Signal Intensity Analysis and Optimization for in Vivo Imaging of Cherenkov and Excited Luminescence.

During external beam radiotherapy (EBRT), in vivo Cherenkov optical emissions can be used as a dosimetry tool or to excite luminescence, termed Cherenkov-excited luminescence (CEL) with microsecond-level time-gated cameras. The goal of this work was to develop a complete theoretical foundation for the detectable signal strength, in order to provide guidance on optimization of the limits of detection and how to optimize near real time imaging. The key parameters affecting photon production, propagation and detection were considered and experimental validation with both tissue phantoms and a murine model are shown. Both the theoretical analysis and experimental data indicate that the detection level is near a single photon-per-pixel for the detection geometry and frame rates commonly used, with the strongest factor being the signal decrease with the square of distance from tissue to camera. Experimental data demonstrates how the SNR improves with increasing integration time, but only up to the point where the dominance of camera read noise is overcome by stray photon noise that cannot be suppressed. For the current camera in a fixed geometry, the signal to background ratio limits the detection of light signals, and the observed in vivo Cherenkov emission is on the order of 100×  stronger than CEL signals. As a result, imaging signals from depths  \u3c 15 mm is reasonable for Cherenkov light, and depths  \u3c 3 mm is reasonable for CEL imaging. The current investigation modeled Cherenkov and CEL imaging of two oxygen sensing phosphorescent compounds, but the modularity of the code allows for easy comparison of different agents or alternative cameras, geometries or tissues

Optical Tracer Size Differences Allow Quantitation of Active Pumping Rate Versus Stokes–Einstein Diffusion in Lymphatic Transport

Lymphatic uptake of interstitially administered agents occurs by passive convective–diffusive inflow driven by interstitial concentration and pressure, while the downstream lymphatic transport is facilitated by active propulsive contractions of lymphatic vessel walls. Near-infrared fluorescence imaging in mice was used to measure these central components of lymphatic transport for the first time, using two different-sized molecules––methylene blue (MB) and fluorescence-labeled antibody immunoglobulin G (IgG)-IRDye 680RD. This work confirms the hypothesis that lymphatic passive inflow and active propulsion rates can be separated based upon the relative differences in Stokes–Einstein diffusion coefficient. This coefficient specifically affects the passive-diffusive uptake when the interstitial volume and pressure are constant. Parameters such as mean time-to-peak signal, overall fluorescence signal intensities, and number of active peristaltic pulses, were estimated from temporal imaging data. While the mean time to attain peak signal representative of diffusion-dominated flow in the lymph vessels was 0.6±0.2  min for MB and 8±6  min for IgG, showing a size dependence, the active propulsion rates were 3.4±0.8  pulses/min and 3.3±0.5  pulses/min, respectively, appearing size independent. The propulsion rates for both dyes decreased with clearance from the interstitial injection-site, indicating intrinsic control of the smooth muscles in response to interstitial pressure. This approach to size-comparative agent flow imaging of lymphatic function can enable noninvasive characterization of diseases related to uptake and flow in lymph networks

Contrast Enhanced-Magnetic Resonance Imaging as a Surrogate to Map Verteporfin Delivery in Photodynamic Therapy

The use of in vivo contrast-enhanced magnetic resonance (MR) imaging as a surrogate for photosensitizer (verteporfin) dosimetry in photodynamic therapy of pancreas cancer is demonstrated by correlating MR contrast uptake to ex vivo fluorescence images on excised tissue. An orthotopic pancreatic xenograft mouse model was used for the study. A strong correlation ([i]r=0.57 ) was found for bulk intensity measurements of T1-weighted gadolinium enhancement and verteporfin fluorescence in the tumor region of interest. The use of contrast-enhanced MR imaging shows promise as a method for treatment planning and photosensitizer dosimetry in human photodynamic therapy (PDT) of pancreas cancer

Deletion of Rb Accelerates Pancreatic Carcinogenesis by Oncogenic Kras and Impairs Senescence in Pre-Malignant Lesions

Rb1 encodes a cell-cycle regulator that is functionally disrupted in most human cancers. Pancreatic ductal adenocarcinomas (PDACs) have a high frequency of mutations in KRAS and INK4A/CDKN2A that might allow cells to bypass the regulatory actions of retinoblastoma (RB). To determine the role of loss of RB function in PDAC progression, we investigated the effects of Rb disruption during pancreatic malignant transformation initiated by oncogenic Kras.We generated mice with pancreas-specific disruption of Rb, in the absence or presence of oncogenic Kras, to examine the role of RB in pancreatic carcinogenesis

Improved Tumor Contrast Achieved by Single Time Point Dual-Reporter Fluorescence Imaging

In this study, we demonstrate a method to quantify biomarker expression that uses an exogenous dual-reporter imaging approach to improve tumor signal detection. The uptake of two fluorophores, one nonspecific and one targeted to the epidermal growth factor receptor (EGFR), were imaged at 1 h in three types of xenograft tumors spanning a range of EGFR expression levels (n  =  6 in each group). Using this dual-reporter imaging methodology, tumor contrast-to-noise ratio was amplified by \u3e6 times at 1 h postinjection and \u3e2 times at 24 h. Furthermore, by as early as 20 min postinjection, the dual-reporter imaging signal in the tumor correlated significantly with a validated marker of receptor density (P  \u3c  0.05, r  =  0.93). Dual-reporter imaging can improve sensitivity and specificity over conventional fluorescence imaging in applications such as fluorescence-guided surgery and directly approximates the receptor status of the tumor, a measure that could be used to inform choices of biological therapies

