Microdose Levels Research Articles

A Miniaturized Platform for Multiplexed Drug Response Imaging in Live Tumors

Simple SummaryWe have developed an implantable microdevice that is placed into a live tumor, and can directly image how effective various chemotherapy drugs are at inducing cell death, without having to remove or process the tumor tissue. Currently drug optimization is performed by assessing tumor shrinkage after treating a patient with systemic doses of a chemotherapy agent; this only evaluates a single treatment at a time and typically takes weeks-months before an optimal treatment strategy is found (if found at all) for a specific patient. In contrast, using the technology presented here, a personalized cancer treatment strategy can potentially be optimized and tailored to a specific patient’s tumor characteristics within several hours, without requiring surgical tissue removal or prolonged trials of potentially ineffective chemotherapies.By observing the activity of anti-cancer agents directly in tumors, there is potential to greatly expand our understanding of drug response and develop more personalized cancer treatments. Implantable microdevices (IMD) have been recently developed to deliver microdoses of chemotherapeutic agents locally into confined regions of live tumors; the tissue can be subsequently removed and analyzed to evaluate drug response. This method has the potential to rapidly screen multiple drugs, but requires surgical tissue removal and only evaluates drug response at a single timepoint when the tissue is excised. Here, we describe a “lab-in-a-tumor” implantable microdevice (LIT-IMD) platform to image cell-death drug response within a live tumor, without requiring surgical resection or tissue processing. The LIT-IMD is inserted into a live tumor and delivers multiple drug microdoses into spatially discrete locations. In parallel, it locally delivers microdose levels of a fluorescent cell-death assay, which diffuses into drug-exposed tissues and accumulates at sites of cell death. An integrated miniaturized fluorescence imaging probe images each region to evaluate drug-induced cell death. We demonstrate ability to evaluate multi-drug response over 8 h using murine tumor models and show correlation with gold-standard conventional fluorescence microscopy and histopathology. This is the first demonstration of a fully integrated platform for evaluating multiple chemotherapy responses in situ. This approach could enable a more complete understanding of drug activity in live tumors, and could expand the utility of drug-response measurements to a wide range of settings where surgery is not feasible.

Implantable sensor for local Cherenkov-excited luminescence imaging of tumor pO2 during radiotherapy.

.Significance: The necessity to use exogenous probes for optical oxygen measurements in radiotherapy poses challenges for clinical applications. Options for implantable probe biotechnology need to be improved to alleviate toxicity concerns in human use and facilitate translation to clinical trial use.Aim: To develop an implantable oxygen sensor containing a phosphorescent oxygen probe such that the overall administered dose of the probe would be below the Federal Drug Administration (FDA)-prescribed microdose level, and the sensor would provide local high-intensity signal for longitudinal measurements of tissue .Approach: PtG4, an oxygen quenched dendritic molecule, was mixed into an agarose matrix at concentration, allowing for local injection into tumors at the total dose of 10 nmol per animal, forming a gel at the site of injection. Cherenkov-excited luminescence imaging (CELI) was used to acquire the phosphorescence and provide intratumoral .Results: Although PtG4 does not form covalent bonds with agarose and gradually leaches out into the surrounding tissue, its retention time within the gel was sufficiently long to demonstrate the capability to measure intratumoral with the implantable gel sensors. The sensor’s performance was first evaluated in vitro in tissue simulation phantoms, and then the sensor was used to measure changes in oxygen in MDA-MB-231 tumors during hypofractionated radiotherapy.Conclusions: Our study demonstrates that implantable oxygen sensors in combination with CELI present a promising approach for quantifying oxygen changes during the course of radiation therapy and thus for evaluating the tumor response to radiation. By improving the design of the gel–probe composition in order to prevent leaching of the probe into the tissue, biosensors can be created that should allow longitudinal oxygen measurements in tumors by means of CELI while using FDA-compliant microdose levels of the probe and thus lowering toxicity concerns.

Microdose lithium reduces cellular senescence in human astrocytes - a potential pharmacotherapy for COVID-19?

Cell senescence is a process that causes growth arrest and the release of a senescence associated secretory phenotype (SASP), characterized by secretion of chemokines, cytokines, cell growth factors and metalloproteases, leading to a tissue condition that may precipitate cancers and neurodegenerative processes. With the recent pandemic of coronavirus, senolytic drugs are being considered as possible therapeutic tools to reduce the virulence of SARS-CoV-2. In the last few years, our research group showed that lithium carbonate at microdose levels was able to stabilize memory and change neuropathological characteristics of Alzheimer’s disease (AD). In the present work, we present evidence that low-dose lithium can reduce the SASP of human iPSCs-derived astrocytes following acute treatment, suggesting that microdose lithium could protect cells from senescence and development of aging-related conditions. With the present findings, a perspective of the potential use of low-dose lithium in old patients from the “high risk group” for COVID-19 (with hypertension, diabetes and chronic obstructive pulmonary disease) is presented.

