Noi riconosciamo il sostegno del National Cancer Institute (NCI) R01 CA128908 e Stanford Medical Research Scholar Fellowship. Nessun altro conflitto di interessi rilevanti per questo articolo è stato segnalato.

1. Modello Tumore <ol><li> Coltura cellule H460 (American Type Culture Collection) in terreno RPMI 1640 supplementato con 10% di siero fetale bovino e 1% di penicillina / streptomicina (Invitrogen Life Technologies). Va notato che le scelte di linee cellulari, terreni di coltura, al luogo di inoculazione, il numero di xenotrapianti per topo, e altre considerazioni sono essere adattati agli obiettivi di uno studio particolare. Qui ci limiteremo a presentare un disegno specifico progetto per servire come illustrazione. </li><li> Mantenere linee cellulari in atmosfera umidificata al 5% di CO 2 a 37 ° C e il cambiamento di mezzo fresco ogni altro giorno. </li><li> Quando un monostrato confluente 75% delle cellule è formata, staccare il monostrato con tripsina e dissociare celle in una singola cella di sospensione per coltura cellulare ulteriormente. </li><li> Sospendere circa 1 x 10 6 cellule H460 in tampone fosfato salino (PBS; Invitrogen) e impianto per via sottocutanea inspalle destra e sinistra di topi nudi femmine (topi nudi atimici (nu / nu), 4 - 6 settimane di età, Charles River Laboratories, Inc.). </li><li> Lasciare i tumori a crescere fino a 150 - 200 mm 3. Ci vogliono circa 2 settimane per H460 xenotrapianti tumorali a crescere a questa dimensione. Standard pinza misurazione viene effettuata per tracciare dimensioni tumorali. </li><li> Quando la malattia raggiunge la dimensione ideale dei topi affetti da tumore sono ora pronti per il trattamento e imaging in vivo sia tramite PET e CLI. </li></ol> 2. PET <ol><li> Eseguire gli studi PET in base a questo programma o qualsiasi variazione di esso a seconda del progetto specifico (Figura 1) 12. Un certo numero di fattori possono influenzare la progettazione del programma, compreso, ma non limitato a, scelta delle linee cellulari tumorali xenotrapianto, farmaci antitumorali, e regimi di dosaggio. Qui ci limiteremo a presentare un programma specifico di imaging. Gli studi CLI devono essere eseguite secondo lapianificazione stessi degli studi PET, con CLI eseguito immediatamente dopo la PET corrispondente. Si deve inoltre notare che l&#39;obiettivo degli studi PET è principalmente per la convalida dei risultati CLI. Per gli utenti comuni che hanno appena desiderano utilizzare gli strumenti di OI per le sonde di imaging radiomarcati, PET non è necessario. Tuttavia, se si fa PET convalida desiderio è opportuno sottolineare che gli strumenti di PET e CLI deve essere situato molto vicino per la convalida di successo a causa della breve emivita di 18 F (109,77 min). </li><li> Dividere i topi in gruppi di trattamento e di controllo (n ≥ 3 ciascuno). Trattare topi nel gruppo di trattamento con 2 iniezioni di bevacizumab di 20 mg / kg nei giorni 0 e 2. Giorno 0 è definito dalla prima iniezione. Si noti che a Giorno -1 una pre-scansione deve essere eseguito sia attraverso PET e CLI. </li><li> Piccoli animali PET di topi portatori di tumore deve essere eseguita con un modello di roditore scanner R4 (Siemens Medical Solutions USAA, Inc.). </li><li> Anestetizzare tutti i mouse con il 2% isoflurano (Aerrane, Baxter) e iniettare con 3&#39;-desossi-3&#39;-18 F-fluorothymidine (18 F-FLT; 7,3-8,0 MBq [198-215 uCi]) attraverso la vena della coda. La sonda PET deve essere diluito in PBS prima dell&#39;iniezione. </li><li> Dopo 1 h, anestetizzare topi ancora e posizionare topi anestetizzati prona e vicino al centro del campo di vista del piccolo animale scanner PET. </li><li> Ottenere scansioni statici di tre minuti e ricostruire le immagini da un 2-dimensionale ordinato-sottoinsiemi algoritmo di attesa massimo. Correzione del fondo non è necessaria. </li><li> Disegnare le regioni di interesse (ROI; 5 pixel per fette coronali e transassiale) sui tumori sul decadimento-corretti immagini coronali tutto il corpo. Ottenere i conteggi massimo per pixel al minuto dal ROI e convertire in conteggi per millilitro