This work was supported by a grant from the Jan Kornelis de Cock foundation.

1 Creare Silicone Stampi per Inclusioni-tumorali simulazione <ol><li> Raccogliere gli elementi solidi della forma desiderata e dimensione che può servire da modello per inclusioni-tumorali simulando, ad esempio, perline o marmi. </li><li> Pulire accuratamente i modelli tumorali. Per garantire una facile rimozione dallo stampo in silicone, i modelli tumorali possono essere spruzzati con spray antiaderente o ricoperti da un sottile strato di vaselina o cera d&#39;api. </li><li> Mettere ogni modello in una piazza fortificata scatola separata sottile (plastica) con una superficie liscia. Se necessario, fissare il modello al fondo della scatola per mantenerlo in posizione. Utilizzare una scatola che è leggermente più grande rispetto al modello tumore stesso per evitare di sprecare quantità eccessive di silicone. </li><li> Versare la quantità necessaria di silicone componente A in una terrina e aggiungere il componente siliconico B in un rapporto di 10: 1 in peso. Mescolare accuratamente i due componenti. Facoltativamente, una pompa a vuoto può essere usato per rimuovere le bolle d&#39;aria dalla miscela di silicone. </li><li> Delicatamente Pour la miscela di silicone nella scatola di plastica per evitare di intrappolare bolle d&#39;aria. La miscela di silicone dovrebbe essere elaborato entro 45 minuti per ottenere risultati ottimali. </li><li> Lasciate che la miscela di silicone solidificare per almeno 6 ore prima di tagliare lo stampo e rimuovere il modello di tumore. Facoltativamente, lo stampo in silicone può essere tagliato a zig-zag per consentire di adattarsi di nuovo insieme in modo pulito. Massima resistenza del silicone è ottenuta dopo 3 giorni. </li></ol> 2. Creare soluzione salina tamponata con Tris <ol><li> Creare una soluzione salina tamponata con Tris (TBS) con l&#39;aggiunta di 6,1 g (50 mm) Tris e 8,8 g (150 mm) NaCl a 800 ml di acqua deionizzata. </li><li> Aggiungere 1,0 g (15 mmol) di NaN 3 per bloccare ossigenazione dell&#39;emoglobina (passo 3.3 e 4.4) e per inibire la crescita batterica. ATTENZIONE: NaN 3 è un veleno grave. Può essere fatale a contatto con la pelle o per ingestione. La tossicità di questo composto è paragonabile a quella di cianuri alcalini solubili e la dose letale per un essere umano adultoè di circa 0,7 g. Seguire sempre le istruzioni di sicurezza come previsto dal costruttore. </li><li> Regolare il pH a 7,4 e portare il volume a 1000 ml con acqua deionizzata. </li></ol> 3 Creare fluorescenti Inclusioni <ol><li> Aggiungere 2 g di agarosio a 50 ml TBS dal punto 2 Il punto di fusione più elevato di agarosio rispetto alla gelatina (punto 4.2) impedirà le inclusioni da sciogliere e perdite colorante fluorescente quando sono immessi in gelatina sciolta. Facoltativamente, la quantità di agarosio aggiunto può essere modificato per 1 o 3 g di ottenere inclusioni tumorali più morbide o palpabili, rispettivamente. </li><li> Riscaldare il liquame agarosio utilizzando un forno a microonde fino a raggiungere il punto di ebollizione. Mescolare accuratamente fino a quando l&#39;agarosio è completamente sciolto. </li><li> Aggiungere 1,1 g (17 mmol) di emoglobina e 5 ml di Intralipid 20% sciolto in 50 ml di TBS alla miscela di agarosio sotto costante agitazione per assomigliare le caratteristiche ottiche del tessuto circostante phantom seno (punto 4). </li><li> Aggiungere 20,0 mg (25.8 Mmol) del colorante fluorescente verde indocianina a 83,8 ml di acqua deionizzata. Assicurarsi che il colorante è completamente sciolto. </li><li> Pipettare 5,0 ml di questa soluzione e aggiungere al composto di agarosio per ottenere una concentrazione finale di 14 mM. Facoltativamente, altri coloranti fluorescenti che ICG possono essere utilizzati, se desiderato con la propria concentrazione ottimale. </li><li> Riempire delicatamente gli stampi in silicone creato nel passaggio 1 con la miscela di agarosio calda con una siringa (Figura 1A). Ripetere questo processo fino a quando tutti gli stampi sono riempiti. </li><li> Lasciate che le inclusioni fluorescenti solidificano a temperatura ambiente per circa un ora. Proteggere le inclusioni di luce coprendo l&#39;intero stampo con un foglio di alluminio. </li><li> Dopo