was increased significantly in BilIN 3 compared to normal tissue (P < 0.0001) and BTC (P < 0.0001). BTC showed no significant difference in DMBT1 expression compared to non-neoplastic biliary tissue (P = 0.315). Absent DMBT1 expression in non-neoplastic biliary tissue of BTC patients was associated with poorer survival (P = 0.027). DMBT1 expression was correlated significantly with patients' age (P < 0.001). CONCLUSION: DMBT1 is expressed differently in cholangiocarcinogenesis and poorer patients' survival rates are associated with absent DMBT1 expression in non-neoplastic biliary tissue, suggesting a tumour-suppressive role of DMBT1 in early cholangiocarcinogenesis.

Protein-based nanotoxicology assessment strategy.

The nanomaterial community calls for standardized in vitro assays to determine nanoparticle toxicity in the effort to reduce the number of in vivo validation experiments. We demonstrate that chip-based protein detection is suitable for assessing toxicity and may complement traditional assays to improve selection of primary hits for subsequent analysis. As nanodrug mimics, we analyzed the effect of transiently transfected siRNAs in MCF7 breast cancer cells and normal MCF12A breast cells, resembling a differential screen. As a measure of cytotoxicity, we determined cell viability as well as protein expression of glyceraldehyde-3-phosphate dehydrogenase, transferrin receptor, and the proliferation marker Ki67. The evaluation of cell lethality and protein expression unraveled cellular effects overseen by one method alone.

Shift of microRNA profile upon glioma cell migration using patient-derived spheroids and serum-free conditions.

Glioblastoma multiforme (GBM) is the most frequent malignant primary brain tumor. A major reason for the overall median survival being only 14.6 months is migrating tumor cells left behind after surgery. Another major reason is tumor cells having a so-called cancer stem cell phenotype being therefore resistant towards traditional chemo- and radiotherapy. A group of novel molecular targets are microRNAs (miRNAs). MiRNAs are small non-coding RNAs exerting post-transcriptional regulation of gene expression. The aim of this study was to identify differentially expressed miRNAs in migrating GBM cells using serum-free stem cell conditions. We used patient-derived GBM spheroid cultures for a novel serum-free migration assay. MiRNA expression of migrating tumor cells isolated at maximum migration speed was compared with corresponding spheroids using an OpenArray Real-Time PCR System. The miRNA profiling revealed 30 miRNAs to be differentially expressed. In total 13 miRNAs were upregulated and 17 downregulated in migrating cells compared to corresponding spheroids. The three most deregulated miRNAs, miR-1227 (up-regulated), miR-32 (down-regulated) and miR-222 (down-regulated), were experimentally overexpressed. A non-significantly increased migration rate was observed after miR-1227 overexpression. A significantly reduced migration rate was observed after miR-32 and miR-222 overexpression. In conclusion a shift in microRNA profile upon glioma cell migration was identified using an assay avoiding serum-induced migration. Both the miRNA profiling and the functional validation suggested that miR-1227 may be associated with increased migration and miR-32 and miR-222 with decreased migration. These miRNAs may represent potential novel targets in migrating glioma cells.

DMBT1 promotes basal and meconium-induced nitric oxide production in human lung epithelial cells in vitro.

Meconium aspiration syndrome (MAS) is characterized by surfactant inactivation and inflammation. As lung epithelial cells up-regulate nitric oxide (NO) in response to inflammation, the NO production following meconium exposition was examined in relation to expression of Deleted in Malignant Brain Tumors 1 (DMBT1), a protein with functions in innate immunity and inflammatory regulation. Here, DMBT1 expression was analyzed by immunohistochemistry in postmortem lung sections from patients with MAS. The lung epithelial cell line A549, stably transfected with a DMBT1 (DMBT1+ cells) expression plasmid or with an empty expression plasmid (DMBT1- cells), was exposed to meconium. NO was determined in dependence of aminoguanidine (inducible NO synthase inhibitor), steroids and lipopolysaccharide (LPS). DMBT1 is highly expressed in lungs with MAS. In the absence of meconium, DMBT1+ cells showed a higher NO production than the DMBT1- cells (p = 0.0090). Meconium led in DMBT1- and DMBT1+ cells to elevated NO levels (p < 0.0001), but with a higher NO level in DMBT1+ cells (p < 0.0001). Aminoguanidine, an iNOS inhibitor, reduced the higher NO production in DMBT1+ cells (p = 0.0476), but NO levels remained above NO production from DMBT1- cells (p = 0.0289). Dexamethasone diminished NO production in DMBT1+ cells after meconium exposition (p = 0.0076). Combined addition of LPS and meconium significantly increased NO production in both cell types (p < 0.0001). In comparison to exposure with only meconium, the combined addition of LPS and meconium to the cells increased NO levels in both DMBT1- cells (p = 0.0030) and DMBT1+ cells (p = 0.0028). In conclusion, basal and meconium-induced NO production in lung epithelial cells is positively regulated by DMBT1.