al minuto per mezzo di una costante di taratura. Con l&#39;assunzione di una densità di tessuto 1 g / ml, convertire il ROI di conteggiper grammo al minuto. Determinare immagine ROI derivati% ID / g valori dividendo conteggi al minuto per grammo di dose iniettata. Correzione dell&#39;attenuazione non è necessaria. </li></ol> 3. CLI <ol><li> CLI deve essere eseguita con un sistema Spectrum IVIS (Caliper Life Sciences). Acquisizione e analisi di immagini devono essere effettuate con Living Immagine software 3.0 (Caliper Life Sciences). Lunghezza d&#39;onda risolto imaging spettrale deve essere eseguita utilizzando un 18-set banda stretta filtro di emissione (490-850 nm). Ancora una volta, per ogni topo, effettuare CLI immediatamente dopo PET per minimizzare la quantità di decadimento radioattivo se gli studi PET sono inclusi nel protocollo. </li><li> Mettere gli animali in una camera a tenuta di luce in anestesia isoflurano. Topi più può essere collocato contemporaneamente per aumentare la velocità. </li><li> Acquisire le immagini utilizzando 3 Tempo di esposizione min (f / stop = 1, binning = 4). Utilizzare le impostazioni di illuminazione stesse (tensione di lampada, filtri, f / stop, i campi di vista, binning) per acquisire tutte le immagini. Utilizzare la zona dorsale pelle per calcolare l&#39;intensità del segnale del tessuto sfondo. Normalizza emissione di fluorescenza di fotoni al secondo per centimetro quadrato per steradiante (p / s / cm 2 / sr). </li></ol> 4. Risultati rappresentativi Confronto visivo tra CLI e immagini PET può essere facilmente eseguito. Dopo unificazione della barra della scala attraverso le immagini dalla modalità e nello stesso luogo CLI e PET immagini affiancate si può vedere in questo gruppo rappresentativo (Figura 2A) che sia CLI e PET ha rivelato i segnali sono diminuiti sensibilmente tra H460 xenotrapianti in topi trattati dal pre-trattamento al giorno 3, suggerendo effetto terapeutico significativo. Come confronto, moderatamente aumentato a segnali invariati sono stati osservati nei topi non trattati durante lo stesso periodo (dati non mostrati). Mediante ispezione visiva da sola si può osservare che vi è una buona consistenza tra contrasti tumorali che sono visivazato da CLI e PET. In realtà, questa correlazione visiva ha una risoluzione sufficiente per mostrare necrosi centrale del tumore secondario al regime di trattamento antitumorale (Confrontare il CLI e immagini PET dal giorno 3). Per convalidare le quantificazioni dei reperti radiologici e analisi di correlazione può essere effettuata. Quantificazioni di CLI e immagini PET e un montaggio semplice tramite regressione lineare ha mostrato che le due modalità effetti aveva una eccellente correlazione (Figura 2B, R 2 = 0,9309 per 18 F-FLT sondato gruppo di trattamento). In particolare, in tutto il nostro CLI e studi di imaging PET con diversi modelli di tumore e diversi farmaci antitumorali pendici delle crisi convulsive sono anche molto vicino, suggerendo così una misura eccellente di regressione lineare anche di tutti i dati sono conglomerati (dati non riportati). Entrambe le immagini rappresentative sono adattati dal nostro 12 precedenti pubblicazioni. er.within-page = &quot;always&quot;&gt; <img src="/files/ftp_upload/4341/4341fig1.jpg" alt="Figura 1"/> Figura 1. Schema di disegno sperimentale degli studi PET e CLI. I tumori sono stati impiantati bilateralmente nella zona della spalla e fatti crescere a 150-200 mm 3, e topi portatori di tumore sono stati sottoposti a imaging in vivo mediante PET e CLI al giorno -1, 1, e 3. Trattamento Bevacizumab è stato eseguito da 2 iniezioni di 20 mg / kg nei giorni 0 e 2. <img src="/files/ftp_upload/4341/4341fig2.jpg" alt="Figura 2"/> Figura 2. (A) in vivo CLI e immagini PET di topi portatori di H460 eterotrapianti trattati con Bevacizumab prima del trattamento (pre-scansione) e dopo il trattamento (giorno 3). (B) corrispondente analisi quantitativa di CLI e PET risultati (n = 3) e le loro correlazioni. Adattato da immagini (6).arge.jpg &quot;target =&quot; _blank &quot;&gt; Clicca qui per ingrandire la figura.