la solidificazione, aprire delicatamente lo stampo e premere l&#39;inclusione (Figura 1B). Facoltativamente, utilizzare la punta della siringa per applicare piccole gocce di miscela agarosio fuso sulla superficie della inclusione. Ripetendo questo processo più volte sulla stessa posizione, piccolo tusperoni mor possono essere creati per simulare tumori infiltranti. </li><li> Proteggere le inclusioni agarosio dalla luce e disidratazione avvolgendoli in un foglio di alluminio e conservare in un contenitore umidificato a 4 ° C. NOTA: L&#39;uso di concentrazioni di colorante fluorescente inferiori o superiori al noto ottimale concentrazione sia risultato in diminuzione di intensità del segnale fluorescente. La riduzione apparentemente controintuitivo dell&#39;intensità del segnale con concentrazioni crescenti di colorante sopra la concentrazione ottimale colorante fluorescente è dovuto ad un fenomeno noto come tempra. Nel valutare la profondità di penetrazione massima di un colorante fluorescente in fantasmi, con la concentrazione ottimale è obbligatoria. </li></ol> 4 Creare Phantoms seno <ol><li> Ottenere uno stampo a forma di coppa per creare fantasmi seno della dimensione desiderata e volume, per esempio, un recipiente di vetro o plastica. Lo stampo deve avere una superficie liscia per evitare la forma gelatina aderendo allo stampo. A volum muffae di 500 ml creerà fantasmi del seno di dimensioni sufficienti. </li><li> Per creare un fantasma seno con un volume di 500 ml, aggiungere 50 g di gelatina 250 bloom a 500 ml TBS (step 2). Riscaldare il liquame di gelatina a 50 ° C sotto costante agitazione. </li><li> Una volta che la gelatina è completamente sciolto, lasciare che la miscela di gelatina raffreddare lentamente e mantenere ad una temperatura costante di 35 ° C utilizzando un bagno di acqua calda. </li><li> Sotto costante agitazione, aggiungere 5,5 g (85 mmol) di emoglobina bovina e 25 ml di Intralipid 20% per simulare l&#39;assorbimento e la dispersione dei fotoni nei tessuti, rispettivamente. </li><li> Prechill lo stampo a tazza a 4 ° C per almeno 1 ora. Successivamente, versare il composto di gelatina nello stampo ad un livello corrispondente alla profondità predefinita del agarosio-tumorale simulando inclusione (Figura 1C). Lasciare la miscela di gelatina solidificare a 4 ° C per 30 minuti per un ora. </li><li> Dopo solidificazione, posizionare una fluorescente inclusione agarosio-tumore simulando sulla superficie di<html> il fantasma temporaneamente fissano l&#39;inserimento con un piccolo ago. Fino a un massimo di tre inclusioni fluorescenti-tumorali simulazione possono essere incorporati in un singolo fantasma seno. Spazio sufficiente (almeno 5 cm) deve essere mantenuto tra i singoli inclusioni-tumorali simulazione (Figura 1D). </li><li> Versare il resto della miscela di gelatina calda nel volume residuo stampo, consentendo adesione dei due strati senza creare artefatti rifrazione. Segnare la posizione delle inclusioni-tumorali simulando fluorescenti sullo stampo. Lasciate che il phantom solidificare O / N a 4 ° C. </li><li> Una volta solidificato, rimuovere gli aghi utilizzati per il fissaggio temporaneo delle inclusioni e rimuovere delicatamente il fantasma seno dal suo stampo (Figura 1E). Proteggere il fantasma del seno dalla luce e disidratazione avvolgendolo in un foglio di alluminio e conservarlo in un contenitore umidificato a 4 ° C. </li></ol><html> ure 1 &quot;fo: content-width =&quot; &quot;src =&quot; 5in / files / ftp_upload / 51776 / 51776fig1highres.jpg &quot;width =&quot; 500 &quot;/&gt; Figura 1. fasi sequenziali di creare fantasmi mammarie contenenti inclusioni-tumorali simulando fluorescenti. Dopo creare stampi in silicone di forma e dimensioni desiderate, gli stampi vengono riempiti con fusa miscela di agarosio utilizzando una siringa (A). -Tumorali simulando inclusioni di forma e dimensione diverse sono state prodotte nel corso di studio (B). Successivamente, uno strato sottile di miscela di gelatina fusa è versata in uno stampo personalizzato seno legno rivestite (C). Dopo solidificazione, le inclusioni-tumorali simulazione sono posizionati, temporaneamente fissata, e rivestito con un altro strato di miscela di gelatina fusa (D). Dopo solidificazione, il fantasma seno viene delicatamente rimosso dal suo stampo (E). Il fantasma può quindi essere applicato per simulare varie applicazioni di imaging NIRF (F).ref = &quot;file / ftp_upload / 51776 / 51776fig1highres.jpg /&quot; target = &quot;_blank&quot;&gt; Clicca qui per visualizzare una versione più grande di questa figura. 