Neuregulin 1 expression is a predictive biomarker for response to AV-203, an ERBB3 inhibitory antibody, in human tumor models.

PURPOSE: ERBB3 is overexpressed in a broad spectrum of human cancers, and its aberrant activation is associated with tumor pathogenesis and therapeutic resistance to various anticancer agents. Neuregulin 1 (NRG1) is the predominant ligand for ERBB3 and can promote the heterodimerization of ERBB3 with other ERBB family members, resulting in activation of multiple intracellular signaling pathways. AV-203 is a humanized IgG1/κ ERBB3 inhibitory antibody that completed a first-in-human phase I clinical trial in patients with advanced solid tumors. The purpose of this preclinical study was to identify potential biomarker(s) that may predict response to AV-203 treatment in the clinic. EXPERIMENTAL DESIGN: We conducted in vivo efficacy studies using a broad panel of xenograft models representing a wide variety of human cancers. To identify biomarkers that can predict response to AV-203, the relationship between tumor growth inhibition (TGI) by AV-203 and the expression levels of ERBB3 and NRG1 were evaluated in these tumor models. RESULTS: A significant correlation was observed between the levels of NRG1 expression and TGI by AV-203. In contrast, TGI was not correlated with ERBB3 expression. The correlation between the levels of NRG1 expression in tumors and their response to ERBB3 inhibition by AV-203 was further validated using patient-derived tumor explant models. CONCLUSIONS: NRG1 is a promising biomarker that can predict response to ERBB3 inhibition by AV-203 in preclinical human cancer models. NRG1 warrants further clinical evaluation and validation as a potential predictive biomarker of response to AV-203.

Dynamic cell adhesion and viscoelastic signatures distinguish normal from malignant human mammary cells using quartz crystal microbalance.

During transformation of a normal cell to a cell capable of forming a cancerous growth, cellular morphology, the cytoskeleton, and focal contacts undergo significant changes. These changes should be capable of being characterized via real-time monitoring of the dynamic cell adhesion process and viscoelastic properties of cells. Here, we describe use of the quartz crystal microbalance (QCM) to distinguish the dynamic cell adhesion signatures of human normal (HMEC) versus malignant (MCF-7) mammary epithelial cells. The significantly reduced QCM responses (changes in frequency [Δf] and motional resistance ΔR) of MCF-7 cells compared with those of HMECs mirror the cancer cells' morphological features as observed via optical microscope. We analyzed the initial 2-h cell adhesion kinetics, suggesting cell-cell cooperativity for HMECs and no or weak cell-cell interactions for MCF-7 cells. We propose that changes of the ΔR/Δf ratio, which we term the cell viscoelastic index (CVI), reflect the establishment of cytoskeleton structure and dynamic viscoelastic properties of living cells. The CVI decreases significantly on initiation of cell to surface interactions as cells establish their cytoskeletal structures. During the cell adhesion process, MCF-7 cells were consistently softer, exhibiting up to a 2.5-fold smaller CVI when compared with HMECs.

Extracellular matrix-derived products modulate endothelial and progenitor cell migration and proliferation in vitro and stimulate regenerative healing in vivo.

Most adult mammals heal without restorative replacement of lost tissue and instead form scar tissue at an injury site. One exception is the adult MRL/MpJ mouse that can regenerate ear and cardiac tissue after wounding with little evidence of scar tissue formation. Following production of a MRL mouse ear hole, 2mm in diameter, a structure rapidly forms at the injury site that resembles the amphibian blastema at a limb amputation site during limb regeneration. We have isolated MRL blastemal cells (MRL-B) from this structure and adapted them to culture. We demonstrate by RT-PCR that even after continuous culturing of these cells they maintain expression of several progenitor cell markers, including DLK (Pref-1), and Msx-1. We have isolated the underlying extracellular matrix (ECM) produced by these MRL-B cells using a new non-proteolytic method and studied the biological activities of this cell-free ECM. Multiplex microELISA analysis of MRL-B cell-free ECM vs. cells revealed selective enrichment of growth factors such as bFGF, HGF and KGF in the matrix compartment. The cell-free ECM, degraded by mild enzyme treatment, was active in promoting migration and proliferation of progenitor cells in vitro and accelerating wound closure in a mouse full thickness cutaneous wound assay in vivo. In vivo, a single application of MRL-B cell matrix-derived products to full thickness cutaneous wounds in non-regenerative mice, B6, induced re-growth of pigmented hair, dermis and epidermis at the wound site whereas scar tissue replaced these tissues at wound sites in mice treated with vehicle alone. These studies suggest that matrix-derived products can stimulate regenerative healing and avert scar tissue formation in adult mammals.

Electropolymerized tyrosine-based thin films: selective cell binding via peptide recognition to novel electropolymerized biomimetic tyrosine RGDY films.

