Monolithic Chip for High-throughput Blood Cell Depletion to Sort Rare Circulating Tumor Cells.

Circulating tumor cells (CTCs) are a treasure trove of information regarding the location, type and stage of cancer and are being pursued as both a diagnostic target and a means of guiding personalized treatment. Most isolation technologies utilize properties of the CTCs themselves such as surface antigens (e.g.

Detection of T790M, the Acquired Resistance EGFR Mutation, by Tumor Biopsy versus Noninvasive Blood-Based Analyses.

Purpose: The T790M gatekeeper mutation in the EGFR is acquired by some EGFR-mutant non-small cell lung cancers (NSCLC) as they become resistant to selective tyrosine kinase inhibitors (TKI). As third-generation EGFR TKIs that overcome T790M-associated resistance become available, noninvasive approaches to T790M detection will become critical to guide management.

Experimental Design: As part of a multi-institutional Stand-Up-To-Cancer collaboration, we performed an exploratory analysis of 40 patients with EGFR-mutant tumors progressing on EGFR TKI therapy.

Microfluidic, marker-free isolation of circulating tumor cells from blood samples.

The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen-independent microfluidic CTC-iChip technology. The CTC-iChip uses deterministic lateral displacement, inertial focusing and magnetophoresis to sort up to 10⁷ cells/s.

Inertial focusing for tumor antigen-dependent and -independent sorting of rare circulating tumor cells.

Circulating tumor cells (CTCs) are shed into the bloodstream from primary and metastatic tumor deposits. Their isolation and analysis hold great promise for the early detection of invasive cancer and the management of advanced disease, but technological hurdles have limited their broad clinical utility. We describe an inertial focusing-enhanced microfluidic CTC capture platform, termed "CTC-iChip," that is capable of sorting rare CTCs from whole blood at 10(7) cells/s.

The effect of enzymatically degradable IPN coatings on peri-implant bone formation and implant fixation.

Short-term osseointegration of orthopedic implants is critical for the long-term stability of the implant-bone interface. To improve initial implant stability, one strategy under consideration involves the presentation of adhesion ligands on the implant surface to stimulate bone regeneration in the peri-implant region. To assess the relative effects of implant surface chemistry and topography on osseointegration within the rat femoral ablation implant model, a nonfouling, enzymatically degradable interpenetrating polymer network (edIPN) of poly(AAm-co-EG/AAc) amenable to presenting the cell signaling domain Arg-Gly-Asp (RGD), was developed.

Peri-implant bone formation and implant integration strength of peptide-modified p(AAM-co-EG/AAC) interpenetrating polymer network-coated titanium implants.

Interpenetrating polymer networks (IPNs) of poly (acrylamide-co-ethylene glycol/acrylic acid) functionalized with an -Arg-Gly-Asp- (RGD) containing 15 amino acid peptides, derived from rat bone sialoprotein (bsp-RGD(15), were grafted to titanium implants in an effort to modulate bone formation in the peri-implant region in the rat femoral ablation model. Bone-implant contact (BIC) and bone formation within the medullary canal were determined using microcomputed tomography at 2 and 4 weeks postimplantation. BIC for bsp-RGD(15)-IPN implants was enhanced relative to hydroxyapatite tricalcium phosphate (HA-TCP) coated implants, but was similar to all other groups.

In vitro characterization of peptide-modified p(AAm-co-EG/AAc) IPN-coated titanium implants.

Interpenetrating polymer networks (IPNs) of poly(acrylamide-co-ethylene glycol/acrylic acid) [p(AAm-co-EG/AAc)] functionalized with an -Arg-Gly-Asp- containing peptide derived from rat bone sialoprotein [bsp-RGD(15)] were grafted to titanium implants in an effort to modulate osteoblast behavior in vitro. Surface characterization data were consistent with the presence of an IPN, and ligand density measurements established that the range of peptide density on the modified implants spanned three orders of magnitude (0.01-20 pmol/cm2).

Ligand density characterization of peptide-modified biomaterials.

A simple fluorescence based characterization method was developed to assess ligand density on peptide-modified biomaterials. The method exploits the exquisite sensitivity of proteolysis for the purpose of liberating a fluorescently labeled probe fragment from an immobilized peptide. The released fragment can then be detected in solution using high-throughput fluorometry.

Patterning, prestress, and peeling dynamics of myocytes.

As typical anchorage-dependent cells myocytes must balance contractility against adequate adhesion. Skeletal myotubes grown as isolated strips from myoblasts on micropatterned glass exhibited spontaneous peeling after one end of the myotube was mechanically detached. Such results indicate the development of a prestress in the cells.

Peptide-modified p(AAm-co-EG/AAc) IPNs grafted to bulk titanium modulate osteoblast behavior in vitro.

Interpenetrating polymer networks (IPNs) of poly(acrylamide-co-ethylene glycol/acrylic acid) (p(AAm-co-EG/AAc) applied to model surfaces prevent protein adsorption and cell adhesion. Subsequently, IPN surfaces functionalized with the RGD cell-binding domain from rat bone sialoprotein (BSP) modulated bone cell adhesion, proliferation, and matrix mineralization. The objective of this study was to utilize the same biomimetic modification strategy to produce functionally similar p(AAm-co-EG/AAc) IPNs on clinically relevant titanium surfaces.

Sequential robust design methodology and X-ray photoelectron spectroscopy to analyze the grafting of hyaluronic acid to glass substrates.

Sequential Robust Design experiments and X-ray photoelectron spectroscopic (XPS) studies were performed to examine the immobilization of hyaluronic acid (HA) on glass substrates chemisorbed with N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS). Numerous reaction conditions were investigated, including the concentrations of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (Sulfo-NHS), and HA, and the reaction buffer type, concentration, and pH. The elemental surface compositions of carbon and silicon (C/Si ratio) were used to assess the extent of HA immobilization, leading to the identification of critical HA-binding reaction conditions and the determination of an optimum surface chemistry.