Soil moisture management and fertilizer micro-dosing on yield and land utilization efficiency of inter-cropping maize-pigeon-pea in sub humid Tanzania

Principally caused by soil water stress and declining soil fertility, low crop productivity results in both food and income insecurity. The effects of nitrogen and phosphorus fertilizer micro-dosing with inter-row rainwater harvesting practices for maize and pigeon-pea inter-cropping on yield and land use efficiency are inadequately documented in sub humid tropics. A field experiment on sandy loam soils in sub humid conditions using a split-split plot design was conducted. Plots used in situ rainwater harvesting practices of tied ridges, open ridges, and flat cultivation. Sub-plots were sole maize, sole pigeon-pea, and 1:1 maize-pigeon pea inter-cropping. The sub-sub plots were control, fertilizer (N and P) application at the micro-dose level, and recommended rates. Tied ridges significantly (p < 0.001) conserved more soil moisture than flat cultivation at 30 cm depth after ten days of rainfall. Ridges increased maize yield by 0.3 t ha−1 over flat cultivation. Fertilizer application significantly (p < 0.001) increased maize yield by 1.12 t ha−1 with micro-dosing and by 1.60 t ha−1 with recommended rates over the control. Combining tied ridges and fertilizer significantly (p < 0.040) increased maize yield by 132–156% compared to flat cultivation without fertilizer. Reflecting a land equivalent ratio, land use efficiency was 67–122% higher in inter-cropping than sole crop. Tied ridges conserved more soil moisture than flat cultivation, enhancing fertilizer use efficiency that improved crop yields and land equivalent ratio under inter-cropping. This strategy could increase food availability and income generation under smallholder farming systems in sub-humid tropic areas.

Measuring microdose ABY-029 fluorescence signal in a primary human soft-tissue sarcoma resection.

Microdose administration of ABY-029, an anti-epidermal growth factor receptor Affibody molecule conjugated to IRDye 800CW, is being studied in a Phase 0 trial for resection of soft-tissue sarcomas. The excised tissue of a single patient in the microdose administration group was imaged with both a wide-field fluorescence surgical system and a flat-bed scanning fluorescence imaging system. Here the resultant fluorescence from a breadloaf section of the primary tumor specimen and six region-specific tissue samples collected from that breadloaf are compared using these two imaging systems - a flatbed, black-box, fluorescence scanning system, the Odyssey CLx, and a open-air, wide-field, pre-clinical surgical imaging system, the Solaris. Florescence signal is compared using a variety of methods including: mean, standard deviation, variance, tumor-to-background ratio, biological-variance ratio, and contrast-to-noise ratio. The images produced from the Odyssey scanner have higher signal variance but more accurately represent the EGFR expression in small tissue sections. The Solaris system has higher depth sensitivity and volume averaging, and as such has lower signal variation and higher contrast-to-noise ratio.

Rapid screening of nine unradiolabeled candidate compounds as PET brain imaging agents using cassette-wave microdosing and LC-MS/MS

The R&D of PET imaging agents is a complex system engineering, simplifying screening steps and increasing screening efficiency have become popular issues. The purpose of this study is to develop a new screening procedure using cassette-wave microdosing and LC-MS/MS to enhance the screening throughput of unradiolabeled candidate compounds as PET imaging agents. Nine compounds were divided into 3 sets and made into 3 cassettes. Fifteen rats were randomly divided into 3 groups, and every animal received three intravenous bolus injections at three different time points; the doses were at microdose levels. This dosing approach takes advantage of temporal and spatial differences and is likened to an input wave; therefore, this approach was named cassette-wave microdosing. The samples of different brain regions such as the hypothalamus, striatum, hippocampus, cortex, cerebellum and the remainder of the brain were detected by LC-MS/MS analysis. The research potential of the compounds as PET imaging agents is evaluated in terms of brain biodistribution data. The screening method is rapid, highly efficient, reliable and reduces animal usage. Additionally, it can shorten the evaluation process of radiopharmaceuticals and enhance the screening throughput of PET radiopharmaceuticals without the use of radioactive agents.

Threshold Analysis and Biodistribution of Fluorescently Labeled Bevacizumab in Human Breast Cancer.