We have created thin films by cyclic voltammetry (CV) electropolymerizations of the following phenolic functional group-based monomers: phenol; tyrosineamide; the tetrapeptide RGDY-containing the integrin membrane adhesion protein recognition tripeptide RGD; RDGY, a nonsense control tetrapeptide; and 1:3 mixtures of tyrosineamide with the two tetrapeptide monomers. The film formation process and description of the film properties were obtained by repetitive CV cycling using the oscillating quartz frequency shift, Deltaf, and motional resistance shift, DeltaR, parameters obtained with the electrochemical quartz crystal microbalance technique. Only the poly(phenol) film exhibited close chain packing-based self-limiting behavior, where all film synthesis ceased after approximately 7 CV cycles. All other films continued to form by electropolymerization with successive CV cycles out to the maximum cycle number (30 cycles) we measured. All of the films exhibited little energy dissipation behavior. Using the quartz crystal microbalance, we next compared the time course of cell attachment with the washed films and demonstrated that cells bound best to films in the following order: RGDY sense peptide:tyrosineamide films>RDGY nonsense peptide:tyrosineamide films=tyrosineamide films>phenol films. Cell enumeration after washing and trypsinization revealed firm protein-based cell attachment to the underlying extracellular matrix for the RGDY-containing films. These sense peptide films bound and retained two- to fivefold as many cells as the other films, with cells exhibiting a normal morphology. These results suggest the operation of specific cell attachment to the electropolymerized films via the RGD binding site for cellular integrin membrane proteins. The electropolymerization method we studied here forms a cassette system for creating electropolymerized films tailored to specific attachment of different cell types by varying the electropolymerized Y(tyrosine)-containing recognition peptide.

Degradation products of extracellular matrix affect cell migration and proliferation.

Biologic scaffolds composed of extracellular matrix (ECM) are utilized in numerous regenerative medicine applications to facilitate the constructive remodeling of tissues and organs. The mechanisms by which the host remodeling response occurs are not fully understood, but recent studies suggest that both constituent growth factors and biologically active degradation products derived from ECM play important roles. The objective of the present study was to determine if degradation of ECM scaffold materials in vitro by methods that are biochemically and physiologically relevant can yield products that possess chemotactic and/or mitogenic activities for fully differentiated mammalian endothelial cells and undifferentiated multipotential progenitor cells. ECM harvested from porcine urinary bladder was degraded enzymatically with pepsin/hydrochloric acid or papain. The ECM degradation products were tested for chemoattractant properties utilizing either 48-well chemotaxis filter migration microchambers or fluorescence-based filter migration assays, and were tested for mitogenic properties in cell proliferation assays. Results showed that ECM degradation products possessed chemotactic and mitogenic activities for multipotential progenitor cells and that the same degradation products inhibited both chemotaxis and proliferation of differentiated endothelial cells. These findings support the concept that degradation products of ECM bioscaffolds are important modulators of the recruitment and proliferation of appropriate cell types during the process of ECM scaffold remodeling.

Biocatalytically oligomerized epicatechin with potent and specific anti-proliferative activity for human breast cancer cells.

Catechins, naturally occurring flavonoids derived from wine and green tea, are known to exhibit multiple health benefits. Epigallocatechin gallate (EGCG) is one of the most widely investigated catechins, but its efficacy in cancer therapy is still inconsistent and limited. The poor stability of EGCG has contributed to the disparity in the reported anti-cancer activity and other beneficial properties. Here we report an innovative enzymatic strategy for the oligomerization of catechins (specifically epicatechin) that yields stable, water-soluble oligomerized epicatechins with enhanced and highly specific anti-proliferative activity for human breast cancer cells. This one-pot oxidative oligomerization is carried out in ambient conditions using Horseradish Peroxidase (HRP) as a catalyst yielding water-soluble oligo(epicatechins). The oligomerized epicatechins obtained exhibit excellent growth inhibitory effects against human breast cancer cells with greater specificity towards growth-inhibiting cancer cells as opposed to normal cells, achieving a high therapeutic differential. Our studies indicate that water-soluble oligomeric epicatechins surpass EGCG in stability, selectivity and efficacy at lower doses.

A comparative study of the cytoskeleton binding drugs nocodazole and taxol with a mammalian cell quartz crystal microbalance biosensor: different dynamic responses and energy dissipation effects.

