PuraMatrix Encapsulation of Cancer Cells

Increasing evidence suggests that culturing cancer cells in three dimensions more accurately recapitulates the complexity of tumor biology. Many of these ...

longitudinal-measurement-extracellular-matrix-rigidity-3d-tumor

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Gastric mucin, a high molecular weight glycoprotein, is responsible for providing the gel-forming properties and protective function of the gastric mucus layer. Bulk rheology measurements in the linear viscoelastic regime show that gastric mucin undergoes a pH-dependent sol-gel transition from a viscoelastic solution at neutral pH to a soft viscoelastic gel in acidic conditions, with the transition occurring near pH 4. In addition to pH-dependent gelation behavior in this system, further rheological studies under nonlinear deformations reveal shear thinning and an apparent yield stress in this material which are also highly influenced by pH.

Rheology of gastric mucin exhibits a pH-dependent sol-gel transition.

Magen Mucin, ein Hochmolekulares Glykoprotein, ist verantwortlich f&uuml;r die Bereitstellung der Gel bildende Eigenschaften und Schutzfunktion der Magen Schleim Schicht. Massen Rheologie Messungen im linearen viskoelastischen Regime zeigen, dass dieser Magen Mucin einen pH-Wert-abh&auml;ngige Sol-Gel-&Uuml;bergang von eine Viskoelastische L&ouml;sung bei neutralem pH-Wert zu einem weichen viskoelastischen Gel unter sauren Bedingungen, mit &Uuml;bergang stattfindet, in der N&auml;he von pH 4 unterzogen wird. Zus&auml;tzlich zu pH-Wert-abh&auml;ngige Gelbildung Verhalten in diesem System enth&uuml;llen weitere Rheologische Untersuchungen unter nichtlineare Verformungen Effilier Schere und eine scheinbare Flie&szlig;spannung in diesem Material, das auch stark vom pH-Wert beeinflusst werden.

Rheologie von Magen Mucin weist einen pH-Wert-abh&auml;ngige Sol-Gel-&Uuml;bergang.

The ulcer-causing gastric pathogen Helicobacter pylori is the only bacterium known to colonize the harsh acidic environment of the human stomach. H. pylori survives in acidic conditions by producing urease, which catalyzes hydrolysis of urea to yield ammonia thus elevating the pH of its environment. However, the manner in which H. pylori is able to swim through the viscoelastic mucus gel that coats the stomach wall remains poorly understood. Previous rheology studies on gastric mucin, the key viscoelastic component of gastric mucus, indicate that the rheology of this material is pH dependent, transitioning from a viscous solution at neutral pH to a gel in acidic conditions. Bulk rheology measurements on porcine gastric mucin (PGM) show that pH elevation by H. pylori induces a dramatic decrease in viscoelastic moduli. Microscopy studies of the motility of H. pylori in gastric mucin at acidic and neutral pH in the absence of urea show that the bacteria swim freely at high pH, and are strongly constrained at low pH. By using two-photon fluorescence microscopy to image the bacterial motility in an initially low pH mucin gel with urea present we show that the gain of translational motility by bacteria is directly correlated with a rise in pH indicated by 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), a pH sensitive fluorescent dye. This study indicates that the helicoidal-shaped H. pylori does not bore its way through the mucus gel like a screw through a cork as has previously been suggested, but instead achieves motility by altering the rheological properties of its environment.

Helicobacter pylori moves through mucus by reducing mucin viscoelasticity.