5 Impostare il sistema di telecamere NIRF <ol><li> È necessaria una sistema di telecamere NIRF per l&#39;applicazione intraoperatoria per la simulazione di imaging NIRF mirato nella chirurgia del cancro al seno. Diversi sistemi di imaging NIRF per imaging in tempo reale NIRF intraoperatorio sono attualmente disponibili per uso sperimentale. Sebbene esistano alcune differenze tra questi dispositivi, tutti contengono una sorgente di luce di eccitazione (per eccitazione delle inclusioni tumorali fluorescenti) e un dispositivo di imaging altamente sensibile per la rilevazione di fotoni emessi. </li><li> Assicurarsi di utilizzare una fonte di luce di eccitazione di una lunghezza d&#39;onda sufficiente. Per le inclusioni-tumorali simulazione contenenti ICG, utilizzare una fonte di luce di eccitazione (ad esempio, laser) che emette fotoni tra 750 e 800 nm. Se si utilizza un colorante fluorescente alternativa, la lunghezza d&#39;onda di eccitazione deve essere regolata conforme alla ma<html> istruzioni di azienda i valori. </li><li> Nel caso in cui il sistema di telecamere NIRF contiene un filtro di emissione per filtrare i segnali di fondo indesiderati, assicurarsi che il filtro corretto viene utilizzato. Per le inclusioni-tumorali simulazione contenenti ICG, utilizzare un filtro di emissione tra 800 e 850 nm. Coloranti fluorescenti alternativi possono richiedere filtri di emissione diversi, a seconda delle istruzioni del fabbricante. NOTA: Accertarsi che non vi è nulla sovrapposizione tra l&#39;eccitazione e le lunghezze d&#39;onda di emissione per evitare immagini troppo saturi. Inoltre, il tempo di acquisizione dell&#39;immagine può essere regolato per ottenere immagini fluorescenti ottimali. Nel caso di inclusioni fluorescenti profondo seduti o segnali fluorescenti deboli, immagine tempo di acquisizione può essere aumentato fino a diversi secondi a min. Nel caso di inclusioni superficiali o segnali fluorescenti forti, tempo di acquisizione può essere diminuita per diversi msec per consentire il video-rate imaging di fluorescenza in tempo reale. </li></ol>e &quot;&gt; 6. Simulazione di applicazioni NIRF Imaging nella chirurgia del cancro al seno <ol><li> Prendete il fantasma del seno di tessuto-simulando dal suo contenitore e collocarlo su una superficie piana nonfluorescent. Successivamente, posizionare il dispositivo di imaging NIRF sopra il fantasma del seno, lasciando una sufficiente distanza di lavoro per escissione delle inclusioni-tumorali simulazione. </li><li> Localizzare l&#39;inclusione fluorescente-tumore simulazione utilizzando NIRF imaging e / o la palpazione del seno fantasma. Nel caso in cui nessun segnale fluorescente può essere rilevato, l&#39;inclusione o è posizionata troppo in profondità nel fantoccio per il rilevamento o il tempo di acquisizione delle immagini deve essere aumentato. </li><li> Una volta che l&#39;inclusione è localizzato, incidere il seno phantom e rimuovere l&#39;inclusione-tumore simulazione in tempo reale NIRF-guida utilizzando strumenti chirurgici convenzionali. In alternativa, l&#39;inserimento può essere asportato guidato esclusivamente mediante ispezione visiva e palpazione del seno phantom per simulare lo standard di cura. </li><li> Direttamente dopo la rimozionedell&#39;inclusione-tumore simulazione, immagine cavità chirurgica per qualsiasi attività fluorescente rimanente indica l&#39;escissione inadeguata. </li><li> In caso di qualsiasi attività fluorescente rimanente, asportare il residuo inclusione sotto la guida NIRF diretta fino a quando non segnale fluorescente è lasciato. </li><li> Immagine frammenti fantasma escisse per simulare valutazione dello stato macroscopico margine NIRF-guidata. Presente regolamento, affettare il tessuto phantom in 3 - 5 placche mm e l&#39;immagine le placche di conseguenza. Segnale di fluorescenza raggiungendo nei margini chirurgici indica l&#39;esistenza di margini chirurgici positivi. </li></ol>

