Cellular heterogeneity profiling by hyaluronan probes reveals an invasive but slow-growing breast tumor subset

Tumor heterogeneity confounds cancer diagnosis and the outcome of therapy, necessitating analysis of tumor cell subsets within the tumor mass. Elevated expression of hyaluronan (HA) and HA receptors, receptor for HA-mediated motility (RHAMM)/HA-mediated motility receptor and cluster designation 44 (CD44), in breast tumors correlates with poor outcome. We hypothesized that a probe for detecting HA–HA receptor interactions may reveal breast cancer (BCa) cell heterogeneity relevant to tumor progression. A fluorescent HA (F-HA) probe containing a mixture of polymer sizes typical of tumor microenvironments (10–480 kDa), multiplexed profiling, and flow cytometry were used to monitor HA binding to BCa cell lines of different molecular subtypes. Formulae were developed to quantify binding heterogeneity and to measure invasion in vivo. Two subsets exhibiting differential binding (HA^−/low vs. HA^high) were isolated and characterized for morphology, growth, and invasion in culture and as xenografts in vivo. F-HA–binding amounts and degree of heterogeneity varied with BCa subtype, were highest in the malignant basal-like cell lines, and decreased upon reversion to a nonmalignant phenotype. Binding amounts correlated with CD44 and RHAMM displayed but binding heterogeneity appeared to arise from a differential ability of HA receptor-positive subpopulations to interact with F-HA. HA^high subpopulations exhibited significantly higher local invasion and lung micrometastases but, unexpectedly, lower proliferation than either unsorted parental cells or the HA^−/low subpopulation. Querying F-HA binding to aggressive tumor cells reveals a previously undetected form of heterogeneity that predicts invasive/metastatic behavior and that may aid both early identification of cancer patients susceptible to metastasis, and detection/therapy of invasive BCa subpopulations.Breast tumors display substantial heterogeneity driven by genetic and epigenetic mechanisms (1–3). These processes select and support tumor cell subpopulations with distinct phenotypes in proliferation, metastatic/invasive proclivity, and treatment susceptibility that contribute to clinical outcomes. Currently, there is a paucity of biomarkers to identify these subpopulations (3–12). Although detection of genetic heterogeneity may itself be a breast cancer (BCa) prognostic marker (3, 13–15), the phenotypes manifested from this diversity are context-dependent. Therefore, phenotypic markers provide additional powerful tools for biological information required to design diagnostics and therapeutics. Glycomic approaches have enormous potential for revealing tumor cell phenotypic heterogeneity because glycans are themselves highly heterogeneous and their complexity reflects the nutritional, microenvironmental, and genetic dynamics of the tumors (16–18).We used hyaluronan (HA) as a model carbohydrate ligand for probing heterogeneity in glycosaminoglycan–BCa cell receptor interactions. We reasoned this approach would reveal previously undetected cellular and functional heterogeneity linked to malignant progression because the diversity of cell glycosylation patterns, which can occur as covalent and noncovalent modifications of proteins and lipids as well as different sizes of such polysaccharides as HA, is unrivaled (16, 17, 19). In particular, tumor and wound microenvironments contain different sizes of HA polymers that bind differentially to cell receptors to activate signaling pathways regulating cell migration, invasion, survival, and proliferation (19–22).More than other related glycosaminoglycans, HA accumulation within BCa tumor cells and peritumor stroma is a predictor of poor outcome (23) and of the conversion of the preinvasive form of BCa, ductal carcinoma in situ, to an early invasive form of BCa (24). HA is a nonantigenic and large, relatively simple, unbranched polymer, but the manner in which it is metabolized is highly complex (19, 25). There are literally thousands of different HA sizes in remodeling microenvironments, including tumors. HA polymers bind to cells via at least six known receptors (16, 19, 20, 26–32). Two of these, cluster designation 44 (CD44) and receptor for HA-mediated motility/HA-mediated motility receptor (RHAMM/HMMR), form multivalent complexes with different ranges of HA sizes (19, 29, 33), and both receptors are implicated in BCa progression (19–21, 23, 29, 30, 33–36). Elevated CD44 expression in the peritumor stroma is associated with increased relapse (37), and in primary BCa cell subsets may contribute to tumor initiation and progression (38–40). Elevated RHAMM expression in BCa tumor subsets is a prognostic indicator of poor outcome and increased metastasis (22, 33, 41). RHAMM polymorphisms may also be a factor in BCa susceptibility (42, 43).We postulated that multivalent interactions resulting from mixture of a polydisperse population of fluorescent HA (F-HA) sizes, typical of those found in remodeling microenvironments of wounds and tumors (19, 20, 29), with cellular HA receptors would uncover a heterogeneous binding pattern useful for sorting tumor cells into distinct subsets. We interrogated the binding of F-HA to BCa lines of different molecular subtypes, and related binding/uptake patterns to CD44 and RHAMM display, and to tumor cell growth, invasion, and metastasis.     

