Showing papers by "Conor L. Evans published in 2016"

Raman technologies in cancer diagnostics

Abstract: Despite significant effort, cancer still remains a leading cause of death worldwide. In order to reduce its burden, the development and improvement of noninvasive strategies for early detection and diagnosis of cancer are urgently needed. Raman spectroscopy, an optical technique that relies on inelastic light scattering arising from molecular vibrations, is one such strategy, as it can noninvasively probe cancerous markers using only endogenous contrast. In this review, spontaneous, coherent and surface enhanced Raman spectroscopies and imaging, as well as the fundamental principles governing the successful use of these techniques, are discussed. Methods for spectral data analysis are also highlighted. Utilization of the discussed Raman techniques for the detection and diagnosis of cancer in vitro, ex vivo and in vivo is described. The review concludes with a discussion of the future directions of Raman technologies, with particular emphasis on their clinical translation.

Promotion of Proapoptotic Signals by Lysosomal Photodamage: Mechanistic Aspects and Influence of Autophagy.

Abstract: Prior studies demonstrated that a low level (LD10-15 ) of lysosomal photodamage can sensitize cells to the apoptotic death that results from subsequent mitochondrial photodamage. We have proposed that this process occurs via a calpain-catalyzed cleavage of the autophagy-associated protein ATG5 to form a proapoptotic fragment. In this report, we provide evidence for the postulated ATG5 cleavage and show that the sequential photodynamic therapy (PDT) protocol can also partly overcome the adverse effect of hypoxia on the initiation of apoptosis. While autophagy can offer cytoprotection after mitochondrial photodamage, this does not appear to apply when lysosomes are the target. This may account for the ability of very low PDT doses directed at lysosomes to evoke ATG5 cleavage. The resulting proapoptotic effect overcomes intrinsic cytoprotection from mitochondrial photodamage along with a further stimulation of phototoxicity.

PLGA nanoparticle encapsulation reduces toxicity while retaining the therapeutic efficacy of EtNBS-PDT in vitro

Abstract: Photodynamic therapy regimens, which use light-activated molecules known as photosensitizers, are highly selective against many malignancies and can bypass certain challenging therapeutic resistance mechanisms. Photosensitizers such as the small cationic molecule EtNBS (5-ethylamino-9-diethyl-aminobenzo[a]phenothiazinium chloride) have proven potent against cancer cells that reside within acidic and hypoxic tumour microenvironments. At higher doses, however, these photosensitizers induce “dark toxicity” through light-independent mechanisms. In this study, we evaluated the use of nanoparticle encapsulation to overcome this limitation. Interestingly, encapsulation of the compound within poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PLGA-EtNBS) was found to significantly reduce EtNBS dark toxicity while completely retaining the molecule’s cytotoxicity in both normoxic and hypoxic conditions. This dual effect can be attributed to the mechanism of release: EtNBS remains encapsulated until external light irradiation, which stimulates an oxygen-independent, radical-mediated process that degrades the PLGA nanoparticles and releases the molecule. As these PLGA-encapsulated EtNBS nanoparticles are capable of penetrating deeply into the hypoxic and acidic cores of 3D spheroid cultures, they may enable the safe and efficacious treatment of otherwise unresponsive tumour regions.

In vivo coherent Raman imaging of the melanomagenesis-associated pigment pheomelanin

Abstract: Melanoma is the most deadly form of skin cancer with a yearly global incidence over 232,000 patients. Individuals with fair skin and red hair exhibit the highest risk for developing melanoma, with evidence suggesting the red/blond pigment known as pheomelanin may elevate melanoma risk through both UV radiation-dependent and -independent mechanisms. Although the ability to identify, characterize, and monitor pheomelanin within skin is vital for improving our understanding of the underlying biology of these lesions, no tools exist for real-time, in vivo detection of the pigment. Here we show that the distribution of pheomelanin in cells and tissues can be visually characterized non-destructively and noninvasively in vivo with coherent anti-Stokes Raman scattering (CARS) microscopy, a label-free vibrational imaging technique. We validated our CARS imaging strategy in vitro to in vivo with synthetic pheomelanin, isolated melanocytes, and the Mc1re/e, red-haired mouse model. Nests of pheomelanotic melanocytes were observed in the red-haired animals, but not in the genetically matched Mc1re/e; Tyrc/c (“albino-red-haired”) mice. Importantly, samples from human amelanotic melanomas subjected to CARS imaging exhibited strong pheomelanotic signals. This is the first time, to our knowledge, that pheomelanin has been visualized and spatially localized in melanocytes, skin, and human amelanotic melanomas.

