Name: isolation of proximal fluids to investigate the tumor microenvironment of pancreatic adenocarcinoma
Uploaded: 2020-11-05T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma at JoVE.com

We thank Roberta Migliore for technical assistance. The research leading to these results has received funding from Associazione Italiana per la ricerca sul cancro (AIRC) under IG2016-ID.18443 project &#8211; P.I. Marchesi Federica. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

For all patients enrolled, peripheral blood and pancreatic juice were collected at the time of surgery according to protocols approved by the Ethical Committee of the Institution. All the patients were enrolled in the study after signed informed consent including collection of biological specimens and clinical data. The study was approved by the Ethical Committee of the Institution (protocol number ICH-595, approval issued on May 2009). Procedures involving mice and their care were conformed to EU and Institutional Guide...

In this study we have described the technique to intraoperatively collect pancreatic juice, a largely unexplored fluid biopsy. We have recently shown that pancreatic juice can be exploited as a source of metabolic markers of disease12. Metabolomic analysis on other liquid biopsies, such as blood5,6,7, urine8, and saliva9, have shown promising results in disc...

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors, and soon to become the second leading cause of death1,2,3. It is well-known for its immunosuppressive microenvironment and for its unresponsiveness to immunotherapy protocols4. Currently, surgical resection is still the only curative option for PDAC, yet there is a high frequency of early relapses and postsurgical complications. The lack of specific symptoms until an advanced stage does not allow for an early diagnosis, contributing to the deadliness of the disease. Furthermore, the overlap of symptoms between PDAC and other benign pancreatic pathologies can hamper the achievement of a prompt and reliable diagnosis with the current diagnostic strategies. The identification of variables associated to specific pancreatic pathologies could facilitate the surgical decision-making process and improve patient profiling.
Promising results in biomarker discovery have been achieved using easily accessible body fluids, such as blood5,6,7, urine8, saliva9 and pancreatic juice10,11,12. Many studies have exploited comprehensive &#8220;omics&#8221; approaches, such as genomic, proteomic and metabolomic techniques, to identify candidate molecules or signatures that could discriminate between PDAC and other benign pancreatic afflictions. We recently demonstrated that pancreatic juice, a relatively unexplored body fluid, can be used to identify metabolic signatures of patients with distinct clinical profiles12. Pancreatic juice is a protein-rich fluid, which accumulates the secretome of pancreatic ductal cells and flows to the main pancreatic duct, and then to the main common bile duct. Due to its proximity to the pancreas, it could be strongly affected by microenvironmental perturbations induced by the tumor mass (Figure 1), and therefore more informative than blood or urine, or tissue-based profiling. Several studies have explored the potential of pancreatic juice to identify novel biomarkers of disease using various approaches, including cytologic analysis13, proteomic analysis performed by mass-spectrometry14,15, assessment of genetic and epigenetic markers such as K-ras and p53 mutations16,17, alterations in DNA methylation18, and miRNAs19. Technically, pancreatic juice can be collected intraoperatively or with minimally invasive procedures, such as endoscopic ultrasound, retrograde cholangio-pancreatography, or by endoscopic collection of duodenal juice secretion20. It is not yet clear to what extent pancreatic juice composition is affected by the collection technique used. We describe here the intraoperative collection procedure and show that pancreatic juice can represent a precious source for PDAC biomarkers.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61687/61687fig01.jpg" /> 
