Cathepsin protease expression in infiltrative soft tissue sarcomas: cathepsin-K correlates with infiltrative tumor growth and clinical outcomes.

Cathepsin proteases, activated in the lysosomes, are upregulated in many cancers. Intraoperative detection systems of microscopic residual tumor using cathepsin-mediated release of fluorescent nanoparticles may guide surgical excisions to improve local control. We sought to define the genetic and proteomic expression of cathepsins and their clinicopathological correlates in myxofibrosarcoma and undifferentiated pleomorphic sarcoma (UPS)-soft tissue sarcomas with high rates of positive resection margins and local recurrence-and to establish a cellular justification for cathepsin-dependent systems to identify residual cancer in the resection bed. Real-time quantitative polymerase chain reaction analysis of 58 fresh-frozen tumor specimens revealed that 56 (97%) had elevated mRNA expression of ≥1 cathepsin, including cathepsin-B (79%), cathepsin-K (59%), cathepsin-L (71%), and -S (71%). Immunohistochemical analysis of these fresh-frozen specimens revealed that 98% of tumors were positive for one or more of cathepsin-B (85%), cathepsin-K (50%), cathepsin-L (63%), and -S (10%). Strong cathepsin-K expression was associated with greater risks of local recurrence (hazard ratio, 3.78; p = 0.044) and disease-specific mortality (hazard ratio, 3.70; p = 0.025). Immunohistochemical analysis of 33 formalin-fixed paraffin-embedded block samples revealed that 97% were positive for cathepsin-B (88%), cathepsin-K (76%), cathepsin-L (52%), or -S (52%) at the tumor periphery; cathepsin-K positivity correlated with a radiographic tail-like sign (p = 0.004) and microscopic infiltrative growth (p = 0.020). We conclude that cathepsins are broadly overexpressed in myxofibrosarcoma and UPS, and cathepsin-K may be an immunohistochemical marker of local infiltration and poorer prognosis that could be used to guide precision surgery.

Methods for registering and calibrating in vivo terahertz images of cutaneous burn wounds.

A method to register THz and visible images of cutaneous burn wounds and to calibrate THz image data is presented. Images of partial and full thickness burn wounds in 9 rats were collected over 435 mins. = 7.25 hours following burn induction. A two-step process was developed to reference the unknown structure of THz imaging contrast to the known structure and the features present in visible images of the injury. This process enabled the demarcation of a wound center for each THz image, independent of THz contrast. Threshold based segmentation enabled the automated identification of air (0% reflectivity), brass (100% reflectivity), and abdomen regions within the registered THz images. Pixel populations, defined by the segmentations, informed unsupervised image calibration and contrast warping for display. The registered images revealed that the largest variation in THz tissue reflectivity occurred superior to the contact region at ~0.13%/min. Conversely the contact region showed demonstrated an ~6.5-fold decrease at ~0.02%/min. Exploration of occlusion effects suggests that window contact may affect the measured edematous response.

THz imaging system for in vivo human cornea.

Terahertz (THz) imaging of corneal tissue water content (CTWC) is a proposed method for early, accurate detection and study of corneal diseases. Despite promising results from ex vivo and in vivo cornea studies, interpretation of the reflectivity data is confounded by the contact between corneal tissue and rigid dielectric window used to flatten the imaging field. This work develops a novel imaging system and image reconstruction methods specifically for nearly spherical targets such as human cornea. A prototype system was constructed using a 650 GHz multiplier source and Schottky diode detector. Resolution and imaging field strength measurement from characterization targets correlate well with those predicted by the quasioptical theory and physical optics analysis. Imaging experiments with corneal phantoms and ex vivo corneas demonstrate the hydration sensitivity of the imaging system and reliable measurement of CTWC. We present successful acquisition of non-contact THz images of in vivo human cornea, and discuss strategies for optimizing the imaging system design for clinical use.

Optical System Design for Noncontact, Normal Incidence, THz Imaging of in vivo Human Cornea.

Reflection mode Terahertz (THz) imaging of corneal tissue water content (CTWC) is a proposed method for early, accurate detection and study of corneal diseases. Despite promising results from ex vivo and in vivo cornea studies, interpretation of the reflectivity data is confounded by the contact between corneal tissue and dielectric windows used to flatten the imaging field. Herein, we present an optical design for non-contact THz imaging of cornea. A beam scanning methodology performs angular, normal incidence sweeps of a focused beam over the corneal surface while keeping the source, detector, and patient stationary. A quasioptical analysis method is developed to analyze the theoretical resolution and imaging field intensity profile. These results are compared to the electric field distribution computed with a physical optics analysis code. Imaging experiments validate the optical theories behind the design and suggest that quasioptical methods are sufficient for designing of THz corneal imaging systems. Successful imaging operations support the feasibility of non-contact in vivo imaging. We believe that this optical system design will enable the first, clinically relevant, in vivo exploration of CTWC using THz technology.