Das Geschw&uuml;r verursacht Magen-Erreger Helicobacter Pylori ist das einzige Bakterium bekannt, dass das harte saure Milieu im menschlichen Magen besiedeln. H. Pylori &uuml;berlebt in sauren Bedingungen durch die Herstellung von Urease, die Harnstoff zu Ammoniak erh&ouml;hen somit den pH-Wert ihrer Umgebung f&uuml;hren katalysiert. Die Weise, in welcher H. Pylori ist jedoch in der Lage, durch das Viskoelastische Schleim Gel zu schwimmen, die M&auml;ntel, die die Magen Wand schlecht bleibt verstanden. Fr&uuml;heren Rheologie Studien &uuml;ber Magen Mucine, die wichtigsten Viskoelastische Komponente des gastrischen Schleims, zeigen, dass die Rheologie dieses Materials pH-Wert abh&auml;ngige, &Uuml;bergang von eine z&auml;hfl&uuml;ssige L&ouml;sung bei neutralem pH-Wert ein Gel in sauren Bedingungen. Massen Rheologie Messungen an Schweinen Magen Mucin (PGM) zeigen, dass die pH-Wert-Erh&ouml;hung von H. Pylori einen dramatischen R&uuml;ckgang der Viskoelastische Modulraum induziert. Mikroskopie Studien &uuml;ber die Motilit&auml;t des H. Pylori im Magen Mucin bei Saurer und neutraler pH-Wert in Ermangelung von Harnstoff zeigen, dass die Bakterien frei an hohen pH-Wert Schwimmen und werden bei niedrigem pH-Wert stark eingeschr&auml;nkt. Mit zwei-Photonen-Mikroskopie Bild die bakterielle Motilit&auml;t in einem urspr&uuml;nglich niedrigen pH Mucin Gel mit Harnstoff vorhanden, wir zeigen, dass die Verst&auml;rkung der translatorischen Motilit&auml;t durch Bakterien direkt, mit einem pH-Wert von 2&#39;,7&#39;-Bis-(2-Carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) angegeben korreliert sind, eine sensible pH-Wert-Leuchtstoff f&auml;rben. Diese Studie zeigt, dass die helicoidal-f&ouml;rmigen H. Pylori seinen Weg durch das Schleim-Gel wie eine Schraube durch einen Korken wie zuvor vorgeschlagen worden, aber stattdessen Motilit&auml;t erreicht, durch die Ver&auml;nderung der rheologischen Eigenschaftendes von seiner Umgebung nicht langweilen.

Helicobacter Pylori bewegt sich durch Schleim durch Reduzierung Mucin Viskoelastizit&auml;t.

Imaging and photodynamic therapy: mechanisms, monitoring, and optimization.

Bildgebung und photodynamische Therapie: Mechanismen, &Uuml;berwachung und Optimierung.

Targeting molecular markers and pathways implicated in cancer cell growth is a promising avenue for developing effective therapies. Although the Ki-67 protein (pKi-67) is a key marker associated with aggressively proliferating cancer cells and poor prognosis, its full potential as a therapeutic target has never before been successfully shown. In this regard, its nuclear localization presents a major hurdle because of the need for intracellular and intranuclear delivery of targeting and therapeutic moieties. Using a liposomally encapsulated construct, we show for the first time the specific delivery of a Ki-67-directed antibody and subsequent light-triggered death in the human ovarian cancer cell line OVCAR-5. Photoimmunoconjugate-encapsulating liposomes (PICEL) were constructed from anti-pKi-67 antibodies conjugated to fluorescein 5(6)-isothiocyanate, as a photoactivatable agent, followed by encapsulation in noncationic liposomes. Nucleolar localization of the PICELs was confirmed by confocal imaging. Photodynamic activation with PICELs specifically killed pKi-67-positive cancer cells both in monolayer and in three-dimensional (3D) cultures of OVCAR-5 cells, with the antibody TuBB-9 targeting a physiologically active form of pKi-67 but not with MIB-1, directed to a different epitope. This is the first demonstration of (a) the exploitation of Ki-67 as a molecular target for therapy and (b) specific delivery of an antibody to the nucleolus in monolayer cancer cells and in an in vitro 3D model system. In view of the ubiquity of pKi-67 in proliferating cells in cancer and the specificity of targeting in 3D multicellular acini, these findings are promising and the approach merits further investigation.

Ki-67 as a molecular target for therapy in an in vitro three-dimensional model for ovarian cancer.

Targeting molekularer Marker und Wege Zellwachstum Krebs in Verbindung gebracht, ist ein vielversprechender Weg f&uuml;r die Entwicklung von wirksamer Therapien. Obwohl das Ki-67 Protein (pKi-67) ein wichtiger Marker, die aggressiv wuchernden Krebszellen und schlechte Prognose, sein volles Potential zugeordnet ist, da ein therapeutisches Ziel nie hat erfolgreich gezeigt. In diesem Zusammenhang seine nukleare Lokalisierung pr&auml;sentiert eine gro&szlig;e H&uuml;rde wegen der Notwendigkeit der intrazellul&auml;ren und intranukle&auml;ren Lieferung des Zielens und therapeutische Polymerharze. Anhand eine liposomally gekapselte Konstruktion zeigen wir erstmals die spezifische Lieferung eines Antik&ouml;rpers unter der Regie von Ki-67 und nachfolgende Lichtgesteuerte Tod in die menschlichen Eierstockkrebs-Zelllinie OVCAR-5. Photoimmunoconjugate-Kapseln Liposomen (PICEL) wurden von Anti-pKi-67-Antik&ouml;rper Konjugation mit Fluoreszein 5 6-Isothiocyanat, als Agent Photoactivatable, gefolgt von Kapselung in Noncationic Liposomen gebaut. Konfokale Bildgebung best&auml;tigte nukleol&auml;res Lokalisierung von der Pixel. Photodynamische Aktivierung mit Pixel get&ouml;tet speziell pKi-67-positiven Krebszellen in Monolage und dreidimensionale (3D) Kulturen OVCAR-5 Zellen, die Antik&ouml;rper TuBB-9 auf eine physiologisch aktive Form von pKi-67 aber nicht mit MIB-1, an ein anderes Epitop gerichtet. Dies ist die erste Demonstration von (a) die Nutzung der Ki-67 als molekulare Zielscheibe f&uuml;r Therapie und (b) bestimmte Lieferung eines Antik&ouml;rpers, der Nukleolus Monolage Krebszellen und eine in-vitro-3D-Modell-System. Angesichts der weiten Verbreitung von pKi-67 in proliferierenden Zellen bei Krebs und die Besonderheit des Zielens in 3D vielzelligen Acini diese Ergebnisse sind vielversprechend, und der Ansatz verdient eine weitere Untersuchung.