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Nessun conflitto di interessi dichiarati.

CLI sta emergendo come una tecnica promettente di imaging molecolare che ha trovato in potenziali applicazioni delle scienze della ricerca di base e molte anche uso clinico 4,5,15,16,17. I vantaggi principali di CLI rispetto alle tradizionali modalità di imaging nucleare, quali le staminali PET dal suo uso di strumenti di OI, che sono più facili da usare, caratterizzati da tempi di acquisizione brevi e ad alto rendimento, molto meno costosi, e più ampiamente a disposizione dei ricercatori. Inoltre, ciò ch...

<table border="1"&gt;<tr&gt;<td<strong&gt; Nome</strong</td&gt;<td<strong&gt; Azienda</strong</td&gt;<td<strong&gt; Numero di catalogo</strong</td&gt;</tr&gt;<tr&gt;<td&gt; H460 linea cellulare</td&gt;<td&gt; American Type Culture Collection</td&gt;<td&gt; ATCC Numero: HTB-177</td&gt;</tr&gt;<tr&gt;<td&gt; RPMI 1640</td&gt;<td&gt; Invitrogen Life Technologies</td&gt;<td&gt; 12633-012</td&gt;</tr&gt;<tr&gt;<td&gt; Siero fetale bovino</td&gt;<td&gt; Invitrogen Life Technologies</td&gt;<td&gt; 10091-148</td&gt;</tr&gt;<tr&gt;<td&gt; Penicillina / streptomicina</td&gt;<td&gt; Invitrogen Life Technologies</td&gt;<td&gt; 15640-055</td&gt;</tr&gt;<tr&gt;<td&gt; PBS</td&gt;<td&gt; Invitrogen Life Technologies</td&gt;<td&gt; 10010-023</td&gt;</tr&gt;<tr&gt;<td&gt; Donne topi nudi atimici</td&gt;<td&gt; Charles River Laboratories, Inc.</td&gt;<td&gt; Codice Strain: 088</td&gt;</tr&gt;<tr&gt;<td&gt; Bevacizumab (Avastin)</td&gt;<td&gt; Genentech / Roche</td&gt;<td&gt; N / A</td&gt;</tr&gt;<tr&gt;<td&gt; MicroPET roditori R4</td&gt;<td&gt; Siemens Medical Solutions USA, Inc.</td&gt;<td&gt; N / A</td&gt;</tr&gt;<tr&gt;<td&gt; Isoflurano (Aerrane)</td&gt;<td&gt; Baxter</td&gt;<td&gt; Baxter Numero: AHN3637</td&gt;</tr&gt;<tr&gt;<td&gt; IVIS Spectrum</td&gt;<td&gt; Caliper Life Sciences</td&gt;<td&gt; N / A</td&gt;</tr&gt;</table

