Earlier Detection and Diagnosis

Imaging In Vivo

Current imaging methods can only detect cancers once they have made a visible change to a tissue, by which time, thousands of cells will have proliferated and perhaps metastasized. And even when visible, the nature of the tumor—malignant or benign—and the characteristics that might make it responsive to a particular treatment must be assessed through tissue biopsies. Furthermore, while some primary malignancies can be determined to be metastatic, tumor pre-seeding of metastatic sites and micro-metastases are extremely difficult to detect with modern imaging modalities, even if the tissue in which they commonly occur are known, a priori. Finally, surgical resection of tumor tissue remains the standard of care for many tumor types and surgeons must weigh the consequences of removing often vital healthy tissue versus the cancerous mass which has grown non-uniformly within. Ultimately, removal of cancer cells at the single cell level is not possible with current surgical techniques.

Nanotechnology based imaging contrast agents being developed and translated today, offer the ability to specifically target and greatly enhance detection of tumor in vivo by way of conventional scanning devices, such as magnetic resonance imaging (MRI), (PET), and computed tomography (CT). Moreover, current nanoscale imaging platforms are enabling novel imaging modalities not traditional utilized for clinical cancer treatment and diagnosis, for example photoacoustic tomography (PAT), Raman spectroscopic imaging and multimodal imaging (i.e., contrast agents specific to several imaging modalities simultaneously). Nanotechnology enables all of these platforms by way of its ability to carry multiple components simultaneously (e.g., cancer cell-specific targeting agents or traditional imaging contrast agents) and nanoscale materials that are themselves the contrast agents of which enable greatly enhanced signal.

NCI-funded research has produced many notable examples over the last several years. For example, researchers at Stanford University and Memorial Sloan Kettering Cancer Center developed multimodal nanoparticles capable of delineating the margins of brain tumors both preoperatively and intraoperatively. These MRI-PAT-Raman nanoparticles are able to be used both to track tumor growth and surgical staging, by way of MRI, but also in the same particle be used during surgical resection of brain tumor to give the surgeon ‘eyes’ down to the single cancer cell level, increasing the potential tumor specific tissue removal.

For metastatic melanoma, researchers at MSKCC and Cornell University have developed silica-hybrid nanoparticles (‘C-dots’) that deliver both PET and optical imaging contrast in the same platform. These nanoparticles are actively targeted to the cancer with cRGDY peptides that target this specific tumor type and have already made it successfully through initial clinical trials.

Another clinical cancer imaging problem being addressed by nanoscale solutions is prostate cancer. Researchers at Stanford University recently have been developing nanotechnologies that give both anatomical size and location of prostate cancer cells (nanobubbles for ultrasound imaging) and functional information to avoid overdiagnosis/treatment as well as to monitor progression (self-assemblying nanoparticles for photoacoustic imaging). The nanoplatforms developed by this group are coupled directly to their recently approved handheld transrectal ultrasound and photoacoustic (TRUSPA) device. Ultimately offering a more effective, integrated and less invasive technique to image and biopsy prostate cancers for diagnosis and prognostication prior to performing common interventions (surgical resection, radiotherapy, etc.).

Similarly, gold nanoparticles are being used to enhance light scattering for endoscopic techniques that can be used during colonoscopies. One really powerful potential that has always been envisioned for nanotechnology in cancer has been the potential to simultaneously image and deliver therapy in vivo and several groups have been pushing forward these ‘theranostic’ nanoscale platforms. One group at Emory University has been developing one of these for ovarian and pancreatic cancers, which are traditionally harder to deliver therapeutics to. Their platform for pancreatic cancer can break through the fibrotic stromal tissue of which these tumors are protected by in the pancreas. After traversing through this barrier, they are composed of magnetic iron cores which allow MRI contrast for diagnosis and deliver small-molecule drugs directly to cancer cells to treat.

Finally, nanotechnology is enabling the visualization of molecular markers that identify specific stages and cancer cell death induced by therapy, allowing doctors to see cells and molecules undetectable through conventional imaging. A group at Stanford has developed the Target-Enabled in Situ Ligand Assembly (TESLA) nanoparticle system. This is based off nanoparticles which form directly in the body after IV-injection of molecular precursors. The precursors contain specific sequences of atoms which can only form larger nanoparticles after being cleaved by enzymes produced by cancer cells during apoptosis (i.e., cell death) and carry various image contrast agents to monitor (PET, MRI, etc.) local tumor response to therapies. Being able to track cancer cell death in vivo and at the molecular level is extremely important for delivering effective dosing regimens and/or precisely administering novel therapies or combinations.

Sensing In Vitro

Nanotechnology-enabled in vitro diagnostic devices offer high sensitivity and selectivity, and capability to perform simultaneous measurements of multiple targets. Well-established fabrication techniques (e.g., lithography) can be used to manufacture integrated, portable devices or point-of-care systems. A diagnostic device or biosensor contains a biological recognition element, which through biochemical reaction can detect the presence, activity or concentration of a specific biological molecule in the solution. This reaction could be associated, for example with: binding of antigen and antibody, hybridization of two single stranded DNA fragments, or binding of capture ligand to the cell surface epitope. A transducer part of the detection device is used to convert the biochemical event into a quantifiable signal which can be measured. The transduction mechanisms can rely on light, magnetic, or electronic effects.