Chitosan-Alginate Scaffold Culture System for Hepatocellular Carcinoma Increases Malignancy and Drug Resistance

Purpose  

Hepatocellular carcinoma (HCC) is a prevalent solid malignancy. Critically needed discovery of new therapeutics has been hindered by lack of an in vitro cell culture system that can effectively represent the in vivo tumor microenvironment. To address this need, a 3D in vitro HCC model was developed using a biocompatible, chitosan-alginate (CA) scaffold cultured with human HCC cell lines.     

Clinical characteristics predict response to antithymocyte globulin in paroxysmal nocturnal haemoglobinuria

Seven patients with paroxysmal nocturnal haemoglobinuria (PNH) were treated with antithymocyte globulin (ATG). Each patient received ATG (20 mg/kg/d) for 8 d and prednisone to prevent or control serum sickness. Three patients experienced a sustained improvement in at least one peripheral blood cytopenia, including one patient who had a complete trilineage response. Several pre-treatment clinical features appeared to be associated with response to ATG. All responding patients had hypoproliferative features including depressed platelet counts (<30×10⁹/l), and a minor degree of chronic haemolysis as indicated by relatively low reticulocyte counts (<100×10⁹/l), lactate dehydrogenase (<1000 U/l) and total bilirubin (<17μmol/l) levels. Responding patients continued to have chronic low-grade haemolysis after their response to immunosuppression that was similar to that observed prior to treatment. The non-responding patients had a classic haemolytic form of PNH characterized by elevated reticulocyte counts (>100×10⁹/l), lactate dehydrogenase (>2000 U/l) and total bilirubin (>17μmol/l) levels.
The impaired haemopoiesis that occurs in hypoproliferative PNH may respond to ATG treatment, but the haemolytic component of the disease, and hence the PNH clone, is not altered by immunosuppressive therapy.     

Two-Dimensional Protein Micropatterning for Sensor Applications Through Chemical Selectivity Technique

Two-dimensional protein micropatterning with immobil-ization of IgG and poly (ethylene glycol) (PEG) on patterned Au and Si surfaces was performed through a new technique. The technique for micropatterning is based on a chemical selectivity method by creating chemical bonding between protein, self-assembled monolayers (SAMs) and substrates rather than physical means. The substrates used in this study are pre-fabricated with silicon wafer patterned with arrays of gold squares. The silicon regions of the substrate are modified with polyethylene glycol (PEG) to resist protein adsorption and cell adhesion. The gold regions on the substrate are first immobilized with bifunctional SAM layers that can covalently bound adhesion proteins for individual cell attachment against a PEG background. The surface coatings are characterized by contact angle measurement, ellips-ometry, and atomic force microscopy (AFM). The patterns of fluorescence-labeled proteins are examined using fluorescence microscopy. Our study demonstrated that the PEG modified silicon region showed an effective protein reduction while the gold regions were successfully covalently bonded with proteins. This technique also demonstrated a combined feature of ensuring the activity, selectivity, and stability of the immobilized proteins. A simple lift-off microfabrication process was introduced in this study to pattern metal on silicon substrates without using expensive metal etching.     

A ligand-mediated nanovector for targeted gene delivery and transfection in cancer cells

As conventional cancer therapies struggle with toxicity issues and irregular remedial efficacy, the preparation of novel gene therapy vectors could offer clinicians the tools for addressing the genetic errors of diseased tissue. The transfer of gene therapy to the clinic has proven difficult due to safety, target specificity, and transfection efficiency concerns. Polyethylenimine (PEI) nanoparticles have been identified as promising gene carriers that induce gene transfection with high efficiency. However, the inherent toxicity of the material and non-selective delivery are the major concerns in applying these particles clinically. Here, a non-viral nanovector has been developed by PEGylation of DNA-complexing PEI in nanoparticles functionalized with an Alexa Fluor 647 near infrared fluorophore, and the chlorotoxin (CTX) peptide which binds specifically to many forms of cancer. With this nanovector, the potential toxicity to healthy cells is minimized by both the reduction of the toxicity of PEI with the biocompatible copolymer and the targeted delivery of the nanovector to cancer cells, as evaluated by viability studies. The nanovector demonstrated high levels of targeting specificity and gene transfection efficiency with both C6 glioma and DAOY medulloblastoma tumor cells. Significantly, with the CTX as the targeting ligand, the nanovector may serve as a widely applicable gene delivery system for a broad array of cancer types.     

