Gli autori hanno ricevuto il sostegno della Avner Pancreatic Cancer Foundation.

Tutte le procedure sono state approvate dal Comitato per la cura degli animali e l'etica dell'Università del Nuovo Galles del Sud (17/109A). Per questo protocollo sono stati utilizzati topi nudi atimici Balb/c femminili, di età compresa tra 8 e 10 settimane del peso di 16-19 g. I topi erano alloggiati in gabbie micro-isolanti e alimentati commercialmente disponibili con cibo e acqua a pellet ad libitum.
1. Impianto di cancro al pancreas ortotopico
<ol><li>Preparare le cellule per l'impianto. In primo luogo, calcolare il numero di cellule necessarie per la procedura (sono necessarie 1 x10 6 cellule AsPC-1 taggate alla luciferasi e 1 x 106 cellule stellate pancreatiche umane associate al cancro [CAhPSC] per ogni animale).<ol>
<li>Mantenere queste cellule in un incubatore di CO2 umidificato a temperatura controllata ed eseguire test di micoplasma di routine. I mezzi di coltura utilizzati per aspc-1 e CAhPSC sono RPMI 1640 (con 300 mg/L L-glutammina, 20% v/v siero bovino fetale, 1% v/v penicillina/streptomicina) e IMDM (con 4 mM L-glutammina, 10% v/v siero bovino fetale, 1% v/v penicillina/streptomicina).</li>
		<li>Utilizzare tecniche standard di coltura cellulare per tripinare le celle in una sospensione cellulare. Neutralizzare la tripina utilizzando il rispettivo supporto di coltura completo con un volume doppio rispetto alla soluzione di tripina utilizzata.</li>
		<li>Lavare queste cellule due volte con saline tamponate di fosfato (PBS) e resospersi in una miscela contenente 1 x 106 cellule AsPC-1 e 1 x 106 CAhPSC in una sospensione cellulare di 50 μL.</li>
		<li>Mantenere questa sospensione sul ghiaccio fino all'uso.</li>
	</ol></li>
	<li>Preparare un armadietto di biosicurezza di classe II per la procedura. Utilizzare un tappetino riscaldante sovrapposto da un drappo di plastica sterile. Per l'ingrandimento durante la procedura, utilizzare una coppia da 2,5x a 3,5 volte lopi chirurgici di ingrandimento.</li>
	<li>Preparare tamponi a corda di borsa tagliando un foro, di 1 cm di diametro, in un tampone di garza. Fissare questo buco con una sutura a corda di borsa. Qualsiasi sutura intrecciata fine può essere utilizzata per questo (ad esempio, sutura di acido poliglicolico 5/0). Si consiglia il materiale di sutura intrecciata in quanto consente al nodo sciolto di rimanere in posizione dopo il serraggio. Ciò è illustrato nella figura 1a.</li>
	<li>Anestetizzare il topo con 80 mg/kg di chetamina e 10 mg/kg di xiazina per iniezione intraperitoneale.</li>
	<li>Somministrare 5 mg/kg di profilassi antibiotica all'enrofloxacina, 2,5 mg/kg di analgesia di flunixina e 1 mL dello 0,9% di saline sottocutaneamente.</li>
	<li>Una volta anestetizzato, posizionare il topo sul campo sterile in posizione supina e applicare povidone-iodio seguito dal 70% di etanolo per la preparazione della pelle.</li>
	<li>Fare un'incisione longitudinale nella pelle del quadrante cranica sinistro dell'addome, quindi entrare nell'addome intasando lo strato muscolare tra le pinza.</li>
	<li>Caricare una siringa per insulina da 29 G con 50 μL di sospensione cellulare, pari a 1 x 106 CAhPSC e 1 x 106 cellule AsPC-1 contrassegnate con luciferasi per iniettato. Montarlo sul dispositivo di iniezione. Il design e la funzione di questo dispositivo di iniezione sono spiegati in dettaglio nella figura 1b e nella sua legenda.</li>
	<li>Posizionare il tampone a corda della borsa sopra l'incisione della laparotomia e quindi esternalizzare la milza e la coda pancreatica attraverso l'apertura di questo tampone. Stringere la corda della borsa per circondare delicatamente il corpo del pancreas, esponendo la coda pancreatica per iniezione. È importante essere abbastanza stretti che la garza contatti il pancreas circonferenzialmente e allo stesso tempo non lo costrizioni.</li>
	<li>Usando un paio di forcep, afferrare la coda del pancreas e posizionare delicatamente la tensione laterale su di esso. Forare la superficie peritoneale ventrale con l'ago ad un angolo poco profondo e quindi iniettare la sospensione cellulare nel pancreas in modo lento e controllato (oltre 10−15 s) con il dispositivo di iniezione.</li>
	<li>Durante il processo di iniezione, osservare attentamente la perdita, sia intorno al sito di iniezione (dal reflusso) che dall'altro lato del lobulo pancreatico (in caso di penetrazione through-and-through). Se si verifica una perdita visibile, interrompere l'iniezione e notare il volume di perdite controllando il volume di iniettato rimanente nella siringa. Se la perdita è di piccolo volume (<10 μL), quindi assorbire eventuali perdite con garza e riposizionare l'ago in un diverso lobulo pancreatico per completare l'iniezione.</li>
	<li>Sostituire la milza e il pancreas e chiudere la parete addominale con una sutura di acido poliglicolico 5/0 in modo continuo. Chiudere la pelle con le clip.</li>
	<li>Monitorare il mouse in una gabbia riscaldata fino a quando non viene recuperato dall'anestetico. Una volta sveglio e vigile, riporta il mouse nella sua gabbia.</li>