Terahertz Imaging of Cutaneous Edema: Correlation With Magnetic Resonance Imaging in Burn Wounds.

OBJECTIVE: In vivo visualization and quantification of edema, or 'tissue swelling' following injury, remains a clinical challenge. Herein, we investigate the ability of reflective terahertz (THz) imaging to track changes in tissue water content (TWC)-the direct indicator of edema-by comparison to depth-resolved magnetic resonance imaging (MRI) in a burn-induced model of edema. METHODS: A partial thickness and full thickness burns were induced in an in vivo rat model to elicit unique TWC perturbations corresponding to burn severity. Concomitant THz surface maps and MRI images of both burn models were acquired with a previously reported THz imaging system and T2-weighted MRI, respectively, over 270 min. Reflectivity was analyzed for the burn contact area in THz images, while proton density (i.e., mobile TWC) was analyzed for the same region at incrementally increasing tissue depths in companion, transverse MRI images. A normalized cross correlation of THz and depth-dependent MRI measurements was performed as a function of time in histologically verified burn wounds. RESULTS: For both burn types, strong positive correlations were evident between THz reflectivity and MRI data analyzed at greater tissue depths (>258 µm). MRI and THz results also revealed biphasic trends consistent with burn edema pathogenesis. CONCLUSION: This paper offers the first in vivo correlative assessment of mobile TWC-based contrast and the sensing depth of THz imaging. SIGNIFICANCE: The ability to implement THz imaging immediately following injury, combined with TWC sensing capabilities that compare to MRI, further support THz sensing as an emerging tool to track fluid in tissue.

Non-invasive terahertz imaging of tissue water content for flap viability assessment.

Accurate and early prediction of tissue viability is the most significant determinant of tissue flap survival in reconstructive surgery. Perturbation in tissue water content (TWC) is a generic component of the tissue response to such surgeries, and, therefore, may be an important diagnostic target for assessing the extent of flap viability in vivo. We have previously shown that reflective terahertz (THz) imaging, a non-ionizing technique, can generate spatially resolved maps of TWC in superficial soft tissues, such as cornea and wounds, on the order of minutes. Herein, we report the first in vivo pilot study to investigate the utility of reflective THz TWC imaging for early assessment of skin flap viability. We obtained longitudinal visible and reflective THz imagery comparing 3 bipedicled flaps (i.e. survival model) and 3 fully excised flaps (i.e. failure model) in the dorsal skin of rats over a postoperative period of 7 days. While visual differences between both models manifested 48 hr after surgery, statistically significant (p < 0.05, independent t-test) local differences in TWC contrast were evident in THz flap image sets as early as 24 hr. Excised flaps, histologically confirmed as necrotic, demonstrated a significant, yet localized, reduction in TWC in the flap region compared to non-traumatized skin. In contrast, bipedicled flaps, histologically verified as viable, displayed mostly uniform, unperturbed TWC across the flap tissue. These results indicate the practical potential of THz TWC sensing to accurately predict flap failure 24 hours earlier than clinical examination.

Engineering A11 Minibody-Conjugated, Polypeptide-Based Gold Nanoshells for Prostate Stem Cell Antigen (PSCA)-Targeted Photothermal Therapy.

Currently, there is no curative treatment for advanced metastatic prostate cancer, and options, such as chemotherapy, are often nonspecific, harming healthy cells and resulting in severe side effects. Attaching targeting ligands to agents used in anticancer therapies has been shown to improve efficacy and reduce nonspecific toxicity. Furthermore, the use of triggered therapies can enable spatial and temporal control over the treatment. Here, we combined an engineered prostate cancer-specific targeting ligand, the A11 minibody, with a novel photothermal therapy agent, polypeptide-based gold nanoshells, which generate heat in response to near-infrared light. We show that the A11 minibody strongly binds to the prostate stem cell antigen that is overexpressed on the surface of metastatic prostate cancer cells. Compared to nonconjugated gold nanoshells, our A11 minibody-conjugated gold nanoshell exhibited significant laser-induced, localized killing of prostate cancer cells in vitro. In addition, we improved upon a comprehensive heat transfer mathematical model that was previously developed by our laboratory. By relaxing some of the assumptions of our earlier model, we were able to generate more accurate predictions for this particular study. Our experimental and theoretical results demonstrate the potential of our novel minibody-conjugated gold nanoshells for metastatic prostate cancer therapy.

