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

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.