</ol>2. Chirurgia della resezione del cancro: pancreatectomia distale e splenectomia
<ol><li>La tempistica della resezione in relazione all'impianto può variare a seconda del protocollo sperimentale. In generale, consentire ai tumori di crescere almeno per 3 settimane prima della resezione, ma ottimizzarlo empiricamente per la particolare linea cellulare tumorale impiantata.</li>
	<li>Il giorno prima dell'intervento di resezione, eseguire l'imaging della bioluminescenza sugli animali per confermare la presenza di un tumore primario localizzato. Si noti che questo studio di imaging viene semplicemente utilizzato per escludere dalla resezione topi con ovvia malattia extra-pancreatica. Né le dimensioni né il flusso radiante dovrebbero essere utilizzati come soglie per determinare l'ammissibilità alla resezione.<ol>
<li>Pesare i topi e iniettare con D-luciferina per via intraperitoneale (150 mg/kg).</li>
		<li>Determinare la tempistica della fase di imaging in relazione all'iniezione di luciferina per ogni esperimento mediante le prestazioni di una curva cinetica luciferina. Il periodo di tempo in cui il flusso radiante è superiore al 90% del suo massimo rappresenta il tempo ottimale per l'imaging a bioluminescenza (in questo esperimento, da 18 a 26 minuti dopo l'iniezione)</li>
		<li>Indurre l'anestesia e mantenere utilizzando isoflurane (rispettivamente 4% e 3% con ossigeno) ed eseguire immagini utilizzando un dispositivo di imaging bioluminescente (ad esempio, IVIS Lumina II). Utilizzare impostazioni di esposizione e binning automatiche (questo può tuttavia essere ottimizzato per il flusso radiante previsto).</li>
	</ol>
</li>
	<li>Preparare l'armadio di biosicurezza di classe II per la procedura. Utilizzare un tappetino riscaldante sovrapposto da un drappo di plastica sterile. Per l'ingrandimento durante la dissezione, utilizzare una coppia da 2,5x a 3,5 volte lochie chirurgiche di ingrandimento.</li>
	<li>Anestetizzare il topo con 80 mg/kg di chetamina e 10 mg/kg di xiazina per iniezione intraperitoneale.</li>
	<li>Somministrare 5 mg/kg di profilassi antibiotica all'enrofloxacina, 2,5 mg/kg di analgesia di flunixina e 1 mL dello 0,9% di saline sottocutaneamente.</li>
	<li>Posizionare il topo sul campo sterile in posizione supina e applicare povidone-iodio seguito dal 70% di etanolo per la preparazione della pelle.</li>
	<li>Effettuare un'incisione longitudinale nella pelle del quadrante cranica sinistro dell'addome, preferibilmente attraverso il precedente sito di incisione.</li>
	<li>Sezionare senza mezzi termini la pelle dalla parete addominale muscolare sottostante, quindi posizionare un riavvolgitore autocontenente Alm per tenere aperta la ferita della pelle.</li>
	<li>Incidere lo strato muscolare tra le forcep solo su un lato della linea di sutura dell'operazione precedente, quindi estendere l'incisione per asportare l'intera linea di sutura precedente.</li>
	<li>Esternalizza la milza e il pancreas distale e ritrailo cranialmente. All'aspetto caudale del pancreas, il colon può essere trovato attaccato da aderenze filmate. Se questo viene trovato, sezionare senza mezzi termini i due punti.</li>
	<li>Passare con cura un paio di forcep dorsali al corpo del pancreas e dei vasi splenici e aprire questo spazio. Questo libera un segmento di pancreas per la successiva legatura.</li>
	<li>Legare il corpo del pancreas prossimale al tumore con una clip di legatura di titanio, quindi trascriire il pancreas distale a questo con cautery. Un modo alternativo per controllare il ceppo pancreatico è quello di legarlo in continuità con la sutura di acido poliglicolico 5/0 prima della trasezione.</li>
	<li>Ritrarre il pancreas caudally e cauterizzare i vasi gastrosplenici tra il palo cranico della milza e lo stomaco.</li>
	<li>Rimuovere il campione e confermare l'emostasi.</li>
	<li>Chiudere la parete addominale con sutura di acido poliglicolico 5/0 in modo continuo. Chiudere la pelle con le clip.</li>
</ol>3. Gestione postoperatoria
<ol><li>Nell'immediato periodo post anestetico (per entrambe le procedure di cui sopra), monitorare il topo in una gabbia riscaldata fino a quando non viene recuperato dall'anestetico. Una volta sveglio e vigile, riporta il mouse nella sua gabbia. Nel periodo postoperatorio, monitorare gli animali alla ricerca di dolore e segni di angoscia. Somministrare 0,05 mg/kg di buprenorfina per iniezione sottocutanea e osservare attentamente gli animali per 12 ore.</li>
	<li>Successivamente, monitorare quotidianamente i topi per peso, assunzione di cibo e attività. Esaminare i siti di incisione e palpato per la dimensione del tumore. Rimuovere le clip della pelle il settimo giorno postoperatorio.</li>
	<li>Eutanasia del mouse se vengono raggiunti endpoint umani. Questi endpoint umani includono: perdita di peso corporeo >20%, caratteristiche di disagio non curabile (tra cui postura curva, mancanza di movimento o toelettatura) e dimensioni tumorali superiori a 1 cm3 come stimato dalla palpazione esterna.</li>
</ol>