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Abbiamo simulato potenziali applicazioni cliniche della BCS NIRF-guidati attraverso l&#39;uso di fantasmi a forma di seno con inclusioni-tumorali simulazione integrate. Localizzazione intraoperatoria del tumore, la resezione del tumore NIRF-guida, la valutazione sulla portata della chirurgia, e la valutazione postoperatoria di margini chirurgici sono stati tutti trovati fattibile utilizzando un sistema di telecamere NIRF custom-costruire. Rilevamento non invasivo di inclusioni-tumorali simulando fluorescenti era fattibi...

Chirurgia conservativa del seno (BCS), seguita da radioterapia è il trattamento standard per i pazienti di cancro al seno con T 1-T 2 carcinoma mammario 1,2. Imprecisioni nella valutazione intraoperatoria della misura di risultato chirurgia dei margini chirurgici positivi in 20 al 40% dei pazienti sottoposti a BCS, che richiede un intervento chirurgico aggiuntivo o radioterapia 3,4,5. Sebbene ampia resezione del tessuto mammario sano adiacente potrebbe ridurre la frequenza dei margini chirurgici positivi, questo sarà anche ostacolare risultato estetico e aumentare comorbidità 6,7. Sono pertanto necessarie nuove tecniche che forniscono un feedback intraoperatoria sulla localizzazione del tumore primario e l&#39;estensione della chirurgia. L&#39;imaging ottico, in particolare fluorescenza nel vicino infrarosso (NIRF) di imaging, potrebbe ridurre la frequenza dei margini chirurgici positivi seguenti BCS, fornendo al chirurgo uno strumento per la localizzazione del tumore pre e intraoperatorio in rEAL-tempo. Recentemente, il nostro gruppo ha riportato sulla prima prova in-umana del tumore-mirato imaging di fluorescenza nei pazienti con tumore ovarico, che mostra la fattibilità di questa tecnica per rilevare i tumori primari e metastasi intraperitoneali ad alta sensibilità 8. Prima di procedere a studi clinici in pazienti con carcinoma mammario, tuttavia, la fattibilità di diverse applicazioni di imaging NIRF tumore-mirato in BCS può già essere valutata preclinico con fantasmi. Il seguente protocollo di ricerca descrive l&#39;utilizzo dell&#39;imaging NIRF in fantasmi del seno di tessuto-simulazione-tumorali contenenti simulando fluorescenti inclusioni 9. I fantasmi forniscono uno strumento economico e versatile per simulare la resezione localizzazione del tumore pre e intraoperatorio, in tempo reale NIRF-guidato tumore, la valutazione dello stato di margine chirurgico, e l&#39;individuazione di malattia residua. I fantasmi gelatinose hanno proprietà elastiche simili a tessuti umani e possono essere tagliati con s convenzionalestrumenti urgical. Durante la procedura chirurgica simulata, il chirurgo è guidato da informazioni tattili (nel caso di inclusioni palpabili) e l&#39;ispezione visiva del campo operatorio. Inoltre, l&#39;imaging NIRF viene applicato per fornire al chirurgo con feedback in tempo reale intraoperatoria sulla misura della chirurgia. Va sottolineato che l&#39;imaging NIRF richiede l&#39;uso di coloranti fluorescenti. Idealmente, coloranti fluorescenti dovrebbero essere usati che emettono fotoni nel campo spettrale del vicino infrarosso (650-900 nm) per ridurre al minimo l&#39;assorbimento e la dispersione dei fotoni da molecole fisiologicamente abbondanti nei tessuti (ad esempio, l&#39;emoglobina, lipidi, elastina, collagene, e l&#39;acqua) 10,11. Inoltre, autofluorescenza (cioè, l&#39;attività di fluorescenza intrinseca nei tessuti a causa di reazioni biochimiche nelle cellule viventi) è minimizzato nella gamma spettrale del vicino infrarosso, con conseguente rapporti ottimali 11 tumorale-a-bassa. Con coniugando NIRF tinge di tumore-targefrazioni TED (ad esempio, anticorpi monoclonali), somministrazione mirata di coloranti fluorescenti possono essere ottenuti per applicazioni di imaging intraoperatorie. Poiché l&#39;occhio umano non è sensibile alla luce nella gamma spettrale del vicino infrarosso, un dispositivo di telecamera ad alta sensibilità è necessaria per l&#39;imaging NIRF. Sono stati sviluppati finora 12 Diversi sistemi di imaging NIRF per uso intraoperatorio. In questo studio, abbiamo utilizzato una generazione personalizzata sistema di imaging NIRF che è stato sviluppato per l&#39;applicazione intraoperatoria in collaborazione con l&#39;Università Tecnica di Monaco di Baviera. Il sistema permette l&#39;acquisizione simultanea di immagini a colori e immagini di fluorescenza. Per migliorare la precisione delle immagini di fluorescenza, uno schema di correzione viene implementato le variazioni di intensità della luce nel tessuto. Una descrizione dettagliata è fornita da Themelis et al. 13