Multiphoton excited hemoglobin fluorescence and third harmonic generation for non-invasive microscopy of stored blood.

Abstract: Red blood cells (RBC) in two-photon excited fluorescence (TPEF) microscopy usually appear as dark disks because of their low fluorescent signal. Here we use 15fs 800nm pulses for TPEF, 45fs 1060nm pulses for three-photon excited fluorescence, and third harmonic generation (THG) imaging. We find sufficient fluorescent signal that we attribute to hemoglobin fluorescence after comparing time and wavelength resolved spectra of other expected RBC endogenous fluorophores: NADH, FAD, biliverdin, and bilirubin. We find that both TPEF and THG microscopy can be used to examine erythrocyte morphology non-invasively without breaching a blood storage bag.

PLGA nanoparticle encapsulation reduces toxicity while retaining the therapeutic efficacy of EtNBS-PDT in vitro

Abstract: Photodynamic therapy regimens, which use light-activated molecules known as photosensitizers, are highly selective against many malignancies and can bypass certain challenging therapeutic resistance mechanisms. Photosensitizers such as the small cationic molecule EtNBS (5-ethylamino-9-diethyl-aminobenzo[a]phenothiazinium chloride) have proven potent against cancer cells that reside within acidic and hypoxic tumour microenvironments. At higher doses, however, these photosensitizers induce “dark toxicity” through light-independent mechanisms. In this study, we evaluated the use of nanoparticle encapsulation to overcome this limitation. Interestingly, encapsulation of the compound within poly(lactic-co-glycolic acid) (PLGA) nanoparticles (PLGA-EtNBS) was found to significantly reduce EtNBS dark toxicity while completely retaining the molecule’s cytotoxicity in both normoxic and hypoxic conditions. This dual effect can be attributed to the mechanism of release: EtNBS remains encapsulated until external light irradiation, which stimulates an oxygen-independent, radical-mediated process that degrades the PLGA nanoparticles and releases the molecule. As these PLGA-encapsulated EtNBS nanoparticles are capable of penetrating deeply into the hypoxic and acidic cores of 3D spheroid cultures, they may enable the safe and efficacious treatment of otherwise unresponsive tumour regions.

Longitudinal, label-free, quantitative tracking of cell death and viability in a 3D tumor model with OCT

Abstract: Three-dimensional in vitro tumor models are highly useful tools for studying tumor growth and treatment response of malignancies such as ovarian cancer. Existing viability and treatment assessment assays, however, face shortcomings when applied to these large, complex, and heterogeneous culture systems. Optical coherence tomography (OCT) is a noninvasive, label-free, optical imaging technique that can visualize live cells and tissues over time with subcellular resolution and millimeters of optical penetration depth. Here, we show that OCT is capable of carrying out high-content, longitudinal assays of 3D culture treatment response. We demonstrate the usage and capability of OCT for the dynamic monitoring of individual and combination therapeutic regimens in vitro, including both chemotherapy drugs and photodynamic therapy (PDT) for ovarian cancer. OCT was validated against the standard LIVE/DEAD Viability/Cytotoxicity Assay in small tumor spheroid cultures, showing excellent correlation with existing standards. Importantly, OCT was shown to be capable of evaluating 3D spheroid treatment response even when traditional viability assays failed. OCT 3D viability imaging revealed synergy between PDT and the standard-of-care chemotherapeutic carboplatin that evolved over time. We believe the efficacy and accuracy of OCT in vitro drug screening will greatly contribute to the field of cancer treatment and therapy evaluation.

Sweating the small stuff.