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+a+novel+aspect+of+pancreatic+stellate+cell+biology.">Cell migration: a novel aspect of pancreatic stellate cell biology.</a> Gut. 52 (5), 677-682 (2003).</li><li>Boj, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer.">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Egberts, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superiority+of+extended+neoadjuvant+chemotherapy+with+gemcitabine+in+pancreatic+cancer:+a+comparative+analysis+in+a+clinically+adapted+orthotopic+xenotransplantation+model+in+SCID+beige+mice.">Superiority of extended neoadjuvant chemotherapy with gemcitabine in pancreatic cancer: a comparative analysis in a clinically adapted orthotopic xenotransplantation model in SCID beige mice.</a> Cancer Biology &amp; Therapy. 6 (8), 1227-1232 (2007).</li></ol>

Gli autori non hanno nulla da rivelare in merito a questo progetto.

Un modello di topo ortotopico resezionale del cancro al pancreas è importante perché consente il test dei trattamenti adiuvanti e neoadiuvanti. Ciò è particolarmente importante nel cancro al pancreas, dove la chirurgia rimane il trattamento più efficace ma è associata ad un alto rischio di recidiva. Questo documento descrive un metodo che produrrà in modo affidabile un cancro al pancreas potenzialmente curabile con resezione, replicando lo scenario clinico in cui è richiesta una terapia neoadiuvante / adiuvante.<...