Polypeptide-Based Gold Nanoshells for Photothermal Therapy.

Targeted killing of cancer cells by engineered nanoparticles holds great promise for noninvasive photothermal therapy applications. We present the design and generation of a novel class of gold nanoshells with cores composed of self-assembled block copolypeptide vesicles with photothermal properties. Specifically, poly(L-lysine)60- block-poly(L-leucine)20 (K60L20) block copolypeptide vesicles coated with a thin layer of gold demonstrate enhanced absorption of light due to surface plasmon resonance (SPR) in the near-infrared range. We show that the polypeptide-based K60L20 gold nanoshells have low toxicity in the absence of laser exposure, significant heat generation upon exposure to near-infrared light, and, as a result, localized cytotoxicity within the region of laser irradiation in vitro. To gain a better understanding of our gold nanoshells in the context of photothermal therapy, we developed a comprehensive mathematical model for heat transfer and experimentally validated this model by predicting the temperature as a function of time and position in our experimental setup. This model can be used to predict which parameters of our gold nanoshells can be manipulated to improve heat generation for tumor destruction. To our knowledge, our results represent the first ever use of block copolypeptide vesicles as the core material of gold nanoshells.

Preliminary results of non-contact THz imaging of cornea.

This paper presents a novel THz optical design that allows the acquisition of THz reflectivity maps of in vivo cornea without the need for a field flattening window and preliminary imaging results of in vivo rabbit cornea. The system's intended use is to sense small changes in corneal tissue water content (CTWC) that can be precursors for a host of diseases and pathologies. Unique beam optics allows the scanning of a curved surface at normal incidence while keeping the source detector and target stationary. Basic system design principles are discussed and image sets of spherical calibration targets and corneal phantom models are presented. The presented design will enable, for the first time, non-contact THz imaging of animal and human cornea.

THz and mm-Wave Sensing of Corneal Tissue Water Content: Electromagnetic Modeling and Analysis.

Terahertz (THz) spectral properties of human cornea are explored as a function of central corneal thickness (CCT) and corneal water content, and the clinical utility of THz-based corneal water content sensing is discussed. Three candidate corneal tissue water content (CTWC) perturbations, based on corneal physiology, are investigated that affect the axial water distribution and total thickness. The THz frequency reflectivity properties of the three CTWC perturbations were simulated and explored with varying system center frequency and bandwidths (Q-factors). The modeling showed that at effective optical path lengths on the order of a wavelength the cornea presents a lossy etalon bordered by air at the anterior and the aqueous humor at the posterior. The simulated standing wave peak-to-valley ratio is pronounced at lower frequencies and its effect on acquired data can be modulated by adjusting the bandwidth of the sensing system. These observations are supported with experimental spectroscopic data. The results suggest that a priori knowledge of corneal thickness can be utilized for accurate assessments of corneal tissue water content. The physiologic variation of corneal thickness with respect to the wavelengths spanned by the THz band is extremely limited compared to all other structures in the body making CTWC sensing unique amongst all proposed applications of THz medical imaging.

THz and mm-Wave Sensing of Corneal Tissue Water Content: In Vivo Sensing and Imaging Results.

A pulsed terahertz (THz) imaging system and millimeter-wave reflectometer were used to acquire images and point measurements, respectively, of five rabbit cornea in vivo. These imaging results are the first ever produced of in vivo cornea. A modified version of a standard protocol using a gentle stream of air and a Mylar window was employed to slightly dehydrate healthy cornea. The sensor data and companion central corneal thickness (CCT) measurements were acquired every 10-15 min over the course of two hours using ultrasound pachymmetry.. Statistically significant positive correlations were established between CCT measurements and millimeter wave reflectivity. Local shifts in reflectivity contrast were observed in the THz imagery; however, the THz reflectivity did not display a significant correlation with thickness in the region probed by the 100 GHz and CCT measurements. This is explained in part by a thickness sensitivity at least 10× higher in the mm-wave than the THz systems. Stratified media and effective media modeling suggest that the protocol perturbed the thickness and not the corneal tissue water content (CTWC). To further explore possible etalon effects, an additional rabbit was euthanized and millimeter wave measurements were obtained during death induced edema. These observations represent the first time that the uncoupled sensing of CTWC and CCT have been achieved in vivo.