In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant and nonmalignant tissues are usually distinguished on fluorescence images by applying empirically determined fluorescence intensity thresholds. Here, we report the development of fSTREAM, a set of analytic methods designed to streamline the analysis of surgically excised breast tissues by collecting and statistically processing hybrid multiscale fluorescence, color, and histology readouts toward precision fluorescence imaging. fSTREAM addresses core questions of how to relate fluorescence intensity to tumor tissue and how to quantitatively assign a normalized threshold that sufficiently differentiates tumor tissue from healthy tissue. Using fSTREAM we assessed human breast tumors stained in vivo with fluorescent bevacizumab at microdose levels. Showing that detection of such levels is achievable, we validated fSTREAM for high-resolution mapping of the spatial pattern of labeled antibody and its relation to the underlying cancer pathophysiology and tumor border on a per patient basis. We demonstrated a 98% sensitivity and 79% specificity when using labeled bevacizumab to outline the tumor mass. Overall, our results illustrate a quantitative approach to relate fluorescence signals to malignant tissues and improve the theranostic application of fluorescence molecular imaging. Cancer Res; 77(3); 623-31. ©2016 AACR.

Toxicity and Pharmacokinetic Profile for Single-Dose Injection of ABY-029: a Fluorescent Anti-EGFR Synthetic Affibody Molecule for Human Use.

ABY-029, a synthetic Affibody peptide, Z03115-Cys, labeled with a near-infrared fluorophore, IRDye® 800CW, targeting epidermal growth factor receptor (EGFR) has been produced under good manufacturing practices for a US Food and Drug Administration-approved first-in-use human study during surgical resection of glioma, as well as other tumors. Here, the pharmacology, phototoxicity, receptor activity, and biodistribution studies of ABY-029 were completed in rats, prior to the intended human use. Male and female Sprague Dawley rats were administered a single intravenous dose of varying concentrations (0, 245, 2449, and 24,490μg/kg corresponding to 10×, 100×, and 1000× an equivalent human microdose level) of ABY-029 and observed for up to 14days. Histopathological assessment of organs and tissues, clinical chemistry, and hematology were performed. In addition, pharmacokinetic clearance and biodistribution of ABY-029 were studied in subgroups of the animals. Phototoxicity and ABY-029 binding to human and rat EGFR were assessed in cell culture and on immobilized receptors, respectively. Histopathological assessment and hematological and clinical chemistry analysis demonstrated that single-dose ABY-029 produced no pathological evidence of toxicity at any dose level. No phototoxicity was observed in EGFR-positive and EGFR-negative glioma cell lines. Binding strength and pharmacokinetics of the anti-EGFR Affibody molecules were retained after labeling with the dye. Based on the successful safety profile of ABY-029, the 1000× human microdose 24.5mg/kg was identified as the no observed adverse effect level following intravenous administration. Conserved binding strength and no observed light toxicity also demonstrated ABY-029 safety for human use.

Fluorescent Affibody Molecule Administered In Vivo at a Microdose Level Labels EGFR Expressing Glioma Tumor Regions

PurposeFluorescence guidance in surgical oncology provides the potential to realize enhanced molecular tumor contrast with dedicated targeted tracers, potentially with a microdose injection level. For most glioma tumors, the blood brain barrier is compromised allowing some exogenous drug/molecule delivery and accumulation for imaging. The aberrant overexpression and/or activation of epidermal growth factor receptor (EGFR) is associated with many types of cancers, including glioblastoma, and so the use of a near-infrared (NIR) fluorescent molecule targeted to the EGFR receptor provides the potential for improving tumor contrast during surgery. Fluorescently labeled affibody molecule (ABY-029) has high EGFR affinity and high potential specificity with reasonably fast plasma clearance. In this study, ABY-29 was evaluated in glioma versus normal brain uptake from intravenous injection at a range of doses, down to a microdose injection level.ProcedureNude rats were inoculated with the U251 human glioma cell line in the brain. Tumors were allowed to grow for 3–4 weeks. ABY-029 fluorescence ex vivo imaging of brain slices was acquired at different time points (1–48 h) and varying injection doses from 25 to 122 μg/kg (from human protein microdose equivalent to five times microdose levels).ResultsThe tumor was most clearly visualized at 1-h post-injection with 8- to 16-fold average contrast relative to normal brain. However, the tumor still could be identified after 48 h. In all cases, the ABY-029 fluorescence appeared to localize preferentially in EGFR-positive regions. Increasing the injected dose from a microdose level to five times, a microdose level increased the signal by 10-fold, and the contrast was from 8 to 16, showing that there was value in doses slightly higher than the microdose restriction. Normal tissue uptake was found to be affected by the tumor size, indicating that edema was a likely factor affecting the expected tumor to normal tissue contrast.ConclusionThese results suggest that the NIR-labeled affibody molecules provide an excellent potential to increase surgical visualization of EGFR-positive tumor regions.