cerenkov luminescence imaging (cli) for cancer therapy monitoring

In imaging molecolare, tomografia ad emissione di positroni (PET) e di imaging ottico (OI) sono due delle modalità più importanti e quindi più utilizzato 1-3. PET è caratterizzato da una eccellente sensibilità e la capacità di quantificazione, mentre OI si distingue per la non-radiazione, relativamente a basso costo, la scansione in tempo breve, ad alto rendimento, e ampia disponibilità ai ricercatori di base. Tuttavia, entrambe le modalità hanno i loro difetti pure. PET soffre di scarsa risoluzione spaziale e di costo elevato, mentre OI principalmente è limitato ad applicazioni preclinici causa della sua limitata penetrazione nel tessuto con segnali ottici prominenti dispersione attraverso lo spessore dei tessuti viventi. Recentemente un ponte tra PET e OI è emerso con la scoperta di luminescenza Imaging Cerenkov (CLI) 4-6. CLI è una nuova modalità di imaging che sfrutta Cerenkov Radiazione (CR) per i radionuclidi di immagini con strumenti OI. Nobel russo Laureate Alekseyevich Cerenkov ei suoi colleghi hanno scoperto CR originariamente nel 1934. Si tratta di una forma di radiazione elettromagnetica emessa quando una particella carica viaggia ad una velocità superluminale in un mezzo dielettrico 7,8. La particella carica, se positroni o elettroni, perturba il campo elettromagnetico del mezzo spostando gli elettroni nei suoi atomi. Dopo aver superato dei fotoni interruzione vengono emessi come gli elettroni sfollati tornare allo stato fondamentale. Per esempio, un decadimento F 18 è stato valutato per produrre una media di 3 fotoni in acqua 5. Dal suo emergere, CLI è stato studiato per il suo utilizzo in una varietà di applicazioni in cui preclinici immagine del tumore in vivo, gene reporter di imaging, radiotracciante sviluppo, multimodalità imaging, tra gli altri 4,5,9,10,11. La ragione più importante per cui CLI ha goduto di molto successo finora è che questa nuova tecnologia si avvale della co bassost e ampia disponibilità di OI di radionuclidi di immagini, che ha usato per essere ripreso solo più costosi e meno disponibili modalità di imaging nucleare come il PET. Qui, presentiamo il metodo di utilizzo di CLI per monitorare la terapia antitumorale. Il nostro gruppo ha recentemente studiato questa nuova applicazione e convalidato la fattibilità di un proof-of-concept studio 12. Abbiamo dimostrato che CLI e PET esposte correlazioni eccellenti in xenotrapianti di tumori diversi e sonde di imaging. Ciò è coerente con il principio generale di CR che CLI visualizza essenzialmente gli stessi radionuclidi PET. Abbiamo selezionato Bevacizumab (Avastin, Genentech / Roche) come il nostro agente terapeutico perché è un noto inibitore dell&#39;angiogenesi 13,14. Maturazione di questa tecnologia nel prossimo futuro può essere immaginato avere un impatto significativo sullo sviluppo di farmaci preclinico, screening, così come il monitoraggio della terapia dei pazienti che ricevono trattamenti.

Watch this Scientific Journal Video about Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring at JoVE.com

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Uso di luminescenza Imaging Cerenkov (CLI) per il monitoraggio del trattamento preclinico cancro è descritto qui. Questo metodo sfrutta la radiazione Cerenkov (CR) e imaging ottico (OI) per visualizzare le sonde radiomarcati e fornisce quindi una valida alternativa al PET in studi preclinici di monitoraggio terapeutico e screening di farmaci.

Luminescenza Imaging Cerenkov (CLI) per il monitoraggio Cancer Therapy

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

cerenkov-luminescence-imaging-cli-for-cancer-therapy-monitoring

Research

JoVE Journal

Medicine

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

13.6K Views.  Stanford University. Uso di luminescenza Imaging Cerenkov (CLI) per il monitoraggio del trattamento preclinico cancro è descritto qui. Questo metodo sfrutta la radiazione Cerenkov (CR) e imaging ottico (OI) per visualizzare le sonde radiomarcati e fornisce quindi una valida alternativa al PET in studi preclinici di monitoraggio terapeutico e screening di farmaci.

Video: Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Monitoraggio metastasi tumorali e lesioni osteolitiche con bioluminescenza e Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

Ricerca

Medicina

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modellazione e imaging 3-Dimensional Invasion cella Collettiva

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Cancer therapies which are less toxic and invasive than their existing counterparts are highly desirable. The use of RF electric-fields that penetrate deep into the body, causing minimal toxicity, are currently being studied as a viable means of non-invasive cancer therapy. It is envisioned that the interactions of RF energy with internalized nanoparticles (NPs) can liberate heat which can then cause overheating (hyperthermia) of the cell, ultimately ending in cell necrosis.
In the case of non-biological systems, we present detailed protocols relating to quantifying the heat liberated by highly-concentrated NP colloids. For biological systems, in the case of in vitro experiments, we describe the techniques and conditions which must be adhered to in order to effectively expose cancer cells to RF energy without bulk media heating artifacts significantly obscuring the data. Finally, we give a detailed methodology for in vivo mouse models with ectopic hepatic cancer tumors.