Bottom up design of nanoparticles for anti-cancer diapeutics: "put the drug in the cancer's food".

The story starts in Basel at CLINAM in 2013, when I asked Pieter about making nanoparticles and he advised me to "try this solvent-exchange method we have developed for making limit sized particles". We are particularly interested in what are "limit size materials" because we want to test the feasibility of an idea: could we design, make, develop, and test the concept for treating metastatic cancer by, "Putting the Drug in the Cancer's Food? "Limit size" is the size of the cancer's food, ? the common Low Density Lipoprotein, (LDL) ~20 nm diameter. In this contribution to Pieter's LTAA we focus on the "bottom" (nucleation) and the "up" (growth) of "bottom-up design" as it applies to homogeneous nucleation of especially, hydrophobic drugs and the 8 physico-chemical stages and associated parameters that determine the initial size, and any subsequent coarsening, of a nanoparticle suspension. We show that, when made by the rapid solvent-exchange method, the same sized particles can be obtained without phospholipid. Furthermore, the obtained size follows the predictions of classic nucleation theory when the appropriate values for the parameters (surface tension and supersaturation) at nucleation are included. Calculations on dissolution time for nanoparticles reveal that a typical fewmicromolar-solubility, hydrophobic, anti-cancer drug (like Lapatinib, Niclosamide, Abiraterone, and Fulvestrant) of 500 nm diameter would take between 3?7 s to dissolve in an infinite sink like the blood stream; and a 50 nm particle would dissolve in less than a second! And so the nanoparticle design requires a highly water-insoluble drug, and a tight, encapsulating, impermeable lipid:cholesterol monolayer. While the "Y" junction can be used to mix an ethanolic solution with anti-solvent, we find that a "no-junction" can give equally good results. A series of nanoparticles (DiI-fluorescently labeled Triolein-cored and drug-cored nanoparticles of Orlistat) were then tested in well-characterized cell lines for uptake and efficacy as well as a PET-imageable nanoparticle in initial PET-imaging studies in animals for EPR uptake and tumor detection. We show that, while free-drug cannot be optimally administered in vivo, a nanoparticle formulation of orlistat could in principle represent a stable parenteral delivery system. The article ends with a brief discussion of what we see as the way forward in Individualized Medicine from the Diagnostic-Therapeutic ("Diapeutic") side, requiring 18FDG detection of metastatic lesions, functional imaging of a protein target (e.g. Fatty Acid Synthase) using 11C acetate, then a PET (or other)-imageable nanoparticle to demonstrate EPR accumulation, and then the administration of the pure-drug nanoparticle taken in by the most aggressive cancer cells in the perivascular space, as they would their "food".

Comprehensive analysis of high-throughput screens with HiTSeekR.

High-throughput screening (HTS) is an indispensable tool for drug (target) discovery that currently lacks user-friendly software tools for the robust identification of putative hits from HTS experiments and for the interpretation of these findings in the context of systems biology. We developed HiTSeekR as a one-stop solution for chemical compound screens, siRNA knock-down and CRISPR/Cas9 knock-out screens, as well as microRNA inhibitor and -mimics screens. We chose three use cases that demonstrate the potential of HiTSeekR to fully exploit HTS screening data in quite heterogeneous contexts to generate novel hypotheses for follow-up experiments: (i) a genome-wide RNAi screen to uncover modulators of TNFα, (ii) a combined siRNA and miRNA mimics screen on vorinostat resistance and (iii) a small compound screen on KRAS synthetic lethality. HiTSeekR is publicly available at http://hitseekr.compbio.sdu.dk It is the first approach to close the gap between raw data processing, network enrichment and wet lab target generation for various HTS screen types.

Shift of microRNA profile upon orthotopic xenografting of glioblastoma spheroid cultures.