Ki-67 als molekulare Ziel f&uuml;r die Therapie in einer in-vitro-dreidimensionales Modell f&uuml;r Eierstockkrebs.

Three-dimensional tumor models have emerged as valuable in vitro research tools, though the power of such systems as quantitative reporters of tumor growth and treatment response has not been adequately explored. We introduce an approach combining a 3-D model of disseminated ovarian cancer with high-throughput processing of image data for quantification of growth characteristics and cytotoxic response. We developed custom MATLAB routines to analyze longitudinally acquired dark-field microscopy images containing thousands of 3-D nodules. These data reveal a reproducible bimodal log-normal size distribution. Growth behavior is driven by migration and assembly, causing an exponential decay in spatial density concomitant with increasing mean size. At day 10, cultures are treated with either carboplatin or photodynamic therapy (PDT). We quantify size-dependent cytotoxic response for each treatment on a nodule by nodule basis using automated segmentation combined with ratiometric batch-processing of calcein and ethidium bromide fluorescence intensity data (indicating live and dead cells, respectively). Both treatments reduce viability, though carboplatin leaves micronodules largely structurally intact with a size distribution similar to untreated cultures. In contrast, PDT treatment disrupts micronodular structure, causing punctate regions of toxicity, shifting the distribution toward smaller sizes, and potentially increasing vulnerability to subsequent chemotherapeutic treatment.

Quantitative imaging reveals heterogeneous growth dynamics and treatment-dependent residual tumor distributions in a three-dimensional ovarian cancer model.

Dreidimensionale Tumor Modelle entstanden als in-vitro-Forschung wertvolle Tools, aber die Macht solcher Systeme als quantitative Reporter von Tumorwachstum und Behandlung Antwort nicht ausreichend erforscht. Wir stellen einen Ansatz, bei dem ein 3D-Modell disseminierte Eierstockkrebs mit hohem Durchsatz Verarbeitung der Bilddaten zur Quantifizierung der Wachstumseigenschaften und zytotoxische Reaktion. Wir entwickelten benutzerdefinierte MATLAB-Routinen um in L&auml;ngsrichtung erworbenen Dunkelfeld-Mikroskopie-Bildern, die mit Tausenden von 3-d Kn&ouml;tchen zu analysieren. Diese Daten zeigen eine reproduzierbare Bimodale Log-Normal Verteilung. Wachstum Verhalten wird durch Migration und Montage, verursacht eine exponentielle Zerfall in r&auml;umliche Dichte mit zunehmender mittlere Gr&ouml;&szlig;e damit einhergehende vorangetrieben. Am 10. Tag werden die Kulturen mit Carboplatin oder photodynamische Therapie (PDT) behandelt. Wir quantifizieren gr&ouml;&szlig;enabh&auml;ngigen zytotoxische Reaktion f&uuml;r jede Behandlung auf Basis des Kn&ouml;tchen von Kn&ouml;tchen mit automatischen Segmentierung kombiniert mit ratiometrisch Batch-Verarbeitung von Calcein und Ethidiumbromid Bromid Fluoreszenz Intensit&auml;t (Hinweis auf lebenden und tote Zellen, beziehungsweise). Beide Behandlungen reduzieren Lebensf&auml;higkeit, obwohl Carboplatin Micronodules weitgehend strukturell intakt mit eine Korngr&ouml;&szlig;enverteilung unbehandelten Kulturen &auml;hnlich l&auml;sst. Im Gegensatz dazu st&ouml;rt PDT-Behandlung Micronodular Struktur, punktierte Regionen der Toxizit&auml;t, verlagern die Verteilung in Richtung der kleineren Gr&ouml;&szlig;en und potentiell zunehmende Anf&auml;lligkeit f&uuml;r nachfolgende chemotherapeutischen Behandlung verursacht.

