Amin Aalipour

Quantification of nanowire penetration into living cells

High-aspect ratio nanostructures such as nanowires and nanotubes are a powerful new tool for accessing the cell interior for delivery and sensing. Controlling and optimizing cellular access is a critical challenge for this new technology, yet even the most basic aspect of this process, whether these structures directly penetrate the cell membrane, is still unknown. Here we report the first quantification of hollow nanowires-nanostraws-that directly penetrate the membrane by observing dynamic ion delivery from each 100-nm diameter nanostraw. We discover that penetration is a rare event: 7.1±2.7% of the nanostraws penetrate the cell to provide cytosolic access for an extended period for an average of 10.7±5.8 penetrations per cell. Using time-resolved delivery, the kinetics of the first penetration event are shown to be adhesion dependent and coincident with recruitment of focal adhesion-associated proteins. These measurements provide a quantitative basis for understanding nanowire-cell interactions, and a means for rapidly assessing membrane penetration.

Bruton tyrosine kinase inhibitors: a promising novel targeted treatment for B cell lymphomas

Constitutive or aberrant signalling of the B cell receptor signalling cascade has been implicated in the propagation and maintenance of a variety of B cell malignancies. Small molecule inhibitors of Bruton tyrosine kinase (BTK), a protein early in this cascade and specifically expressed in B cells, have emerged as a new class of targeted agents. There are several BTK inhibitors, including ONO‐WG‐307, LFM‐A13, dasatinib, CC‐292, and PCI‐32765 (ibrutinib), in preclinical and/or clinical development of which ibrutinib is currently in phase III trials. Recent clinical data suggest significant activity of ibrutinib as a first in class oral inhibitor of BTK. This review provides an overview of ongoing clinical studies of BTK inhibitors.

Towards clinically translatable in vivo nanodiagnostics

Nanodiagnostics as a field makes use of fundamental advances in nanobiotechnology to diagnose, characterize and manage disease at the molecular scale. As these strategies move closer to routine clinical use, a proper understanding of different imaging modalities, relevant biological systems and physical properties governing nanoscale interactions is necessary to rationally engineer next-generation bionanomaterials. In this Review, we analyse the background physics of several clinically relevant imaging modalities and their associated sensitivity and specificity, provide an overview of the materials currently used for in vivo nanodiagnostics, and assess the progress made towards clinical translation. This work provides a framework for understanding both the impressive progress made thus far in the nanodiagnostics field as well as presenting challenges that must be overcome to obtain widespread clinical adoption.

Determining the Time Window for Dynamic Nanowire Cell Penetration Processes.

Nanowire (NW) arrays offer opportunities for parallel, nondestructive intracellular access for biomolecule delivery, intracellular recording, and sensing. Spontaneous cell membrane penetration by vertical nanowires is essential for these applications, yet the time- and geometry-dependent penetration process is still poorly understood. In this work, the dynamic NW-cell interface during cell spreading was examined through experimental cell penetration measurements combined with two mechanical models based on substrate adhesion force or cell traction forces. Penetration was determined by comparing the induced tension at a series of given membrane configurations to the critical membrane failure tension. The adhesion model predicts that penetration occurs within a finite window shortly after initial cell contact and adhesion, while the traction model predicts increasing penetration over a longer period. NW penetration rates determined from a cobalt ion delivery assay are compared to the predicted results from the two models. In addition, the effects of NW geometry and cell properties are systematically evaluated to identify the key factors for penetration.

Bruton’s tyrosine kinase inhibitors and their clinical potential in the treatment of B-cell malignancies: focus on ibrutinib:

Aberrant signaling of the B-cell receptor pathway has been linked to the development and maintenance of B-cell malignancies. Bruton’s tyrosine kinase (BTK), a protein early in this pathway, has emerged as a new therapeutic target in a variety of such malignancies. Ibrutinib, the most clinically advanced small molecule inhibitor of BTK, has demonstrated impressive tolerability and activity in a range of B-cell lymphomas which led to its recent approval for relapsed mantle cell lymphoma and chronic lymphocytic leukemia. This review focuses on the preclinical and clinical development of ibrutinib and discusses its therapeutic potential.