Corticotropin-releasing factor acts on the brain to reduce gastric contractility

Various stressors (cold restraint, electric shock, etc.) applied to rats increase gastric contractility and are associated with gastric erosions. Intracisternal (i.c.) thyrotropin-releasing hormone (TRH) increases contractility, gastric acid secretion and the incidence of erosion formation. Corticotropin-releasing hormone (CRF) is released by stress, and acting centrally produces autonomic and endocrine changes. We have studied the role of i.c. CRF on gastric contractility in anesthetized rats (n = 6). Contractility was measured by extraluminal force transducers sutured to the gastric corpus. Frequency, amplitude of contractions and a motility index were analyzed by computer. Baseline measures, after recovery from surgery were obtained in 24-hour fasted rats. Contractility was stimulated by intravenous carbachol (100 mg/kg/h) or i.c. injection of the TRH analog, RX 77368. Contractility thus induced was inhibited by intravenous atropine (1 mg/kg). CRF (30-1,000 ng i.c.) produced a dose-dependent suppression of RX 77368 (p less than 0.05) but had no effect on that induced by intravenous carbachol; saline i.c. had none either. Intravenous CRF was 1/10th as potent in suppressing contractions produced by i.c. RX 77368. Diminution of gastric contractions after i.c. CRF (1 mg) occurred within 5 min of administration, and lasted for at least 60 min. These data show that i.c. CRF injection acts centrally to inhibit gastric contractions stimulated centrally (i.e. by i.c. RX 77368) but not peripherally (i.e. by carbachol), and by inference reduces the risk of erosion formation induced by some stressors.     

Design and fabrication of magnetic nanoparticles for targeted drug delivery and imaging

Magnetic nanoparticles (MNPs) represent a class of non-invasive imaging agents that have been developed for magnetic resonance (MR) imaging. These MNPs have traditionally been used for disease imaging via passive targeting, but recent advances have opened the door to cellular-specific targeting, drug delivery, and multi-modal imaging by these nanoparticles. As more elaborate MNPs are envisioned, adherence to proper design criteria (e.g. size, coating, molecular functionalization) becomes even more essential. This review summarizes the design parameters that affect MNP performance in vivo, including the physicochemical properties and nanoparticle surface modifications, such as MNP coating and targeting ligand functionalizations that can enhance MNP management of biological barriers. A careful review of the chemistries used to modify the surfaces of MNPs is also given, with attention paid to optimizing the activity of bound ligands while maintaining favorable physicochemical properties.     

Nucleic acid-mediated intracellular protein delivery by lipid-like nanoparticles

Intracellular protein delivery has potential biotechnological and therapeutic application, but remains technically challenging. In contrast, a plethora of nucleic acid carriers have been developed, with lipid-based nanoparticles (LNPs) among the most clinically advanced reagents for oligonucleotide delivery. Here, we validate the hypothesis that oligonucleotides can serve as packaging materials to facilitate protein entrapment within and intracellular delivery by LNPs. Using two distinct model proteins, horseradish peroxidase and NeutrAvidin, we demonstrate that LNPs can yield efficient intracellular protein delivery in vitro when one or more oligonucleotides have been conjugated to the protein cargo. Moreover, in experiments with NeutrAvidin in vivo, we show that oligonucleotide conjugation significantly enhances LNP-mediated protein uptake within various spleen cell populations, suggesting that this approach may be particularly suitable for improved delivery of protein-based vaccines to antigen-presenting cells.     

Guided cell patterning on gold-silicon dioxide substrates by surface molecular engineering

We report an effective approach to patterning cells on gold-silicon dioxide substrates with high precision, selectivity, stability, and reproducibility. This technique is based on photolithography and surface molecular engineering and requires no cell positioning or delivery devices, thus significantly reducing the potential damage to cells. The cell patterning was achieved by activating the gold regions of the substrate with functionalized thiols that covalently bind proteins onto the gold regions to guide subsequent cell adhesion while passivating the silicon dioxide background with polyethylene glycol to resist cell adhesion. Fourier transform infrared reflectance spectroscopy verified the successful immobilization of proteins on gold surfaces. Protein patterns were visualized by tagging proteins with Rhodamine fluorescent probes. Time-of-flight secondary ion mass spectrometry was used to characterize the chemistry of both the cell-adhesive and cell-resistant regions of surfaces after each key chemical reaction occurring during the molecular surface engineering. The ability of the engineered surfaces to guide cell adhesion was illustrated by differential interference contrast (DIC) reflectance microscopy. The cell patterning technique introduced in this study is compatible with micro- and photo-electronics, and may have many medical, environmental, and defense applications.