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 Guidelines (protocol ID 121/2016-PR).
1. Isolation of pancreatic juice
NOTE: The withdrawal of pancreatic juice is executed in the context of an open procedure of pancreatic resection (e.g., pancreaticoduodenectomy, total pancreatectomy, distal pancreatectomy) by an equipe of expert pancreatic surgeons.
<ol>
	<li>Patient selection
	<ol>
		<li>Consider for the procedure any patient scheduled for an open pancreatic resection.</li>
		<li>Confirm inclusion if main pancreatic duct size is deemed sufficient to allow pancreatic juice retrieval. The minimum limit in diameter of the main pancreatic duct is considered 2 mm at contrast-enhanced CT imaging.</li>
	</ol>
	</li>
	<li>Pre-operative study of pancreatic duct at contrast-enhanced CT imaging to help the planning of pancreatic juice retrieval
	<ol>
		<li>Tridimensionally localize the main pancreatic duct within the gland at the level of the pancreatic neck: measure the distance of the main pancreatic duct from the anterior, superior and inferior pancreatic margin on cross-sectional slides, and coronal and sagittal renderings. Once in the operating theatre, use these measurements to approximate the correct place where to puncture the pancreas to cannulate the Wirsung duct and to retrieve pancreatic juice.</li>
	</ol>
	</li>
	<li>Preparation of material
	<ol>
		<li>Sterile material: Open the sterile envelope of one 25 G needle and one 3 mL syringe, and position them in the sterile field with the cooperation of the scrub nurse.</li>
		<li>Unsterile material: Keep a 3 mL K2EDTA vacuum test tube ready at hand in the operating room for the storage of the fluid.</li>
	</ol>
	</li>
	<li>Preparation of the patient
	<ol>
		<li>Position the patient on the operating room bed. Induce mixed general anesthesia, using Remifentanil, Sevorane and Rocuronium, then intubate and start ventilating the patient. Position the patient in supine decubitus with the right arm tucked to the body and the left arm abducted to 90&#176; degrees secured on an armboard.</li>
		<li>Disinfect the skin of the abdomen at the site of the incision. Create and maintain a sterile field on the abdomen draping the patient.</li>
	</ol>
	</li>
	<li>Surgical procedure
	<ol>
		<li>Perform a subcostal incision and gain access to the abdominal cavity. Position a Rochard abdominal retraction for organ&#160;exposure.</li>
		<li>Expose and mobilize the pancreas through Kocher maneuver, opening of the gastrocolic ligament, incision of the retroperitoneal tissue along superior and inferior border of the pancreas creating a dissection plane between pancreatic neck and superior mesenteric vein located posteriorly.</li>
		<li>Once the pancreas is mobilized and exposed, proceed to pancreatic juice withdrawal before sectioning of pancreatic neck.</li>
	</ol>
	</li>
	<li>Identification and localization of the pancreatic duct
	<ol>
		<li>Estimate the location of the pancreatic duct using the measurement taken at imaging and then palpate the anterior surface of the pancreas to identify its precise location.</li>
	</ol>
	</li>
	<li>Collection of pancreatic juice
	<ol>
		<li>Hold the pancreatic head and the duodenum from underneath and elevate it with the left hand, marking the location of the pancreatic duct with the first digit.</li>
		<li>Take hold with the right hand of the 3 mL syringe with the 25 G needle mounted on.</li>
		<li>Use the right hand to insert the needle in 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.</li>
		<li>Withdraw the juice with the syringe. If it is not possible to retrieve the juice, relocate the needle in the four directions trying to cannulate the pancreatic duct.</li>
		<li>Once the pancreatic juice is retrieved, move it outside the sterile field and transfer it to the 3 mL K2EDTA vacuum test tube. Keep at 4 &#176;C until the sample is transferred to the lab and proceed to further processing as early as possible. 
		NOTE: The volume of pancreatic juice that can be recovered with this procedure varies greatly, ranging approximately from 0.2 mL to 3 mL in our experience. The amount of juice retrieved is highly dependent on the patient: the dimension of the Wirsung duct and the functional status of the pancreas (functioning versus atrophic gland). In our experience there is no expedient that can be used to increase the amount of pancreatic juice retrieved.</li>
	</ol>
	</li>
</ol>
2. Processing of pancreatic juice
<ol>
	<li>Centrifuge pancreatic juice at 400 x g for 10 min at 4 &#176;C to remove any cells or debris. 
	NOTE: Pancreatic juice should be clear and transparent in color before centrifugation. Blood contamination during surgery can sometimes occur, making the sample appear murkier and redder in color. Consider excluding such samples from further analyses.</li>
	<li>Recover the supernatant, aliquot and store at -80 &#176;C until further analyses.</li>
</ol>
3. Induction of subcutaneous tumors
NOTE: The murine Panc02 and DT6606 cell lines were obtained from Prof. Lorenzo Piemonti (San Raffaele Diabetes Institute, Milan, Italy) and Prof. Francesco Novelli (Center for Experimental Research and Medical Studies, Torino, Italy) respectively, as previously described12.
<ol>
	<li>Growth of tumor cells
	<ol>
		<li>Culture Panc02 and DT6606 cells in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (FBS), 2mM L-Glutamine and 1% penicillin-streptomycin antibiotic.</li>
		<li>Thaw frozen cells 1-2 weeks before tumor injection, according to the growth rate of the cell line.</li>
