Ashley M. Laughney

Predicting therapeutic nanomedicine efficacy using a companion magnetic resonance imaging nanoparticle

Magnetic nanoparticles predict the efficacy of drug-loaded polymeric nanoparticles in vivo, helping select for tumors more responsive to nanomedicine. Particle prediction One particle, it seems, can predict the behavior of another. Thankfully, this is not the beginning of a lesson on quantum physics; instead, it is the basis for potentially designing targeted clinical trials in nanomedicine, by knowing if a tumor is likely to respond to a particular therapeutic nanoparticle. Miller et al. hypothesized that if a tumor readily takes up magnetic nanoparticles (MNP), it will also accumulate other nanoparticles carrying a deadly payload. The authors injected MNPs and a fluorescent version of the therapeutic nanoparticles into mice and followed their biodistribution using imaging. Both types of nanoparticles had similar pharmacokinetics and uptake in tumor-associated host cells owing to the enhanced permeability and retention effect. In mice with human tumors, Miller and colleagues found that the tumors with high MNP uptake were significantly more responsive than those with medium or low uptake to nanoparticles delivering chemotherapeutics. Thus, MNPs can be used as companion imaging agents during nanomedicine trials to predict the therapeutic effect of their nanosized counterparts. Therapeutic nanoparticles (TNPs) have shown heterogeneous responses in human clinical trials, raising questions of whether imaging should be used to identify patients with a higher likelihood of NP accumulation and thus therapeutic response. Despite extensive debate about the enhanced permeability and retention (EPR) effect in tumors, it is increasingly clear that EPR is extremely variable; yet, little experimental data exist to predict the clinical utility of EPR and its influence on TNP efficacy. We hypothesized that a 30-nm magnetic NP (MNP) in clinical use could predict colocalization of TNPs by magnetic resonance imaging (MRI). To this end, we performed single-cell resolution imaging of fluorescently labeled MNPs and TNPs and studied their intratumoral distribution in mice. MNPs circulated in the tumor microvasculature and demonstrated sustained uptake into cells of the tumor microenvironment within minutes. MNPs could predictably demonstrate areas of colocalization for a model TNP, poly(d,l-lactic-co-glycolic acid)-b-polyethylene glycol (PLGA-PEG), within the tumor microenvironment with >85% accuracy and circulating within the microvasculature with >95% accuracy, despite their markedly different sizes and compositions. Computational analysis of NP transport enabled predictive modeling of TNP distribution based on imaging data and identified key parameters governing intratumoral NP accumulation and macrophage uptake. Finally, MRI accurately predicted initial treatment response and drug accumulation in a preclinical efficacy study using a paclitaxel-encapsulated NP in tumor-bearing mice. These approaches yield valuable insight into the in vivo kinetics of NP distribution and suggest that clinically relevant imaging modalities and agents can be used to select patients with high EPR for treatment with TNPs.

Spectral discrimination of breast pathologies in situ using spatial frequency domain imaging

IntroductionNationally, 25% to 50% of patients undergoing lumpectomy for local management of breast cancer require a secondary excision because of the persistence of residual tumor. Intraoperative assessment of specimen margins by frozen-section analysis is not widely adopted in breast-conserving surgery. Here, a new approach to wide-field optical imaging of breast pathology in situ was tested to determine whether the system could accurately discriminate cancer from benign tissues before routine pathological processing.MethodsSpatial frequency domain imaging (SFDI) was used to quantify near-infrared (NIR) optical parameters at the surface of 47 lumpectomy tissue specimens. Spatial frequency and wavelength-dependent reflectance spectra were parameterized with matched simulations of light transport. Spectral images were co-registered to histopathology in adjacent, stained sections of the tissue, cut in the geometry imaged in situ. A supervised classifier and feature-selection algorithm were implemented to automate discrimination of breast pathologies and to rank the contribution of each parameter to a diagnosis.ResultsSpectral parameters distinguished all pathology subtypes with 82% accuracy and benign (fibrocystic disease, fibroadenoma) from malignant (DCIS, invasive cancer, and partially treated invasive cancer after neoadjuvant chemotherapy) pathologies with 88% accuracy, high specificity (93%), and reasonable sensitivity (79%). Although spectral absorption and scattering features were essential components of the discriminant classifier, scattering exhibited lower variance and contributed most to tissue-type separation. The scattering slope was sensitive to stromal and epithelial distributions measured with quantitative immunohistochemistry.ConclusionsSFDI is a new quantitative imaging technique that renders a specific tissue-type diagnosis. Its combination of planar sampling and frequency-dependent depth sensing is clinically pragmatic and appropriate for breast surgical-margin assessment. This study is the first to apply SFDI to pathology discrimination in surgical breast tissues. It represents an important step toward imaging surgical specimens immediately ex vivo to reduce the high rate of secondary excisions associated with breast lumpectomy procedures.