The quartz crystal microbalance (QCM) was used to create piezoelectric whole-cell biosensors utilizing either living endothelial cells (ECs) or the metastatic human mammary cancer cell line MDA-MB-231 adhering to the gold QCM surface under in vitro growth conditions. We utilized the whole-cell QCM biosensors for the detection of the effects of varying concentrations of the microtubule binding drugs taxol and nocodazole by measuring changes in the QCM steady state frequency (Deltaf) and motional resistance (DeltaR), shift values. Using 0.11-50 microM nocodazole, we observed the Deltaf shift values of the biosensors, consisting of 20,000 ECs, to decrease significantly in magnitude (nearly 100%) to a limiting value, in a dose-dependent fashion, over a 5- to 6-h incubation period following drug addition. This effect is consistent with nocodazole's known disruption of intracellular microtubules. On the other hand, 10 microM taxol caused little alteration in Deltaf over the same time period, consistent with its microtubule hyperstabilization effect. When the EC QCM biosensor Deltaf shift values were normalized by the number of ECs found firmly attached to the QCM surface via trypsin removal and electronic counting, the dose curve was shifted to lower nocodazole concentrations, resulting in a more sensitive drug biosensor. The kinetics of the Deltaf decrease with increasing nocodazole concentrations measured by the EC QCM biosensor was found to be similar at all drug concentrations and was well fit by a single first-order exponential decay equation. For all nocodazole doses, t(0.5) was invariant, averaging t(0.5)=0.83+/-0.14 h. These data demonstrate that a single dynamic sensing system within the cell, the microtubules, is disrupted by the addition of nocodazole and this process is sensed by the cell QCM biosensor. This interpretation of the data was confirmed by a fluorescence light microscopy investigation of ECs undergoing treatment with increasing nocodazole doses using a fluorescent antibody to alpha-tubulin. These studies revealed a corresponding loss of the spread morphology of the cells, concomitant with a rearrangement of the extended native microtubules into increasingly large aggregates with the cells eventually lifting from the surface in significant numbers at 50 microM. At 6 microM nocodazole, partial reversibility of the EC QCM biosensor was demonstrated. These results indicate that the EC QCM biosensor can be used to detect and study EC cytoskeleton alterations and dynamics. We suggest the potential of this cellular biosensor for the real-time identification or screening of all classes of biologically active drugs or biological macromolecules that affect cellular attachment and cellular spreading, regardless of their molecular mechanism of action.

Quartz crystal microbalance biosensor study of endothelial cells and their extracellular matrix following cell removal: Evidence for transient cellular stress and viscoelastic changes during detachment and the elastic behavior of the pure matrix.

A quartz crystal microbalance (QCM) cell biosensor utilizing living endothelial cells (ECs) or human breast cancer cells (MCF-7) adhering to the gold QCM surface was used to study the relative contributions of the cells and their underlying extracellular matrix (ECM) to the measured QCM Deltaf and DeltaR shifts. The ECM represents a natural biomaterial that is synthesized by the cells to enable their attachment to surfaces. We followed the detachment of the ECs or MCF-7 cells from their ECM using a nonproteolytic method and were able to apportion the total frequency, Deltaf, decrease of the biosensor into contributions from cell attachment and from the intact underlying ECM. We also demonstrated that the Deltaf shift remaining after EC removal corresponds to ECM as determined by light microscopic visualization of the stained protein. During the process of cell detachment, we observed a novel transient increase in viscoelastic behavior expressed as a transient increase in the motional resistance, DeltaR, parameter. Then we showed via a simulation experiment using ECs stained with fluorescent rhodamine-labeled phalloidin, an actin stain, that the transient viscoelastic increase correlated with cellular stress exhibited by the cells during removal with ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'- tetraacetic acid. Prior to cells lifting from their ECM, the attached ECs rearrange their actin microfilaments first into peripheral stress fibers and second into internal aggregates, to maintain cell-cell connectivity, retain their spread morphology, and attempt to adhere more tightly to their underlying ECM. The decrease in DeltaR following its transient rise corresponds to cells finally losing their attachment focal points and lifting from the ECM. We also characterized the normalized f shifts, -Delta(Deltaf)(ECM)/attached cell and -Delta(Deltaf)(cells)/attached cell, as a function of varying the number of adherent cells. Finally, we demonstrate that the underlying native ECM biomaterial, from which all cells have been removed, does not exhibit any significant level of energy dissipation, in contrast to the cells when they are attached to the ECM.

Detection of apoptosis and drug resistance of human breast cancer cells to taxane treatments using quartz crystal microbalance biosensor technology.

Taxanes are used for the treatment of many human cancers, as first- and second-line chemotherapeutics. In the course of treatment many patients develop resistance or hypersensitivity to one form of taxane and require a different taxane to rescue the therapeutic benefit of the drug. There is currently no method to reliably predict tumor responses to taxanes prior to therapy or when resistance or hypersensitivity develops. We adapted the quartz crystal microbalance (QCM) biosensor technique to study responses of human mammary epithelial tumor cells to taxanes. Studies indicate that stable frequency and resistance levels are reached at 24 h. Cells in the QCM can then be treated with taxanes and responses monitored in real time via frequency and resistance changes reflecting alterations of cell mass distribution and viscoelastic properties. Distinct shifts in frequency and resistance accurately predicted apoptosis or resistance to treatment, as determined in parallel convention assays. QCM analysis accurately predicted docetaxel was more effective than paclitaxel and MCF-7 cells were more resistant to taxanes compared to MDA-MB-231 cells. These studies suggest "signature" patterns for taxane responsivity could be compared to those of patient biopsy samples to predict therapy outcome prior to treatment for initial therapy or to rescue therapy efficacy.