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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors, and soon to become the second leading cause of death^1,2,3. 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 blood^5,6,7, urine⁸, saliva⁹ and pancreatic juice^10,11,12. Many studies have exploited comprehensive “omics” 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-spectrometry^14,15, assessment of genetic and epigenetic markers such as K-ras and p53 mutations^16,17, 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.

Figure 1: Schematic representation of pancreatic juice collection. (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. Please click here to view a larger version of this figure.

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 plasma^22,23,24. Several studies have proven TIF to be a suitable sample for high-throughput proteomic approaches, such as mass spectrometry techniques^23,24,25, 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 ultrafiltration^28,29,30,31 and microdialysis^32,33,34,35) and ex vivo methods (tissue centrifugation^22,36,37,38 and tissue elution^39,40,41,42). These techniques have been reviewed in extensive detail^43,44. 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 literature^24,38, 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).

Figure 2: Schematic representation of tumor interstitial fluid isolation methods. Schematic illustration of the techniques described in detail in the protocol, namely tissue centrifugation (A) and tissue elution (B). Please click here to view a larger version of this figure.

Protokół

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.

1. Patient selection
   1. Consider for the procedure any patient scheduled for an open pancreatic resection.
   2. 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.
2. Pre-operative study of pancreatic duct at contrast-enhanced CT imaging to help the planning of pancreatic juice retrieval
   1. 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.
3. Preparation of material
   1. 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.
   2. Unsterile material: Keep a 3 mL K2EDTA vacuum test tube ready at hand in the operating room for the storage of the fluid.
4. Preparation of the patient
   1. 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° degrees secured on an armboard.
   2. Disinfect the skin of the abdomen at the site of the incision. Create and maintain a sterile field on the abdomen draping the patient.
5. Surgical procedure
   1. Perform a subcostal incision and gain access to the abdominal cavity. Position a Rochard abdominal retraction for organ exposure.
   2. 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.
   3. Once the pancreas is mobilized and exposed, proceed to pancreatic juice withdrawal before sectioning of pancreatic neck.
6. Identification and localization of the pancreatic duct
   1. 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.
7. Collection of pancreatic juice
   1. 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.
   2. Take hold with the right hand of the 3 mL syringe with the 25 G needle mounted on.
   3. 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.
   4. 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.
   5. 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 °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.

2. Processing of pancreatic juice

1. Centrifuge pancreatic juice at 400 x g for 10 min at 4 °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.
2. Recover the supernatant, aliquot and store at -80 °C until further analyses.

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.

1. Growth of tumor cells
   1. 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.
   2. Thaw frozen cells 1-2 weeks before tumor injection, according to the growth rate of the cell line.
   3. Grow cells at 37 °C with 5% CO₂ and 95% humidity in sterile conditions.
   4. Detach cells with 0.025% Trypsin/EDTA solution for 5 min at 37 °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.
2. Injection of tumor cells in vivo
   1. 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.
   2. 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 μL to inject in each mouse. Prepare the cells in excess. Keep the cells at 4 °C or on ice until the end of the procedure.
   3. Group the animals (8-week old female C57BL/6J mice) in different cages according to different cell lines or treatments.
   4. 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.
   5. Shave the site of injection, usually a flank above a leg, with an electric shaver and carefully clean the site of injection with alcohol.
   6. 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.
   7. 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.
   8. Slowly inject 100 μL 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.
   9. Return the mouse back to its cage and monitor the recovery from anesthesia.
   10. 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.

4. Isolation of tumor interstitial fluid (TIF)

1. Excision of subcutaneous tumors
   1. 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 scissors, clamps and eventually a scalpel.
   2. Weigh the tumor and keep it in a clean tube on ice until following isolation of TIF.
2. Isolation of TIF by centrifugation
   1. 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.
   2. Immediately transfer the tumor into a 20 μm nylon cell strainer affixed atop a 50 mL conical tube.
   3. Centrifuge the tube at 400 x g for 10 min at 4 °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.
   4. Recover the TIF from the bottom of the tube, eventually aliquot it and immediately freeze it on dry ice and store at -80 °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.
3. Isolation of TIF by elution
   1. Cut the tumor into small pieces (≈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.
   2. Transfer the tumor pieces into a 1.5 mL tube and add 500 μL of PBS with a protease inhibitor cocktail to avoid degradation of analytes. Incubate for 1 hour at 37 °C and 5% CO₂.
   3. Recover the supernatant and transfer it to a new 1.5 mL tube. Centrifuge at 1,000 x g for 5 min at 4 °C to remove any cells from the sample.
   4. Transfer the supernatant to a new tube and centrifuge again at 2,000 x g for 8 min at 4 °C.
   5. Transfer the supernatant to a new tube and centrifuge again at 20,000 x g for 30 min at 4 °C to remove any debris. Recover the supernatant. Immediately aliquot and freeze TIF sample on dry ice and store at -80 °C until further analysis.

Wyniki

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...

Dyskusje

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 blood^5,6,7, urine⁸, and saliva⁹, have shown promising results in disc...

Materiały

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