Quantitative Bildgebung offenbart heterogene Wachstumsdynamik und Behandlung-abh&auml;ngige residual Tumor Distributionen in einem dreidimensionalen Eierstockkrebs-Modell.

Metastatic ovarian cancer (OvCa) frequently recurs due to chemoresistance, highlighting the need for nonoverlapping combination therapies that mechanistically synergize to eradicate residual disease. Photodynamic therapy (PDT), a photochemistry-based cytotoxic modality, sensitizes ovarian tumors to platinum agents and biologics and has shown clinical promise against ovarian carcinomatosis. We introduce a three-dimensional (3D) model representing adherent ovarian micrometastases and high-throughput quantitative imaging methods to rapidly screen the order-dependent effects of combining benzoporphyrin-derivative (BPD) monoacid A-based PDT with low-dose carboplatin. 3D ovarian micronodules grown on Matrigel were subjected to BPD-PDT either before or after carboplatin treatment. We developed custom fluorescence image analysis routines to quantify residual tumor volume and viability. Carboplatin alone did not eradicate ovarian micrometastases at a dose of 400 mg/m2, leaving surviving cores that were nonsensitive or impermeable to chemotherapy. BPD-PDT (1.25 Î¼mol/LÂ·J/cm2) created punctate cytotoxic regions within tumors and disrupted micronodular structure. Treatment with BPD-PDT prior to low-dose carboplatin (40 mg/m2) produced a significant synergistic reduction [P<0.0001, analysis of covariance (ANCOVA)] in residual tumor volume [0.26; 95% confidence interval (95% CI), 0.19-0.36] compared with PDT alone (0.76; 95% CI, 0.63-0.92) or carboplatin alone (0.95; 95% CI, 0.83-1.09), relative to controls. This synergism was not observed with the reverse treatment order. Here, we demonstrate for the first time the use of a 3D model for micrometastatic OvCa as a rapid and quantitative reporter to optimize sequence and dosing regimens of clinically relevant combination strategies. This approach combining biological modeling with high-content imaging provides a platform to rapidly screen therapeutic strategies for a broad array of metastatic tumors.

Synergistic enhancement of carboplatin efficacy with photodynamic therapy in a three-dimensional model for micrometastatic ovarian cancer.

Metastasierendem Ovarialkarzinom (OvCa) nimmt h&auml;ufig aufgrund von Chemoresistance, was die Notwendigkeit f&uuml;r nicht &uuml;berlappende Kombinationstherapien, die f&uuml;r die Beseitigung der r&eacute;siduelle mechanistisch synergize heute. Photodynamische Therapie (PDT), Photochemie-basierte zytotoxischen Modalit&auml;t, Eierstockkrebs Platin-Agenten und Biologics sensibilisiert und klinische Versprechen gegen Eierstock-Neoplastica gezeigt hat. Wir stellen ein dreidimensionales (3D)-Modell, adh&auml;rente Ovarien Micrometastases und Hochdurchsatz-quantitative Sicherungsmethoden rasch die Reihenfolge-abh&auml;ngige Effekte der Kombination Benzoporphyrin-Derivat (BPD) Monoacid A-basierte PDT mit niedrig dosierten Carboplatin Bildschirm darstellt. 3D ovariellen Micronodules auf Matrigel angebaut wurden BPD-PDT vor oder nach der Carboplatin-Behandlung unterzogen. Wir entwickelten benutzerdefinierte Fluoreszenz Bild-Analyse-Routinen zur Quantifizierung der residual Tumorvolumen und Lebensf&auml;higkeit. Carboplatin alleine nicht ausrotten ovariellen Micrometastases in einer Dosis von 400 mg/m2, verlassen, Kerne, die Personalisierungscookies oder undurchl&auml;ssige zur Chemotherapie zu &uuml;berleben. BPD-PDT (1,25 &mu;mol/GmbHJ/cm2) erstellt punctata zytotoxischen Regionen innerhalb der Tumoren und Micronodular Struktur gest&ouml;rt. Behandlung mit BPD-PDT vor niedrig dosierte Carboplatin (40 mg/m2) produziert eine signifikante Reduktion der synergistische [P &lt; 0,0001, Analyse Kovarianzanalyse (ANCOVA)] in residual Tumor Band [0,26; 95 % Konfidenzintervall (95 % CI), 0.19-0.36] verglichen mit PDT alleine (0,76; 95 % CI, 0.63-0.92) oder Carboplatin alleine (0.95; 95 % CI, 0,83-1.09), im Verh&auml;ltnis zu Steuerelementen. Dieser Synergismus wurde nicht mit der Bestellung der umgekehrte Behandlung beobachtet. Hier zeigen wir erstmals die Verwendung von 3D-Modellen f&uuml;r Micrometastatic OvCa als schnelle und quantitative Reporter, Reihenfolge und Dosierung bestehend aus klinisch relevante Kombination Strategien zu optimieren. Dieser Ansatz kombiniert biologische Modellierung mit hoch-Inhalt-Bildgebung bietet eine Plattform, um schnell Bildschirm therapeutische Strategien f&uuml;r eine breite Palette von metastasierenden Tumoren.