Glioblastomas always recur despite surgery, radiotherapy and chemotherapy. A key player in the therapeutic resistance may be immature tumor cells with stem-like properties (TSCs) escaping conventional treatment. A group of promising molecular targets are microRNAs (miRs). miRs are small non-coding RNAs exerting post-transcriptional regulation of gene expression. In this study we aimed to identify over-expressed TSC-related miRs potentially amenable for therapeutic targeting. We used non-differentiated glioblastoma spheroid cultures (GSCs) containing TSCs and compared these to xenografts using a NanoString nCounter platform. This revealed 19 over-expressed miRs in the non-differentiated GSCs. Additionally, non-differentiated GSCs were compared to neural stem cells (NSCs) using a microarray platform. This revealed four significantly over-expressed miRs in the non-differentiated GSCs in comparison to the NSCs. The three most over-expressed miRs in the non-differentiated GSCs compared to xenografts were miR-126, -137 and -128. KEGG pathway analysis suggested the main biological function of these over-expressed miRs to be cell-cycle arrest and diminished proliferation. To functionally validate the profiling results suggesting association of these miRs with stem-like properties, experimental over-expression of miR-128 was performed. A consecutive limiting dilution assay confirmed a significantly elevated spheroid formation in the miR-128 over-expressing cells. This may provide potential therapeutic targets for anti-miRs to identify novel treatment options for GBM patients.

Elevated DMBT1 levels in neonatal gastrointestinal diseases.

Deleted in malignant brain tumor 1 (DMBT1) is involved in innate immunity and epithelial differentiation. Previous studies in adults indicated a strong intestinal expression of DMBT1 and an important role in inflammatory bowel diseases. Here, we analyzed the DMBT1 expression in the fetal gastrointestinal system depending on gestational age and in patients with necrotizing enterocolitis (NEC), volvulus, intestinal perforation (IP), or herniation, representing typical diseases of preterm and term infants. We used immunohistochemistry and RNA in situ hybridization to detect DMBT1 protein and mRNA in fetal tissues, supplemented by postmortem analysis of DMBT1 expression in died newborns and analysis of surgically removed tissues. DMBT1 expression is detectable in the early developmental stages of the gastrointestinal system. In NEC, volvulus, IP, or herniation, characterized by high systemic inflammatory responses, DMBT1 expression is strongly increased. High DMBT1 expression was also found in the bile ducts of older infants with sepsis or cholestasis. The study shows that DMBT1 expression is observed in the developing gastrointestinal system and up-regulated in infants with NEC, volvulus, IP, and herniation. DMBT1 may play a role in epithelial differentiation and local innate immunity during neonatal inflammatory bowel processes.

Development of a cell-based bioassay for phospholipase A2-triggered liposomal drug release.

The feasibility of exploiting secretory phospholipase A2 (sPLA2) enzymes, which are overexpressed in tumors, to activate drug release from liposomes precisely at the tumor site has been demonstrated before. Although the efficacy of the developed formulations was evaluated using in vitro and in vivo models, the pattern of sPLA2-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA2-triggered release of luciferin from liposomes. To this end, we engineered breast cancer cells to produce both luciferase and sPLA2 enzymes, where the latter is secreted to the extracellular medium. We report on setting up a robust and reproducible bioassay for testing sPLA2-sensitive, luciferin remote-loaded liposomal formulations, using 1,2-distearoyl-sn-glycero-3-phosphatidylcholine/1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPC/DSPG) 7:3 and DSPC/DSPG/cholesterol 4:3:3 as initial test systems. Upon their addition to the cells, the liposomes were degraded almost instantaneously by sPLA2 releasing the encapsulated luciferin, which provided readout from the luciferase-expressing cells. Cholesterol enhanced the integrity of the formulation without affecting its susceptibility to sPLA2. PEGylation of the liposomes only moderately broadened the release profile of luciferin. The provided bioassay represents a useful tool for monitoring active drug release in situ in real time as well as for testing and optimizing of sPLA2-sensitive lipid formulations. In addition, the bioassay will pave the way for future in-depth in vitro and in vivo studies.

Deleted in malignant brain tumors 1 (DMBT1) elicits increased VEGF and decreased IL-6 production in type II lung epithelial cells.