L'adenocarcinoma duttale pancreatico (tumore al pancreas [PC]) è associato a una prognosi infausta1. La resezione chirurgica rimane l'unico trattamento potenzialmente curativo per PC e deve essere presa in considerazione per i pazienti che presentano una malattia in fase iniziale. Sfortunatamente, anche con la resezione R0 (cioè margini di resezione liberi da tumore), il tasso di recidiva (locale o da malattia metastatica non rilevata) èalto 2,3. Pertanto, la terapia adiuvante sistemica è indicata in quasi tutti i pazienti sottoposti a resezione4. Inoltre, mentre la terapia neoadiuvante è ora raccomandata solo per i tumori resectabili al limite, le sue indicazioni si stanno espandendo in modo tale che il suo uso di routine sia al centro di moltericerche cliniche 5,6,7,8. Al fine di sviluppare nuovi approcci terapeutici per PC che coinvolgono la resezione, questi approcci devono essere prima valutati in modelli preclitici che riascindono accuratamente le impostazioni cliniche.
I modelli di mouse ortotopici di PC sono stati frequentemente utilizzati in passato per testare i trattamentifarmacologici 9,10. Molti di questi sono stati prodotti per iniezione di cellule tumorali da sole nel pancreas del topo, con conseguente tumori che mancavano dell'importante stroma che è caratteristico del PC. Più recentemente, i modelli ortotopici a co-iniezione, come quello che abbiamo sviluppato per la prima volta iniettando una miscela di PC umano e cellule stellate pancreatiche umane (PSC, i produttori primari dello stroma collagenoso nel PC), sono venuti in usoregolare 11,12. I tumori prodotti da tale co-iniezione di cancro e cellule stromali mostrano (i) sia gli elementi tumorali che la componente stromale caratteristica (desmoplastica) del PC, e (ii) maggiore proliferazione e metastasi delle cellule tumorali11. Pertanto, questo modello assomiglia molto al PC umano. Mentre un certo numero di modelli resezionali di PC ortotopico sono statidescritti 13,14,15,16, nessuno ha riflesso le realtà cliniche della resezione pancreatica nell'uomo accurato come questo modello, e quindi sono stati non ottimale per testare trattamenti adiuvanti o neoadiuvanti.
Gli obiettivi del modello di topo presentato erano di dimostrare come: i) impiantare con successo il cancro al pancreas ortotopico riducendo al minimo la diffusione peritoneale involontaria e (ii) successivamente resect completamente il cancro. Il documento evidenzia suggerimenti e potenziali insidie di questa tecnica.