Synergistische Verbesserung von Carboplatin Wirksamkeit mit photodynamische Therapie in ein dreidimensionales Modell f&uuml;r Micrometastatic ovarian Krebs.

An outstanding problem in cancer therapy is the battle against treatment-resistant disease. This is especially true for ovarian cancer, where the majority of patients eventually succumb to treatment-resistant metastatic carcinomatosis. Limited perfusion and diffusion, acidosis, and hypoxia play major roles in the development of resistance to the majority of front-line therapeutic regimens. To overcome these limitations and eliminate otherwise spared cancer cells, we utilized the cationic photosensitizer EtNBS to treat hypoxic regions deep inside in vitro 3D models of metastatic ovarian cancer. Unlike standard regimens that fail to penetrate beyond âˆ¼150 Âµm, EtNBS was found to not only penetrate throughout the entirety of large (>200 Âµm) avascular nodules, but also concentrate into the nodules' acidic and hypoxic cores. Photodynamic therapy with EtNBS was observed to be highly effective against these hypoxic regions even at low therapeutic doses, and was capable of destroying both normoxic and hypoxic regions at higher treatment levels. Imaging studies utilizing multiphoton and confocal microscopies, as well as time-lapse optical coherence tomography (TL-OCT), revealed an inside-out pattern of cell death, with apoptosis being the primary mechanism of cell killing. Critically, EtNBS-based photodynamic therapy was found to be effective against the model tumor nodules even under severe hypoxia. The inherent ability of EtNBS photodynamic therapy to impart cytotoxicity across a wide range of tumoral oxygenation levels indicates its potential to eliminate treatment-resistant cell populations.

Killing hypoxic cell populations in a 3D tumor model with EtNBS-PDT.

Eine herausragende Problem in der Krebstherapie ist der Kampf gegen die Behandlung-best&auml;ndige Krankheit. Dies gilt insbesondere f&uuml;r Eierstock-Krebs, die meisten Patienten schlie&szlig;lich Behandlung-best&auml;ndig metastasierendem Neoplastica erliegen. Begrenzte Perfusion und Diffusion, Azidose und Hypoxie spielen wichtige Rollen in der Entwicklung von Resistenzen, die Mehrheit von therapeutischen Front-Line-Therapien. Diese Beschr&auml;nkungen &uuml;berwinden und beseitigen sonst verschont Krebszellen, wir genutzt die kationischen Photosensibilisator EtNBS zur Behandlung von Hypoxie Regionen tief in in-vitro-3D-Modelle von metastasierendem Ovarialkarzinom. Im Gegensatz zu standard-Therapien, die nicht &uuml;ber &sim;150 &micro;m zu durchdringen, EtNBS wurde festgestellt, dass nicht nur in der Gesamtheit der gro&szlig;en durchdringen (&gt; 200 &micro;m) avaskul&auml;re Kn&ouml;tchen, sondern auch Konzentrat in die Kn&ouml;tchen sauer und Hypoxie Kerne. Photodynamische Therapie mit EtNBS wurde hochwirksames Mittel gegen diese Hypoxie Regionen auch bei geringen therapeutischen Dosen werden beobachtet und war in der Lage, Normoxic und Hypoxie Regionen auf h&ouml;heren Ebenen der Behandlung zu zerst&ouml;ren. Imaging-Studien unter Verwendung multiphoton und konfokalen Mikroskopien sowie Zeitraffer Optische Koh&auml;renztomografie ergab (TL-Okt), eine Inside-Out-Muster des Zelle Todes, mit Apoptosis wird der prim&auml;re Mechanismus der Zelle T&ouml;tung. Kritisch, photodynamische Therapie auf der Basis von EtNBS erwies sich wirksam gegen die Modell-Tumor-Kn&ouml;tchen auch unter schweren Hypoxie. Die angeborene F&auml;higkeit des EtNBS photodynamische Therapie Zytotoxizit&auml;t in einem breiten Spektrum von tumorale Sauerstoffversorgung Ebenen vermitteln zeigt sein Potenzial zur Behandlung-best&auml;ndige Zelle Bev&ouml;lkerungen zu beseitigen.