Several devices have been designed for detection of various biological signatures from serum or tissue. Few examples of diagnostic devices relying on nanotechnology or nanoparticles are given in Figure #. The bio-barcode assay was designed as a sandwich immunoassay in the laboratory of Chad Mirkin at Northwestern University. It utilizes magnetic nanoparticles (MMPs) which are functionalized with monoclonal antibodies specific to the target protein of interest and then mixed with the sample to promote capture of target proteins. The MMP-protein hybrid structures are then combined with gold nanoparticle (Au-NP) probes which carry DNA-barcodes. Target protein-specific DNA barcodes are released into solution and detected using the scanometric assay with sensitivities in femto-picomolar range.

James Heath’s laboratory at Caltech designed sandwich immunoassay devices which rely on DNA-encoded antibody libraries (DEAL). DEAL technique uses DNA-directed immobilization of antibodies in microfluidic channels allowing to convert a pre-patterned single stranded (ss) DNA barcode microarray into an antibody microarray. ssDNA oligomers attached onto the sensor surface are robust and can withstand elevated temperatures of channel fabrication. Subsequent flow-through of the DNA-antibody conjugates in channels transforms the DNA microarray into an antibody microarray and allow to perform multiplex surface-bound sandwich immunoassays. These devices allow for on-chip blood separation and measurement of large protein panels directly from blood.

Diagnostic Magnetic Resonance (DMR) sensor platform was designed in the laboratory of Ralph Weissleder at Massachusetts General Hospital. The DMR mechanism exploits changes in the transverse relaxation signal of water molecules in a magnetic field as a sensing mechanism for magnetic nanoparticle labeled analytes. Highly integrated systems including microfluidic processing circuits and nuclear magnetic resonance (NMR) detection head with high signal to noise ratio were built and are capable of to detect presence of cells, vesicles, and proteins in clinical samples.

Shan Wang’s laboratory at Stanford University designed Giant Magnetoresistive (GMR) biosensors for protein detection. These nanosensors operate by changing their electrical resistance in response to changes in the local magnetic field. They were adapted to detection of biological signatures in solution by implementing a traditional sandwich assay directly on GMR nanosensors. Antibodies are immobilized on the GMR sensor surface and are used as capture probes for the sample containing target proteins. A magnetic particle is used to label the biomolecule of interest in the sample and GMR sensor is used for signal transduction. These sensors were used to measure protein levels in complex sample mixtures and also were employed to assess kinetics of protein interactions.

The devices described above are capable of analyzing large panels of biological signatures at the same time providing for high level of multiplexing. The data analysis can establish correlations among different biomarker levels and map correlations of network signaling and thus provide tools for patient stratification based on their response to different treatments and ultimately improve therapeutic efficacy of the one selected. New advancements in microfluidic technologies opened opportunities to integrate sample preparation and sample processing with biosensors and to realize fully integrated devices that directly deliver full data for a medical diagnosis from a single sample.

Measuring Response to Therapy and the Liquid Biopsy

Measurement of an individual patient’s response to therapeutics during the course of their disease is the basis for precise and prognostic medical care. Accurate and disease relevant monitoring can allow for optimized treatment regimens (e.g., therapeutic course correction, drug combinations, and dose attenuation), preemptive clinical decision making (e.g., therapeutic responders vs. non-responders, and more), and patient stratification for clinical trials. Beyond the more traditional gold standards of in vivo imaging, tissue biopsy and in vitro diagnostics available for this purpose, the “liquid biopsy” offers the ability to measure response to therapy by way of simple and serial blood draws. Traditional biopsies involve resection of small volumes of the tumor tissue directly, and thus, remain invasive procedures that cannot offer the sampling necessitated to track disease progression relative to the course of therapy or the dynamics of its evolving biology. Liquid biopsies rely on the fact that tumors shed material (e.g., cells, DNA, other cancer-specific biomolecules) into circulation, over time and in response to therapy. Although, the amount of materials shed by any given tumor and / or stage is typically at incredibly low concentrations relative to the rest of the blood’s constituents (e.g., erythrocytes, leukocytes, thrombocytes, plasma, etc.). This requires specific and sensitive tools to detect, capture, and purify the circulating tumor material relative to the rest. Nanotechnology is enabling these tools to become reality.

Recent technological advances in the coupling of complex microfluidics and nanoscale materials have allowed the high-purity capture and downstream functional characterization of circulating tumor cells (CTCs), cell-free tumor DNA, microemboli, exosomes, proteins, neoantigens, and more. Recent examples include, capture and subsequent release of CTCs within microfluidic systems to maintain viable cells for downstream whole genome sequencing, ex vivo expansion, RNA sequencing, and more. Of these examples, one type of device uses magnetic nanoparticles to enrich whole blood prior to magnetic separation within the microfluidic and the other device uses thermoresponsive nanopolymers that specifically capture CTCs as they flow through the microfluidic then release upon a change in temperature once blood processing is complete. In both cases, the detection sensitivities are very high (e.g., for enumeration >95%) and capture purity is much higher than other non-nanomaterial based devices. Furthermore, the processing times are increasing every year as the technology evolves, currently averaging 10 mL blood per 30 minutes.