Earlier Detection and Diagnosis

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Researchers at the Emory/Georgia Tech Center of Cancer Nanotechnology Excellence synthesize, by vapor-solid process, aligned ZnO nanowire arrays as shown in the scanning electron microscopy (SEM) image.

Credit: National Cancer Institute

In the fight against cancer, half of the battle is won based on its early detection. Nanotechnology provides new molecular contrast agents and materials to enable earlier and more accurate initial diagnosis as well as in continual monitoring of cancer patient treatment.

Although not yet deployed clinically for cancer detection or diagnosis, nanoparticles are already on the market in numerous medical screens and tests, with the most widespread use that of gold nanoparticles in home pregnancy tests. Nanoparticles are also at the heart of the Verigene® system from Nanosphere and the T2MR system from T2 Biosystems, currently used in hospitals for a variety of indications.

For cancer, nanodevices are being investigated for the capture of blood borne biomarkers, including cancer-associated proteins circulating tumor cells, circulating tumor DNA, and tumor-shed exosomes. Nano-enabled sensors are capable of high sensitivity, specificity and multiplexed measurements. Next generation devices couple capture with genetic analysis to further elucidate a patient’s cancer and potential treatments and disease course.

Already clinically established as contrast agents for anatomical structure, nanoparticles are being developed to act as molecular imaging agents, reporting on the presence of cancer-relevant genetic mutations or the functional characteristics of tumor cells. This information can be used to choose a treatment course or alter a therapeutic plan. Bioactivatable nanoparticles that change properties in response to factors or processes within the body act as dynamic reporters of in vivo states and can provide both spatial and temporal information on disease progression and therapeutic response.

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.

Principle of a triple-modality MRI-photoacoustic-Raman nanoparticle for clinical use. The nanoparticle is injected intravenously. In contrast to small molecule contrast agents that wash out of the tumor quickly, the nanoparticles are stably internalized within the brain tumor cells, allowing the whole spectrum from preoperative MRI for surgical planning to intraoperative imaging to be performed with a single injection. T1-weighted MRI depicts the outline of the tumor due to the T1-shortening effect of the gadolinium. During the surgery, photoacoustic imaging with its greater depth penetration and 3D imaging capabilities can be used to guide the gross resection steps, while Raman imaging can guide the resection of the microscopic tumor at the resection margins. Raman would be used for rapid conformation of clean margins in the operating room instead of the time-consuming analysis of frozen sections.

Credit: National Cancer Institute

Present and future of NanoOncology Image-guided Surgical Suite. Preoperative conventional imaging tools are used to screen for disease and inform optically- driven minimally-invasive and open surgical procedures. Clinically available particle platforms can be monitored in real-time using portable multichannel camera systems. Representative translational probes and devices for future clinical use are also shown. In the future, the operating surgeon will select suitable probe-device combinations for specific indications, and be provided with structural, functional, and/or molecular-level data regarding ssue status for further treatment management.

Credit: National Cancer Institute

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.

The image shows the DEAL (DNA-encoded antibody library) barcode assay, a high density information test for human blood proteins designed to show the individual identities of every human disease, allowing for personalized medicine.

Credit: National Cancer Institute

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.

 

Here, when cancer cells (cell nuclei in blue) were treated with antibody-conjugated nanoparticles, the antibodies (red) and the nanoparticle cores (green) separated into different cellular compartments. Such knowledge may lead to improved methods of cancer detection in vivo as well as better nanoparticle-based treatments.

Credit: National Cancer Institute

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

Imaged here is a microfluidic magneto-nano chip with 8 by 8 sensors arrays and 8 microfluidic channels. These chips are being developed to monitor protein profiles in blood samples from cancer patients to improve therapeutic effectiveness.

Credit: National Cancer Institute

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.

Here a fluorescent nanosensor (cyan) and a chemical with anti-cancer activity (dichloroacetic acid) were injected into the blood vessels (red) of a mouse implanted with human breast cancer (green). This image shows that the nanosensor detects the presence of dichloroacetic acid near the tumor.

Credit: National Cancer Institute

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.

Selected References
  1. Weissleder et al. Imaging approaches to optimize molecular therapies. Science Trans Med. 2016

  2. Phillips et al. Clinical translation of an ultrasmall inorganic optical-PET imaging nanoparticle probe. Science Trans Med. 2016

  3. Ye et al. Bioorthogonal cyclization-mediated in situ self-assembly of small-molecule probes for imaging caspase activity in vivo. Nature Chemistry 2014

  4. Kircher et al. A brain tumor molecular imaging strategy using a new triple-modality MRI-photoacoustic-Raman nanoparticle. Nature Med 2012

  5. Nam JM, Thaxton CS, Mirkin CA. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science. 2003; 26;301:1884-6.

  6. Fan R, Heath JR et al. Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood. Nat Biotechnol. 2008;26:1373-8

  7. Haun JB, Castro CM, Wang R, Peterson VM, Marinelli BS, Lee H, Weissleder R. Micro-NMR for rapid molecular analysis of human tumor samples. Sci Transl Med. 2011;3:71ra16

  8. Gaster RS, Hall DA, Wang SX. nanoLAB: an ultraportable, handheld diagnostic laboratory for global health. Lab Chip. 2011; 7;11:950-6

  9. Park et al., Molecular profiling of single circulating tumor cells from lung cancer patients. PNAS 2016

  10. Lin et al. Nanostructure Embedded Microchips for Detection, Isolation, and Characterization of Circulating Tumor Cells. Accounts of Chemical Research 2014

  11. Jiang et al. A comparison of isolated circulating tumor cells and tissue biopsies using whole-genome sequencing in prostate cancer. Oncotarget 2015

  12. Shuhendler et al. Molecular Magnetic Resonance Imaging of Tumor Response to Therapy. Scientific Reports 2015

  • Posted: August 8, 2017

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