T&ouml;tung hypoxic Zelle Bev&ouml;lkerungen in einem 3D-Tumor-Modell mit EtNBS-PDT.

Pancreatic cancer is notoriously difficult to treat and resistant to virtually all therapeutics including gemcitabine, the standard front line agent for palliative chemotherapy. Early clinical studies point to a potential role for photodynamic therapy (PDT) in the management of this deadly disease. Here we examine PDT with verteporfin for treatment of cells that are nonresponsive to gemcitabine and identify intracellular and extracellular factors that govern sensitivity to each modality.

Verteporfin-based photodynamic therapy overcomes gemcitabine insensitivity in a panel of pancreatic cancer cell lines.

Bauchspeicheldr&uuml;senkrebs ist notorisch schwierig zu behandeln und resistent gegen praktisch alle Therapeutika einschlie&szlig;lich Gemcitabin, der standard Front-Line-Agent f&uuml;r palliative Chemotherapie. Fr&uuml;he klinische Studien weisen auf eine m&ouml;gliche Rolle f&uuml;r photodynamische Therapie (PDT) bei der Verwaltung dieser t&ouml;dlichen Krankheit. Hier untersuchen wir PDT mit AMD f&uuml;r die Behandlung der Zellen, die Frontblende reagieren nicht Gemcitabin und intrazellul&auml;ren und extrazellul&auml;re Faktoren, die Sensibilit&auml;t f&uuml;r jede Modalit&auml;t Regeln feststellen.

AMD-basierte photodynamische Therapie &uuml;berwindet Gemcitabin Unempfindlichkeit in einem Panel von Bauchspeicheldr&uuml;senkrebs Zelllinien.

Photodynamic therapy (PDT) is a light-based treatment modality in which wavelength specific activation of a photosensitizer (PS) generates cytotoxic response in the irradiated region. PDT response is critically dependent on several parameters including light dose, PS dose, uptake time, fluence rate, and the mode of light delivery. While the systematic optimization of these treatment parameters can be complex, it also provides multiple avenues for enhancement of PDT efficacy under diverse treatment conditions, provided that a rational framework is established to quantify the impact of parameter selection upon treatment response. Here we present a theranostic technique, combining the inherent ability of the PS to serve simultaneously as a therapeutic and imaging agent, with the use of image-based treatment assessment in three dimensional (3D) in vitro tumor models, to comprise a platform to evaluate the impact of PDT parameters on treatment outcomes. We use this approach to visualize and quantify the uptake, localization, and photobleaching of the PS benzoporphyrin derivative monoacid ring-A (BPD) in a range of treatment conditions with varying uptake times as well as continuous and fractionated light delivery regimens in 3D cultures of AsPC-1 and PANC-1 cells. Informed by photobleaching patterns and correlation with cytotoxic response, asymmetric fractionated light delivery at 4 hours BPD uptake was found to be the most effective regimen assessed. Quantification of the spatial profile of cell killing within multicellular nodules revealed that these conditions also achieve the highest depth of cytotoxicity along the radial axis of 3D nodules. The framework introduced here provides a means for systematic assessment of PDT treatment parameters in biologically relevant 3D tumor models with potential for broader application to other systems.

Image-Based Quantification of Benzoporphyrin Derivative Uptake, Localization, and Photobleaching in 3D Tumor Models, for Optimization of PDT Parameters.

The successful implementation of photodynamic therapy (PDT)-based regimens depends on an improved understanding of the dosimetric and biological factors that govern therapeutic variability. Here, the kinetics of tumor destruction and regrowth are characterized by systematically varying benzoporphyrin derivative (BPD)-light combinations to achieve fixed PDT doses (M × J cm(-2)). Three endpoints were used to evaluate treatment response: (1) Viability evaluated every 24 h for 5 days post-PDT; (2) Photobleaching assessed immediately post-PDT; and (3) Caspase-3 activation determined 24 h post-PDT. The specific BPD-light parameters used to construct a given PDT dose significantly impact not only acute cytotoxic efficacy, but also treatment durability. For each dose, PDT with 0.25 μM BPD produces the most significant and sustained reduction in normalized viability compared to 1 and 10 μM BPD. Percent photobleaching correlates with normalized viability for a range of PDT doses achieved within BPD concentrations. To produce a cytotoxic response with 10 μM BPD that is comparable to 0.25 and 1 μM BPD a reduction in irradiance from 150 to 0.5 mW cm(-2) is required. Activated caspase-3 does not correlate with normalized viability. The parameter-dependent durability of outcomes within fixed PDT doses provides opportunities for treatment customization and improved therapeutic planning.