<table>
	<tbody>
		<tr>
			<td>Animals, Materials and Equipment for Implantation Procedure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AsPC-1 human pancreatic cancer cell line, luciferase tagged (luc+ gene from Promega PGL3 Basic plasmid)</td>
			<td>American Type Culture Collection, Manassas, VA, USA</td>
			<td></td>
			<td>supplied by Professor Takashi Murakami, Saitama Medical University, Saitama, Japan</td>
		</tr>
		<tr>
			<td>Autoclip wound clips, 9 mm</td>
			<td>Becton Dickson Pty Ltd, North Ryde, NSW, Australia</td>
			<td>500346</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Dressing Pack</td>
			<td>Multigate Medical Products Pty Ltd, Villawood, NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cancer associated human pancreatic stellate cells</td>
			<td>Pancreatic Research Group cell bank</td>
			<td></td>
			<td>In house cell bank</td>
		</tr>
		<tr>
			<td>Cryogenic tubes, 1.0 mL</td>
			<td>Thermo Fisher Scientific Australia Pty Ltd, Scoresby, VIC, Australia</td>
			<td>366656</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable stainless-steel scalpel blade with handle, size 15</td>
			<td>Livingstone International, Mascot, NSW,</td>
			<td>SCP15</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal bovine serum (FBS)</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilles fine tooth forceps 12 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Heated mats to maintain body temperature during surgery and postoperative recovery</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Homozygous athymic nude mice: Strain BALB/c-Fox1nu/Ausb, female</td>
			<td>Australian Bioresources, Moss Vale, NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#39;s modified Dulbecco&#39;s medium (IMDM) with 4mM L-glutamine and no phenol red</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>21056023</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewellers forceps 11.5 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro needle holder (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro scissors (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Penicillin 10,000 U/mL, streptomycin 10,000 &#956;g/mL</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyglycolic acid suture, size USP 5/0 on 13mm half-circle round-bodied needle</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>C1049407</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable weighing scale</td>
			<td>Precision balances, Bradford, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex clip applier and clip remover</td>
			<td>World Precision Instruments, Sarasota, FL, USA</td>
			<td>500345</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640 with phenol red and 300 mg/L Lglutamine</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>11875085</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bodied vessel dilator 15 cm, 0.1 mm tip</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Trypsin 0.05%, EDTA 0.02%</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>25300054</td>
			<td>For pancreatic stellate cells</td>
		</tr>
		<tr>
			<td>Trypsin 0.25%, EDTA 0.02%</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>25200056</td>
			<td>For ASPC-1 cells</td>
		</tr>
		<tr>
			<td>U-100 insulin syringes, 0.5 mL with 29 G (0.33 mm) &#215; 13 mm needle</td>
			<td>Terumo Medical Corporation, Elkton, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment for Resection Procedure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alm self-retaining retractor</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Autoclip wound clips 9 mm</td>
			<td>Becton Dickson Pty Ltd, North Ryde, NSW</td>
			<td>500346</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Dressing Pack</td>
			<td>Multigate Medical Products Pty Ltd, Villawood, NSW, Australia</td>
			<td>08-559NP</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable stainless-steel scalpel blade with handle, size 15</td>
			<td>Livingstone International, Mascot, NSW,</td>
			<td>SCP15</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilles fine tooth forceps 12 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Hand-held high temperature fine tip cautery</td>
			<td>Bovie Medical Corporation, Melville, NY, USA</td>
			<td>AA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated mats to maintain body temperature during surgery and postoperative recovery</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina II Bioluminescent Imaging Device</td>
			<td>Caliper Life Sciences, Hopkinton, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jewellers forceps 11.5 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro needle holder (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro scissors (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Polyglycolic acid suture, size USP 5/0 on 13mm half-circle round-bodied needle</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>C1049407</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable weighing scale</td>
			<td>Precision balances, Bradford, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex wound clip applier and clip remover</td>
			<td>World Precision Instruments, Sarasota, FL, USA</td>
			<td>500345</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bodied vessel dilator 15 cm, 0.1 mm tip</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Titanium &#8220;Weck style&#8221; Ligaclip, small</td>
			<td>HZMIM, Hangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium Ligaclip applier for open surgery, small</td>
			<td>HZMIM, Hangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Volatile anaesthetic machine, including vapouriser and induction chamber</td>
			<td>Generic</td>
			<td></td>
			<td>Generic vapouriser and induction chamber</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drugs for Procedures</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% w/w ethanol solution</td>
			<td>Sigma-Aldrich Pty Ltd, Castle Hill, NSW, Australia</td>
			<td></td>
			<td>Applied topically as surgical skin preparation</td>
		</tr>
		<tr>
			<td>Buprenorphine 0.3 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 0.05 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>D-Luciferin (1 U/g)</td>
			<td>PerkinElmer, Inc., Waltham, MA, USA</td>
			<td>122799</td>
			<td>diluted in PBS to 15 mg/mL. Dose: 150 mg/kg i.p</td>
		</tr>
		<tr>
			<td>Enrofloxacin 50 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 5 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>Flunixin 50 mg/mL</td>
			<td>Norbrook Laboratories Australia, Tullamarine, VIC, Australia</td>
			<td></td>
			<td>Dose: 2.5 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis Australia Pty Ltd., Rhodes, NSW, Australia</td>
			<td></td>
			<td>Dose (vapourised with oxygen): 4% induction, 3% maintenance</td>
		</tr>
		<tr>
			<td>Ketamine 100 mg/mL</td>
			<td>Maylab, Slacks Creek, QLD, Australia</td>
			<td></td>
			<td>Dose: 80 mg/kg i.p.</td>
		</tr>
		<tr>
			<td>Povidone-Iodine 10% w/v solution</td>
			<td>Perrigo Australia, Balcatta, WA, Australia</td>
			<td>RIO00802F</td>
			<td>Applied topically to the anterior abdomen as surgical skin preparation</td>
		</tr>
		<tr>
			<td>Refresh eye ointment (liquid paraffin 42.5% w/w, soft white paraffin 57.3% w/w)</td>
			<td>Allergan Australia Pty Ltd, Gordon, NSW, Australia</td>
			<td></td>
			<td>Applied to both eyes</td>
		</tr>
		<tr>
			<td>Sodium chloride 0.9% w/v</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>9481P</td>
			<td>Dose: 900 &#956;L s.c.</td>
		</tr>
		<tr>
			<td>Water for injections BP</td>
			<td>Pfizer Australia, Sydney, NSW, Australia</td>
			<td></td>
			<td>For dilution of drugs</td>
		</tr>
		<tr>
			<td>Xylazine 20 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 10 mg/kg i.p.</td>
		</tr>
	</tbody>
</table>