We describe the protocols used to investigate the interactions of 13.56 MHz radiofrequency (RF) electric-fields with gold nanoparticle colloids in both non-biological and biological systems (in vitro/vivo). These interactions are being investigated for applications in cancer therapy.

Protocolli per la valutazione radiofrequenza interazioni con nanoparticelle di oro e Sistemi Biologici per la non-invasiva Ipertermia Cancer Therapy

Cancer therapies  which are less toxic and invasive than their existing counterparts are highly  desirable. The use of RF electric-fields that penetrate ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

Cerenkov Luminescenza Imaging di interscapolare Brown Tessuto adiposo

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Modello murino per l&#39;imaging non invasivo per rilevare e monitorare Ovarian Cancer Ricorrenza

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Dynamic Lung Tumor Tracking for Stereotactic Ablative Body Radiation Therapy

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable risks for normal tissue injury and the hazards of physical tumor miss. A first-of-its-kind SBRT platform achieves high-precision ablative radiation treatment through a combination of versatile real-time imaging solutions and sophisticated tumor tracking capabilities. It uses dual-diagnostic kV x-ray units for stereoscopic open-loop feedback of cancer target intrafraction movement occurring as a consequence of respiratory motions and heartbeat. Image-guided feedback drives a gimbaled radiation accelerator (maximum 15 x 15 cm field size) capable of real-time &#177;4 cm pan-and-tilt action. Robot-driven &#177;60&#176; pivots of an integrated &#177;185&#176; rotational gantry allow for coplanar and non-coplanar accelerator beam set-up angles, ultimately permitting unique treatment degrees of freedom. State-of-the-art software aids real-time six dimensional positioning, ensuring irradiation of cancer targets with sub-millimeter accuracy (0.4 mm at isocenter). Use of these features enables treating physicians to steer radiation dose to cancer tumor targets while simultaneously reducing radiation dose to normal tissues. By adding respiration correlated computed tomography (CT) and 2-[18F] fluoro-2-deoxy-&#7429;-glucose (18F-FDG) positron emission tomography (PET) images into the planning system for enhanced tumor target contouring, the likelihood of physical tumor miss becomes substantially less1. In this article, we describe new radiation plans for the treatment of moving lung tumors.

Stereotactic ablative body radiation therapy (SBRT) involves the precise delivery of high-dose radiation to cancer tumor targets. A novel SBRT platform offers a first-of-its-kind gimbaled radiation accelerator mounted within a pivoting O-ring gantry capable of dynamic image-guided tumor tracking. Here, we describe dynamic tumor tracking for lung targets.

Polmone dinamico Tumore inseguimento per stereotassica ablativo corpo Radioterapia

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Un sistema organotipica High Throughput per la caratterizzazione di droga sensibilità di primaria multicampo cellule del mieloma

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Imaging fluorescente combinata vicino infrarosso e la tomografia micro-computerizzata per Direttamente Visualizzazione cerebrale tromboembolie

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Visualizzazione quantitativa di leucociti infiltrati in un modello di murina della miocardite fulminante mediante microscopia a foglio chiaro

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The appearance of a mass on certain magnetic resonance (MR) sequences can help differentiate a thrombus from competing diagnoses such as a tumor. T1 and T2 signal characteristics of a thrombus are related to the evolution of hemoglobin properties. A thrombus typically does not enhance following contrast administration, which also helps differentiation from a tumor. We also highlight the emerging role of T1 mapping in the evaluation of a thrombus, which can add another level of support in diagnosis. Prior to any CMR exam, patient screening and interviews are critical to ensure safety and to optimize patient comfort. Effective communication during the exam between the technologist and the patient promotes proper breath holding technique and higher quality images. Volumetric post processing and structured reporting are helpful to ensure that the radiologist answers the ordering services&#39; question and communicates these results effectively. Optimal pre-MR safety evaluation, CMR exam execution, and post exam processing and reporting allow for delivery of high quality radiological service in the evaluation of a suspected cardiac thrombus.

The goal of this article is to describe how cardiac magnetic resonance can be used for the evaluation and diagnosis of a suspected cardiac thrombus. The method presented will describe data acquisition as well as the pre-procedure and post-procedure protocol.

Risonanza magnetica cardiaca per la valutazione di un trombo cardiaco sospetto: tecniche convenzionali ed emergenti

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...