PDT dose parameters impact tumoricidal durability and cell death pathways in a 3D ovarian cancer model.

It has become increasingly widely recognized that the stroma plays several vital roles in tumor growth and development and that tumor-stroma interactions can in many cases account poor therapeutic response. Inspired by an emerging body of literature, we consider the potential role of photodynamic therapy (PDT) for targeting interactions with stromal fibroblasts and mechano-sensitive signaling with the extracellular matrix as a means to drive tumors toward a more therapeutically responsive state and synergize with other treatments. This concept is particularly relevant for cancer of the pancreas, which is characterized by tumors with a profoundly dense, rigid fibrous stroma. Here we introduce new in vitro systems to model interactions between pancreatic tumors and their mechanical microenvironment and restore signaling with stromal fibroblasts. Using one such model as a test bed it is shown here that PDT treatment is able to destroy fibroblasts in an in vitro 3D pancreatic tumor-fibroblast co-culture. These results and the literature suggest the further development of PDT as a potential modality for stromal depletion.

Stromal interactions as regulators of tumor growth and therapeutic response: A potential target for photodynamic therapy?

Seventy-five percent of patients with epithelial ovarian cancer present with advanced-stage disease that is extensively disseminated intraperitoneally and prognosticates the poorest outcomes. Primarily metastatic within the abdominal cavity, ovarian carcinomas initially spread to adjacent organs by direct extension and then disseminate via the transcoelomic route to distant sites. Natural fluidic streams of malignant ascites triggered by physiological factors, including gravity and negative subdiaphragmatic pressure, carry metastatic cells throughout the peritoneum. We investigated the role of fluidic forces as modulators of metastatic cancer biology in a customizable microfluidic platform using 3D ovarian cancer nodules. Changes in the morphological, genetic, and protein profiles of biomarkers associated with aggressive disease were evaluated in the 3D cultures grown under controlled and continuous laminar flow. A modulation of biomarker expression and tumor morphology consistent with increased epithelial-mesenchymal transition, a critical step in metastatic progression and an indicator of aggressive disease, is observed because of hydrodynamic forces. The increase in epithelial-mesenchymal transition is driven in part by a posttranslational up-regulation of epidermal growth factor receptor (EGFR) expression and activation, which is associated with the worst prognosis in ovarian cancer. A flow-induced, transcriptionally regulated decrease in E-cadherin protein expression and a simultaneous increase in vimentin is observed, indicating increased metastatic potential. These findings demonstrate that fluidic streams induce a motile and aggressive tumor phenotype. The microfluidic platform developed here potentially provides a flow-informed framework complementary to conventional mechanism-based therapeutic strategies, with broad applicability to other lethal malignancies.

Flow induces epithelial-mesenchymal transition, cellular heterogeneity and biomarker modulation in 3D ovarian cancer nodules.

The bacterium Helicobacter pylori (H. pylori), has evolved to survive in the highly acidic environment of the stomach and colonize on the epithelial surface of the gastric mucosa. Its pathogenic effects are well known to cause gastritis, peptic ulcers, and gastric cancer. In order to infect the stomach and establish colonies on the mucus epithelial surface, the bacterium has to move across the gel-like gastric mucus lining of the stomach under acidic conditions. In this review we address the question of how the bacterium gets past the protective mucus barrier from a biophysical perspective. We begin by reviewing the molecular structure of gastric mucin and discuss the current state of understanding concerning mucin polymerization and low pH induced gelation. We then focus on the viscoelasticity of mucin in view of its relevance to the transport of particles and bacteria across mucus, the key first step in H. pylori infection. The second part of the review focuses on the motility of H. pylori in mucin solutions and gels, and how infection with H. pylori in turn impacts the viscoelastic properties of mucin. We present recent microscopic results tracking the motion of H. pylori in mucin solutions and gels. We then discuss how the biochemical strategy of urea hydrolysis required for survival in the acid is also relevant to the mechanism that enables flagella-driven swimming across the mucus gel layer. Other aspects of the influence of H. pylori infection such as, altering gastric mucin expression, its rate of production and its composition, and the influence of mucin on factors controlling H. pylori virulence and proliferation are briefly discussed with references to relevant literature.

The Influence of Mucus Microstructure and Rheology in Helicobacter pylori Infection.