Figure 1: Schematic representation of pancreatic juice collection.&#160;(A) Schematic representation depicting the secretion of pancreatic juice into the pancreatic duct and its collection during surgery. The inset shows a close-up of the tumor microenvironment: pancreatic juice collects molecules released by tumor and stromal cells in the pancreatic ducts. <a href="https://www.jove.com/files/ftp_upload/61687/61687fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The collection of pancreatic juice in genetic and orthotopic mouse models of PDAC would be appreciated in the perspective to exploit this biofluid in preclinical mechanistic studies; however, this procedure can be technically very challenging and is not feasible for simpler models such as subcutaneous tumors. For this reason, we identified tumor interstitial fluid (TIF) as an alternative source to pancreatic juice, for its similar characteristic of acting as an indicator of surrounding perturbations. Interstitial fluid (IF) is the extracellular liquid, found outside blood and lymphatic vessels, which bathes tissue cells21. IF composition is affected by both blood circulation to the organ and local secretion; in fact, surrounding cells actively produce and secrete proteins in the IF21. The interstitium reflects microenvironmental changes of surrounding tissues and could therefore represent a valuable source for biomarker discovery in several pathological contexts, such as tumors. The high concentration of locally secreted proteins in TIF can be used to identify candidate molecules to be tested as prognostic or diagnostic biomarkers in plasma22,23,24. Several studies have proven TIF to be a suitable sample for high-throughput proteomic approaches, such as mass spectrometry techniques23,24,25, as well as multiplex ELISA approaches26, and microRNA profiling27.
Several approaches have been proposed for the isolation of IF in tumors, which can be broadly categorized as in vivo (capillary ultrafiltration28,29,30,31 and microdialysis32,33,34,35) and ex vivo methods (tissue centrifugation22,36,37,38 and tissue elution39,40,41,42). These techniques have been reviewed in extensive detail43,44. The choice of the appropriate method should take into account issues such as the downstream analyses and applications and the volume recovered. We recently used this approach as a proof of principle to demonstrate the different metabolic activity of tumors from two murine pancreatic adenocarcinoma cell lines12. Based on literature24,38, we chose to use the low speed centrifugation method to avoid cell breakage and dilution from intracellular content. Both the amount of glucose and lactate in TIF reflected the different glycolytic characteristics of the two different cell lines. Here we describe in detail the protocol for the two most commonly used methods for the isolation of TIF: tissue centrifugation and tissue elution (Figure 2).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61687/61687fig02.jpg" /> 
Figure 2: Schematic representation of tumor interstitial fluid isolation methods.&#160;Schematic illustration of the techniques described in detail in the protocol, namely tissue centrifugation (A) and tissue elution (B). <a href="https://www.jove.com/files/ftp_upload/61687/61687fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>1 mL syringe</td>
			<td>BD Biosciences</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf tube</td>
			<td>Greiner BioOne</td>
			<td>GR616201</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;m nylon cell strainer</td>
			<td>pluriSelect</td>
			<td>43-50020-03</td>
			<td></td>
		</tr>
		<tr>
			<td>25G needle</td>
			<td>BD Biosciences</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL K2EDTA vacutainer</td>
			<td>BD Biosciences</td>
			<td>366473</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>BD Biosciences</td>
			<td>309656</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>Corning</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamps</td>
			<td>Medicon</td>
			<td>06.20.12</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable scalpel</td>
			<td>Medicom</td>
			<td>9000-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Microtech</td>
			<td>MG10432</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat-tipped forceps</td>
			<td>Medicon</td>
			<td>06.00.10</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Lonza</td>
			<td>ECB3001D</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>34044100</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium</td>
			<td>Euroclone</td>
			<td>ECB9006L</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Medicon</td>
			<td>02.04.09</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA 1x</td>
			<td>Lonza</td>
			<td>BE17-161F</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultraglutamine 100x</td>
			<td>Lonza</td>
			<td>BE17-605E/U1</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of proximal fluids to investigate the tumor microenvironment of pancreatic adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

We followed the procedure described above to obtain pancreatic juice from patients with PDAC (n=31) and other benign pancreatic afflictions (non-PDAC, n=9), including pancreatitis (n=2), papillary-ampulla tumors (n=4), neuroendocrine tumors (n=2), intraductal papillary mucinous neoplasia (IPMN; n=1)12. The pancreatic juice samples were then subjected to metabolomic analysis using nuclear magnetic resonance (1H-NMR)12. By filtering the broad NMR signa...

