Advanced optical microscopy toolkits for non-invasive imaging in oncology

Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, 2018.Cataloged from PDF version of thesis.Includes bibliographical references (pages 245-271).Despite significant advances in the fields of biophotonics and oncology alike, several challenges persist in the study, assessment, and treatment of cancer, ranging from the accurate identification and examination of potential risk factors, early diagnosis of dysplastic lesions, and monitoring of the complex heterogeneity of cellular populations within tumors. To study such dynamics at the microscale, non-invasive optical toolkits offer the potential to identify, characterize, and visualize key molecules and their interactions in their native biological context, ranging from in vitro cell cultures to in vivo studies in both animal models and humans. In the present thesis, examples of such applications of optical tools will be presented, including: (1) the assessment of cellular oxidative stress in ex vivo human skin cultures by imaging endogenous and exogenous fluorescent compounds using two-photon excitation fluorescence (TPEF) and fluorescence lifetime imaging microscopy (FLIM); (2) visualizing water and lipid distribution as well as cellular morphology using coherent Raman scattering (CRS) imaging techniques in the stratum corneum, the most superficial layer of the epidermis; (3) using photoconvertible labels to optically tag cell sub-populations of interest in situ for long-term monitoring of heterogeneous cell cultures from in vitro monolayers to in vivo xenograft models; (4) visualizing melanin species in the context of melanoma with coherent anti-Stokes Raman scattering (CARS) and sum-frequency absorption (SFA) microscopies. Altogether, development of such advanced microscopy toolkits will serve to improve both understanding of cancer pathology, as well as to validate clinical diagnostic and therapeutic strategies.by Sam Osseiran.Ph. D. in Medical Engineering and Medical Physic

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

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

Enhanced quantification of metabolic activity for individual adipocytes by label-free FLIM

Fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) allows for label-free quantification of metabolic activity of individual cells over time and in response to various stimuli, which is not feasible using traditional methods due to their destructive nature and lack of spatial information. This study uses FLIM to measure pharmacologically induced metabolic changes that occur during the browning of white fat. Adipocyte browning increases energy expenditure, making it a desirable prospect for treating obesity and related disorders. Expanding from the traditional two-lifetime model of NADH to a four-lifetime model using exponential fitting and phasor analysis of the fluorescence decay results in superior metabolic assessment compared to traditional FLIM analysis. The four lifetime components can also be mapped to specific cellular compartments to create a novel optical ratio that quantitatively reflects changes in mitochondrial and cytosolic NADH concentrations and binding states. This widely applicable approach constitutes a powerful tool for studies where monitoring cellular metabolism is of key interest

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

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

Visualization 2: Multiphoton excited hemoglobin fluorescence and third harmonic generation for non-invasive microscopy of stored blood

THG of flowing RBCs Originally published in Biomedical Optics Express on 01 September 2016 (boe-7-9-3449

Using elongated microparticles to enhance tailorable nanoemulsion delivery in excised human skin and volunteers

This study demonstrates, for the first time, clinical testing of elongated silica microparticles (EMP) combined with tailorable nanoemulsions (TNE) to enhance topical delivery of hydrophobic drug surrogates. Likewise, this is the first report of 6-carboxyfluorescein (a model molecule for topically delivered hydrophobic drugs) AM1 & DAMP4 (novel short peptide surfactants) used in volunteers. The EMP penetrates through the epidermis and stop at the dermal-epidermal junction (DEJ). TNE are unusually stable and useful because the oil core allows high drug loading levels and the surface properties can be easily controlled. At first, we chose alginate as a crosslinking agent between EMP and TNE. We initially incorporated a fluorescent lipophilic dye, DiI, as a hydrophobic drug surrogate into TNE for visualization with microscopy. We compared four different coating approaches to combine EMP and TNE and tested these formulations in freshly excised human skin. The delivery profile characterisation was imaged by dye- free coherent anti-Stoke Raman scattering (CARS) microscopy to detect the core droplet of TNE that was packed with pharmaceutical grade lipid (glycerol) instead of DiI. These data show the EMP penetrating to the DEJ followed by controlled release of the TNE. Freeze-dried formulations with crosslinking resulted in a sustained release profile, whereas a freeze-dried formulation without crosslinking showed an immediate burst-type release profile. Finally, we tested the crosslinked TNE coated EMP formulation in volunteers using multiphoton microscopy (MPM) and fluorescence-lifetime imaging microscopy (FLIM) to document the penetration depth characteristics. These forms of microscopy have limitations in terms of image acquisition speed and imaging area coverage but can detect fluorescent drug delivery through the superficial skin in volunteers. 6-Carboxyfluorescein was selected as the fluorescent drug surrogate for the volunteer study based on the similarity of size, charge and hydrophobicity characteristics to small therapeutic drugs that are difficult to deliver through skin. The imaging data showed a 6-carboxyfluorescein signal deep in volunteer skin supporting the hypothesis that EMP can indeed enhance the delivery of TNE in human skin. There were no adverse events recorded at the time of the study or after the study, supporting the use of 6-carboxyfluorescein as a safe and detectable drug surrogate for topical drug research. In conclusion, dry formulations, with controllable release profiles can be obtained with TNE coated EMP that can effectively enhance hydrophobic payload delivery deep into the human epidermis

NNT mediates redox-dependent pigmentation via a UVB- and MITF-independent mechanism

Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes.Fil: Allouche, Jennifer. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Rachmin, Inbal. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Adhikari, Kaustubh. Colegio Universitario de Londres; Reino Unido. The Open University (ou); Reino UnidoFil: Pardo, Luba M.. Erasmus Medical Center; Países BajosFil: Lee, Ju Hee. Yonsei University College of Medicine; Corea del SurFil: McConnell, Alicia M.. Howard Hughes Medical Institute; Estados UnidosFil: Kato, Shinichiro. Nagoya University Graduate School of Medicine; Japón. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Fan, Shaohua. Fudan University; ChinaFil: Kawakami, Akinori. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Suita, Yusuke. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Wakamatsu, Kazumasa. Fujita Health University; JapónFil: Igras, Vivien. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Zhang, Jianming. Shanghai Jiao Tong University School of Medicine; ChinaFil: Navarro, Paula P.. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Lugo, Camila Makhlouta. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Noonan, Haley R.. Howard Hughes Medical Institute; Estados Unidos. Boston Children’s Hospital; Estados UnidosFil: Christie, Kathleen A.. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Itin, Kaspar. Hospital Universitario de Basilea; SuizaFil: Mujahid, Nisma. University of Boston. School of Medicine; Estados Unidos. University of Utah; Estados Unidos. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Lo, Jennifer A.. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Won, Chong Hyun. Ulsan University College of Medicine; Corea del SurFil: Evans, Conor L.. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Weng, Qing Yu. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Wang, Hequn. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Osseiran, Sam. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Lovas, Alyssa. Harvard Medical School. Department of Medicine. Massachusetts General Hospital; Estados UnidosFil: Németh, István. University of Szeged; HungríaFil: Cozzio, Antonio. Kantonsspital St. Gallen; SuizaFil: Navarini, Alexander A.. Hospital Universitario de Basilea; SuizaFil: Gonzalez-Jose, Rolando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Centro Nacional Patagónico. Instituto Patagónico de Ciencias Sociales y Humanas; Argentin