Label-free enrichment of rare unconventional circulating neoplastic cells using a microfluidic dielectrophoretic sorting device.

Cellular circulating biomarkers from the primary tumor such as circulating tumor cells (CTCs) and circulating hybrid cells (CHCs) have been described to harbor tumor-like phenotype and genotype. CHCs are present in higher numbers than CTCs supporting their translational potential. Methods for isolation of CHCs do not exist and are restricted to low-throughput, time consuming, and biased methodologies. We report the development of a label-free dielectrophoretic microfluidic platform facilitating enrichment of CHCs in a high-throughput and rapid fashion by depleting healthy peripheral blood mononuclear cells (PBMCs). We demonstrated up to 96.5% depletion of PBMCs resulting in 18.6-fold enrichment of cancer cells. In PBMCs from pancreatic adenocarcinoma patients, the platform enriched neoplastic cells identified by their KRAS mutant status using droplet digital PCR with one hour of processing. Enrichment was achieved in 75% of the clinical samples analyzed, establishing this approach as a promising way to non-invasively analyze tumor cells from patients.

Relevance of circulating hybrid cells as a non-invasive biomarker for myriad solid tumors.

Metastatic progression defines the final stages of tumor evolution and underlies the majority of cancer-related deaths. The heterogeneity in disseminated tumor cell populations capable of seeding and growing in distant organ sites contributes to the development of treatment resistant disease. We recently reported the identification of a novel tumor-derived cell population, circulating hybrid cells (CHCs), harboring attributes from both macrophages and neoplastic cells, including functional characteristics important to metastatic spread. These disseminated hybrids outnumber conventionally defined circulating tumor cells (CTCs) in cancer patients. It is unknown if CHCs represent a generalized cancer mechanism for cell dissemination, or if this population is relevant to the metastatic cascade. Herein, we detect CHCs in the peripheral blood of patients with cancer in myriad disease sites encompassing epithelial and non-epithelial malignancies. Further, we demonstrate that in vivo-derived hybrid cells harbor tumor-initiating capacity in murine cancer models and that CHCs from human breast cancer patients express stem cell antigens, features consistent with the potential to seed and grow at metastatic sites. Finally, we reveal heterogeneity of CHC phenotypes reflect key tumor features, including oncogenic mutations and functional protein expression. Importantly, this novel population of disseminated neoplastic cells opens a new area in cancer biology and renewed opportunity for battling metastatic disease.

Gas-Stabilizing Sub-100 nm Mesoporous Silica Nanoparticles for Ultrasound Theranostics.

Recent studies have demonstrated that gas-stabilizing particles can generate cavitating micron-sized bubbles when exposed to ultrasound, offering excellent application potential, including ultrasound imaging, drug delivery, and tumor ablation. However, the majority of the reported gas-stabilizing particles are relatively large (>200 nm), and smaller particles require high acoustic pressures to promote cavitation. Here, this paper reports the preparation of sub-100 nm gas-stabilizing nanoparticles (GSNs) that can initiate cavitation at low acoustic intensities, which can be delivered using a conventional medical ultrasound imaging system. The highly echogenic GSNs (F127-hMSN) were prepared by carefully engineering the surfaces of ∼50 nm mesoporous silica nanoparticles. It was demonstrated that the F127-hMSNs could be continuously imaged with ultrasound in buffer or biological solutions or agarose phantoms for up to 20 min. Also, the F127-hMSN can be stored in phosphate-buffered saline for at least a month with no loss in ultrasound responsiveness. The particles significantly degraded when diluted in simulated body fluids, indicating possible biodegradation of the F127-hMSNs in vivo. Furthermore, at ultrasound imaging conditions, F127-hMSNs did not cause detectable cell death, supporting the potential safety of these particles. Finally, strong cavitation activity generation by the F127-hMSNs under high-intensity focused ultrasound insonation was demonstrated and applied to effectively ablate cancer cells.

Silica cloaking of adenovirus enhances gene delivery while reducing immunogenicity.

Viral gene therapy is a means of delivering genes to replace malfunctioning ones, to kill cancer cells, or to correct genetic mutations. This technology is emerging as a powerful clinical tool; however, it is still limited by viral tropism, uptake and clearance by the liver, and most importantly an immune response. To overcome these challenges, we sought to merge the robustness of viral gene expression and the versatility of nanoparticle technology. Here, we describe a method for cloaking adenovirus (Ad) in silica (SiAd) as a nanoparticle formulation that significantly enhances transduction. Intratumoral injections in human glioma xenografts revealed SiAd expressing luciferase improved tumor transduction while reducing liver uptake. In immune-competent mice SiAd induced no inflammatory cytokines and reduced production of neutralizing antibodies. Finally, SiAd expressing TNF-related apoptosis-inducing ligand inhibited tumor growth of glioma xenografts. These results reveal that silica cloaking of Ad can enhance viral gene delivery while reducing immunogenicity.

Optimized Scintillator YAG:Pr Nanoparticles for X-ray Inducible Photodynamic Therapy.

We describe a sol-gel synthetic method for the production of praseodymium-doped yttrium aluminum garnet (YAG) nanoparticles suitable for X-ray inducible photodynamic therapy (X-PDT). Our sol-gel based approach was optimized by varying temperature and time of calcination, resulting in nanoparticles that were smooth, spherical, and 50-200 nm in crystallite size. The powders were uniformly coated with a thin (10 nm) layer of silica to facilitate surface conjugation with functional moieties. Measurements of photon flux revealed that coated and uncoated powders emitted a similar photon emission spectrum in response to 50 keVp X-rays. We also determined that the presence of silica did not significantly reduce flux and the emission peak had a maximum at approximately 320 nm. Thus, these YAG:Pr powders are suitable candidates for future in vivo X-PDT studies.

Selection of DNA nanoparticles with preferential binding to aggregated protein target.

High affinity and specificity are considered essential for affinity reagents and molecularly-targeted therapeutics, such as monoclonal antibodies. However, life's own molecular and cellular machinery consists of lower affinity, highly multivalent interactions that are metastable, but easily reversible or displaceable. With this inspiration, we have developed a DNA-based reagent platform that uses massive avidity to achieve stable, but reversible specific recognition of polyvalent targets. We have previously selected these DNA reagents, termed DeNAno, against various cells and now we demonstrate that DeNAno specific for protein targets can also be selected. DeNAno were selected against streptavidin-, rituximab- and bevacizumab-coated beads. Binding was stable for weeks and unaffected by the presence of soluble target proteins, yet readily competed by natural or synthetic ligands of the target proteins. Thus DeNAno particles are a novel biomolecular recognition agent whose orthogonal use of avidity over affinity results in uniquely stable yet reversible binding interactions.