		<li>Grow cells at 37 &#176;C with 5% CO2 and 95% humidity in sterile conditions.</li>
		<li>Detach cells with 0.025% Trypsin/EDTA solution for 5 min at 37 &#176;C when they reach 80% confluency and eliminate trypsin by centrifugation. 
		NOTE: DT6606 are primary, not immortalized, cells, derived from the LSL-KrasG12D-Pdx1-Cre mouse, and should not be passaged more than 3 times before injection in vivo in order to maintain their original characteristics. It is recommended to thaw DT6606 cells 7-10 days prior to injection.</li>
	</ol>
	</li>
	<li>Injection of tumor cells in vivo
	<ol>
		<li>Trypsinize cells (see step 3.1.4) and wash them once with Phosphate-Buffered Saline (PBS). Eliminate PBS by centrifugation and resuspend cells in fresh PBS before counting.</li>
		<li>Count the cells and resuspend them in PBS at a concentration of 0.5-1 x 107 cells/mL in order to have a final concentration of 0.5-1 x 106 cells/100 &#956;L to inject in each mouse. Prepare the cells in excess. Keep the cells at 4 &#176;C or on ice until the end of the procedure.</li>
		<li>Group the animals (8-week old female C57BL/6J mice) in different cages according to different cell lines or treatments.</li>
		<li>Restrain the animals manually and anaesthetize them using a mixture of Ketamine (80 mg/kg) and Xylazine (10 mg/kg) or according to locally-approved procedures.</li>
		<li>Shave the site of injection, usually a flank above a leg, with an electric shaver and carefully clean the site of injection with alcohol.</li>
		<li>Pipette up and down the cell suspension with a 1 mL syringe, removing any air bubbles by moving the piston up and down. Attach a 25 G needle to the syringe and push the piston upwards until the cell suspension reaches the needle opening.</li>
		<li>Pinch the skin of the flank with flat-tipped forceps and carefully insert the needle at the base of skin fold between the forceps without puncturing the peritoneal cavity or the musculature. To verify the correct position of the needle, gently try to move the tip of the needle sideways under the skin. The needle should move freely.</li>
		<li>Slowly inject 100 &#956;L of cell suspension (containing 0.5-1 x 106 cells), clamp gently the site of injection for a few seconds and slowly withdraw the needle without any sideways movement.</li>
		<li>Return the mouse back to its cage and monitor the recovery from anesthesia.</li>
		<li>Check tumor growth using a caliper for 3-4 weeks. Euthanize the animals when tumors reach approximately 0.5-1 cm3 using CO2 or according to locally-approved procedures.</li>
	</ol>
	</li>
</ol>
4. Isolation of tumor interstitial fluid (TIF)
<ol>
	<li>Excision of subcutaneous tumors
	<ol>
		<li>Block the limbs of the animals with paper tape and clean the skin with alcohol. Cut the skin open on the abdomen in order to separate it from the peritoneum and go on up to the limbs. Excise the tumor grown under the skin of the flank with the help of&#160;scissors, clamps and eventually a scalpel.</li>
		<li>Weigh the tumor and keep it in a clean tube on ice until following isolation of TIF.</li>
	</ol>
	</li>
	<li>Isolation of TIF by centrifugation
	<ol>
		<li>Cut the tumor in half, rinse the two parts quickly in PBS and blot them gently on filter paper to remove excess of PBS. Carry out these steps as fast as possible to avoid evaporation from the tumor.</li>
		<li>Immediately transfer the tumor into a 20 &#956;m nylon cell strainer affixed atop a 50 mL conical tube.</li>
		<li>Centrifuge the tube at 400 x g for 10 min at 4 &#176;C. 
		NOTE: This low speed centrifugation preserves cell integrity avoiding contamination of TIF by intracellular compartment. Intracellular content leakage can be tested in downstream applications for example by assessing the presence of intracellular housekeeping proteins, such as ribosomal proteins25.</li>
		<li>Recover the TIF from the bottom of the tube, eventually aliquot it and immediately freeze it on dry ice and store at -80 &#176;C until further analysis. 
		Optional: Based on the downstream proteomic analysis to be performed, dilute the sample in PBS with a protease inhibitor cocktail to avoid degradation of specific molecules. 
		NOTE: Depending on the composition of the tumor, in some cases very small tumors do not yield any fluid.</li>
	</ol>
	</li>
	<li>Isolation of TIF by elution
	<ol>
		<li>Cut the tumor into small pieces (&#8776;1-3 mm3) with scissors or a scalpel and rinse carefully with cold PBS. 
		NOTE: In this step it is very important to work fast and perform minimum manipulation to avoid cell damage.</li>
		<li>Transfer the tumor pieces into a 1.5 mL tube and add 500 &#956;L of PBS with a protease inhibitor cocktail to avoid degradation of analytes. Incubate for 1 hour at 37 &#176;C and 5% CO2.</li>
		<li>Recover the supernatant and transfer it to a new 1.5 mL tube. Centrifuge at 1,000 x g for 5 min at 4 &#176;C to remove any cells from the sample.</li>
		<li>Transfer the supernatant to a new tube and centrifuge again at 2,000 x g for 8 min at 4 &#176;C.</li>
		<li>Transfer the supernatant to a new tube and centrifuge again at 20,000 x g for 30 min at 4 &#176;C to remove any debris. Recover the supernatant. Immediately aliquot and freeze TIF sample on dry ice and store at -80 &#176;C until further analysis.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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 &amp;micro;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 ...