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Bovine hemoglobin</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>H2500</td>
			<td>Simulates absorption of photons in tissue&#160;</td>
		</tr>
		<tr>
			<td>Intralipid 20%</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>I141</td>
			<td>Simulates scattering of photons in tissue</td>
		</tr>
		<tr>
			<td>Silicone A translucent 40 (2-components poly-addition silicone)</td>
			<td>NedForm, Geleen, The Netherlands</td>
			<td>N/A</td>
			<td>Package consists of components A and B, that should be mixed one on one (A:B=10:1).&#160; Link to manufacturers page: http://tinyurl.com/ncjq7jx</td>
		</tr>
		<tr>
			<td>Gelatine 250 Bloom</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>48724</td>
			<td>Construction of breast-shaped phantoms</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Hispanagar, Burgos, Spain</td>
			<td>N/A</td>
			<td>Construction of tumor-simulating inclusions</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>T1503&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hcl</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>258148</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich, Zwijndrecht, The Netherlands</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH3</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>822335</td>
			<td>CAUTION: severe poison. The toxicity of this compound is comparable to that of soluble alkali cyanides and the lethal dose for an adult human is about 0.7 grams.</td>
		</tr>
		<tr>
			<td colspan="2">Examples of NIRF imaging devices for intraoperative application:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T2 NIRF imaging platform&#160;</td>
			<td>SurgVision BV, Heerenveen, The Netherlands</td>
			<td>N/A</td>
			<td>Customized NIRF imaging system used in the current study. More details available at www.surgvision.com</td>
		</tr>
		<tr>
			<td>Photodynamic Eye</td>
			<td>Hamamatsu Photonics Deutschland GmbH, Herrsching am Ammersee, Germany</td>
			<td>PC6100</td>
			<td>www.iht-ltd.com</td>
		</tr>
		<tr>
			<td>FLARE imaging system kit</td>
			<td>The FLARE Foundation Inc, Wayland, MA, USA</td>
			<td>N/A</td>
			<td>www.theflarefoundation.org</td>
		</tr>
		<tr>
			<td>Fluobeam</td>
			<td>Fluoptics, Grenoble, France</td>
			<td>N/A</td>
			<td>www.fluoptics.com</td>
		</tr>
		<tr>
			<td>Artemis handheld camera</td>
			<td>Quest Medical Imaging BV, Middenmeer, the Netherlands</td>
			<td>N/A</td>
			<td>www.quest-mi.com</td>
		</tr>
		<tr>
			<td colspan="2">Examples of NIRF fluorescent dyes for intraoperative application:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Indocyanine green</td>
			<td>ICG-PULSION,&#160; Feldkirchen, Germany</td>
			<td>PICG0025DE&#160;&#160;</td>
			<td>Clinical grade fluorescent dye for NIRF imaging used in the current study. More details available at www.pulsion.com</td>
		</tr>
		<tr>
			<td>IRDye 800CW NHS Ester</td>
			<td>LI-COR Biosciences, Lincoln, NE, USA</td>
			<td>929-70021</td>
			<td>www.licor.com</td>
		</tr>
	</tbody>
</table>

tissue-simulating phantoms for assessing potential near-infrared fluorescence imaging applications in breast cancer surgery