While it is increasingly recognized that three-dimensional (3D) cell culture models recapitulate drug responses of human cancers with more fidelity than monolayer cultures, a lack of quantitative analysis methods limit their implementation for reliable and routine assessment of emerging therapies. Here, we introduce an approach based on computational analysis of fluorescence image data to provide high-content readouts of dose-dependent cytotoxicity, growth inhibition, treatment-induced architectural changes and size-dependent response in 3D tumour models. We demonstrate this approach in adherent 3D ovarian and pancreatic multiwell extracellular matrix tumour overlays subjected to a panel of clinically relevant cytotoxic modalities and appropriately designed controls for reliable quantification of fluorescence signal. This streamlined methodology reads out the high density of information embedded in 3D culture systems, while maintaining a level of speed and efficiency traditionally achieved with global colorimetric reporters in order to facilitate broader implementation of 3D tumour models in therapeutic screening.

An imaging-based platform for high-content, quantitative evaluation of therapeutic response in 3D tumour models.

ABSTRACT. Common methods to characterize treatment efficacy based on morphological imaging may misrepresent outcomes and exclude effective therapies. Using a three-dimensional model of ovarian cancer, two functional treatment response metrics are used to evaluate photodynamic therapy (PDT) efficacy: total volume, calculated from viable and nonviable cells, and live volume, calculated from viable cells. The utility of these volume-based metrics is corroborated using independent reporters of photodynamic activity: viability, a common fluorescence-based ratiometric analysis, and photosensitizer photobleaching, which is characterized by a loss of fluorescence due in part to the production of reactive species during PDT. Live volume correlated with both photobleaching and viability, suggesting that it was a better reporter of PDT efficacy than total volume, which did not correlate with either metric. Based on these findings, live volume and viability are used to probe the susceptibilities of tumor populations to a range of PDT dose parameters administered using 0.25, 1, and 10 μM benzoporphyrin derivative (BPD). PDT with 0.25 μM BPD produces the most significant reduction in live volume and viability and mediates a substantial shift toward small nodules. Increasingly sophisticated bioengineered models may complement current treatment planning approaches and provide unique opportunities to critically evaluate key parameters including metrics of therapeutic response.

ArticleTotal : 2	Year
Publication title Cited by 31	2009
Publication title Cited by 20	2014

Article	Year
Rheology of gastric mucin exhibits a pH-dependent sol-gel transition. Biomacromolecules\| PubMed ID: 17402780	2007
Helicobacter pylori moves through mucus by reducing mucin viscoelasticity. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 19706518	2009
Imaging and photodynamic therapy: mechanisms, monitoring, and optimization. Chemical reviews\| PubMed ID: 20353192	2010
Ki-67 as a molecular target for therapy in an in vitro three-dimensional model for ovarian cancer. Cancer research\| PubMed ID: 21045152	2010
Quantitative imaging reveals heterogeneous growth dynamics and treatment-dependent residual tumor distributions in a three-dimensional ovarian cancer model. Journal of biomedical optics\| PubMed ID: 21054077	2010
Synergistic enhancement of carboplatin efficacy with photodynamic therapy in a three-dimensional model for micrometastatic ovarian cancer. Cancer research\| PubMed ID: 21062986	2010
Killing hypoxic cell populations in a 3D tumor model with EtNBS-PDT. PloS one\| PubMed ID: 21876751	2011
Verteporfin-based photodynamic therapy overcomes gemcitabine insensitivity in a panel of pancreatic cancer cell lines. Lasers in surgery and medicine\| PubMed ID: 22057484	2011
Image-Based Quantification of Benzoporphyrin Derivative Uptake, Localization, and Photobleaching in 3D Tumor Models, for Optimization of PDT Parameters. Theranostics\| PubMed ID: 23082096	2012
PDT dose parameters impact tumoricidal durability and cell death pathways in a 3D ovarian cancer model. Photochemistry and photobiology\| PubMed ID: 23442192	2013
Stromal interactions as regulators of tumor growth and therapeutic response: A potential target for photodynamic therapy? Israel journal of chemistry\| PubMed ID: 23457416	2012
Flow induces epithelial-mesenchymal transition, cellular heterogeneity and biomarker modulation in 3D ovarian cancer nodules. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 23645635	2013
The Influence of Mucus Microstructure and Rheology in Helicobacter pylori Infection. Frontiers in immunology\| PubMed ID: 24133493	2013
An imaging-based platform for high-content, quantitative evaluation of therapeutic response in 3D tumour models. Scientific reports\| PubMed ID: 24435043	2014
Impact of treatment response metrics on photodynamic therapy planning and outcomes in a three-dimensional model of ovarian cancer. Journal of biomedical optics\| PubMed ID: 24802230	2013

Jonathan P. Celli

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