Abstract: skin cancer in Germany. Br J Dermatol 2016; 174:778–85. 4 Rudolph C, Schnoor M, Eisemann N, Katalinic A. Incidence trends of nonmelanoma skin cancer in Germany from 1998 to 2010. J Dtsch Dermatol Ges 2015; 13:788–97. 5 Papandreou D, Hamid ZT. The role of vitamin D in diabetes and cardiovascular disease: an updated review of the literature. Dis Markers 2015; 2015:580474. 6 Green AC, Baade P, Coory M et al. Population-based 20-year survival among people diagnosed with thin melanomas in Queensland, Australia. J Clin Oncol 2012; 30:1462–7. 7 Whiteman DC, Baade PD, Olsen CM. More people die from thin melanomas (≤1 mm) than from thick melanomas (>4 mm) in Queensland, Australia. J Invest Dermatol 2015; 135:1190–3. 8 Youlden DR, Soyer HP, Youl PH et al. Incidence and survival for Merkel cell carcinoma in Queensland, Australia, 1993–2010. JAMA Dermatol 2014; 150:864–72. 9 Lemos BD, Storer BE, Iyer JG et al. Pathologic nodal evaluation improves prognostic accuracy in Merkel cell carcinoma: analysis of 5823 cases as the basis of the first consensus staging system. J Am Acad Dermatol 2010; 63:751–61.

Special Section Guest Editorial:Translational biophotonics

Abstract: Author(s): Aguirre, Aaron D; Apiou-Sbirlea, Gabriela; Roblyer, Darren; Tromberg, Bruce J | Abstract: This guest editorial introduces the Special Section on Translational Biophotonics.

Photoacoustic imaging sounds the alarm on liver fibrosis.

Abstract: Liver fibrosis is a "silent" disease. Repeated liver injury can develop without symptoms for decades before detection. This progressive scarring can be caused by inflammatory processes related to hepatitis B or C infections, obesity, and long-term alcohol abuse. Although short-term inflammatory injuries to the liver are largely reversible, long-term chronic conditions can drive liver fibrosis toward cirrhosis or even liver failure. Blood tests alone are not reliable for detecting the disease, leaving biopsies as the gold standard for diagnosis. An inexpensive, noninvasive screening method that could be carried out in a physician's office would revolutionize the detection and prevention of liver fibrosis and advanced liver disease. Now, van den Berg et al. describe point-of-care imaging technology that might one day find utility in the clinical diagnosis of liver fibrosis. The research team made use of the molecular imaging tool known as photoacoustic (PA) imaging to visualize and detect fibrotic conditions. In the PA process, light absorbed by endogenous chromophores, such as hemoglobin, triggers the generation of acoustic waves at ultrasonic frequencies. These waves can be detected using ultrasound (US) transducers to build three-dimensional images. The same transducer can be operated alongside PA for combined standard US imaging. The authors created an inexpensive handheld PA/US tool based on pulsed light emitting diodes (LEDs), a significant simplification over the standard pulsed laser systems found in most laboratories. Using a mouse model of chronic liver injury, the authors were able to follow the remodeling of the liver tissue and correlate their PA/US findings with gold standard histological parameters of liver fibrosis. This finding proves the potential for such a device in the preclinical setting and hopefully sets the stage for future clinical instruments in the future. Several hurdles will need to be overcome to scale this technology to humans, including improving the delivery of light to the liver and building registration methods to revisit liver locations for long-term monitoring. P. J. van den Berg et al ., Preclinical detection of liver fibrosis using dual-modality photoacoustic/ultrasound system. Biomed. Opt. Express 7 , 5081–5091 (2016). [[Full Text]][1] [1]: https://www.osapublishing.org/boe/abstract.cfm?uri=boe-7-12-5081

Peering inside the mind: Imaging brain activity with advanced diffuse optical tomography