Imprecisioni nella localizzazione del tumore intraoperatoria e la valutazione dei risultati chirurgici stato dei margini in esito ottimale della chirurgia conservativa del seno (BCS). L&#39;imaging ottico, in particolare l&#39;imaging di fluorescenza nel vicino infrarosso (NIRF), potrebbe ridurre la frequenza dei margini chirurgici positivi seguente BCS, fornendo al chirurgo uno strumento per la localizzazione del tumore pre-e intraoperatoria in tempo reale. In questo studio, il potenziale di NIRF guidata BCS è valutata utilizzando fantasmi del seno di tessuto-simulazione per motivi di fini di normalizzazione e di formazione. Fantasmi del seno con caratteristiche ottiche paragonabili a quelle del tessuto mammario normale sono stati utilizzati per simulare intervento conservativo della mammella. -Tumore simulando inclusioni contenenti il ​​colorante fluorescente verde indocianina (ICG) sono stati inglobati nei fantasmi in luoghi predefiniti e ripreso per la localizzazione del tumore pre e intraoperatorio, la resezione del tumore in tempo reale NIRF-guidata, NIRF-guidatovalutazione sulla portata della chirurgia, e la valutazione postoperatoria dei margini chirurgici. Una telecamera NIRF misura è stato utilizzato come prototipo clinico per scopi di imaging. Fantasmi del seno contenenti inclusioni-tumorali simulando offrono uno strumento semplice, economico e versatile per simulare e valutare l&#39;imaging tumorale intraoperatorio. I fantasmi gelatinose hanno proprietà elastiche simili a tessuti umani e possono essere tagliati con strumenti chirurgici convenzionali. Inoltre, i fantasmi contengono emoglobina e Intralipid per imitare l&#39;assorbimento e la dispersione di fotoni rispettivamente, creando uniformi proprietà ottiche simili al tessuto mammario umano. Lo svantaggio principale delle immagini NIRF è la profondità di penetrazione limitato di fotoni quando propagazione attraverso il tessuto, che ostacola (non invasiva), l&#39;imaging dei tumori profondi con le strategie epi-illuminazione.

I risultati di questo studio sono stati precedentemente riportati altrove 9. I nostri dati mostrano che l&#39;imaging NIRF può essere applicata per rilevare inclusioni-tumorali simulando fluorescenti in fantasmi del seno di tessuto-simulazione, simulazione di chirurgia conservativa del seno NIRF-guidata in pazienti con carcinoma mammario. Utilizzando il nostro modello phantom, abbiamo trovato la localizzazione intraoperatoria del tumore, la resezione del tumore NIRF-guida, la val...

Watch this Scientific Journal Video about Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery at JoVE.com

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.

Tissue-simulando Phantoms per valutare il potenziale vicino infrarosso applicazioni di imaging di fluorescenza nella chirurgia del cancro al seno

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

tissue-simulating-phantoms-for-assessing-potential-near-infrared

Research

JoVE Journal

Medicine

12.0K Views.  University Medical Center Groningen. Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes. 

Video: Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

In vivo Near Infrared fluorescenza (NIRF) intravascolare Imaging Molecolare della placca infiammatoria, un approccio multimodale per Imaging di aterosclerosi

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

Ricerca

Medicina

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multi-modal Imaging di angiogenesi in un modello di ratto Nudo di metastasi ossee cancro al seno utilizzando la risonanza magnetica, tomografia computerizzata volumetrica e Ultrasuoni

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Profiling di microRNA regolati dagli estrogeni in cellule di carcinoma mammario

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Voluntary Breath-hold Technique for Reducing Heart Dose in Left Breast Radiotherapy

Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these techniques, patients hold their breath while radiotherapy is delivered, pushing the heart down and away from the radiotherapy field. Despite clear dosimetric benefits, these techniques are not yet in widespread use. One reason for this is that commercially available solutions require specialist equipment, necessitating not only significant capital investment, but often also incurring ongoing costs such as a need for daily disposable mouthpieces. The voluntary breath-hold technique described here does not require any additional specialist equipment. All breath-holding techniques require a surrogate to monitor breath-hold consistency and whether breath-hold is maintained. Voluntary breath-hold uses the distance moved by the anterior and lateral reference marks (tattoos) away from the treatment room lasers in breath-hold to monitor consistency at CT-planning and treatment setup. Light fields are then used to monitor breath-hold consistency prior to and during radiotherapy delivery.