Maximum-Intensity-Projection and Computer-Aided-Detection Algorithms as Stand-Alone Reader Devices in Lung Cancer Screening Using Different Dose Levels and Reconstruction Kernels.

The objective of our study was to evaluate lung nodule detection rates on standard and microdose chest CT with two different computer-aided detection systems (SyngoCT-CAD, VA 20, Siemens Healthcare [CAD1]; Lung CAD, IntelliSpace Portal DX Server, Philips Healthcare [CAD2]) as well as maximum-intensity-projection (MIP) images. We also assessed the impact of different reconstruction kernels. Standard and microdose CT using three reconstruction kernels (i30, i50, i70) was performed with an anthropomorphic chest phantom. We placed 133 ground-glass and 133 solid nodules (diameters of 5 mm, 8 mm, 10 mm, and 12 mm) in 55 phantoms. Four blinded readers evaluated the MIP images; one recorded the results of CAD1 and CAD2. Sensitivities for CAD and MIP nodule detection on standard dose and microdose CT were calculated for each reconstruction kernel. Dose for microdose CT was significantly less than that for standard-dose CT (0.1323 mSv vs 1.65 mSv; p < 0.0001). CAD1 delivered superior results compared with CAD2 for standard-dose and microdose CT (p < 0.0001). At microdose level, the best stand-alone sensitivity (97.6%) was comparable with CAD1 sensitivity (96.0%; p = 0.36; both with i30 reconstruction kernel). Pooled sensitivities for all nodules, doses, and reconstruction kernels on CAD1 ranged from 88.9% to 97.3% versus 49.6% to 73.9% for CAD2. The best sensitivity was achieved with standard-dose CT, i50 kernel, and CAD1 (97.3%) versus 96% with microdose CT, i30 or i50 kernel, and CAD1. MIP images and CAD1 had similar performance at both dose levels (p = 0.1313 and p = 0.48). Submillisievert CT is feasible for detecting solid and ground-glass nodules that require soft-tissue kernels for MIP and CAD systems to achieve acceptable sensitivities. MIP reconstructions remain a valuable adjunct to the interpretation of chest CT for increasing sensitivity and have the advantage of significantly lower false-positive rates.

Vision 20/20: Molecular-guided surgical oncology based upon tumor metabolism or immunologic phenotype: Technological pathways for point of care imaging and intervention.

Surgical guidance with fluorescence has been demonstrated in individual clinical trials for decades, but the scientific and commercial conditions exist today for a dramatic increase in clinical value. In the past decade, increased use of indocyanine green based visualization of vascular flow, biliary function, and tissue perfusion has spawned a robust growth in commercial systems that have near-infrared emission imaging and video display capabilities. This recent history combined with major preclinical innovations in fluorescent-labeled molecular probes, has the potential for a shift in surgical practice toward resection guidance based upon molecular information in addition to conventional visual and palpable cues. Most surgical subspecialties already have treatment management decisions partially based upon the immunohistochemical phenotype of the cancer, as assessed from molecular pathology of the biopsy tissue. This phenotyping can inform the surgical resection process by spatial mapping of these features. Further integration of the diagnostic and therapeutic value of tumor metabolism sensing molecules or immune binding agents directly into the surgical process can help this field mature. Maximal value to the patient would come from identifying the spatial patterns of molecular expression in vivo that are well known to exist. However, as each molecular agent is advanced into trials, the performance of the imaging system can have a critical impact on the success. For example, use of pre-existing commercial imaging systems are not well suited to image receptor targeted fluorophores because of the lower concentrations expected, requiring orders of magnitude more sensitivity. Additionally the imaging system needs the appropriate dynamic range and image processing features to view molecular probes or therapeutics that may have nonspecific uptake or pharmacokinetic issues which lead to limitations in contrast. Imaging systems need to be chosen based upon objective performance criteria, and issues around calibration, validation, and interpretation need to be established before a clinical trial starts. Finally, as early phase trials become more established, the costs associated with failures can be crippling to the field, and so judicious use of phase 0 trials with microdose levels of agents is one viable paradigm to help the field advance, but this places high sensitivity requirements on the imaging systems used. Molecular-guided surgery has truly transformative potential, and several key challenges are outlined here with the goal of seeing efficient advancement with ideal choices. The focus of this vision 20/20 paper is on the technological aspects that are needed to be paired with these agents.