Abstract: Brain imaging has become central to a host of research and clinical applications, from magnetic resonance imaging (MRI) investigations of consciousness and thought to traumatic brain injury diagnostics. Functional brain imaging can interrogate activity and quantify processes in a spatiotemporal manner throughout the brain and is therefore useful across many clinical and research settings. Functional MRI has been a core research tool in this field, giving researchers and clinicians the ability to map activity throughout the adult brain. Diffuse optical tomography (DOT) is an optical spectroscopy technology that uses light to monitor brain activity. Light can undergo multiple scattering events after entering the tissue, with some photons redirected back towards the tissue surface. The distance this light travels, as well as changes in its intensity and spectrum, carries information that can be decoded to measure events (e.g., absorption) and map their locations within tissue. Some of the first applications using this toolkit to capture brain activity measured hemodynamics via the detection of oxygenated and deoxygenated hemoglobin through the intact skull using red and near-infrared light. Now, Chitnis et al. introduced a new DOT method for imaging brain activity that makes use of a fine meshwork of multiple miniature LED and detector modules. The combination of four modules yields 128 channels over a wide area of the scalp, providing the spatial resolution to map the 3D localization of hemodynamic changes. When subjects performed a thumb-to-finger extension movement, the module system was able to visualize the activated region within the motor cortex by detecting changes in hemoglobin oxygenation. The authors noted that while this measurement was made with four modules, their current method could be scaled theoretically to 75 modules for imaging over the entire scalp. Unlike functional MRI machines, this imaging system is lightweight and highly portable, which has the potential to enable wearable, functional brain imaging in office, field, and low-resource settings. D. Chitnis et al ., Functional imaging of the human brain using a modular, fibre-less, high-density diffuse optical tomography system. Biomed. Opt. Express 7 , 4275–4288 (2016). [[Full Text]][1] [1]: https://www.osapublishing.org/boe/fulltext.cfm?uri=boe-7-10-4275&id=350433

Non-invasive visualization and quantification of natural pigments

Abstract: A system for visualizing melanin present in tissue can include an imaging system to record a signal based on a presence of melanin in tissue and a display device to display an image based on the signal. A first laser source can emit a Stokes pulse train and a second laser source can emit a pump pulse train. Both the first laser source and the second laser source comprise a tunable center wavelength or frequency. An energy difference between a frequency of the Stokes pulse train and a frequency of the pump pulse train is from 1750 cm -1 to 2250 cm -1 . The Stokes and the pump pulse train overlap in space and time. A scanning mechanism focuses the combined Stokes pulse train and pump pulse train within the tissue and scans across the tissue. A detector detects the signal based on a presence of melanin within the tissue.

In vivo coherent Raman imaging of the melanomagenesis-associated pigment pheomelanin

Abstract: Melanoma is the most deadly form of skin cancer with a yearly global incidence over 232,000 patients. Individuals with fair skin and red hair exhibit the highest risk for developing melanoma, with evidence suggesting the red/blond pigment known as pheomelanin may elevate melanoma risk through both UV radiation-dependent and -independent mechanisms. Although the ability to identify, characterize, and monitor pheomelanin within skin is vital for improving our understanding of the underlying biology of these lesions, no tools exist for real-time, in vivo detection of the pigment. Here we show that the distribution of pheomelanin in cells and tissues can be visually characterized non-destructively and noninvasively in vivo with coherent anti-Stokes Raman scattering (CARS) microscopy, a label-free vibrational imaging technique. We validated our CARS imaging strategy in vitro to in vivo with synthetic pheomelanin, isolated melanocytes, and the Mc1re/e, red-haired mouse model. Nests of pheomelanotic melanocytes were observed in the red-haired animals, but not in the genetically matched Mc1re/e; Tyrc/c (“albino-red-haired”) mice. Importantly, samples from human amelanotic melanomas subjected to CARS imaging exhibited strong pheomelanotic signals. This is the first time, to our knowledge, that pheomelanin has been visualized and spatially localized in melanocytes, skin, and human amelanotic melanomas.

Theranostic nanoparticles give the best of both worlds

Abstract: The delivery of therapeutics into tumors poses a number of challenges, including leaky neovascular networks that feed cancer cells, dense stroma associated with many tumors, and acidic interstitial spaces that act to reduce drug delivery. Theranostics—agents that have both therapeutic and

The nature of multiphoton fluorescence from red blood cells

Abstract: We report on the nature of multiphoton excited fluorescence observed from human erythrocytes (red blood cells RBC's) and their "ghosts" following 800nm sub-15 fs excitation. The detected optical signal is assigned as two-photon excited fluorescence from hemoglobin. Our findings are supported by wavelength-resolved fluorescence lifetime decay measurements using time-correlated single photon counting system from RBC's, their ghosts as well as in vitro samples of various fluorophores including riboflavin, NADH, NAD(P)H, hemoglobin. We find that low-energy and short-duration pulses allow two-photon imaging of RBC’s, but longer more intense pulses lead to their destruction.