The current priority in breast cancer radiotherapy is to reduce cardiac doses without compromising target tissue coverage. The voluntary breath-hold technique described here is a simple, inexpensive solution to this problem and capable of being instituted widely without the need for specialized equipment.

Volontariato Tecnica Breath-hold per la riduzione Cuore Dose in Radioterapia della mammella sinistra

Breath-holding techniques reduce the amount of radiation received by cardiac structures during tangential-field left breast radiotherapy. With these ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

In vivo ed ex vivo approcci per studiare il cancro ovarico metastatico colonizzazione delle strutture Lattea Spot in peritoneale adiposo

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.

This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.

Generazione di Media e Applicazioni organo condizionata per lo studio Influenze organo-specifiche sul cancro al seno metastatico Comportamento

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused ...

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 ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

Studiare il ruolo dei macrofagi alveolari in Breast Cancer metastasi

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

A Quantitative Sensory Testing Paradigm to Obtain Measures of Pain Processing in Patients Undergoing Breast Cancer Surgery

Chronic pain following surgery, persistent postsurgical pain, is an important highly prevalent condition contributing to significant symptom burden and lower quality of life. Persistent postsurgical pain is relatively refractory to treatment hence generating a high need for preventive strategies and treatments. Therefore, the identification of patients at risk of developing persistent pain is an area of active ongoing research. Recently it was demonstrated that peri-operative disruptions in central pain processing may be able to predict persistent postsurgical pain at long term follow-up in breast cancer patients. The aim of the current report is to present a short protocol to obtain pain thresholds to different stimuli at multiple sites and a measure of endogenous analgesia in breast cancer patients. We have used this method successfully in a clinical context and detail some representative results from a clinical study.

Persistent postsurgical pain may be related to changes in pain processing. By assessing pain in response to standardized stimuli, changes in pain processing can be elucidated. We present methods to obtain pain thresholds to different stimuli and a measure of endogenous analgesia in patients undergoing breast cancer surgery.

Un paradigma di test sensoriale quantitativo per ottenere misure di elaborazione nei pazienti sottoposti a chirurgia del cancro al seno dolore

Chronic pain following surgery, persistent postsurgical pain, is an important highly prevalent condition contributing to significant symptom burden and ...

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with 'One Stop Shop' Near-infrared Spectroscopy

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic demand. Reactive hyperemia (in response to a brief period of tissue ischemia) is an independent predictor of cardiovascular events and provides important insight into vascular health and vasodilatory capacity. Skeletal muscle oxidative capacity is equally important in health and disease, as it determines the energy supply for myocellular processes. Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess each of these major clinical endpoints (reactive hyperemia, neurovascular coupling, and muscle oxidative capacity) during a single clinic or laboratory visit. Unlike Doppler ultrasound, magnetic resonance images/spectroscopy, or invasive catheter-based flow measurements or muscle biopsies, our approach is less operator-dependent, low-cost, and completely non-invasive. Representative data from our lab taken together with summary data from previously published literature illustrate the utility of each of these end-points. Once this technique is mastered, application to clinical populations will provide important mechanistic insight into exercise intolerance and cardiovascular dysfunction.

Skeletal Muscle Neurovascular Coupling, Oxidative Capacity, and Microvascular Function with &#039;One Stop Shop&#039; Near-infrared Spectroscopy

Here, we describe a simple, non-invasive approach using near-infrared spectroscopy to assess reactive hyperemia, neurovascular coupling and skeletal muscle oxidative capacity in a single clinic or laboratory visit.

Muscolo scheletrico Neurovascular accoppiamento, capacità ossidativa e funzione microvascolare con 'One Stop Shop' spettroscopia nel vicino infrarosso

Exercise represents a major hemodynamic stress that demands a highly coordinated neurovascular response in order to match oxygen delivery to metabolic ...