Molecular profiling of single circulating tumor cells from lung cancer patients

Significance There exists an urgent need for minimally invasive molecular analysis tools for cancer assessment and management, particularly in advanced-stage lung cancer, when tissue procurement is challenging and gene mutation profiling is crucial to identify molecularly targeted agents for treatment. High-throughput compartmentalization and multigene profiling of individual circulating tumor cells (CTCs) from whole-blood samples using modular gene panels may facilitate highly sensitive, yet minimally invasive characterization of lung cancer for therapy prediction and monitoring. We envision this nanoplatform as a compelling research tool to investigate the dynamics of cancer disease processes, as well as a viable clinical platform for minimally invasive yet comprehensive cancer assessment. Circulating tumor cells (CTCs) are established cancer biomarkers for the “liquid biopsy” of tumors. Molecular analysis of single CTCs, which recapitulate primary and metastatic tumor biology, remains challenging because current platforms have limited throughput, are expensive, and are not easily translatable to the clinic. Here, we report a massively parallel, multigene-profiling nanoplatform to compartmentalize and analyze hundreds of single CTCs. After high-efficiency magnetic collection of CTC from blood, a single-cell nanowell array performs CTC mutation profiling using modular gene panels. Using this approach, we demonstrated multigene expression profiling of individual CTCs from non–small-cell lung cancer (NSCLC) patients with remarkable sensitivity. Thus, we report a high-throughput, multiplexed strategy for single-cell mutation profiling of individual lung cancer CTCs toward minimally invasive cancer therapy prediction and disease monitoring.

Plasma Membrane and Actin Cytoskeleton as Synergistic Barriers to Nanowire Cell Penetration

Nanowires are a rapidly emerging platform for manipulation of and material delivery directly into the cell cytosol. These high aspect ratio structures can breach the lipid membrane; however, the yield of penetrant structures is low, and the mechanism is largely unknown. In particular, some nanostructures appear to defeat the membrane transiently, while others can retain long-term access. Here, we examine if local dissolution of the lipid membrane, actin cytoskeleton, or both can enhance nanowire penetration. It is possible that, during cell contact, membrane rupture occurs; however, if the nanostructures do not penetrate the cytoskeleton, the membrane may reclose over a relatively short time frame. We show with quantitative analysis of the number of penetrating nanowires that the lipid bilayer and actin cytoskeleton are synergistic barriers to nanowire cell access, yet chemical poration through both is still insufficient to increase long-term access for adhered cells.

An intravascular magnetic wire for the high-throughput retrieval of circulating tumour cells in vivo

The detection and analysis of rare blood biomarkers is necessary for early diagnosis of cancer and to facilitate the development of tailored therapies. However, current methods for the isolation of circulating tumour cells (CTCs) or nucleic acids present in a standard clinical sample of only 5–10 ml of blood provide inadequate yields for early cancer detection and comprehensive molecular profiling. Here, we report the development of a flexible magnetic wire that can retrieve rare biomarkers from the subject’s blood in vivo at a much higher yield. The wire is inserted and removed through a standard intravenous catheter and captures biomarkers that have been previously labelled with injected magnetic particles. In a proof-of-concept experiment in a live porcine model, we demonstrate the in vivo labelling and single-pass capture of viable model CTCs in less than 10 s. The wire achieves capture efficiencies that correspond to enrichments of 10–80 times the amount of CTCs in a 5-ml blood draw, and 500–5,000 times the enrichments achieved using the commercially available Gilupi CellCollector.A magnetic wire for the intravascular recovery of labelled circulating tumour cells improves cell capture in anaesthetized pigs by up to two orders of magnitude with respect to a standard blood draw.