BACKGROUND: Deleted in malignant brain tumors 1 (DMBT1) is an innate defence protein expressed in the lungs of preterm infants and adults. Recent studies showed that DMBT1 is important in angiogenesis and can bind to different growth factors including VEGF. We aimed at examining relationships between VEGF and IL-6 levels to DMBT1 expression in the lungs of preterm and term infants and in lung epithelial cells in vitro. METHODS: We examined by ELISA VEGF levels in 120 tracheal aspirates of 57 preterm and term infants and tested for correlation with different perinatal factors as well as with DMBT1 levels. To examine the effect of DMBT1 on VEGF and IL-6 expression we compared type II lung epithelial A549 cells stably transfected with a DMBT1 expression plasmid (DMBT1+ cells) to A549 cells stably transfected with an empty expression plasmid (DMBT1- cells). The concentrations of VEGF and IL-6 were determined via ELISA in the supernatant of the unstimulated cells and after stimulation with LPS, TNFα and Phorbol-12-myristate-13-acetate (PMA). RESULTS: The VEGF levels in the tracheal aspirates of preterm and term infants were significantly correlated with DMBT1 levels (p = 0.0032), the postnatal age (p = 0.0073) and the presence of neonatal infection/sepsis (p = 0.0002). Unstimulated DMBT1+ A549 cells showed significantly higher VEGF expression (p = 0.0017) than DMBT1- cells. Significantly elevated VEGF levels were also confirmed for DMBT1+ cells after stimulation with TNFα (p = 0.0008), LPS (p = 0.0232) and PMA (p = 0.0025). The IL-6 levels were comparable in DMBT1+ versus DMBT1- cells without stimulation (p = 0.6028), but they were significantly reduced in DMBT1+ cells after stimulation with TNFα (p = 0.0003), LPS (p = 0.0088) and PMA (p = 0.0039). CONCLUSIONS: The data indicate that DMBT1 promotes VEGF and suppresses IL-6 production in alveolar tissues, which could point to DMBT1 having a possible role in the transition from inflammation to regeneration and being a potentially useful clinical marker.

Clonal expansion and linear genome evolution through breast cancer progression from pre-invasive stages to asynchronous metastasis.

Evolution of the breast cancer genome from pre-invasive stages to asynchronous metastasis is complex and mostly unexplored, but highly demanded as it may provide novel markers for and mechanistic insights in cancer progression. The increasing use of personalized therapy of breast cancer necessitates knowledge of the degree of genomic concordance between different steps of malignant progression as primary tumors often are used as surrogates of systemic disease. Based on exome sequencing we performed copy number profiling and point mutation detection on successive steps of breast cancer progression from one breast cancer patient, including two different regions of Ductal Carcinoma In Situ (DCIS), primary tumor and an asynchronous metastasis. We identify a remarkable landscape of somatic mutations, retained throughout breast cancer progression and with new mutational events emerging at each step. Our data, contrary to the proposed model of early dissemination of metastatic cells and parallel progression of primary tumors and metastases, provide evidence of linear progression of breast cancer with relatively late dissemination from the primary tumor. The genomic discordance between the different stages of tumor evolution in this patient emphasizes the importance of molecular profiling of metastatic tissue directing molecularly targeted therapy at recurrence.

Tunable CD44-specific cellular retargeting with hyaluronic acid nanoshells.

PURPOSE: In this work we specifically investigate the molecular weight (Mw) dependent combinatorial properties of hyaluronic acid (HA) for exhibiting stealth and targeting properties using different Mw HA nanoshells to tune nanoparticle retargeting to CD44-expressing cancer cells. METHODS: HA of different Mw was covalently grafted onto model polystyrene nanoparticles and advanced surface analysis by X-ray photoelectron spectroscopy performed to quantify and evaluate the effect of the coating procedure. Specific CD44-mediated retargeting was investigated by flow cytometry and confocal microscopy using isogenic D44-deficient and CD44-expressing MCF-7 breast adenocarcinoma cells. RESULTS: Surface analysis demonstrated effective surface coating with 33, 260 and 900 kDa HA resulting in increased colloidal stability and highly negative surface charge due to presentation of up to 4.7% carboxyl groups that indicates an extended and non-constricted HA polymer surface. Reduced non-specific particle interaction in CD44(-) cells was shown for all HA nanoshells but CD44-dependent cellular retargeting and internalization in CD44(+) cells was highly dependent on the coating HA Mw properties. CONCLUSION: The combination of advanced surface characterization and evaluation of particle interactions in isogenic cells with and without CD44 receptor demonstrates direct evidence for the dual capacity of HA for stealth and CD44-mediated retargeting tunable by the HA molecular weight.