an orthotopic resectional mouse model of pancreatic cancer

Mancano modelli animali soddisfacenti per studiare la terapia adiuvante e/o neoadiuvante nei pazienti presi in considerazione per la chirurgia del cancro al pancreas (PC). Per affrontare questa carenza, descriviamo un modello di topo che prevede l'impianto ortotopico del PC seguito da pancreatectomia distale e splenectomia. Il modello ha dimostrato di essere sicuro e adeguatamente flessibile per lo studio di vari approcci terapeutici in contesti adiuvanti e neoadiuvanti.
In questo modello, un tumore al pancreas viene generato per la prima volta impiantando una miscela di cellule tumorali pancreatiche umane (AsPC-1 taggate con luciferasi) e cellule stellate pancreatiche associate al cancro umano nel pancreas distale dei topi nudi atimici Balb / c. Dopo tre settimane, il cancro viene reinsediato da re-laparotomia, pancreatectomia distale e splenectomia. In questo modello, l'imaging a bioluminescenza può essere utilizzato per seguire i progressi dello sviluppo del cancro e gli effetti della resezione / trattamenti. Dopo la resezione, può essere somministrata una terapia adiuvante. In alternativa, il trattamento neoadiuvante può essere somministrato prima della resezione.
Vengono presentati i dati rappresentativi di 45 topi. Tutti i topi hanno subito una pancreatectomia/splenectomia distale di successo senza problemi di emostasi. Un margine pancreatico prossimale macroscopico superiore a 5 mm è stato raggiunto in 43 (96%) topi. Il tasso di successo tecnico della resezione pancreatica è stato del 100%, con una mortalità precoce e una morbilità dello 0%. Nessuno degli animali è morto durante la settimana successiva alla resezione.
In sintesi, descriviamo una tecnica robusta e riproducibile per un modello di resezione chirurgica del cancro al pancreas nei topi che imita lo scenario clinico. Il modello può essere utile per il test di trattamenti sia adiuvanti che neoadiuvanti.

Cinquantanove topi consecutivi sono stati sottoposti a un intervento chirurgico di impianto. Perdite lorde si sono verificate in otto (14%) topi. Il grado di perdita al momento dell'iniezione è stimato come descritto sopra nella sezione del protocollo. Dopo tre settimane per consentire la crescita di questi tumori impiantati, è stata eseguita l'imaging a bioluminescenza pre-resezione per escludere i topi con malattia metastatica grave prima della resezione. Quarantacinque (76%) topi sono stati sottoposti a resezione ch...

Watch this Scientific Journal Video about An Orthotopic Resectional Mouse Model of Pancreatic Cancer at JoVE.com

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

Nel contesto clinico, i pazienti con cancro al pancreas localizzato saranno sottoposti a pancreatectomia seguita da un trattamento adiuvante. Questo protocollo qui riportato mira a stabilire un metodo sicuro ed efficace per modellare questo scenario clinico nei topi nudi, attraverso l'impianto ortotopico del cancro al pancreas seguito da pancreatectomia distale e splenectomia.

Un modello ortotopico di topo resezionale del cancro al pancreas

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

an-orthotopic-resectional-mouse-model-of-pancreatic-cancer

Research

JoVE Journal

Cancer Research

10.0K Views.  University of New South Wales. Nel contesto clinico, i pazienti con cancro al pancreas localizzato saranno sottoposti a pancreatectomia seguita da un trattamento adiuvante. Questo protocollo qui riportato mira a stabilire un metodo sicuro ed efficace per modellare questo scenario clinico nei topi nudi, attraverso l'impianto ortotopico del cancro al pancreas seguito da pancreatectomia distale e splenectomia.

Video: An Orthotopic Resectional Mouse Model of Pancreatic Cancer

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Fluorescente ortotopico modello murino di cancro al pancreas

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Ricerca

Ricerca sul cancro

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Isolamento delle cellule tumorali circolanti in un modello di topo ortotopico del tumore del colon-retto

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

Un modello murino di cancro del pancreas Syngeneic per studiare gli effetti dell'elettroporazione irreversibile

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Immunophenotyping di Orthotopic omotrapianto (Syngeneic) dell'Adenocarcinoma Ductal pancreatico murino primario KPC tramite flusso Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.

This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

Un modello di cancro endometriale ortotopico con retroperitoneale Lmphadenopatia fatta da cellule VX2 in vivo propagate e coltivate

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

Un modello di topo ortotopico oncogenico indotto dall'epatocito del cancro epatocellulare in aumento nell'impostazione dell'infiammazione epatica e della fibrosi

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Studiare il cancro al seno triplo negativo utilizzando il modello di cancro al seno ortotopico

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

Impianto ortotopico a ultrasuoni dell'adenocarcinoma dugalese Murine

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

Generazione di tumori pancreatici ortotopici e caratterizzazione ex vivo della citotossicità delle cellule T infiltranti di tumore

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Generazione di uno xenotrapianto ortotopico di cellule tumorali pancreatiche mediante iniezione guidata da ultrasuoni

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...