Deactivated CRISPR Associated Protein 9 for Minor-Allele Enrichment in Cell-Free DNA

BACKGROUND Cell-free DNA (cfDNA) diagnostics are emerging as a new paradigm of disease monitoring and therapy management. The clinical utility of these diagnostics is relatively limited by a low signal-to-noise ratio, such as with low allele frequency (AF) mutations in cancer. While enriching for rare alleles to increase their AF before sample analysis is one strategy that can greatly improve detection capability, current methods are limited in their generalizability, ease of use, and applicability to point mutations. METHODS Leveraging the robust single-base-pair specificity and generalizability of the CRISPR associated protein 9 (Cas9) system, we developed a deactivated Cas9 (dCas9)-based method of minor-allele enrichment capable of efficient single-target and multiplexed enrichment. The dCas9 protein was complexed with single guide RNAs targeted to mutations of interest and incubated with cfDNA samples containing mutant strands at low abundance. Mutation-bound dCas9 complexes were isolated, dissociated, and the captured DNA purified for downstream use. RESULTS Targeting the 3 most common epidermal growth factor receptor mutations (exon 19 deletion, T790M, L858R) found in non-small cell lung cancer (NSCLC), we achieved >20-fold increases in AF and detected mutations by use of qPCR at an AF of 0.1%. In a cohort of 18 NSCLC patient-derived cfDNA samples, our method enabled detection of 8 out of 13 mutations that were otherwise undetected by qPCR. CONCLUSIONS The dCas9 method provides an important application of the CRISPR/Cas9 system outside the realm of genome editing and can provide a step forward for the detection capability of cfDNA diagnostics.

Abstract 3796: An intravascular magnetic wire for high-throughputin vivoenrichment of rare circulating cancer biomarkers

Background: Liquid biopsies have long promised to enable earlier cancer diagnosis and tailored therapy. However, circulating tumor cells (CTCs) are extremely rare (1-10 cells per mL blood), limiting their clinical utility. There are too few CTCs in a standard 5-10 mL blood sample for culture and drug susceptibility testing, or for comprehensively profiling a molecularly heterogeneous cancer and its metastases for drug resistance mutations. To achieve large-scale CTC enrichment, new strategies are needed that can rapidly and effectively interrogate large blood volumes. Aim: To design a flexible magnetic wire capable of high-throughput intravascular enrichment and retrieval of rare biomarkers, including CTCs, from the entire circulating blood volume to attain a much higher biomarker yield for earlier cancer detection and personalized treatment. Methods: We present a promising platform for in vivo enrichment of rare biomarkers, the Magnetic Wire for Intravascular Retrieval and Enrichment (MagWIRE): a flexible, self-contained magnetic wire consisting of a string of small magnets with alternating polarities, achieving high local field gradients along its entire length to capture magnetically labeled targets from a large surrounding volume. The platform is proposed to work as follows: Blood biomarkers are immunomagnetically labeled by injecting a patient with antibody-coated magnetic particles (MPs), similar to FDA-approved Feraheme®. The MagWIRE is then inserted through a standard IV catheter into a superficial vein in the arm or through an existing chemotherapy port to magnetically capture passing MP-bound biomarkers. Within an hour, ~5 liters has circulated through a 2-3-mm-diameter human vein, allowing most of the patient’s blood volume to be sampled. The magnets can then be displaced from the MagWIRE sheath to elute the bound targets into buffer for downstream analysis. We performed proof-of-concept demonstrations in: 1) a closed-loop blood circulation system consisting of a pump, tubing, and a blood reservoir, and 2) in vivo within a porcine ear vein model. To model CTC capture, we targeted H1650 lung cancer cells with 1-μm superparamagnetic iron oxide particles coated with antibodies against epithelial cell adhesion molecule (EpCAM), a commonly used CTC marker. We couple the MagWIRE with a unique approach for rapid ( Results: Considerable gains are achievable by sampling from large volumes, even at relatively low capture efficiencies. In our closed-loop setup, the MagWIRE demonstrated capture efficiencies in whole blood of 56.14+/-15.80% for pre-labeled cells and 10.17+/-5.41% for cells labeled in flow. In a porcine ear model, we captured cells with efficiencies up to 8%, corresponding to 80-fold enrichment when integrated over a 5-liter blood volume compared with a 5 mL blood draw. Citation Format: Ophir Vermesh, Amin Aalipour, Tianjia J. Ge, Yamil Saenz, Yue Guo, Seung-min Park, Yoshiaki Mitsutake, Michael Bachmann, Chin Chun Ooi, Kerstin Mueller, Hamed Arami, Alfredo Green, Shan X. Wang, Sanjiv S. Gambhir. An intravascular magnetic wire for high-throughput in vivo enrichment of rare circulating cancer biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3796. doi:10.1158/1538-7445.AM2017-3796