Single-cell pharmacokinetic imaging reveals a therapeutic strategy to overcome drug resistance to the microtubule inhibitor eribulin

Single-cell pharmacokinetic analysis of a fluorescent eribulin derivative in vivo revealed drug resistance mediated by MDR1-driven efflux, which was overcome by a nanoencapsulated MDR1 inhibitor. Single-Cell Imaging of Cancer Drug Resistance Resistance to drugs is common in cancer and is often caused by the multidrug resistance protein 1 (MDR1). To get to the bottom of this mechanism of drug resistance—and hopefully develop better therapeutics from this knowledge—Laughney et al. created a fluorescent cancer drug and probed tissue distribution and kinetics at both the single-cell and population levels. The authors developed a mouse model of drug-resistant human cancer, with tumors that were heterogeneously resistant (various levels of MDR1 expression). By tracking the fluorescent drug eribulin in vivo, they saw that MDR1-expressing cells accumulated less drug than their wild-type counterparts—and this was a function of distance from blood vessels. MDR1 inhibitors did not appear to increase drug uptake in MDR1-overexpressing cells in vivo, so the authors redesigned the MDR inhibitor as a nanoparticle. In mice, inhibitor loaded in nanoparticles demonstrated bioactivity in the tumor. The combination of single-cell pharmacokinetics, intravital imaging, and drug reengineering suggests a new platform for understanding and overcoming resistance in human cancer. Eribulin mesylate was developed as a potent microtubule-targeting cytotoxic agent to treat taxane-resistant cancers, but recent clinical trials have shown that it eventually fails in many patient subpopulations for unclear reasons. To investigate its resistance mechanisms, we developed a fluorescent analog of eribulin with pharmacokinetic (PK) properties and cytotoxic activity across a human cell line panel that are sufficiently similar to the parent drug to study its cellular PK and tissue distribution. Using intravital imaging and automated tracking of cellular dynamics, we found that resistance to eribulin and the fluorescent analog depended directly on the multidrug resistance protein 1 (MDR1). Intravital imaging allowed for real-time analysis of in vivo PK in tumors that were engineered to be spatially heterogeneous for taxane resistance, whereby an MDR1-mApple fusion protein distinguished resistant cells fluorescently. In vivo, MDR1-mediated drug efflux and the three-dimensional tumor vascular architecture were discovered to be critical determinants of drug accumulation in tumor cells. We furthermore show that standard intravenous administration of a third-generation MDR1 inhibitor, HM30181, failed to rescue drug accumulation; however, the same MDR1 inhibitor encapsulated within a nanoparticle delivery system reversed the multidrug-resistant phenotype and potentiated the eribulin effect in vitro and in vivo in mice. Our work demonstrates that in vivo assessment of cellular PK of an anticancer drug is a powerful strategy for elucidating mechanisms of drug resistance in heterogeneous tumors and evaluating strategies to overcome this resistance.

System analysis of spatial frequency domain imaging for quantitative mapping of surgically resected breast tissues

Abstract. The feasibility of spatial frequency domain imaging (SFDI) for breast surgical margin assessment was evaluated in tissue-simulating phantoms and in fully intact lumpectomy specimens at the time of surgery. Phantom data was evaluated according to contrast-detail resolution, quantitative accuracy and model-data goodness of fit, where optical parameters were estimated by minimizing the residual sum of squares between the measured modulation amplitude and its solutions, modeled according to diffusion and scaled-Monte Carlo simulations. In contrast-detail phantoms, a 1.25-mm-diameter surface inclusion was detectable for scattering contrast >28%; a fraction of this scattering contrast (7%) was detectable for a 10 mm surface inclusion and at least 33% scattering contrast was detected up to 1.5 mm below the phantom surface, a probing depth relevant to breast surgical margin assessment. Recovered hemoglobin concentrations were insensitive to changes in scattering, except for overestimation at visible wavelengths for total hemoglobin concentrations <15  μM. The scattering amplitude increased linearly with scattering concentration, but the scattering slope depended on both the particle size and number density. Goodness of fit was comparable for the diffusion and scaled-Monte Carlo models of transport in spatially modulated, near-infrared reflectance acquired from 47 lumpectomy tissues, but recovered absorption parameters varied more linearly with expected hemoglobin concentration in liquid phantoms for the scaled-Monte Carlo forward model. SFDI could potentially reduce the high secondary excision rate associated with breast conserving surgery; its clinical translation further requires reduced image reconstruction time and smart inking strategies.

Scatter spectroscopic imaging distinguishes between breast pathologies in tissues relevant to surgical margin assessment