Watch this Scientific Journal Video about Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma at JoVE.com

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

The method described here to isolate proximal fluid both in human and in mice, can be applied to study the microenvironment of pancreatic cancer to identify new biomarkers of disease. Using this straightforward protocol, it is possible to translate clinical findings from pancreatic juice to murine models of PDAC to perform mechanistic studies. Pancreatic juice is a largely unexplored body fluid which has shown promise as a source of precious biomarkers to help pre-operative diagnosis and patient profiling.I will demonstrate the first part of the protocol, and Marialuisa Barbagallo, a postdoc from our laboratory, will demonstrate the second part of the protocol. Estimate the location of the pancreatic duct using measurements acquired during imaging. Then palpate the anterior surface of the pancreas to identify its precise location.Hold the pancreatic head in the duodenum from underneath, and elevate it with the left hand, marking the location of the pancreatic duct with the first digit. Use the right hand to insert a three milliliter syringe with a 25-gauge needle into the pancreas, just distal to the left thumb. Decide the depth of penetration and the degree of inclination of the needle based on preoperative measurements, and on the perception of having penetrated the duct wall.Withdraw the juice with the syringe. If it is not possible to retrieve the juice, relocate the needle in an attempt to cannulate the pancreatic duct. Once the pancreatic juice is retrieved, move it outside the sterile field, and transfer it to a three milliliter EDTA vacuum test tube.Keep the tube at four degree Celsius until the sample is transferred to the lab. To process the pancreatic juice, centrifuge it at 400 G and four degree Celsius for 10 minutes to remove any cells or debris. Recover the supernatant, aliquot it, and store it at 80 degree Celsius until further analyses.To isolate tumor interstitial fluid or TIF, by centrifugation, cut the tumor in half, rinse the two parts in PBS, and blot them gently on filter paper, moving as fast as possible to avoid evaporation from the tumor. Immediately transfer the tumor into a 20 micrometer nylon cell strainer, affixed to a 50 milliliter conical tube. Centrifuge the tube at 400 G for 10 minutes at four degrees Celsius.Recover the TIF from the bottom of the tube, then aliquot it and immediately freeze it on dry ice. Store at 80 degrees Celsius until further analysis. To isolate the TIF using elution, cut the tumor into small pieces with scissors or a scalpel, and rinse them carefully with cold PBS.Transfer the tumor pieces into a 1.5 milliliter tube, and add 500 microliters of PBS with a Protease Inhibitor Cocktail to avoid degradation of analytes. Incubate the tissue for one hour at 37 degree Celsius and 5%carbon dioxide. Transfer the supernatant to a new 1.5 milliliter tube, and perform a series of centrifugation steps at four degree Celsius to remove any cells from the sample.Recovering the supernatant after each centrifugation and transferring it to a new tube. After the last centrifugation, immediately aliquot and freeze the TIF sample on dry ice. Store it at 80 degrees Celsius until further analysis.This protocol was used to obtain the pancreatic juice from patients with PDAC and other benign pancreatic afflictions. After filtering the broad NMR signals of macromolecules, the signals from small molecular weight metabolites were apparent. Supervised OPLS-DA analysis showed that the metabolic profile in pancreatic juice was able to discriminate between PDAC and non-PDAC patients with an accuracy of 82.4%Interestingly, metabolomic analysis performed on plasma samples from the same patients, did not yield the same discriminative power.To demonstrate that tumor interstitial fluid content reliably reflects changes in the tumor microenvironment, two pancreatic cancer cell lines with opposite glycolytic rates were subcutaneously injected into mice. After excising the tumors, the concentrations of glucose and lactate were quantified. Tumor interstitial fluid from highly glycolytic tumors contained less glucose and more lactate compared to low glycolytic tumors.In this study, we performed metabolomics analysis. However, this proximal fluids are suitable for many other downstream application, such as mass spectrometry and microRNA profiling.

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