isolation-proximal-fluids-to-investigate-tumor-microenvironment

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Cancer Research

4.2K Views.  Humanitas Clinical and Research Center-IRCCS. Pancreassap is een kostbare bron van biomarkers voor menselijke alvleesklierkanker. We beschrijven hier een methode voor intraoperatieve incassoprocedure. Om de uitdaging van het aannemen van deze procedure in muizenmodellen te overwinnen, stellen we een alternatief monster voor, tumor interstitiële vloeistof, en beschrijven we hier twee protocollen voor de isolatie ervan.

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

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.

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

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

Developmental Biology

Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the 3-dimensional (3D) modeling of this disease have been described. Patient-derived organoid (PDO) models can be isolated from both surgical specimens as well as small biopsies and form rapidly in culture. Importantly, organoid models preserve the pathogenic genetic alterations detected in the patient&#39;s tumor and are predictive of the patient&#39;s treatment response, thus enabling translational studies. Here, we provide comprehensive protocols for adapting tissue culture workflow to study 3D, matrix embedded, organoid models. We detail methods and considerations for isolating and propagating primary PDAC organoids. Furthermore, we describe how bespoke organoid media is prepared and quality controlled in the laboratory. Finally, we describe assays for downstream characterization of the organoid models such as isolation of nucleic acids (DNA and RNA), and drug testing. Importantly we provide critical considerations for implementing organoid methodology in a research laboratory.

Patient-derived organoid cultures of pancreatic ductal adenocarcinoma are a rapidly established 3-dimensional model that represent epithelial tumor cell compartments with high fidelity, enabling translational research into this lethal malignancy. Here, we provide detailed methods to establish and propagate organoids as well as to perform relevant biological assays using these models.

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

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.

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.

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

Pancreatic Tissue Dissection to Isolate Viable Single Cells

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for approximately 90% of the pancreas and includes cells that produce and secrete digestive enzymes. The digestive enzymes are produced in the pancreatic acinar cells, stored in vesicles called zymogens, and are then released into the duodenum via the pancreatic duct to initiate metabolic processes. The enzymes produced by the acinar cells can kill cells or degrade cell-free RNA. In addition, acinar cells are fragile, and common dissociation protocols result in a large number of dead cells and cell-free proteases and RNases. Therefore, one of the biggest challenges in pancreatic tissue digestion is recovering intact and viable cells, especially acinar cells. The protocol presented in this article shows a two-step method that we developed to meet this need. The protocol can be used to digest normal pancreata, pancreata that include pre-malignant lesions, or pancreatic tumors that include a large number of stromal and immune cells.

Pancreatic metaplastic cells are precursors of malignant cells that give rise to pancreatic tumors. However, isolating intact viable pancreatic cells is challenging. Here, we present an efficient method for pancreatic tissue dissociation. The cells can then be used for single-cell RNA sequencing (scRNA-seq) or for two- or three-dimensional co-culturing.

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Biology

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing.
This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 &#181;m thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling.
OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

This protocol describes the preparation of organotypic slice cultures (OTSCs). This technique facilitates the ex vivo cultivation of intact multicellular tissue. OTSCs can be used immediately to test for their respective response to drugs in a multicellular environment.

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

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