Purpose: A new approach to spectroscopic imaging was developed to detect and discriminate microscopic pathologies in resected breast tissues; diagnostic performance of the prototype system was tested in 27 tissues procured during breast conservative surgery. Experimental Design: A custom-built, scanning in situ spectroscopy platform sampled broadband reflectance from a 150-μm-diameter spot over a 1 × 1 cm2 field using a dark field geometry and telecentric lens; the system was designed to balance sensitivity to cellular morphology and imaging the inherent diversity within tissue subtypes. Nearly 300,000 broadband spectra were parameterized using light scattering models and spatially dependent spectral signatures were interpreted using a cooccurrence matrix representation of image texture. Results: Local scattering changes distinguished benign from malignant pathologies with 94% accuracy, 93% sensitivity, 95% specificity, and 93% positive and 95% negative predictive values using a threshold-based classifier. Texture and shape features were important to optimally discriminate benign from malignant tissues, including pixel-to-pixel correlation, contrast and homogeneity, and the shape features of fractal dimension and Euler number. Analysis of the region-based diagnostic performance showed that spectroscopic image features from 1 × 1 mm2 areas were diagnostically discriminant and enabled quantification of within-class tissue heterogeneities. Conclusions: Localized scatter-imaging signatures detected by the scanning spectroscopy platform readily distinguished benign from malignant pathologies in surgical tissues and showed new spectral-spatial signatures of clinical breast pathologies. Clin Cancer Res; 18(22); 6315–25. ©2012 AACR.

Automated classification of breast pathology using local measures of broadband reflectance

We demonstrate that morphological features pertinent to a tissues pathology may be ascertained from localized measures of broadband reflectance, with a mesoscopic resolution (100-μm lateral spot size) that permits scanning of an entire margin for residual disease. The technical aspects and optimization of a k-nearest neighbor classifier for automated diagnosis of pathologies are presented, and its efficacy is validated in 29 breast tissue specimens. When discriminating between benign and malignant pathologies, a sensitivity and specificity of 91 and 77% was achieved. Furthermore, detailed subtissue-type analysis was performed to consider how diverse pathologies influence scattering response and overall classification efficacy. The increased sensitivity of this technique may render it useful to guide the surgeon or pathologist where to sample pathology for microscopic assessment.

Measurement of pressure-displacement kinetics of hemoglobin in normal breast tissue with near-infrared spectral imaging

Applying localized external displacement to the breast surface can change the interstitial fluid pressure such that regional transient microvascular changes occur in oxygenation and vascular volume. Imaging these dynamic responses over time, while different pressures are applied, could provide selective temporal contrast for cancer relative to the surrounding normal breast. In order to investigate this possibility in normal breast tissue, a near-infrared spectral tomography system was developed that can simultaneously acquire data at three wavelengths with a 15 s time resolution per scan. The system was tested first with heterogeneous blood phantoms. Changes in regional blood concentrations were found to be linearly related to recovered mean hemoglobin concentration (Hb(T)) values (R(2)=0.9). In a series of volunteer breast imaging exams, data from 17 asymptomatic subjects were acquired under increasing and decreasing breast compression. Calculations show that a 10 mm displacement applied to the breast results in surface pressures in the range of 0-55 kPa depending on breast density. The recovered human data indicate that Hb(T) was reduced under compression and the normalized change was significantly correlated to the applied pressure with a p value of 0.005. The maximum Hb(T) decreases in breast tissue were associated with body mass index (BMI), which is a surrogate indicator of breast density. No statistically valid correlations were found between the applied pressure and the changes in tissue oxygen saturation (S(t)O(2)) or water percentage (H(2)O) across the range of BMI values studied.

In Vivo Imaging of Multidrug Resistance Using a Third Generation MDR1 Inhibitor

Cellular up-regulation of multidrug resistance protein 1 (MDR1) is a common cause for resistance to chemotherapy; development of third generation MDR1 inhibitors—several of which contain a common 6,7-dimethoxy-2-phenethyl-1,2,3,4-tetrahydroisoquinoline substructure—is underway. Efficacy of these agents has been difficult to ascertain, partly due to a lack of pharmacokinetic reporters for quantifying inhibitor localization and transport dynamics. Some of the recent third generation inhibitors have a pendant heterocycle, for example, a chromone moiety, which we hypothesized could be converted to a fluorophore. Following synthesis and teasing of a small set of analogues, we identified one lead compound that can be used as a cellular imaging agent that exhibits structural similarity and behavior akin to the latest generation of MDR1 inhibitors.

Dark-field scanning in situ spectroscopy platform for broadband imaging of resected tissue

A dark-field geometry spectral imaging system is presented to raster scan thick tissue samples in situ in 1.5 cm square sections, recovering full spectra from each 100 μm diameter pixel. This spot size provides adequate resolution for wide field scanning, while also facilitating scatter imaging without requiring sophisticated light-tissue transport modeling. The system is demonstrated showing accurate estimation of localized scatter parameters and the potential to recover absorption-based contrast from broadband reflectance data measured from 480 nm up to 750 nm in tissue phantoms. Results obtained from xenograft pancreas tumors show the ability to quantitatively image changes in localized scatter response in this fast-imaging geometry. The polychromatic raster scan design allows the rapid scanning necessary for use in surgical/clinical applications where timely decisions are required about tissue pathology.

A photoactivatable drug–caged fluorophore conjugate allows direct quantification of intracellular drug transport

We report here a method that utilizes a photoactivatable drug-caged fluorophore conjugate to quantify intracellular drug trafficking processes at single cell resolution. Photoactivation is performed in labeled cellular compartments to visualize intracellular drug exchange under physiological conditions, without the need for washing, facilitating its translation into in vivo cancer models.