Spatial Frequency Analysis of Anisotropic Drug Transport in Tumor Samples

Directional Fourier spatial frequency analysis was used on standard histological sections to identify salient directional bias in the spatial frequencies of stromal and epithelial patterns within tumor tissue. This directional bias is shown to be correlated to the pathway of reduced fluorescent tracer transport. Optical images of tumor specimens contain a complex distribution of randomly oriented aperiodic features used for neoplastic grading that varies with tumor type, size, and morphology. The internal organization of these patterns in frequency space is shown to provide a precise fingerprint of the extracellular matrix complexity, which is well known to be related to the movement of drugs and nanoparticles into the parenchyma, thereby identifying the characteristic spatial frequencies of regions that inhibit drug transport. The innovative computational methodology and tissue validation techniques presented here provide a tool for future investigation of drug and particle transport in tumor tissues, and could potentially be used a priori to identify barriers to transport, and to analyze real-time monitoring of transport with respect to therapeutic intervention

PV1 Down-Regulation via shRNA Inhibits the Growth of Pancreatic Adenocarcinoma Xenografts

PV1 is an endothelial-specific protein with structural roles in the formation of diaphragms in endothelial cells of normal vessels. PV1 is also highly expressed on endothelial cells of many solid tumours. On the basis of in vitro data, PV1 is thought to actively participate in angiogenesis. To test whether or not PV1 has a function in tumour angiogenesis and in tumour growth in vivo, we have treated pancreatic tumour-bearing mice by single-dose intratumoural delivery of lentiviruses encoding for two different shRNAs targeting murine PV1. We find that PV1 down-regulation by shRNAs inhibits the growth of established tumours derived from two different human pancreatic adenocarcinoma cell lines (AsPC-1 and BxPC-3). The effect observed is because of down-regulation of PV1 in the tumour endothelial cells of host origin, PV1 being specifically expressed in tumour vascular endothelial cells and not in cancer or other stromal cells. There are no differences in vascular density of tumours treated or not with PV1 shRNA, and gain and loss of function of PV1 in endothelial cells does not modify either their proliferation or migration, suggesting that tumour angiogenesis is not impaired. Together, our data argue that down-regulation of PV1 in tumour endothelial cells results in the inhibition of tumour growth via a mechanism different from inhibiting angiogenesis

Cherenkov Excited Short-Wavelength Infrared Fluorescence Imaging in vivo with External Beam Radiation

Cherenkov emission induced by external beam radiation therapy from a clinical linear accelerator (LINAC) can be used to excite phosphors deep in biological tissues. As with all luminescence imaging, there is a desire to minimize the spectral overlap between the excitation light and emission wavelengths, here between the Cherenkov and the phosphor. Cherenkov excited short-wavelength infrared (SWIR, 1000 to 1700 nm) fluorescence imaging has been demonstrated for the first time, using long Stokes-shift fluorophore PdSe quantum dots (QD) with nanosecond lifetime and an optimized SWIR detection. The 1  /  λ2 intensity spectrum characteristic of Cherenkov emission leads to low overlap of this into the fluorescence spectrum of PdSe QDs in the SWIR range. Additionally, using a SWIR camera itself inherently ignores the stronger Cherenkov emission wavelengths dominant across the visible spectrum. The SWIR luminescence was shown to extend the depth sensitivity of Cherenkov imaging, which could be used for applications in radiotherapy sensing and imaging in human tissue with targeted molecular probes

Collagen Complexity Spatially Defines Microregions of Total Tissue Pressure in Pancreatic Cancer.

The poor efficacy of systemic cancer therapeutics in pancreatic ductal adenocarcinoma (PDAC) is partly attributed to deposition of collagen and hyaluronan, leading to interstitial hypertension collapsing blood and lymphatic vessels, limiting drug delivery. The intrinsic micro-regional interactions between hyaluronic acid (HA), collagen and the spatial origins of mechanical stresses that close off blood vessels was investigated here. Multiple localized pressure measurements were analyzed with spatially-matched histochemical images of HA, collagen and vessel perfusion. HA is known to swell, fitting a linear elastic model with total tissue pressure (TTP) increasing above interstitial fluid pressure (IFP) directly with collagen content. However, local TTP appears to originate from collagen area fraction, as well as increased its entropy and fractal dimension, and morphologically appears to be maximized when HA regions are encapsulated by collagen. TTP was inversely correlated with vascular patency and verteporfin uptake, suggesting interstitial hypertension results in vascular compression and decreased molecular delivery in PDAC. Collagenase injection led to acute decreases in total tissue pressure and increased drug perfusion. Large microscopic variations in collagen distributions within PDAC leads to microregional TPP values that vary on the hundred micron distance scale, causing micro-heterogeneous limitations in molecular perfusion, and narrows viable treatment regimes for systemically delivered therapeutics